CN114686429B - Tumor immunotherapy method for activating NK cells - Google Patents

Abstract

The invention provides a tumor immunotherapy method for activating NK cells. The invention provides a use of a Nucleobase (NUCB) or an enhancer thereof for the preparation of a medicament or formulation for (a) promoting expansion of NK cells, (b) treating a tumor; and/or (c) killing or inhibiting tumor cells. The NK cells are activated by the method provided by the invention, and the obtained NK cells have broad-spectrum anti-tumor effect, do not need to identify tumor specific antigens, and have a quick immune system 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 development of society and technology, the advent of drugs such as antibiotics, which have serious threat to human diseases such as infectious diseases in the last century and before, has significantly reduced the harm. However, chronic diseases such as cancer, cardiovascular diseases, neurodegenerative diseases and metabolic diseases become the main health threat facing the human society at present. In 2018, 1810 ten thousand cancer patients are newly increased worldwide, and 960 ten thousand patients die from cancer. The cancer seriously affects the health and life quality of human body, and brings great economic pressure to society.

Tumor immunotherapy is undoubtedly the most important step in tumor treatment in the last decades, and the physiological and medical prize of nobel in 2018 is obtained, which is honored as the third anticancer drug revolution after chemotherapy and targeted therapy. Most tumor immunotherapeutic approaches, whether by immunocheckpoint inhibition, chimeric antigen T cells or bispecific antibodies, are focused on enhancing the activity of T cells. While these approaches have met with great success, only a small percentage of cancer patients benefit from them.

Natural killer cells (NK cells), which are one of the main members of the natural immune system, play a key role in the immune surveillance of the body and early anti-infection and tumor processes. NK cells directly kill tumor cells by releasing lytic particles comprising perforin and granzyme, and can also indirectly play a role by recruiting and regulating the natural immune and acquired immune systems by secreting cytokines and chemokines such as IFN-gamma, TNF-alpha, GM-CSF, etc. These functions are achieved by the activation or inhibition of receptors on the surface of NK cells for various germline-encoded (germline-encoded). However, to date, many of these receptors have not found corresponding ligands, which is detrimental to the development of immunotherapeutic approaches associated with activating NK cells.

In view of the foregoing, there is a great need in the art to develop a new method for activating NK cells efficiently and rapidly.

Disclosure of Invention

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

In a first aspect of the present invention there is provided the use of a Nucleobase (NUCB) or an enhancer thereof for the preparation of a medicament or formulation (a) for the promotion of NK cell expansion, (b) for the treatment of a tumour; and/or (c) killing or inhibiting tumor cells.

In another preferred embodiment, the medicament or formulation is comprised as a tumoricidal agent.

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

In another preferred embodiment, the NUCB protein is of mammalian origin, preferably from rodents (rats, mice) and primates (e.g. humans), more preferably from humans.

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

In another preferred embodiment, the NUCB1 protein has an amino acid sequence as shown in SEQ ID NO. 3 or has an identity of greater than or equal to 85% (preferably greater than or equal to 90%, more preferably greater than or equal to 95% or greater than or equal to 98%) with SEQ ID NO. 3.

In another preferred embodiment, the NUCB2 protein has an amino acid sequence as shown in SEQ ID No. 4 or has an identity of greater than or equal to 85% (preferably greater than or equal to 90%, more preferably greater than or equal to 95% or greater than or equal to 98%) with SEQ ID No. 4.

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

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

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

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 a NUCB active fragment comprising said critical segment.

In another preferred embodiment, the critical segment of the NUCB protein is the amino acid sequence at positions 40-85 of NUCB1 or NUCB 2.

In another preferred embodiment, the sequence of the critical segment of NUCB1 is shown as SEQ ID NO. 1.

In another preferred embodiment, the sequence of the critical segment of NUCB2 is shown as SEQ ID NO. 2.

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

In another preferred embodiment, the promoter comprises a promoter miRNA, a promoter transcriptional regulator, or a promoter targeting small molecule compound.

In another preferred embodiment, 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.

In another preferred embodiment, the medicament or formulation is administered by a mode of administration selected from the group consisting of: intravenous, intratumoral, intracavity, subcutaneous, or hepatic arterial administration (e.g., injection, instillation, etc.).

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

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 a promoter thereof; and

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

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

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

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

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

In another preferred embodiment, 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) A polypeptide derived from (a) having said NUCB activity, which is formed by substitution, deletion or addition of one or more (e.g., 1 to 10) amino acid residues to an amino acid sequence as set forth in SEQ ID No. 4 or 5; or (b)

(c) A polypeptide having said NUCB activity having an amino acid sequence which 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%) homologous to the amino acid sequence shown in SEQ ID No. 4 or 5.

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

(c) A polypeptide having the LY49H activity, wherein the amino acid sequence has a homology of 80% or more (preferably 90% or more, more preferably 95% or more, or 98% or more) with the amino acid sequence shown in SEQ ID No. 8.

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

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

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

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

(b) The complex content H1 in the test group is detected and compared to the complex content H0 in the control group, wherein when H1 is significantly higher than H0, it is indicative that the test substance is a drug or compound that promotes NK cell expansion.

In another preferred embodiment, said significantly higher than H1/H0 is ≡2, preferably ≡3, more preferably ≡4.

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

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

In another preferred embodiment, the non-human mammal comprises 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 cells are surface receptor LY49H positive NK cells.

In another preferred embodiment, the NK cells naturally contain receptors for NUCB or are engineered to obtain surface receptors for NUCB.

In a seventh aspect of the invention there is provided the use of a LY49H protein or an active fragment thereof for the preparation of a pharmaceutical composition for the treatment of a disorder associated with NUCB protein excess.

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

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

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

(c) A polypeptide having the LY49H activity, wherein the amino acid sequence has a homology of 80% or more (preferably 90% or more, more preferably 95% or more, or 98% or more) with the amino acid sequence shown in SEQ ID No. 9.

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

In another preferred embodiment, the disorder associated with elevated NUCB protein is selected from the group consisting of: polycystic ovary syndrome (Polycystic Ovary Syndrome), hypertension (hypertension), anxiety (Anxiety), epilepsy (epiepsy), or combinations thereof.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows that NUCBs specifically bind LY49H. LY49H molecule and NUCBs molecular structure pattern (wherein SS is signal sequence) and EF is EF-hand domain); B. interaction method pattern diagram (LY 49 family plasmid and NUCBs expression plasmid co-transfected into 293T cells, secreted into cell culture medium); results of co-immunoprecipitation experiments with LY49H molecule and NUCB1 protein, wherein NUCB1 protein specifically binds LY49H in the LY49 family; results of co-immunoprecipitation experiments with LY49H molecule and NUCB2 protein, NUCB2 protein specifically binds LY49H in LY49 family; E. LY49H binds eukaryotic 293T cells expressing purified NUCBs proteins; LY49H binds to E.coli to express purified NUCBs protein (where lp: fc is the immunoprecipitation Fc tag, input is the Input of both proteins in the immunoprecipitation experiment, HA is the HA tag, FC is the FC tag, flag is the Flag tag).

FIG. 2 shows that NUCBs specifically bind to the C-type lectin-like domain of LY49H. A cut-off pattern of the extracellular portion of LY49H; the C-type lectin-like domain of LY49H is essential for binding to NUCB 1; the C-type lectin-like domain of LY49H is essential for binding to NUCB 2; alignment of LY49 family C-type lectin-like domain sequences; a LY49 family C-type lectin-like domain sequence relatedness guide tree; leucine residue 196 and histidine residue 216 of the C-type lectin-like domain of F, G.LY49H are the critical amino acids determining its binding specificity with NUCB1/NUCB 2; pattern of binding sites of nucbs on LY49H, wherein CTLD is a C-lectin-like domain.

FIG. 3 shows the binding of amino acids 40-80 of NUCBs to LY49H. C-terminal truncation pattern diagram of NUCBs protein; the 3C-terminal truncations of nucbs did not affect binding to LY 49H; n-terminal truncation pattern diagram of nucbs protein; NUCB1 (lack 44-80) and NUCB2 (lack 44-80) significantly affect binding to LY49H.

Figure 4 shows that overexpression of NUCBs inhibited tumor cell growth in mice. A. Constructing a NUCBs protein over-expression MC38 cell pattern diagram; B. spreading the same cell number, collecting a culture medium after 24 hours, and detecting the overexpression of NUCBs by using Western Blot; overexpression of nucbs protein did not affect proliferation of MC38 cells themselves; d.wild type cells of mc38 and NUCB1 or NUCB2 overexpressing MC38 cells were inoculated subcutaneously with C57 mouse pattern; nucb1 and NUCB2 over-expressing MC38 cells were significantly reduced in mouse subcutaneously grown tumors; mice transplanted with nucb1 and NUCB2 overexpressing MC38 cells had significantly prolonged survival.

Figure 5 shows that injection of NUCBs peptide fragments inhibited tumor cell growth. Protein sequence information of A-C.NUCB1 and NUCB2, sequence information of synthesized NUCBs peptide fragment and sequence information of reference peptide fragment; D. injecting NUCBs peptide fragment experimental model pictures; E. the injection of the NUCB1 peptide fragment or the NUCB2 peptide fragment obviously reduces the size of the tumor; F. NUCB peptide human-mouse sequence comparison information.

FIG. 6 shows the identification of nuCBs knockout mice. Nucb1 gene knockout strategy and sequencing identification; nucb1 knockout mouse genome PCR and identification of mRNA expression (lung tissue); e, F, NUCB2 gene knockout strategy and sequencing identification; genomic PCR identification of G, h.nucb2 gene knockdown, WB identification. Wherein marker is control, and actin is actin.

FIG. 7 shows that NUCBs knockout mice promote tumor growth. Nucb1&2 knockout male mice promote tumor growth, shorten the survival time of mice, but single knockout male mice have no change; D-F. nuCB1&2 knockout female mice promote tumor growth, shorten the survival time of mice, but single knockout female mice have no change (WT: wild type; KO: gene knockout).

FIG. 8 shows that NUCBs promote expansion of LY 49H-positive NK cells. A. Flow cytometric analysis of LY49H positive NK cells; the NUCB1/2 overexpression reduces the tumor size, and promotes the expansion of mouse spleen LY49H positive NK cells (2 NUCB1 overexpressing tumors in 7 NUCB1/2 overexpressing tumors and 5 NUCB2 overexpressing tumors); nucb1&2 gene knockout increased tumor weight, reduced number of mouse spleen LY49H positive NK cells; D. intraperitoneal injection of NUCB2 peptide reduces tumor volume and increases the number of LY 49H-positive NK cells (%) are the proportion of splenocytes CD45+CD3-cells, shown as A); patterns of nucbs promoting LY49H positive NK cell proliferation.

Detailed Description

The present inventors have studied extensively and intensively, and have unexpectedly found for the first time that a Nucleobase (NUCB) can bind to LY49H in nature, thereby effectively promoting NK cell expansion (or proliferation). Specifically, experiments such as knocking out NUCB genes, over-expressing NUCB proteins, injecting key peptide fragments, combining in vitro proteins and the like prove that secreted proteins NUCB1 and NUCB2 are physiological ligands of an NK cell surface receptor LY49H, and binary complexes formed by the NUCB proteins and the LY49H promote amplification of LY49H receptor positive NK cells. Therefore, the Nuleobindin (NUCB) and the promoter thereof can be used for preparing a medicament or preparation for treating tumor and promoting the expansion of NK cells. The present invention has been completed on the basis of this finding.

Terminology

In the present invention, "the inventive fibronectin", "the NUCBs protein", "the NUCB1 and the NUCB2 protein" are used interchangeably, and refer to secreted proteins that bind to NK cell surface receptor LY49H to promote NK cell expansion.

Natural killer cells (NK cells)

NK cells, one of the major components of the natural immune system, play a key role in the monitoring of body immunity and early anti-infection and tumor processes. Many preclinical studies indicate that NK cells play an important role in limiting tumor growth and metastasis. Clinical data also show that NK cell activity has a negative correlation with carcinogenesis and infiltration of NK cells in some tumor microenvironments has a positive correlation with patient survival. Unlike T cells, NK cells possess certain advantages. Firstly, NK cells belong to an innate immune system, and almost all tumor cells or tumor cells with recurrence and metastasis are preferentially attacked by the NK cells when going to the forefront of immune response, so that the NK cells have broad-spectrum anti-tumor effect; secondly, tumor specific antigen recognition or amplification is not needed, and the immune system is started quickly; third, the safety is better and the migration restrictions are less.

NK cells directly kill tumor cells by releasing lytic particles comprising perforin and granzyme, and can also indirectly play a role by recruiting and regulating the natural immune and acquired immune systems by secreting cytokines and chemokines such as IFN-gamma, TNF-alpha, GM-CSF, etc. These functions are achieved by the activation or inhibition of receptors on the surface of NK cells for various germline-encoded (germline-encoded). However, many of these receptors have not found corresponding ligands so far.

Nuclear chain protein (NUCB)

NUCBs (Nucleobindins) is a family of calcium-ion-binding secreted proteins, including NUCB1 and NUCB2. The function of NUCB2 protein was reported to suppress appetite and reduce weight earlier in 2006, but later studies showed that NUCB2 was not appetite and weight regulating under physiological conditions. The biological function of NUCB1 has not been clarified so far. The biological function and mechanism of the NUCBs family is not known. NUCB1 and NUCB2 proteins are conserved in evolution and have high homology, e.g., human and rat NUCB2 proteins are 87.4% similar, and rat and mouse NUCB2 proteins are 95.7% similar.

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

It will be appreciated that there may be an optional tag sequence (e.g., a 6His tag to facilitate purification) at the N-or C-terminus of the NUCB proteins of the invention. Furthermore, NUCB proteins of the invention may comprise wild type and mutant forms, wherein the mutant forms retain at least 30%, preferably at least 50% of the corresponding biological activity of the wild type.

It is to be understood that while NUCB proteins of the invention are derived from mice, 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 proteins are also within the contemplation of the invention. For example, NUCB1 of murine origin (without signal peptide) and human NUCB1 of 89% similarity and NUCB2 of murine origin (without signal peptide) and human NUCB2 of 87% similarity. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

The sequences of NUCB proteins involved in the present invention are shown below:

mouse NUCB1 (SEQ ID NO: 4)

MPTSVPRGAPFLLLPPLLMLSAVLAVPVDRAAPPQEDSQATETPDTGLYYH RYLQEVINVLETDGHFREKLQAANAEDIKSGKLSQELDFVSHNVRTKLDELKRQ EVSRLRMLLKAKMDAKQEPNLQVDHMNLLKQFEHLDPQNQHTFEARDLELLIQTATRDLAQYDAAHHEEFKRYEMLKEHERRRYLESLGEEQRKEAERKLQEQQ RRHREHPKVNVPGSQAQLKEVWEELDGLDPNRFNPKTFFILHDINSDGVLDEQELEALFTKELEKVYDPKNEEDDMREMEEERLRMREHVMKNVDTNQDRLVTLEE FLASTQRKEFGDTGEGWKTVEMSPAYTEEELKRFEEELAAREAELNARAQRLSQETEALGRSQDRLEAQKRELQQAVLQMEQRKQQLQEQSAPPSKPDGQLQFRA DTDDAPVPAPAGDQKDVPASEKKVPEQPPELPQLDSQHL

Mouse NUCB2 (SEQ ID NO: 5)

MRWRIIQVQYCFLLVPCMLTALEAVPIDVDKTKVHNTEPVENARIEPPD TGLYYDEYLKQVIEVLETDPHFREKLQKADIEEIRSGRLSQELDLVSHKVRTR LDELKRQEVGRLRMLIKAKLDALQDTGMNHHLLLKQFEHLNHQNPNTFESRDLDMLIKAATADLEQYDRTRHEEFKKYEMMKEHERREYLKTLSEEKRKEEE SKFEEMKRKHEDHPKVNHPGSKDQLKEVWEETDGLDPNDFDPKTFFKLHDVNNDGFLDEQELEALFTRELEKVYNPQNAEDDMIEMEEERLRMREHVMSEID NNKDRLVTLEEFLRATEKKEFLEPDSWETLDQQQLFTEDELKEYESIIAIQEN ELKKRAEELQKQKEDLQRQHDHLEAQKQEYHQAVQHLEQKKLQQGIAPSGPAGELKFEPHT

Human NUCB1 (SEQ ID NO: 6)

MPPSGPRGTLLLLPLLLLLLLRAVLAVPLERGAPNKEETPATESPDTGLY YHRYLQEVIDVLETDGHFREKLQAANAEDIKSGKLSRELDFVSHHVRTKLDELKRQEVSRLRMLLKAKMDAEQDPNVQVDHLNLLKQFEHLDPQNQHTFEAR DLELLIQTATRDLAQYDAAHHEEFKRYEMLKEHERRRYLESLGEEQRKEAE RKLEEQQRRHREHPKVNVPGSQAQLKEVWEELDGLDPNRFNPKTFFILHDINSDGVLDEQELEALFTKELEKVYDPKNEEDDMREMEEERLRMREHVMKNVD TNQDRLVTLEEFLASTQRKEFGDTGEGWETVEMHPAYTEEELRRFEEELAAREAELNAKAQRLSQETEALGRSQGRLEAQKRELQQAVLHMEQRKQQQQQQQ GHKAPAAHPEGQLKFHPDTDDVPVPAPAGDQKEVDTSEKKLLERLPEVEVP QHL

Human NUCB2 (SEQ ID NO: 7)

MRWRTILLQYCFLLITCLLTALEAVPIDIDKTKVQNIHPVESAKIEPPDTG LYYDEYLKQVIDVLETDKHFREKLQKADIEEIKSGRLSKELDLVSHHVRTKLDELKRQEVGRLRMLIKAKLDSLQDIGMDHQALLKQFDHLNHLNPDKFESTD LDMLIKAATSDLEHYDKTRHEEFKKYEMMKEHERREYLKTLNEEKRKEEES KFEEMKKKHENHPKVNHPGSKDQLKEVWEETDGLDPNDFDPKTFFKLHDVNSDGFLDEQELEALFTKELEKVYDPKNEEDDMVEMEEERLRMREHVMNEV DTNKDRLVTLEEFLKATEKKEFLEPDSWETLDQQQFFTEEELKEYENIIALQENELKKKADELQKQKEELQRQHDQLEAQKLEYHQVIQQMEQKKLQQGIPPSG PAGELKFEPHI

Furthermore, the term "NUCB derived protein" also includes the protein of SEQ ID NO:4 or 5. These variants include (but are not limited to): deletions, insertions and/or substitutions of 1-3 (typically 1-2, more preferably 1) amino acids, and additions or deletions of one or several (typically within 3, preferably within 2, more preferably within 1) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus generally does not alter the structure or function of the protein. Furthermore, the term also includes polypeptides of the invention in monomeric and multimeric form. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

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

A preferred class of reactive derivatives refers to polypeptides having up to 3, preferably up to 2, more preferably up to 1 amino acid replaced by amino acids of similar or similar nature, compared to the amino acid sequence set forth in SEQ ID No. 4 or 5. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

Initial residues	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 invention also provides analogues of NUCB proteins. These analogs may differ from the polypeptides shown in SEQ ID No. 4 or 5 by the amino acid sequence, by modified forms that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acid (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 (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., 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 improve their proteolytic resistance or to optimize solubility.

LY49 receptor family

The LY49 receptor family is one of the mouse NK cell receptors, and can transmit activating or inhibitory signals to regulate NK cell killing effect. The 10 members of the LY49 receptor family protein are LY49A, LY49B, LY49C, LY49D, LY49E, LY49F, LY49G, LY49H, LY49I, LY49Q. Among them, physiological ligands for NK cell surface receptor LY49H have not been found. Corresponding to the LY49 receptor family in human NK cells is the KIR receptor family, comprising 8 inhibitory receptors: KIR2DL1-5, KIR3DL1-3, and 6 activated receptors, KIR2DS1-5, KIR3DS1, wherein a plurality of receptors do not currently find their ligands.

In the present invention, 5 truncated forms of plasmids were made at the extracellular end of receptor LY49H, and binding experiments of these fragments to NUCBs found that the C-type lectin-like domain (CTLD, amino acids 143-266) was necessary for binding to NUCBs. Alignment of the C-lectin-like domain (C-tpye domain) amino acid sequences of LY49 family molecules revealed that LY49H was most similar to LY49F, LY49C, LY49I, with 3 amino acid residues specific to LY49H, specifically L196, H216 and T232.

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

MSEQEVTFPTMRFHKSSGLNSQVRLEGTQRSRKAGLRVCSVPWQLIVIALGI LCSLRLVIVAVFVTKFFQYSQHKQEINETLNHRHNCSNMQRDFNLKEEMLTNKSIDCRPSYELLEYIKREQERWDSETKSVSDSSRDTGRGV KYWFCYGTKCYYFIMNKTTWSGCKANCQHYSVPIVKIEDEDELKFLQRHVILESYWIGLSYDKKKKEWAWIHNGQSK LDMKIKKMNFTSRGCVFLSKARIEDTDCNTPYYCICGKKLDKFPD

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

The extracellular peptide segment of LY49H is SEQ ID NO:9 (67-266 segments of SEQ ID NO: 8): KFFQYSQHKQEINETLNHRHNCSNMQRDFNLKEEMLTNKSIDCRPSYELLEY IKREQERWDSETKSVSDSSRDTGRGVKYWFCYGTKCYYFIMNKTTWSGCKANCQHYSVPIVKIEDEDELKFLQRHVILESYWIGLSYDKKKKEWAWIHNGQSK LDMKIKKMNFTSRGCVFLSKARIEDTDCNTPYYCICGKKLDKFPD

Accelerator and formulation

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

The NUCB protein promoter of the present invention, when administered (dosed) therapeutically, promotes the expression and/or activity of NUCB protein, and thus promotes the expansion and/or activity of NK cells, thereby killing tumors. In another preferred embodiment, the NUCB promoter comprises NUCB gene expression products, a promoter miRNA, a promoter transcription regulator, or a promoter targeting small molecule compound.

Typically, NUCB proteins of the invention or promoters thereof may be formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending upon the nature of the substance 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 formulation comprising a safe and effective amount of the NUCB protein of the invention or an enhancer thereof 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 formulation should be compatible with the mode of administration. The pharmaceutical preparations of the invention can be prepared in the form of injections, for example by conventional methods using physiological saline or aqueous solutions containing glucose and other auxiliaries. The formulation according to the invention is selected from the group consisting of: tablets, capsules, injections, granules, sprays and freeze-dried preparations. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram-10 milligrams per kilogram of body weight per day. The medicament or the preparation is administrated by a medication mode selected from the following groups: intravenous, intratumoral, intracavity, subcutaneous, or hepatic arterial administration (e.g., injection, instillation, etc.).

The invention also provides a pharmaceutical composition comprising a safe and effective amount of the LY49H protein or an active fragment thereof of the invention and a pharmaceutically acceptable carrier or excipient for use in preparing a medicament for treating a disorder associated with excessive NUCB protein. The related diseases of the NUCB protein excess are selected from the following groups: polycystic ovary syndrome (Polycystic Ovary Syndrome), hypertension (hypertension), anxiety (Anxiety), epilepsy (epiepsy), or combinations thereof.

The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredients 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 is matched with the administration mode, and the dosage forms of the pharmaceutical composition are injection, freeze-dried preparation, stem cell preparation and aerosol inhalation preparation. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The pharmaceutical compositions are preferably manufactured under sterile conditions.

Composite material

In the present invention, there is provided a complex which is a binary complex formed by binding NUCB protein to LY 49H.

The complex of the invention can be used for screening drugs or compounds for promoting NK cell expansion, wherein substances for promoting the formation of the complex have the potential for promoting NK cell expansion.

Method for promoting NK cell expansion

The invention provides a method for promoting NK cell expansion and NK cell growth in vivo and in vitro, comprising the following steps:

(1) In vitro: providing a NUCB protein or a promoter thereof; in the presence of the NUCB protein or a promoter thereof, the NK cells are mixed and cultured with the NK cells containing the receptor thereof under proper culture conditions, so that the NK cells are promoted to be amplified in large quantity.

(2) In vivo: providing a NUCB protein or a promoter thereof; after inputting a functional NK cell (the NK cell can naturally contain a receptor of NUCB protein or is artificially modified to contain the receptor of NUCB protein) in vivo, the NUCB protein or an accelerant thereof is then given in vivo, so that the imported NK cell is promoted to be amplified in vivo and a large amount of NK cell exists for a long time; enhancing therapeutic efficacy of infused NK cells.

The main advantages of the invention include:

(1) The invention discovers that secreted proteins NUCB1 and NUCB2 are physiological ligands of 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 provided by the invention, and the obtained NK cells have broad-spectrum anti-tumor effect, do not need to identify tumor specific antigens, and have a quick immune system starting time.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Universal materials and methods

1. Gene cloning

The full length or fragment of the mouse NUCB1, NUCB2 gene is cloned to a pCMV-HA eukaryotic expression vector and a pLVX-DsRed-3 x flag-HA-T2A-Puro lentiviral packaging vector, and the full length or fragment of the LY49 family gene is cloned to a pFUSE-hIgG1-Fc2 vector. The point mutation of the gene is completed through corresponding point mutation primer.

2. Cell culture

HEK 293t, mc38 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ with CO2 concentration of 5%. Cells were plated 18-24 hours prior to transfection, with a total plasmid amount of 2 μg/well for the 6-well plate, and with LacZ when the amount of transfected plasmid was insufficient. The transfection reagent was Lipo3000 (Invitrogen). Corresponding experiments were performed 24-30 hours after transfection.

3. Co-immunoprecipitation

Culture solutions of 293T cell cotransfection NUCB1 or NUCB2 and LY49 family plasmids are collected, centrifuged at 12000rpm for 10 minutes, supernatant plus protein A/G beads are taken and spun at 4 ℃ for 2 hours to immunoprecipitate FC-tagged proteins, the supernatant is sucked off after 3 times of washing, 40 μl of 2 x loading is added and boiled for 10 minutes, and the mixture is preserved at-20 ℃.

4.Western Blot

SDS-PAGE gel with concentration of 10% was prepared and added to protein samples for electrophoretic separation. Protein in electrotransfer gel was transferred to NC (nitrocellulose) membrane 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, each for 5 minutes, with the addition of the corresponding HRP-conjugated secondary antibody, and incubated for 1 hour at room temperature. After three washes, a developer is added for scanning. NUCB2 (N6789) antibody was purchased from Sigma Aldrich, HA (16B 12) antibody was purchased from Covance, flag antibody, his antibody was purchased from Sigma Aldrich, HRP-conjugated Goat anti-human IgG antibody was purchased from Sangon Biotech (Shanghai).

5. Extraction of tissue RNA and Q-PCR

The mice were euthanized and the corresponding tissues were rapidly stripped and placed in a homogenization tube containing steel beads and Trizol. RNA was extracted by phenol-chloroform-isopropanol method after disruption, reverse transcribed into cDNA using superscript TMIII first strand synthesis system (Invitrogen) kit, and quantitative PCR reaction was performed using Quantitative SYBR green PCR kit (TaKara) kit and ABI-Quantum studio 6real time PCR instrument.

6. A mouse

C57BL/6 mice were purchased from Ling Chang Co, fed to SPF-grade animal houses from Biochemical cell sites, turned off for 12 hours (19:00-07:00), and turned on for 12 hours (07:00-19:00). And constructing and obtaining NUCB1 and NUCB2 single-gene knockout mice and NUCB1 and NUCB2 double-knockout mice by a CRISPR-Cas9 method, mating the mice with a purchased wild-type mouse for 5 generations, and performing propagation amplification to obtain the wild-type and corresponding gene knockout mice for experiments. All experimental mice were 7-11 weeks old.

Construction of monoclonal strains in which MC38 cells overexpress NUCB1 or NUCB2

The full length of NUCB1 and NUCB2 genes (containing signal peptide) of mice were obtained by PCR amplification, cloned into pvvx-DsRed-3 x flag-HA-T2A-Puro virus packaging vector, and transfected 293T cell packaging lentivirus together with packaging plasmids pSPAX2, pMD 2G. The obtained virus infects MC38 cells, and monoclonal is selected to obtain MC38 cells which stably and highly express NUCB1 or NUCB 2.

8. Cell proliferation assay

Wild-type MC38 cells and NUCB1 or NUCB2 overexpressing MC38 cell proliferation assays were performed using the CellTiter-Glo fluorescent cell viability assay (Promega) kit and were assayed according to the instructions steps.

9. Subcutaneous transplantation tumor model

Culturing the digested wild type MC38 cells or NUCB1, NUCB2 over-expressing MC38 cells, washing with PBS for 3 times, filtering with 100um filter membrane, and counting to 5×10 ⁶ Per ml of cells, 100ul (containing 5 x 10) ⁵ Cells), the length and width of the tumor was measured after the corresponding time using vernier calipers, and tumor volume was calculated by the formula: volume = length x width/2. When the tumor volume is larger than 2000mm during survival statistics ³ At this time, the mice were euthanized, defined as dead.

10. Polypeptide synthesis

The NUCB1 peptide fragment is amino acid 45-81 of NUCB1 protein, the NUCB2 peptide fragment is amino acid 49-85 of NUCB2 protein, and the reference peptide fragment is identical with the amino acid 49-85 of NUCB2 peptide fragment, but the sequence is randomly arranged. 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:

TGDREKDTYYVQEPKAKLIEQDLERIFSYEEDHLLVI(SEQ ID NO:3)。

11. intraperitoneal injection

Purchased 6 week old wild type C57 mice were acclimatized 1 week later and subcutaneously inoculated with MC38 cells 5 x 10 ⁵ Tumor volumes were measured 5 days later and intraperitoneal injection of 3 peptide fragments was started, 2 times per day, 100 ug/each.

12. Flow cell

Spleens were removed within 5 minutes after euthanasia of the mice and placed in pre-chilled phenol red free DMEM medium. After erythrocyte lysis, 106 cells were counted, following the procedure: dying Live (Live/Dead Fixable Near-IR Dead Cell Stain Kit, thermo Fisher), blocking (anti-CD 16/32, bioLegend), membrane antibodies (anti-CD 45, brilliant Violet 510, bioLegend; anti-CD3, brilliant Violet 605, bioLegend; anti-NK1.1, brilliant Violet 421, bioLegend; anti-LY49H, FITC, eBioscience), on-line analysis (Beckman CytoFlex LX flow cytometry).

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 specific binding of secreted proteins NUCB1 and NUCB2 to NK cell surface receptor LY49H

The inventors found by mass spectrometry that LY49H could be a binding protein for NUCBs, cloned 10 members of the LY49 receptor family proteins (LY 49A, LY49B, LY49C, LY49D, LY49E, LY49F, LY49G, LY49H, LY49I, LY 49Q) in C57BL/6 mice, the outer cell membrane fragments of which were into the expression vector PFUSE-hIgG1-Fc2 (IL 2 ss), because this vector contained the IL2 signal peptide, the gene-expressed protein cloned into this vector would be secreted into the cell culture medium. The protein expression sequences of NUCB1 and NUCB2 of the full-length mice are cloned into eukaryotic expression vector pCMV-HA at the same time, because NUCBs gene itself HAs secretion signal peptide to be secreted into cell culture medium. Simultaneously, both LY49 family plasmids and NUCBs expression plasmids are secreted into the cell culture medium after being transfected into 293T cells (FIGS. 1A and 1B), and the system can well detect the interaction of proteins. Cell culture media was collected 24-30 hours after co-transfected 293T cells, and co-immunoprecipitation found that both NUCB1 and NUCB2 specifically bound LY49H of the LY49 family (FIGS. 1C, 1D). NUCB1 and NUCB2 proteins expressed by eukaryotic 293T cells (fig. 1E) and prokaryotic escherichia coli (fig. 1F) were next purified, confirming the direct interaction of NUCB1 and NUCB2 with LY 49H.

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

EXAMPLE 2 specific binding of LY49H to NUCBs by C-type lectin-like domains

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

The results show that L196, H216 is the key amino acid residue that determines its specific binding to NUCBs (fig. 2f,2 g). Thus, as shown in FIG. 2H, NUCBs bind to the C-type lectin-like domain of LY49H, with leucine 196 and histidine 216 being the critical amino acids.

Example 3 amino acids 40-80 of NUCBs are the critical segment for binding LY49H

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

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

These truncations and full length ratios, NUCB1 (lacks 44-80) and NUCB2 (lacks 44-80) significantly affected binding to LY49H (fig. 3D).

The results indicate that amino acids 40-80 of NUCBs are the critical segment for binding to LY 49H.

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

Through slow virus infection of MC38 cells, monoclonal is selected to obtain stable cell strains with high expression of NUCB1 or NUCB2, and cell culture liquid is collected to detect that the stable cell strains secrete a large amount of NUCB1 or NUCB2 protein into the cell culture liquid (figures 4A and B). Cell proliferation assays, overexpression of NUCB1 or NUCB2 did not affect proliferation of the cells themselves (fig. 4C). Will be 5X 10 ⁵ Wild-type or NUCBs overexpressionThe strain was transplanted subcutaneously into C57 male mice, compared to wild-type cells.

As shown in FIGS. 4D-F, the growth rate of MC38 tumor over-expressing NUCB1 or NUCB2 was significantly reduced, and the survival rate of mice was significantly prolonged.

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

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

NUCB1 peptide fragment (amino acids 45-81):

DTGLYYHRYLQEVINVLETDGHFREKLQAANAEDIKS(SEQ ID NO:1)，

NUCB2 peptide fragment (amino acids 49-85):

DTGLYYDEYLKQVIEVLETDPHFREKLQKADIEEIRS(SEQ ID NO:2)，

control peptide:

TGDREKDTYYVQEPKAKLIEQDLERIFSYEEDHLLVI(SEQ ID NO:3)。

wild-type mice of C57 were inoculated subcutaneously with 5X 10 ⁵ And divided into 3 groups, and intraperitoneal injection of the polypeptide was started after 5 days. The control peptide, NUCB1 peptide and NUCB2 peptide were injected twice daily, at 100 ug/dose. Tumor size was measured from day 5, once every 2 days.

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

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

Example 6 construction of NUCB1 and NUCB2 single knockout mice and NUCB1 and NUCB2 simultaneous knockout double knockout mice

The NUCB1 gene of C57BL/6 mice shares 13 exons, with coding exons 2 to 13. The CRISPR-Cas9 method is adopted, and the sgRNA is designed at the coding signal peptide sequence of the exon 2 so as to achieve the purpose of knockout. Sequencing confirmed that exon 2 of NUCB1 gene knocked out 10 bases in the signal peptide, achieving the purpose of frame shift (FIGS. 6A, B). Both genomic PCR and fluorescent quantitative PCR at the mRNA level confirmed the knockout of NUCB1 (FIGS. 6C, D).

The NUCB2 gene shares 14 exons, with exons 3 to 14 being coding exons. 2 sgRNAs are designed at the coding signal peptide sequence of the No. 3 exon by using a CRISPR-Cas9 method so as to achieve the purpose of knocking out in a certain length. Sequencing confirmed that the NUCB2 gene exon 3 knocked out 83 bases including part of the signal peptide coding sequence, and also achieved the purpose of frame shift (fig. 6e, f). Successful construction of NUCB2 knockout mice was determined at both genomic and protein levels, respectively (fig. 6g, h). After NUCB1 and NUCB2 monogenic editing mice are obtained respectively, the two mice are hybridized to obtain double-gene editing mice, and NUCB1 and NUCB2 double-gene homozygous knockout mice (NUCB 1& 2) are obtained.

Example 7 nucbs knockout mice promoted tumor growth

The NUCBs family includes two members of very high homology, NUCB1 and NUCB2. The biochemical experiment shows that: both NUCB1 and NUCB2 are capable of binding LY49H.

The mouse experiment shows that: the cell over-expression of NUCB1 or NUCB2 has obvious tumor inhibiting effect; the addition of NUCB1 peptide or NUCB2 peptide can also significantly inhibit tumor growth. Using the NUCBs knockout mice obtained, 5×10 mice were inoculated subcutaneously, respectively ⁵ MC38 cells of (C) in NUCB1 and NUCB2 knockout mice alone and NUCB1&2 double knockout mice.

The results show that: double knockout of NUCB1&2 promoted tumor growth, shortening survival, both in male (fig. 7A-C) and female (fig. 7D-F). However, single NUCB1 or NUCB2 knockout had no effect, suggesting that both genes of the NUCBs family have compensatory effects, and silencing one function may be compensated by the other homologous gene.

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

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

FIG. 8A is a loop gate flow chart of flow cytometric analysis of LY49H positive cells. Wild type C57 male mice were subcutaneously inoculated with MC38 cells and NUCB1/2 overexpressing MC38 cells, the tumor volume of the overexpressing cells was much smaller than that of the wild type on day 20, and spleen flow analysis was collected to find that the LY49H positive NK cell proportion was significantly increased in nuCB1/2 overexpressing mice (FIG. 8B). Meanwhile, on day 15, wild-type female mice bearing tumor (MC 38) and NUCB1&2 double knockout female mice were compared, and the tumor weight of the knockout mice was significantly higher than that of the wild-type mice, and flow analysis showed that the LY49H positive NK cell proportion of the spleen of the knockout mice was significantly reduced (fig. 8C). C57 wild type male mice were subcutaneously vaccinated with MC38 cells, NUCB2 peptide and peptide solvent PBS were intraperitoneally injected for 8 consecutive days starting on day 2, NUCB2 peptide group tumor volumes were significantly reduced at day 14, and LY the proportion of LY49H positive NK cells in the spleen was increased (fig. 8D). FIG. 8E is a graph of patterns of NUCBs promoting LY 49H-positive NK cell proliferation.

The result shows that the over-expression of NUCB protein reduces the tumor size and promotes the amplification of LY49H positive NK cells in the spleen of mice (8B); 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, increasing the number of LY49H positive NK cells (8D).

The results in both the positive and negative aspects show that an important physiological effect of NUCBs protein after binding LY49H is to promote the amplification of LY49H positive NK cells, thereby reducing tumors.

Discussion of the invention

Effect of NUCBs on NK cells and receptors in humans

NUCBs are highly conserved in the evolution of human mice and show significant tumor treatment effects in mice. The subsequent aspect will detect the role and mechanism of NUCBs proteins on human NK cells by the human NK cell line NK92 and isolating primary human NK cells. Since the corresponding 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, many of which are not currently found as ligands. These are the most important receptors for potential NUCBs. Subsequent second aspect the inventors will first verify the KIR receptor family and the NKG2C/CD94 receptor, finding the human NK cell NUCBs receptor. The receptor is used as a potential target of NK cells, and has important significance for the immunotherapy of tumors.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

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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> Homo sapiens (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> mice (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> mice (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 (14)

1. Use of a Nucleobase (NUCB) for the preparation of a medicament or formulation (a) for promoting expansion of NK cells, (b) for the treatment of a tumor; and/or (c) killing or inhibiting tumor cells; the NUCB protein is NUCB1 protein and/or NUCB2 protein, and the NUCB protein is derived from mice;

The tumor is colon cancer.

2. The use according to claim 1, wherein said NK cells naturally contain receptors for NUCB or surface receptors for NUCB obtained by artificial engineering.

3. The use of claim 1, wherein the NUCB1 protein has the amino acid sequence set forth in SEQ ID No. 4.

4. The use of claim 1, wherein the NUCB2 protein has the amino acid sequence set forth in SEQ ID No. 5.

5. The use of claim 1, wherein the NUCB protein is a full length NUCB protein, or a critical segment of a NUCB protein, the critical segment of a NUCB protein being amino acid sequence 40-85 of NUCB1 or NUCB 2.

6. The use of claim 1, wherein the NUCB protein specifically binds NK cell surface receptor LY49H.

7. The use of claim 5, wherein the critical segment of the NUCB protein is the critical segment of NUCB2 having the sequence shown in SEQ ID No. 2.

8. The use of claim 5, wherein the critical segment of the NUCB protein is the critical segment of NUCB1 having the sequence set forth in SEQ ID No.: 1.

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

(a) Providing a NUCB protein; and

(b) Culturing NK cells in the presence of said NUCB protein, thereby promoting NK cell expansion; the NUCB protein is NUCB1 protein and/or NUCB2 protein, and the NUCB protein is derived from mice.

10. The method of claim 9, wherein the NK cells are surface receptor LY49H positive NK cells.

11. An isolated complex, wherein the complex is a binary complex formed by binding NUCB protein to LY 49H; the NUCB proteins include NUCB1 proteins and/or NUCB2 proteins, which are derived from mice.

12. The complex of claim 11, wherein the NUCB1 protein has the amino acid sequence set forth in SEQ ID No. 4.

13. The complex of claim 11, wherein the NUCB2 protein has the amino acid sequence set forth in SEQ ID No.: 5.

14. The complex of claim 11, wherein the amino acid sequence of LY49H is set forth in SEQ ID No.: 8.