WO2022143938A1 - 一种激活nk细胞的肿瘤免疫治疗方法 - Google Patents

一种激活nk细胞的肿瘤免疫治疗方法 Download PDF

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WO2022143938A1
WO2022143938A1 PCT/CN2021/143241 CN2021143241W WO2022143938A1 WO 2022143938 A1 WO2022143938 A1 WO 2022143938A1 CN 2021143241 W CN2021143241 W CN 2021143241W WO 2022143938 A1 WO2022143938 A1 WO 2022143938A1
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protein
nucb
cells
ly49h
nucb1
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周云夫
李林
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中国科学院分子细胞科学卓越创新中心
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to the field of biological immunity, in particular, to a tumor immunotherapy method for activating NK cells.
  • Tumor immunotherapy is undoubtedly the most important step in tumor treatment in recent decades. It won the Nobel Prize in Physiology and Medicine in 2018 and is hailed as the third anti-cancer drug revolution after chemotherapy and targeted therapy.
  • NK cells natural killer cells
  • NK cells play a key role in the body's immune surveillance and early anti-infection and tumor processes.
  • NK cells directly kill tumor cells by releasing lytic granules containing perforin and granzyme, and can also recruit and regulate other innate immunity and acquisition by secreting cytokines and chemokines such as IFN- ⁇ , TNF- ⁇ , GM-CSF, etc.
  • the sexual immune system plays an indirect role. These functions are achieved through a variety of germline-encoded activating or inhibitory receptors on the surface of NK cells. But so far, the corresponding ligands for many of these receptors have not been found, which is not conducive to the development of immunotherapeutic methods related to the activation of NK cells.
  • the purpose of the present invention is to provide a new method for activating NK cells efficiently and rapidly.
  • nucleobindin Nucleobindin, NUCB
  • the medicine or preparation (a) is used for promoting the expansion of NK cells , (b) treating tumors; and/or (c) killing or inhibiting tumor cells.
  • the medicament or preparation includes a tumoricidal agent.
  • the NUCB protein is of non-viral origin.
  • the NUCB protein is derived from mammals, preferably from rodents (rats, mice) and primates (such as humans), more preferably from humans.
  • the NUCB protein includes NUCB1 protein and/or NUCB2 protein.
  • amino acid sequence of the NUCB1 protein is shown in SEQ ID NO.:4 or the identity with SEQ ID NO.:4 is ⁇ 85% (preferably ⁇ 90%, more preferably ⁇ 95%) % or ⁇ 98%).
  • amino acid sequence of the NUCB2 protein is shown in SEQ ID NO.: 5 or the identity with SEQ ID NO.: 5 is ⁇ 85% (preferably ⁇ 90%, more preferably ⁇ 95%) % or ⁇ 98%).
  • the NUCB1 protein is human NUCB1 (SEQ ID NO.: 6).
  • the NUCB2 protein is human NUCB2 (SEQ ID NO.: 7).
  • the NUCB protein specifically binds to the NK cell surface receptor LY49H.
  • the NUCB protein includes full-length NUCB protein, mature NUCB protein, a key segment of NUCB that binds to LY49H, or a NUCB active fragment containing the key segment.
  • the key segment of the NUCB protein is the amino acid sequence of positions 40-85 of NUCB1 or NUCB2.
  • sequence of the key segment of NUCB1 is shown in SEQ ID NO.: 1.
  • sequence of the key segment of NUCB2 is shown in SEQ ID NO.: 2.
  • sequence of the key segment of NUCB1 is positions 45-81 of mouse NUCB1 protein (SEQ ID NO. 4).
  • sequence of the key segment of NUCB2 is positions 49-85 of mouse NUCB2 protein (SEQ ID NO. 5).
  • the NUCB protein includes a key segment of NUCB that binds to KIR3DS1.
  • sequence of the key segment of NUCB1 is positions 46-82 of human NUCB1 protein (SEQ ID NO. 6).
  • sequence of the key segment of NUCB2 is positions 49-85 of human NUCB2 protein (SEQ ID NO. 7).
  • sequence of the key segment of NUCB1 is positions 45-138, preferably positions 45-63 of human NUCB1 protein (SEQ ID NO. 6).
  • the NK cells are NK cells positive for the surface receptor LY49H.
  • the promoter includes a promoter-type miRNA, a promoter-type transcriptional regulator, or a promoter-type targeted small molecule compound.
  • 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 .
  • the drug or preparation is administered by a mode of administration selected from the group consisting of intravenous, intratumoral, intracavitary, subcutaneous or hepatic artery administration (eg, injection, drip, etc.).
  • the preparation is selected from the group consisting of tablets, capsules, injections, granules, sprays, and freeze-dried preparations.
  • the preparation is an injection.
  • a method for promoting NK cell expansion in vitro comprising the steps of:
  • the NK cells are NK cells positive for the surface receptor LY49H.
  • the method is an in vitro method.
  • an isolated complex is provided, and the complex is a binary complex formed by the combination of NUCB protein and LY49H.
  • the NUCB protein includes NUCB1 protein and/or NUCB2 protein.
  • amino acid sequence of the NUCB protein is selected from the following group:
  • (a) has the amino acid sequence shown in SEQ ID NO.:4 or 5;
  • amino acid sequence shown in SEQ ID NO.: 4 or 5 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the NUCB activity is composed of (a) a derived polypeptide; or
  • amino acid sequence of the LY49H is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 8 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the LY49H activity is formed by (i) ) derived polypeptide; or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 8 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95% or ⁇ 98%), and a polypeptide having the LY49H activity .
  • the NK cells are preferably NK cells positive for the surface receptor LY49H.
  • a method for screening a drug or compound for promoting NK cell expansion comprising the steps of:
  • test substance is a drug that promotes NK cell expansion or compound.
  • the significantly higher ratio refers to H1/H0 ⁇ 2, preferably, ⁇ 3, more preferably, ⁇ 4.
  • a method of treating tumors by administering a therapeutically effective amount of NUCB protein or its promoter to a subject in need thereof.
  • the subject is a human or a non-human mammal.
  • non-human mammals include rodents (eg, mice, rats, rabbits), primates (eg, monkeys).
  • the method further comprises administering NK cells to a subject in need.
  • the NK cells are NK cells positive for the surface receptor LY49H.
  • the NK cells naturally contain NUCB receptors or obtain NUCB surface receptors through artificial transformation.
  • LY49H protein or its active fragment is provided for preparing a pharmaceutical composition for treating diseases related to excessive NUCB protein.
  • sequence of the LY49H protein or its active fragment is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 9 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the LY49H activity is formed by (a) ) derived polypeptide; or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 9 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95% or ⁇ 98%), and a polypeptide having the LY49H activity .
  • the LY49H protein or its active fragment is the extracellular segment of LY49H (SEQ ID NO.: 9).
  • the related diseases with excessive NUCB protein are selected from the group consisting of polycystic ovary syndrome (Polycystic Ovary Syndrome), hypertension (Hypertention), anxiety (Anxiety), epilepsy (Epilepsy) or its combination.
  • Figure 1 shows that NUCBs specifically bind LY49H.
  • A Schematic diagram of the molecular structure of LY49H molecule and NUCBs (where SS is the signal peptide (signal sequence), EF is the EF-hand domain);
  • B The schematic diagram of the interaction method (LY49 family plasmid and NUCBs expression plasmid were co-transfected into 293T After cells, it is secreted into the cell culture medium);
  • C Results of co-immunoprecipitation experiment of LY49H molecule and NUCB1 protein, in which NUCB1 protein specifically binds to LY49H in the LY49 family; D.
  • NUCB2 protein specifically binds to LY49H in the LY49 family; E.LY49H binds to NUCBs protein expressed and purified by eukaryotic 293T cells; F.LY49H binds to E. coli expressed and purified NUCBs protein (wherein, lp:Fc is co-immunoprecipitation Fc tag, Input is the input amount of the two proteins in the co-immunoprecipitation experiment, HA is the HA tag, FC is the FC tag, and Flag is the Flag tag).
  • Figure 2 shows that NUCBs specifically bind to the C-type lectin-like domain of LY49H. Cut-out pattern of the extracellular segment of LY49H; B. The C-type lectin-like domain of LY49H is required for binding to NUCB1; C. The C-type lectin-like domain of LY49H is required for binding to NUCB2; D. LY49 family C-type lectin-like domain sequence alignment; E. LY49 family C-type lectin-like domain sequence association guide tree; F, G.
  • Leucine 196 of the C-type lectin-like domain of LY49H Residues and 216 histidine residues are the key amino acids that determine their binding specificity with NUCB1/NUCB2; H. NUCBs binding site pattern map on LY49H, in which CTLD is a C-type lectin-like domain.
  • Figure 3 shows that amino acids 40-80 of NUCBs bind LY49H.
  • A C-terminal truncation pattern of NUCBs protein;
  • B The three C-terminal truncations of NUCBs do not affect the binding to LY49H;
  • C N-terminal truncation pattern of NUCBs protein;
  • D NUCB1 (deficient in 44-80 ) and NUCB2 (44-80 deficiency) significantly affected the binding to LY49H.
  • Figure 4 shows that overexpression of NUCBs inhibits tumor cell growth in mice.
  • A Construct the model diagram of MC38 cells overexpressing NUCBs protein;
  • B Spread the same number of cells, harvest the medium 24 hours later, and detect the overexpression of NUCBs by Western Blot;
  • C Overexpression of NUCBs protein does not affect the proliferation of MC38 cells;
  • D MC38 wild-type cells and NUCB1 or NUCB2 overexpressing MC38 cells subcutaneously inoculate C57 mice model diagram;
  • E NUCB1 and NUCB2 overexpressing MC38 cells in mice subcutaneously grow significantly smaller tumors;
  • F NUCB1 and NUCB2 overexpressing MC38 Cell-transplanted mice had significantly longer survival.
  • Figure 5 shows that injection of NUCBs peptides inhibited tumor cell growth.
  • A-C NUCB1 and NUCB2 protein sequence information, synthetic NUCBs peptide sequence information and control peptide sequence information;
  • D Schematic diagram of injection of NUCBs peptide;
  • E Injection of NUCB1 peptide or NUCB2 peptide significantly reduced tumor size ;
  • F NUCB peptide fragment human-mouse sequence comparison information.
  • Figure 6 shows the identification of NUCBs knockout mice.
  • A B. NUCB1 knockout strategy and sequencing identification
  • C D. NUCB1 knockout mouse genome PCR and mRNA expression identification (lung tissue);
  • E F. NUCB2 knockout strategy and sequencing identification;
  • G H . Genome PCR identification of NUCB2 knockout, WB identification.
  • marker is the control
  • actin is actin.
  • Figure 7 shows that NUCBs knockout mice promote tumor growth.
  • C NUCB1&2 knockout male mice promoted tumor growth and shortened the survival time of mice, but single knockout male mice did not change;
  • D-F NUCB1&2 knockout female mice promoted tumor growth and shortened the mice's survival time The survival time of single knockout female mice did not change (WT: wild type; KO: knockout).
  • Figure 8 shows that NUCBs promote the expansion of LY49H-positive NK cells.
  • A Flow cytometric analysis of LY49H-positive NK cells in the gating process;
  • B NUCB1/2 overexpression reduces tumor size and promotes the expansion of LY49H-positive NK cells in mouse spleen (7 NUCB1/2-overexpressing tumors in 2 were NUCB1-overexpressing tumors, and 5 were NUCB2-overexpressing tumors);
  • C NUCB1&2 gene knockout increased tumor weight and decreased the number of LY49H-positive NK cells in the spleen of mice; D.
  • NUCB2 peptides decreased Tumor volume, increasing the number of LY49H-positive NK cells (LY49H-positive NK cells (%): the proportion of CD45+CD3- cells in splenocytes, as shown in A); E. NUCBs promote LY49H-positive NK cell proliferation pattern.
  • Figures 9A-C show that NUCB1 protein in humans binds to the activating receptor KIR3DS1 in the KIR family; D. Human NUCB1 is also highly specific compared to other receptors on the surface of human NK cells, such as NKG2D, TIGIT, DNAM1, NKP30 and NKP46 Binding of KIR3DS1; E. The predicted fragmentation of the secondary mechanism of human NUCB1 shows that positions 45-138 of NUCB1 are the segment that mainly binds KIR3DS1; F. Further breakdown of the core amino acid sequence of human NUCB1, showing positions 45-63 of human NUCB1 Amino acids are central to binding KIR3DS1 and KIR3DL1.
  • nucleobindin can actually bind to LY49H, thereby effectively promoting the expansion (or proliferation) of NK cells.
  • experiments such as knocking out the NUCB gene from mice, overexpressing the NUCB protein, injecting key peptides and in vitro protein binding have proved that the secreted proteins NUCB1 and NUCB2 are the physiological ligands of the NK cell surface receptor LY49H.
  • the formed binary complex promotes the expansion of LY49H receptor-positive NK cells. Therefore, nucleobindin (NUCB) and its promoter can be used to prepare a drug or preparation for treating tumors and promoting the expansion of NK cells.
  • the present invention has been completed on this basis.
  • nucleonectin of the present invention refers to NK cell surface receptor LY49H Binds to a secreted protein that promotes NK cell expansion.
  • NK Cells Natural Killer Cells
  • NK cells play a key role in the body's immune surveillance and early anti-infection and tumor processes. Numerous preclinical studies have shown that NK cells play an important role in limiting tumor growth and metastasis. Clinical data also show that NK cell activity is negatively correlated with cancer development, and that infiltration of NK cells in some tumor microenvironments is positively correlated with patient survival. Unlike T cells, NK cells have certain advantages. First, NK cells belong to the innate immune system and are at the forefront of the immune response.
  • NK cells directly kill tumor cells by releasing lytic granules containing perforin and granzyme, and can also recruit and regulate other innate immunity and acquisition by secreting cytokines and chemokines such as IFN- ⁇ , TNF- ⁇ , GM-CSF, etc.
  • the sexual immune system plays an indirect role. These functions are achieved through a variety of germline-encoded activating or inhibitory receptors on the surface of NK cells. But for many of these receptors, no corresponding ligand has been found so far.
  • NUCB Nucleonectin
  • NUCBs are a family of calcium-binding secreted proteins, including NUCB1 and NUCB2.
  • the function of NUCB2 protein was first reported in 2006 to suppress appetite and reduce weight, but later studies showed that NUCB2 has no appetite and weight regulation functions under physiological conditions.
  • the biological function of NUCB1 is still unclear. Therefore, the biological functions and mechanisms of the NUCBs family are still unclear.
  • NUCB1 and NUCB2 proteins are evolutionarily conserved and have high homology, such as 87.4% similarity between human and rat NUCB2 protein, and 95.7% similarity between rat and mouse NUCB2 protein.
  • NUCBs protein expression exists in a variety of tissues and cells. Under conditions such as stress or inflammation, NUCBs proteins are induced to express. It is currently believed that cells of the tumor and microenvironment induce NUCBs expression in a variety of stressful situations, including in tumor situations, to activate NK cells to deal with stressful situations.
  • the NUCB proteins of the present invention may include wild type and mutant types, wherein the mutant type retains at least 30%, preferably at least 50% of the corresponding biological activity of the wild type.
  • NUCB proteins of the present invention are derived from mice, those from other animals (such as humans) are highly homologous (such as having more than 85%, such as 85%, 90%, 95% or even 98% sequence) with the mouse NUCB protein Other proteins that are identical) are also contemplated by the present invention.
  • human NUCB1 which is 89% similar to murine NUCB1 (without signal peptide)
  • human NUCB2 which is 87% similar to murine NUCB2 (without signal peptide).
  • Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
  • NUCB protein The sequence of the NUCB protein involved in the present invention is as follows:
  • Mouse NUCB1 (SEQ ID NO: 4)
  • derived protein of NUCB also includes a variant form of the sequence shown in SEQ ID NO: 4 or 5, which has the function of binding LY49H.
  • variants include (but are not limited to): deletions, insertions and/or substitutions of 1-3 (usually 1-2, more preferably 1) amino acids, as well as C-terminal and/or N-terminal additions or One or several (usually within 3, preferably within 2, more preferably within 1) amino acids are deleted.
  • substitution with amino acids of similar or similar properties generally does not alter the function of the protein.
  • addition or deletion of one or several amino acids at the C-terminus and/or N-terminus generally does not alter the structure and function of the protein.
  • the term also includes monomeric and multimeric forms of the polypeptides of the invention.
  • the term also includes linear as well as nonlinear polypeptides (eg, cyclic peptides).
  • NUCB-acting derivative proteins of the present invention also include active fragments, derivatives and analogs thereof.
  • fragment refers to polypeptides that substantially retain the function or activity of binding to LY49H.
  • polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more A polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusion of a NUCB protein or a derivative thereof with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol), or (iv) A polypeptide formed by fusing an additional amino acid sequence with this polypeptide sequence (a fusion protein formed by fusing with a leader sequence, a secretory sequence, or a tag sequence such as 6His).
  • a class of preferred active derivatives refers to those with at most 3, preferably at most 2, more preferably at most 1 amino acids with similar or similar properties compared with the amino acid sequence shown in SEQ ID NO.: 4 or 5. Amino acids are replaced to form polypeptides. These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the present invention also provides analogs of the NUCB protein.
  • the differences between these analogs and the polypeptides shown in SEQ ID NO: 4 or 5 can be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
  • Modified (generally without altering the primary structure) forms include chemically derivatized forms such as acetylation or carboxylation of the polypeptide in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modifications can be accomplished by exposing the polypeptide to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize their solubility properties.
  • the LY49 receptor family is one of the mouse NK cell receptors, which can transmit activating or inhibitory signals and regulate the killing effect of NK cells.
  • the 10 members of the LY49 receptor family of proteins are LY49A, LY49B, LY49C, LY49D, LY49E, LY49F, LY49G, LY49H, LY49I, LY49Q. Among them, the physiological ligand of NK cell surface receptor LY49H has not been found.
  • KIR receptor family Corresponding to the LY49 receptor family in human NK cells is the KIR receptor family, including 8 inhibitory receptors: KIR2DL1-5, KIR3DL1-3, and 6 activating receptors, KIR2DS1-5, KIR3DS1, many of which The receptor has not yet found its ligand.
  • C-type lectin-like domain C-tpye lectin-like domain
  • LY49H has three amino acid residues that are unique to LY49H compared to LY49F, LY49C and LY49I, which are the most similar. , specifically L196, H216 and T232.
  • amino acid sequence of the LY49H is shown in SEQ ID NO: 8:
  • the underlined part is the CTLD part in LY49H (143-266 of SEQ ID NO:8).
  • the LY49H extracellular telopeptide segment is SEQ ID NO:9 (segments 67-266 of SEQ ID NO:8):
  • the NUCB protein promoter of the present invention when administered (administered) therapeutically, can promote the expression and/or activity of the NUCB protein, thereby promoting the expansion and/or activity of NK cells, thereby killing tumors.
  • the NUCB promoter includes a NUCB gene expression product, a promoter-type miRNA, a promoter-type transcriptional regulator, or a promoter-type targeted small molecule compound.
  • the NUCB protein of the present invention or its promoter can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, Although pH can vary depending on the nature of the substance being formulated and the condition being treated.
  • the formulated formulation or pharmaceutical composition can be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
  • the present invention also provides a pharmaceutical preparation, which contains a safe and effective amount of the NUCB protein or its promoter of the present invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical preparation of the present invention can be prepared in the form of injection, for example, prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the preparation of the present invention is selected from the group consisting of tablets, capsules, injections, granules, sprays, and freeze-dried preparations.
  • the active ingredient is administered in a therapeutically effective amount, e.g., about 1 microgram to 10 mg/kg body weight per day.
  • the medicaments or preparations of the present invention are administered by means of administration selected from the group consisting of intravenous, intratumoral, intracavitary, subcutaneous or hepatic arterial administration (eg, injection, drip, etc.).
  • the present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the LY49H protein of the present invention or an active fragment thereof and a pharmaceutically acceptable carrier or excipient, for preparing a kind of disease related to the treatment of excessive NUCB protein .
  • the disease associated with excessive NUCB protein is selected from the group consisting of Polycystic Ovary Syndrome, Hypertention, Anxiety, Epilepsy or a combination thereof.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is injection, freeze-dried preparation, stem cell preparation, and atomized inhalation preparation. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical compositions are preferably manufactured under sterile conditions.
  • a complex is provided, which is a binary complex formed by the combination of NUCB protein and LY49H.
  • the complexes of the present invention can be used to screen for drugs or compounds that promote NK cell expansion, wherein substances that can promote the formation of the complexes have the potential to promote NK cell expansion.
  • the present invention provides a method for promoting the expansion of NK cells in vitro or in vivo and the long-term existence of NK cells in vivo, comprising:
  • the present invention finds for the first time that the secreted proteins NUCB1 and NUCB2 are the physiological ligands of the NK cell surface receptor LY49H.
  • the method for activating NK cells of the present invention is a brand-new tumor immunotherapy approach.
  • NK cells are activated by the method of the present invention, and the obtained NK cells have broad-spectrum anti-tumor effects, do not need to recognize tumor-specific antigens, and activate the immune system quickly.
  • the point mutation of the gene is accomplished by the corresponding site-directed mutagenesis primers.
  • HEK 293T and MC38 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO2 concentration. The cells were divided into plates 18-24 hours before transfection, and the total amount of plasmid in the 6-well plate was 2 ⁇ g/well. When the amount of transfected plasmid was insufficient, LacZ was used to make up.
  • the transfection reagent was Lipo3000 (Invitrogen). Corresponding experiments were performed 24-30 hours after transfection.
  • SDS-PAGE gel with a concentration of 10% was prepared, and protein samples were added for electrophoresis separation.
  • the proteins were electrotransferred to NC (nitrocellulose) membranes and blocked with 0.5% skim milk for 1 hour. After washing 3 times with TBST for 5 min each, the corresponding primary antibody was added and incubated for 2 h at room temperature. After washing 3 times with TBST for 5 minutes each time, the corresponding HRP-conjugated secondary antibody was added and incubated for 1 hour at room temperature. After washing three times, add developer to scan.
  • NUCB2 (N6789) antibody was purchased from Sigma Aldrich company
  • HA (16B12) antibody was purchased from Covance company
  • Flag antibody, His antibody were purchased from Sigma Aldrich company
  • HRP-conjugated Goat anti-human IgG antibody was purchased from Sangon Biotech (Shanghai).
  • mice C57BL/6 mice were purchased from Lingchang Company, and were kept in the SPF animal room of the Institute of Biochemical Cells. The lights were turned off for 12 hours (19:00-07:00) and on for 12 hours (07:00-19:00).
  • NUCB1 and NUCB2 single-gene knockout mice and NUCB1 and NUCB2 double-knockout mice were obtained by CRISPR-Cas9 method. Wild-type and corresponding knockout mice. Mice used in all experiments were 7-11 weeks old.
  • the full-length mouse NUCB1 and NUCB2 genes (including signal peptides) were obtained by PCR amplification, cloned into pLVX-DsRed-3*Flag-HA-T2A-Puro virus packaging vector, and transfected into 293T cells together with the packaging plasmids pSPAX2 and pMD2G. Virus. The obtained virus was used to infect MC38 cells, and single clones were picked to obtain MC38 cells with stable high expression of NUCB1 or NUCB2.
  • the proliferation of MC38 wild-type cells and MC38 cells overexpressing NUCB1 or NUCB2 was detected using the CellTiter-Glo Fluorescent Cell Viability Assay (Promega) kit, and detected according to the instructions.
  • the NUCB1 peptide segment is amino acids 45-81 of the NUCB1 protein, and the NUCB2 peptide segment is the amino acids 49-85 of the NUCB2 protein.
  • the control peptide segment is the same as the NUCB2 peptide segment (49-85), but the sequence is randomly arranged. . Synthesized by Shanghai Jill Biochemical Company, the purity is more than 95%. The specific sequence is:
  • NUCB2 peptides NUCB2 peptides:
  • the purchased 6-week-old wild-type C57 mice were subcutaneously inoculated with 5*10 5 MC38 cells/mice after 1 week of adaptation. After 5 days, the tumor volume was measured and 3 kinds of peptides were injected intraperitoneally, 2 times a day, 100ug each time /Only.
  • the spleen was removed within 5 minutes after the mice were euthanized and placed in pre-cooled phenol red-free DMEM medium. After the red blood cells were lysed, count 106 cells, and follow the procedure: live/dead fixable (Live/Dead Fixable Near-IR Dead Cell Stain Kit, Thermo Fisher), blocking (anti-CD16/32, BioLegend), membrane antibody (anti-CD45, Brilliant Violet) 510, BioLegend; anti-CD3, Brilliant Violet 605, BioLegend; anti-NK1.1, Brilliant Violet 421, BioLegend; anti-LY49H, FITC, eBioscience), on-board analysis (Beckman CytoFlex LX flow cytometer).
  • LY49H may be the binding protein of NUCBs, and cloned 10 members of the LY49 receptor family protein in C57BL/6 mice (LY49A, LY49B, LY49C, LY49D, LY49E, LY49F, LY49G, LY49H, LY49I, LY49Q) into the expression vector PFUSE-hIgG1-Fc2 (IL2ss), because this vector contains the IL2 signal peptide, the protein expressed by the gene cloned into this vector will be secreted into the cell culture medium.
  • IL2ss PFUSE-hIgG1-Fc2
  • the cell culture medium of the co-transfected 293T cells was collected 24-30 hours later, and co-immunoprecipitation found that both NUCB1 and NUCB2 specifically bound LY49H of the LY49 family (Fig. 1C, 1D).
  • Fig. 1E eukaryotic 293T cell-expressed
  • Fig. 1F prokaryotic E. coli-expressed
  • NUCB1 and NUCB2 proteins were purified, verifying the direct interaction of NUCB1 and NUCB2 with LY49H.
  • NUCBs are potential ligands of LY49H.
  • Example 2 The C-type lectin-like domain of LY49H specifically binds NUCBs
  • Point mutations were made to the three amino acid positions of LY49H, respectively, to the amino acid residues corresponding to their similar receptors (L196P, leucine at position 196 was mutated to proline; H216D, histidine at position 216 was mutated). mutated to aspartic acid; T232K, threonine at position 232 was mutated to lysine).
  • NUCBs were gradually truncated from the C-terminus to obtain three truncated forms: NUCB1(1-320), NUCB1(1-230), NUCB1(1-160), NUCB2(1-320), NUCB2(1-230), NUCB2(1-160). All of these truncations bound LY49H well (Fig. 3B). Therefore, amino acids 1-160 of NUCBs may be the segment responsible for binding LY49H.
  • NUCB1 Missing 25-44
  • NUCB1 missing 44-80
  • NUCB1 missing 80-120
  • NUCB1 missing 120-159
  • NUCB2 missing 24-44
  • NUCB2 missing 44-80
  • NUCB2 missing 80-125
  • NUCB2 missing 125-159
  • NUCB1 44-80 deficiency
  • NUCB2 44-80 deficiency
  • MC38 cells were infected with lentivirus, and a single clone was picked to obtain a stable cell line with high expression of NUCB1 or NUCB2.
  • the cell culture medium was collected and it was detected that the stable line cells secreted a large amount of NUCB1 or NUCB2 protein into the cell culture medium ( Figure 4A, B).
  • Cell proliferation assay, overexpression of NUCB1 or NUCB2 did not affect the proliferation of the cells themselves (Fig. 4C). 5 ⁇ 10 5 wild-type or NUCBs overexpressing strains were subcutaneously transplanted into C57 male mice and compared with wild-type cells.
  • NUCB1 peptide (45-81 amino acids):
  • mice C57 wild-type mice were inoculated with 5 ⁇ 10 5 MC38 cells subcutaneously and divided into 3 groups, and the polypeptide was injected intraperitoneally after 5 days.
  • the control peptides, NUCB1 peptides and NUCB2 peptides were injected respectively, twice a day in the morning and evening, with a dose of 100ug per animal each time. Tumor size was measured from day 5 and every 2 days.
  • NUCB1 peptide or NUCB2 peptide can significantly reduce the tumor size.
  • Human and mouse NUCBs proteins are highly conserved in evolution. Comparing the functional peptide sequences, the similarity of human and mouse NUCB1 peptides is 97.3%, and the similarity of NUCB2 peptides is 91.9% (Fig. 5F). The results suggest that human NUCB peptides may also have potential tumor-inhibiting effects.
  • the NUCB1 gene of C57BL/6 mice has a total of 13 exons, of which the 2nd to 13th are coding exons.
  • the sgRNA was designed at the exon 2 encoding signal peptide sequence to achieve the purpose of knockout. Sequencing confirmed that exon 2 of the NUCB1 gene knocked out 10 bases in the signal peptide, achieving the purpose of frameshifting (Fig. 6A,B). Both genomic PCR and real-time PCR at the mRNA level confirmed the knockdown of NUCB1 (Fig. 6C,D).
  • the NUCB2 gene has a total of 14 exons, of which exons 3 to 14 are coding exons.
  • two sgRNAs were designed at the exon 3 encoding signal peptide sequence to achieve a certain length of knockout. Sequencing confirmed that 83 bases including part of the signal peptide coding sequence were knocked out in exon 3 of NUCB2 gene, and the purpose of frameshift was also achieved (Fig. 6E, F).
  • the successful construction of NUCB2 knockout mice was confirmed at the genomic and protein levels, respectively (Fig. 6G,H).
  • NUCB1 and NUCB2 single-gene-edited mice were crossed to obtain double-gene-edited mice, and then NUCB1 and NUCB2 double-gene homozygous knockout mice (NUCB1&2) were obtained.
  • the NUCBs family includes two members with high homology, namely NUCB1 and NUCB2. Biochemical experiments showed that both NUCB1 and NUCB2 could bind LY49H.
  • mice show that cells overexpressing NUCB1 or NUCB2 can significantly inhibit tumor growth; the addition of NUCB1 peptide or NUCB2 peptide can also significantly inhibit tumor growth.
  • NUCBs knockout mice 5 ⁇ 10 5 MC38 cells were subcutaneously inoculated into NUCB1 and NUCB2 knockout mice alone and NUCB1&2 double knockout mice, respectively.
  • the knockout mice further demonstrated the antitumor effect of NUCBs.
  • NUCBs protein promotes the expansion of LY49H positive NK cells
  • Figure 8A is a flow cytometry flow cytometry flow cytometry analysis of LY49H positive cells.
  • Wild-type C57 male mice were subcutaneously inoculated with MC38 cells and NUCB1/2-overexpressing MC38 cells. On day 20, the tumor volume of the over-expressed cells was much smaller than that of the wild-type.
  • Flow analysis of the spleens showed that the proportion of LY49H-positive NK cells was significantly higher than that of NUCB1/2-overexpressed NK cells.
  • Significantly increased in mice (Figure 8B).
  • FIG. 8C C57 wild-type male mice were subcutaneously inoculated with MC38 cells. From day 2, NUCB2 peptides and peptide solvent PBS were intraperitoneally injected for 8 consecutive days. On day 14, the tumor volume in the NUCB2 peptide group was significantly reduced, and the proportion of LY49H-positive NK cells in the spleen increased. (Fig. 8D).
  • Figure 8E is a graph showing the pattern of NUCBs promoting the proliferation of LY49H-positive NK cells.
  • NUCB protein overexpression reduced tumor size and promoted the expansion of LY49H-positive NK cells in mouse spleen (8B); NUCB1&2 gene knockout increased tumor weight and decreased the number of LY49H-positive NK cells in mouse spleen (8C); Intraperitoneal injection of NUCB2 peptides reduced tumor volume and increased the number of LY49H-positive NK cells (8D).
  • NUCB1 in humans binds to NK cell surface receptors KIR3DS1 and KIR3DL1
  • Figures 9A-C show that NUCB1 protein in humans binds KIR3DS1, an activating receptor in the KIR family (KIR2DS1-5 in B is an activating receptor in the KIR family, KIR2DL1-5 in C, and KIR3DL1-3 are inhibitory receptors in the KIR family ).
  • KIR2DS1-5 in B is an activating receptor in the KIR family
  • KIR2DL1-5 in C is inhibitory receptors in the KIR family
  • KIR3DL1-3 are inhibitory receptors in the KIR family
  • human NUCB1 also binds KIR3DS1 with high specificity (Fig. 9D). Fragmentation according to the secondary mechanism prediction of human NUCB1 showed that positions 45-138 of NUCB1 were the segment that mainly bound KIR3DS1 (Fig. 9E).
  • NUCBs are highly conserved between humans and mice in evolution, and have shown significant tumor-treating effects in mice.
  • the effect and mechanism of NUCBs protein on human NK cells will be detected by the human NK cell line NK92 and the isolation of primary human blood NK cells.
  • the corresponding LY49 receptor family in human NK cells is the KIR receptor family, including 8 inhibitory receptors: KIR2DL1-5, KIR3DL1-3, and 6 activating receptors, KIR2DS1-5, KIR3DS1, many of which The receptor has not yet found its ligand. These are the most important potential receptors for NUCBs.
  • the inventors will firstly verify the KIR receptor family and the NKG2C/CD94 receptor, and find the human NK cell NUCBs receptor. As potential targets of NK cells, the above receptors are of great significance for tumor immunotherapy.

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Abstract

提供了一种激活NK细胞的肿瘤免疫治疗方法。还提供了一种核连蛋白(Nucleobindin，NUCB)或其促进剂的用途，用于制备一种药物或制剂，所述药物或制剂用于促进NK细胞的扩增，***和/或杀伤或抑制肿瘤细胞。通过所述方法激活NK细胞，获得的NK细胞具有广谱的抗肿瘤作用，不需要识别肿瘤特异性抗原，免疫***启动时间快。

Description

一种激活NK细胞的肿瘤免疫治疗方法 技术领域
本发明涉及生物免疫领域，具体地，涉及一种激活NK细胞的肿瘤免疫治疗方法。
背景技术
随着社会的进步和科技的发展，抗生素等药物的出现，上世纪及以前严重威胁人类的疾病如传染性疾病，其危害有了显著的降低。而癌症，心血管疾病，神经退行性疾病和代谢性疾病等慢性病成为了目前人类社会面临的主要健康威胁。全球在2018年新增1810万癌症患者，有960万人死于癌症。癌症严重的影响了人体健康和生活质量，同时也给社会带来巨大的经济压力。
肿瘤免疫疗法毫无疑问是最近几十年来肿瘤治疗最重要的一步，获得了2018年的诺贝尔生理与医学奖，被誉为化疗和靶向治疗后的第三次抗癌药物革命。绝大多数肿瘤免疫治疗方法，不管是免疫检验点抑制，嵌合抗原T细胞或者双特异性抗体等，都焦距于增强T细胞的活性。虽然这些方法取得了巨大的成功，但是只有很少一部分癌症患者病人能从中获益。
自然杀伤细胞(NK细胞)作为天然免疫***中的主要成员之一，在机体免疫监视和早期的抗感染和肿瘤等过程中发挥着关键的作用。NK细胞通过释放包含穿孔素和颗粒酶的裂解颗粒直接杀死肿瘤细胞，还可以通过分泌IFN-γ、TNF-α、GM-CSF等细胞因子和趋化因子招募和调控别的天然免疫与获得性免疫***间接发挥作用。这些功能的实现是通过NK细胞表面多种生殖系编码(germline-encoded)的激活性或抑制性受体。但是到目前为止，这些受体中的很多还没有找到相应的配体，不利于开发与激活NK细胞相关的免疫治疗方法。
综上所述，本领域迫切需要开发一种高效、快速的激活NK细胞的新方法。
发明内容
本发明的目的在于提供一种高效、快速的激活NK细胞的新方法。
在本发明的第一方面，提供了一种核连蛋白(Nucleobindin，NUCB)或其促进剂的用途，用于制备药物或制剂，所述药物或制剂(a)用于促进NK细胞的扩增，(b)***；和/或(c)杀伤或抑制肿瘤细胞。
在另一优选例中，所述的药物或制剂包括为杀肿瘤剂。
在另一优选例中，所述NUCB蛋白为非病毒来源。
在另一优选例中，所述NUCB蛋白来源于哺乳动物，较佳地，来自啮齿动物(大 鼠、小鼠)和灵长动物(例如人)，更佳地，来自人。
在另一优选例中，所述NUCB蛋白包括NUCB1蛋白和/或NUCB2蛋白。
在另一优选例中，所述NUCB1蛋白的氨基酸序列如SEQ ID NO.:4所示或与SEQ ID NO.:4的相同性≥85％(较佳地≥90％，更佳地≥95％或≥98％)。
在另一优选例中，所述NUCB2蛋白的氨基酸序列如SEQ ID NO.:5所示或与SEQ ID NO.:5的相同性≥85％(较佳地≥90％，更佳地≥95％或≥98％)。
在另一优选例中，所述NUCB1蛋白为人源NUCB1(SEQ ID NO.:6)。
在另一优选例中，所述NUCB2蛋白为人源NUCB2(SEQ ID NO.:7)。
在另一优选例中，所述NUCB蛋白特异性结合NK细胞表面受体LY49H。
在另一优选例中，所述NUCB蛋白包括全长的NUCB蛋白、成熟的NUCB蛋白、NUCB的与LY49H结合的关键区段、或含所述关键区段的NUCB活性片段。在另一优选例中，所述NUCB蛋白的关键区段为NUCB1或NUCB2的第40-85位氨基酸序列。
在另一优选例中，所述NUCB1的关键区段的序列如SEQ ID NO.:1所示。
在另一优选例中，所述NUCB2的关键区段的序列如SEQ ID NO.:2所示。
在另一优选例中，所述NUCB1的关键区段的序列为小鼠NUCB1蛋白(SEQ ID NO.4)的45-81位。
在另一优选例中，所述NUCB2的关键区段的序列为小鼠NUCB2蛋白(SEQ ID NO.5)的49-85位。
在另一优选例中，所述NUCB蛋白包括NUCB的与KIR3DS1结合的关键区段。
在另一优选例中，所述NUCB1的关键区段的序列为人源NUCB1蛋白(SEQ ID NO.6)的46-82位。
在另一优选例中，所述NUCB2的关键区段的序列为人源NUCB2蛋白(SEQ ID NO.7)的49-85位。
在另一优选例中，所述NUCB1的关键区段的序列为人源NUCB1蛋白(SEQ ID NO.6)的45-138位，较佳地为45-63位。
在另一优选例中，所述NK细胞为表面受体LY49H阳性的NK细胞。
在另一优选例中，所述的促进剂包括促进型miRNA、促进型转录调控因子、或促进型靶向小分子化合物。
在另一优选例中，所述肿瘤选自下组：结肠癌、乳腺癌、肺癌、胃癌、肝癌、多发性骨髓瘤、肾癌、胰腺癌、黑色素瘤、淋巴瘤、甲状腺癌、或其组合。
在另一优选例中，所述的药物或制剂通过选自下组的用药方式进行给药：静脉内、瘤内、腔内、皮下或肝动脉给药(如注射、滴注等)。
在另一优选例中，所述的制剂选自下组：片剂、胶囊剂、注射剂、颗粒剂、喷雾剂、冻干剂。
在另一优选例中，所述制剂为注射剂。
在本发明的第二方面，提供了一种体外促进NK细胞扩增的方法，包括步骤：
(a)提供一NUCB蛋白或其促进剂；和
(b)在所述NUCB蛋白或其促进剂的存在下，培养NK细胞，从而促进NK细胞扩增。
在另一优选例中，所述NK细胞为表面受体LY49H阳性的NK细胞。
在另一优选例中，所述方法为体外方法。
在本发明的第三方面，提供了一种分离的复合物，所述复合物为NUCB蛋白与LY49H相结合所形成的二元复合物。
在另一优选例中，所述NUCB蛋白包括NUCB1蛋白和/或NUCB2蛋白。
在另一优选例中，所述NUCB蛋白的氨基酸序列选自下组：
(a)具有SEQ ID NO.:4或5所示的氨基酸序列；
(b)将如SEQ ID NO.:4或5所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述NUCB活性的由(a)衍生的多肽；或
(c)氨基酸序列与SEQ ID NO.:4或5所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述NUCB活性的多肽。
在另一优选例中，所述LY49H的氨基酸序列选自下组：
(a)具有SEQ ID NO.:8所示氨基酸序列；
(b)将如SEQ ID NO.:8所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述LY49H活性的由(i)衍生的多肽；或
(c)氨基酸序列与SEQ ID NO.:8所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述LY49H活性的多肽。
在本发明的第四方面，提供了一种在本发明的第三方面所述的复合物的用途，用于筛选促进NK细胞扩增的药物或化合物。
在另一优选例中，所述NK细胞优选为表面受体LY49H阳性的NK细胞。
在本发明的第五方面，提供了一种筛选促进NK细胞扩增药物或化合物的方法，包括步骤：
(a)在测试组中，在待测物质存在下，培养NK细胞，并且设置无待测物质的对照组；
(b)检测测试组中所述复合物的含量H1并与对照组中的复合物含量H0进行比较，其中当H1显著高于H0，则表示所述测试物为促进NK细胞扩增的药物或化合物。
在另一优选例中，所述显著高于指H1/H0≥2，较佳地，≥3，更佳地，≥4。
在本发明的第六方面，提供了一种***的方法，向需要的对象施用治疗有效量的NUCB蛋白或其促进剂。
在另一优选例中，所述的对象是人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠、兔)、灵长类动物(如猴)。
在另一优选例中，所述方法还包括给有需要的对象施用NK细胞。
在另一优选例中，所述NK细胞为表面受体LY49H阳性的NK细胞。
在另一优选例中，所述NK细胞天然含有NUCB的受体或通过人为改造获得NUCB的表面受体。
在本发明的第七方面，提供了一种LY49H蛋白或其活性片段的用途，用于制备一种治疗NUCB蛋白过高的相关疾病的药物组合物。
在另一优选例中，所述LY49H蛋白或其活性片段的序列选自下组：
(a)具有SEQ ID NO.:9所示的氨基酸序列；
(b)将如SEQ ID NO.:9所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述LY49H活性的由(a)衍生的多肽；或
(c)氨基酸序列与SEQ ID NO.:9所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述LY49H活性的多肽。
在另一优选例中，所述LY49H蛋白或其活性片段为LY49H的胞外段(SEQ ID NO.:9)。
在另一优选例中，所述NUCB蛋白过高的相关疾病选自下组：多囊性卵巢综合征(Polycystic Ovary Syndrome)、高血压(Hypertention)、焦虑(Anxiety)、癫痫(Epilepsy)或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了NUCBs特异性结合LY49H。A.LY49H分子和NUCBs分子结构模式图(其中SS为信号肽(signal sequence)，EF为EF-hand结构域)；B.相互作用方 法模式图(LY49家族质粒和NUCBs表达质粒共转染到293T细胞后，分泌到细胞培养基)；C.LY49H分子和NUCB1蛋白的免疫共沉淀实验的结果图，其中NUCB1蛋白特异性结合LY49家族中的LY49H；D.LY49H分子和NUCB2蛋白的免疫共沉淀实验的结果图，NUCB2蛋白特异性结合LY49家族中的LY49H；E.LY49H结合真核293T细胞表达纯化的NUCBs蛋白；F.LY49H结合大肠杆菌表达纯化的NUCBs蛋白(其中，lp:Fc为免疫共沉淀Fc标签，Input为免疫共沉淀实验中两个蛋白的输入量，HA为HA标签，FC为FC标签，Flag为Flag标签)。
图2显示了NUCBs特异结合LY49H的C-型凝集素样结构域(C-type lectin-like domain)。LY49H细胞外段的切短模式图；B.LY49H的C-型凝集素样结构域是结合NUCB1必需的；C.LY49H的C-型凝集素样结构域是结合NUCB2必需的；D.LY49家族C-型凝集素样结构域序列比对；E.LY49家族C-型凝集素样结构域序列关连性引导树；F,G.LY49H的C-型凝集素样结构域的196位亮氨酸残基和216位组氨酸残基是决定其和NUCB1/NUCB2结合特异性的关键氨基酸；H.NUCBs在LY49H上的结合位点模式图，其中CTLD为C-型凝集素样结构域。
图3显示了NUCBs的第40-80位氨基酸结合LY49H。A.NUCBs蛋白的C端截短模式图；B.NUCBs的3个C端截短型不影响与LY49H的结合；C.NUCBs蛋白的N端截短模式图；D.NUCB1(缺44-80)与NUCB2(缺44-80)显著的影响与LY49H的结合。
图4显示了过表达NUCBs抑制肿瘤细胞在小鼠体内的生长。A.构建NUCBs蛋白过表达MC38细胞模式图；B.铺相同细胞数量，24小时后收培养基，Western Blot检测NUCBs的过表达；C.NUCBs蛋白的过表达不影响MC38细胞本身的增殖；D.MC38的野生型细胞和NUCB1或NUCB2过表达MC38细胞皮下接种C57小鼠模式图；E.NUCB1和NUCB2过表达MC38细胞在小鼠皮下长的瘤明显减小；F.NUCB1和NUCB2过表达MC38细胞移植的小鼠生存率显著延长。
图5显示了注射NUCBs肽段抑制肿瘤细胞生长。A-C.NUCB1和NUCB2蛋白序列信息及合成NUCBs肽段序列信息及对照肽段序列信息；D.注射NUCBs肽段实验模式图；E.注射NUCB1肽段或NUCB2肽段都显著的减小肿瘤的大小；F.NUCB肽段人鼠序列比较信息。
图6显示了NUCBs基因敲除小鼠的鉴定。A,B.NUCB1基因敲除策略和测序鉴定；C,D.NUCB1敲除小鼠基因组PCR和mRNA表达的鉴定(肺组织)；E,F.NUCB2基因敲除策略和测序鉴定；G,H.NUCB2基因敲除的基因组PCR鉴定，WB鉴定。其中，marker为对照，actin为肌动蛋白。
图7显示了NUCBs基因敲除小鼠促进肿瘤生长。C.NUCB1&2基因敲除雄鼠促进肿瘤的生长，减短了小鼠的生存期，但单敲除基因雄鼠没有变化；D-F. NUCB1&2基因敲除雌鼠促进肿瘤的生长，减短了小鼠的生存期，但单敲除基因雌鼠没有变化(WT:野生型；KO：基因敲除)。
图8显示了NUCBs促进LY49H阳性NK细胞的扩增。A.流式细胞分析LY49H阳性NK细胞的圈门流程；B.NUCB1/2过表达减小了肿瘤大小，促进了小鼠脾脏LY49H阳性NK细胞的扩增(7个NUCB1/2过表达肿瘤中2个为NUCB1过表达瘤，5个为NUCB2过表达瘤)；C.NUCB1&2基因敲除增加了肿瘤的重量，降低了小鼠脾脏LY49H阳性NK细胞的数量；D.腹腔注射NUCB2肽段减小肿瘤体积，增加LY49H阳性NK细胞的数量(LY49H阳性NK细胞(％)为：占脾细胞CD45+CD3-细胞的比例，如A所示)；E.NUCBs促进LY49H阳性NK细胞增殖模式图。
图9A-C显示人中NUCB1蛋白结合KIR家族中的激活型受体KIR3DS1；D.相比较于人NK细胞表面其他受体，如NKG2D,TIGIT,DNAM1,NKP30和NKP46，人NUCB1也高特异性的结合KIR3DS1；E.人NUCB1的二级机构预测分片段显示NUCB1的45-138位是主要结合KIR3DS1的区段；F.人NUCB1核心氨基酸序列的进一步细分，显示人NUCB1的45-63位氨基酸是结合KIR3DS1和KIR3DL1的核心。
具体实施方式
本发明人经过广泛而深入地研究，首次意外发现一种核连蛋白(Nucleobindin，NUCB)居然可与LY49H结合，进而有效地促进NK细胞扩增(或增殖)。具体地，从小鼠体内敲除NUCB基因、过表达NUCB蛋白、注射关键肽段和体外蛋白结合等实验证明，分泌蛋白NUCB1和NUCB2是NK细胞表面受体LY49H的生理性配体，NUCB蛋白与LY49H形成的二元复合物促进LY49H受体阳性NK细胞的扩增。因此，核连蛋白(Nucleobindin，NUCB)及其促进剂可用于制备一种***促进NK细胞的扩增的药物或制剂。在此基础上完成了本发明。
术语
在本发明中，“本发明的核连蛋白”、“核连蛋白”、“NUCBs蛋白”、“NUCB蛋白”、“NUCB1和NUCB2蛋白”可互换使用，皆指与NK细胞表面受体LY49H结合促进NK细胞扩增的分泌蛋白。
自然杀伤细胞(NK细胞)
NK细胞作为天然免疫***中的主要成员之一，在机体免疫监视和早期的抗感染和肿瘤等过程中发挥着关键的作用。许多的临床前研究表明NK细胞发挥着限制肿瘤生长和转移的重要的功能。临床数据也显示NK细胞的活性与癌症发 生有着负相关，一些肿瘤微环境NK细胞的浸润与病人生存有着正相关。与T细胞不同，NK细胞拥有一定的优势。第一，NK细胞属于先天免疫***，走在免疫反应的最前方，几乎所有的肿瘤细胞或者复发转移的肿瘤细胞都会优先受到NK细胞的攻击，具有广谱的抗肿瘤作用；第二，不需要肿瘤特异性抗原识别或扩增，免疫***启动时间快；第三，安全性更好，移植限制更少。
NK细胞通过释放包含穿孔素和颗粒酶的裂解颗粒直接杀死肿瘤细胞，还可以通过分泌IFN-γ、TNF-α、GM-CSF等细胞因子和趋化因子招募和调控别的天然免疫与获得性免疫***间接发挥作用。这些功能的实现是通过NK细胞表面多种生殖系编码(germline-encoded)的激活性或抑制性受体。但是这些受体中的很多到目前为止还没有找到相应的配体。
核连蛋白(NUCB)
NUCBs(Nucleobindins)是钙离子结合的分泌蛋白家族，包括NUCB1和NUCB2。NUCB2蛋白的功能最早于2006年被报道为抑制食欲和减轻体重，但后面的研究显示生理条件下NUCB2是没有食欲和体重调节功能的。NUCB1生物学功能到目前还不明确。所以NUCBs家族的生物学功能和机制还不清楚。NUCB1和NUCB2蛋白在进化中保守，而且同源性很高，例如人和大鼠的NUCB2蛋白相似性87.4％，大鼠和小鼠的NUCB2蛋白相似性95.7％。
NUCBs蛋白的表达存在于多种组织和细胞。在压力或炎症等条件下，NUCBs蛋白会被诱导表达。目前认为肿瘤和微环境的细胞在多种压力情况下(包括在肿瘤情况下)诱导NUCBs表达，以激活NK细胞来处理压力情况。
应理解，在本发明NUCB蛋白的N端或C端可以还有任选的标签序列(如便于纯化的6His标签)。此外，本发明的NUCB蛋白可包括野生型和突变型，其中所述突变型保留至少30％，较佳地至少50％野生型的相应生物活性。
应理解，虽然本发明的NUCB蛋白来自小鼠，但是来自其它动物(如人)的与小鼠NUCB蛋白高度同源(如具有85％以上，如85％、90％、95％甚至98％序列相同性)的其它蛋白也在本发明考虑的范围之内。例如，与鼠源NUCB1(不含信号肽)与相似性为89％的人源NUCB1，与鼠源NUCB2(不含信号肽)与相似性为87％的人源NUCB2。比对序列相同性的方法和工具也是本领域周知的，例如BLAST。
本发明中涉及的NUCB蛋白的序列如下所示：
小鼠NUCB1(SEQ ID NO：4)
Figure PCTCN2021143241-appb-000001
Figure PCTCN2021143241-appb-000002
小鼠NUCB2(SEQ ID NO：5)
Figure PCTCN2021143241-appb-000003
人NUCB1(SEQ ID NO：6)
Figure PCTCN2021143241-appb-000004
人NUCB2(SEQ ID NO：7)
Figure PCTCN2021143241-appb-000005
Figure PCTCN2021143241-appb-000006
此外，所述术语“NUCB的衍生蛋白”还包括具有结合LY49H功能的、SEQ ID NO：4或5所示序列的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明作用NUCB的衍生蛋白还包括其活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持与LY49H结合功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)NUCB蛋白或其衍生蛋白与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与SEQ ID NO.:4或5所示的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
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
本发明还提供NUCB蛋白的类似物。这些类似物与SEQ ID NO:4或5所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
LY49受体家族
LY49受体家族为小鼠NK细胞受体之一，可传递活化或抑制性信号，调节NK细胞杀伤效应。LY49受体家族蛋白的10个成员为LY49A，LY49B，LY49C，LY49D，LY49E，LY49F，LY49G，LY49H，LY49I，LY49Q。其中，NK细胞表面受体LY49H的生理性配体一直没有找到。在人NK细胞中对应LY49受体家族的为KIR受体家族，包括8个抑制性受体：KIR2DL1-5，KIR3DL1-3，以及6个激活型受体，KIR2DS1-5，KIR3DS1，其中多个受体目前没有发现其配体。
在本发明中，对受体LY49H的胞外端作了5个截短形式的质粒，这些片段与NUCBs的结合实验发现其C-型凝集素样结构域(CTLD，143-266位氨基酸)是结合NUCBs必需的。比对LY49家族分子的C-型凝集素样结构域(C-tpye  lectin-like domain)氨基酸序列发现，LY49H相比最为相近的LY49F，LY49C，LY49I，有3个氨基酸残基是LY49H所特有的，具体为L196，H216和T232。
在本发明一个优选实施例中，所述LY49H的氨基酸序列如SEQ ID NO:8所示：
Figure PCTCN2021143241-appb-000007
其中，划线部分为LY49H中CTLD部分(SEQ ID NO:8的143-266位)。
LY49H胞外端肽段为SEQ ID NO:9(SEQ ID NO:8的67-266段)：
Figure PCTCN2021143241-appb-000008
促进剂和制剂
利用本发明蛋白，通过各种常规筛选方法，可筛选出与NUCB蛋白发生相互作用的物质，尤其是促进剂等。
本发明NUCB蛋白的促进剂，当在治疗上进行施用(给药)时，可促进NUCB蛋白的表达和/或活性，进而促进NK细胞的扩增和/或活性，从而杀伤肿瘤。在另一优选例中，所述的NUCB促进剂包括NUCB基因表达产物、促进型miRNA、促进型转录调控因子、或促进型靶向小分子化合物。
通常，可将本发明NUCB蛋白或其促进剂配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的制剂或药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、肌内、腹膜内、静脉内、皮下、皮内、或局部给药。
本发明还提供了一种药物制剂，它含有安全有效量的本发明NUCB蛋白或其促进剂以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物制剂可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。本发明所述的制剂选自下组：片剂、胶囊剂、注射剂、颗粒剂、喷雾剂、冻干剂。活性成分的给药量是治疗有 效量，例如每天约1微克-10毫克/千克体重。本发明所述的药物或制剂通过选自下组的用药方式进行给药：静脉内、瘤内、腔内、皮下或肝动脉给药(如注射、滴注等)。
本发明还提供了一种药物组合物，它含有安全有效量的本发明LY49H蛋白或其活性片段以及药学上可接受的载体或赋形剂，用于制备一种治疗NUCB蛋白过高的相关疾病。所述NUCB蛋白过高的相关疾病选自下组：多囊性卵巢综合征(Polycystic Ovary Syndrome)、高血压(Hypertention)、焦虑(Anxiety)、癫痫(Epilepsy)或其组合。
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、冻干制剂、干细胞制剂、雾化吸入制剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。
复合物
在本发明中，提供了一种复合物，所述复合物为NUCB蛋白与LY49H相结合所形成的二元复合物。
本发明的复合物可用于筛选促进NK细胞扩增的药物或化合物，其中，可促进所述复合物形成的物质，具有促进NK细胞扩增的潜力。
促进NK细胞扩增的方法
本发明提供了一种体外或体内促进NK细胞扩增和体内NK细胞长时存在的方法，包括：
(1)体外：提供一NUCB蛋白或其促进剂；在所述NUCB蛋白或其促进剂的存在下，在恰当的培养条件下与含其受体的NK细胞混合培养，促使NK细胞大量扩增。
(2)体内：提供一NUCB蛋白或其促进剂；体内在输入某种功能性NK细胞后(此类NK细胞可天然有NUCB蛋白的受体或人为改造使含有NUCB蛋白的受体)，再体内给予所述NUCB蛋白或其促进剂，促进输入的此类NK细胞在体内扩增和大量长期存在；增强输入的NK细胞的治疗功效。
本发明的主要优点包括：
(1)本发明首次发现分泌蛋白NUCB1和NUCB2是NK细胞表面受体LY49H的生理性配体。
(2)本发明的激活NK细胞的方法是一种全新的肿瘤免疫治疗途径。
(3)通过本发明的方法激活NK细胞，获得的NK细胞具有广谱的抗肿瘤作用，不需要识别肿瘤特异性抗原，免疫***启动时间快。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
通用材料与方法
1.基因克隆
小鼠NUCB1，NUCB2基因全长或片段克隆到pCMV-HA真核表达载体和pLVX-DsRed-3*Flag-HA-T2A-Puro慢病毒包装载体，LY49家族基因全长或片段克隆到pFUSE-hIgG1-Fc2载体。基因的点突变通过相应的定点突变引物完成。
2.细胞培养
HEK 293T，MC38细胞在含10％胎牛血清的DMEM培养液中培养，37℃，CO2浓度为5％。细胞在转染前18-24小时分盘，6孔盘总的质粒量为2μg/孔，转染质粒量不足时用LacZ补足。转染试剂为Lipo3000(Invitrogen)。转染后24-30小时做相应的实验。
3.免疫共沉淀
收集293T细胞共转染NUCB1或NUCB2与LY49家族质粒的培养液，12000rpm离心10分钟，取上清加ProteinA/G beads在4℃冷库旋转2小时免疫沉淀带FC标签的蛋白，清洗3次后吸掉上清，加入40μL的2*loading煮10分钟，-20℃保存。
4.Western Blot
配制浓度10％的SDS-PAGE胶，加入蛋白样品电泳分离。电转胶中蛋白到NC(硝酸纤维素)膜，用0.5％的脱脂牛奶封闭1小时。TBST洗涤3次，每次5分钟，再加入相应的一抗室温孵育2小时。TBST洗涤3次，每次5分钟，加入相应HRP偶联的二抗，室温孵育1小时。洗涤三次后，加入显影液扫描。NUCB2(N6789)抗体购自Sigma Aldrich公司，HA(16B12)抗体购自Covance公司，Flag抗体，His抗体购自Sigma Aldrich公司，HRP-conjugated Goat anti-human IgG抗体购自Sangon Biotech(上海)。
5.组织RNA的抽提和Q-PCR
小鼠被安乐死后快速剥离出相应的组织，放入含钢珠和Trizol的匀浆管中。破碎后用酚-氯仿-异丙醇法提取RNA，利用superscriptTMIII第一链合成体系 (Invitrogen)试剂盒反转录为cDNA，使用Quantitative SYBR green PCR kit(TaKara)试剂盒和ABI-QuantStudio 6Realtime PCR仪做定量PCR反应。
6.小鼠
C57BL/6小鼠购买自灵畅公司，饲养于生化细胞所SPF级别动物房，12小时关灯(19:00-07:00)，12小时开灯(07:00-19:00)。通过CRISPR-Cas9的方法构建获得NUCB1和NUCB2单基因敲除小鼠和NUCB1与NUCB2双敲除小鼠，与购买的野生型小鼠交配5代纯化背景后，用于繁殖扩增获得实验用的野生型和相应的基因敲除小鼠。所有实验用的小鼠为7-11周龄。
7.MC38细胞过表达NUCB1或NUCB2的单克隆株构建
PCR扩增获得小鼠NUCB1和NUCB2基因全长(含信号肽)，克隆到pLVX-DsRed-3*Flag-HA-T2A-Puro病毒包装载体，与包装质粒pSPAX2，pMD2G一起转染293T细胞包装慢病毒。获得的病毒感染MC38细胞，挑取单克隆获得稳定高表达NUCB1或NUCB2的MC38细胞。
8.细胞增殖测定
MC38的野生型细胞和NUCB1或NUCB2过表达MC38细胞增殖检测使用CellTiter-Glo荧光细胞活力测定(Promega)试剂盒，并按照说明书步骤检测。
9.皮下移植瘤模型
培养消化后MC38的野生型细胞或NUCB1，NUCB2过表达MC38细胞，PBS洗3次后100um滤膜过滤，计数调整到5*10 6/ml细胞，皮下接种100ul(含5*10 5细胞)，相应时间后使用游标卡尺测量肿瘤的长度和宽度，通过公式计算肿瘤体积：体积＝长*宽*宽/2。生存统计时，当肿瘤体积大于2000mm 3时，安乐死小鼠，定义为死亡。
10.多肽合成
NUCB1肽段为NUCB1蛋白的第45-81位氨基酸，NUCB2肽段为NUCB2蛋白的第49-85位氨基酸，对照肽段与NUCB2肽段(49-85)的氨基酸一致，但顺序随机打乱排列。由上海吉尔生化公司合成，纯度大于95％。具体序列为：
NUCB1肽段:
Figure PCTCN2021143241-appb-000009
NUCB2肽段:
Figure PCTCN2021143241-appb-000010
对照肽段:
Figure PCTCN2021143241-appb-000011
11.腹腔注射
购买的6周龄野生型C57小鼠，适应1周后，皮下接种MC38细胞5*10 5个/只，5天后测量肿瘤体积并开始腹腔注射3种肽段，每天注射2次，每次100ug/只。
12.流式细胞
小鼠安乐死后5分钟内摘取脾脏放于预冷的无酚红DMEM培养液中。红细胞裂解后，计数106细胞，按照流程：染死活(Live/Dead Fixable Near-IR Dead Cell Stain Kit,Thermo Fisher)，封闭(anti-CD16/32,BioLegend)，膜抗体(anti-CD45,Brilliant Violet 510,BioLegend；anti-CD3,Brilliant Violet 605,BioLegend；anti-NK1.1,Brilliant Violet 421,BioLegend；anti-LY49H,FITC,eBioscience)，上机分析(Beckman CytoFlex LX流式细胞分析仪)。
13.数据统计分析
数据以平均值±标准误(means±s.e.m)的方式呈现。P值通过t检验(student′s test)或者ANOVA分析得出，显著性表示为*：P<0.05，**：P<0.01，***：P<0.001。
实施例1.分泌蛋白NUCB1和NUCB2特异性结合NK细胞表面受体LY49H
通过质谱本发明人发现LY49H可能是NUCBs的结合蛋白，克隆了C57BL/6小鼠中LY49受体家族蛋白的10个成员(LY49A，LY49B，LY49C，LY49D，LY49E，LY49F，LY49G，LY49H，LY49I，LY49Q)的细胞膜外段至表达载体PFUSE-hIgG1-Fc2(IL2ss)中，因为此载体含有IL2信号肽，克隆到此载体的基因表达的蛋白将分泌到细胞培养基中。同时克隆全长小鼠NUCB1和NUCB2的蛋白表达序列到真核表达载体pCMV-HA中，因为NUCBs基因自身有分泌信号肽也将分泌到细胞培养基中。同时一起转染LY49家族质粒和NUCBs表达质粒到293T细胞后都将分泌到细胞培养基中(图1A,1B)，此***能很好的检测蛋白的相互作用。收集共转染的293T细胞24-30小时后的细胞培养基，免疫共沉淀发现NUCB1和NUCB2都特异性地结合LY49家族的LY49H(图1C,1D)。接下来纯化了真核293T细胞表达(图1E)和原核大肠杆菌表达(图1F)的NUCB1和NUCB2蛋白，验证了NUCB1和NUCB2与LY49H的直接相互作用。
实验结果表明，NUCBs是潜在的LY49H的配体。
实施例2.LY49H的C-型凝集素样结构域特异结合NUCBs
对受体LY49H的胞外端作了5个截短形式的质粒，这些片段与NUCBs的结合实验发现其C-型凝集素样结构域(CTLD，143-266位氨基酸)是结合NUCBs必需的(图2A-C)。比对LY49家族分子的C-型凝集素样结构域氨基酸序列发现，LY49H相比最为相近的LY49F，LY49C，LY49I，有3个氨基酸残基是LY49H所特有的，具体为L196，H216和T232(图2D,2E)。对LY49H这三个氨基酸位点做点突变，分别突变成与其相近受体所对应的氨基酸残基(L196P，196位的亮氨酸突变成脯氨酸；H216D，216位的组氨酸突变成天冬氨酸；T232K，232位的 苏氨酸突变成赖氨酸)。
结果显示，L196，H216是决定其和NUCBs特异结合的关键氨基酸残基(图2F,2G)。因此，如图2H所示，NUCBs结合在LY49H的C-型凝集素样结构域，其中第196位亮氨酸与216位组氨酸是关键氨基酸。
实施例3.NUCBs的第40-80位氨基酸是结合LY49H的关键区段
参见图3。如图3A所示，从C端逐渐截短NUCBs，分别得到3种截短形式：NUCB1(1-320)，NUCB1(1-230)，NUCB1(1-160)，NUCB2(1-320)，NUCB2(1-230)，NUCB2(1-160)。这些截短型都能很好的结合LY49H(图3B)。因此，NUCBs的1-160位氨基酸可能才是负责结合LY49H的区段。
如图3C，从N端逐渐截短NUCBs的1-160位，分别得到NUCB1(缺25-44)，NUCB1(缺44-80)，NUCB1(缺80-120)，NUCB1(缺120-159)，NUCB2(缺24-44)，NUCB2(缺44-80)，NUCB2(缺80-125),NUCB2(缺125-159)。
这些截短型和全长比，NUCB1(缺44-80)与NUCB2(缺44-80)显著的影响与LY49H的结合(图3D)。
结果表明，NUCBs的第40-80位氨基酸是结合LY49H的关键区段。
实施例4.过表达NUCBs蛋白抑制肿瘤细胞在小鼠体内生长
通过慢病毒感染MC38细胞，挑取单克隆获得了高表达NUCB1或NUCB2的稳定细胞株，收取细胞培养液检测到稳定株细胞大量分泌NUCB1或NUCB2蛋白到细胞培养液(图4A,B)。细胞增殖检测，过表达NUCB1或NUCB2不影响细胞本身的增殖(图4C)。将5×10 5野生型或NUCBs过表达株皮下移植到C57雄鼠，与野生型细胞比较。
结果如图4D-F所示，过表达NUCB1或NUCB2的MC38瘤生长速度显著降低，小鼠的生存率明显延长。
实施例5.注射NUCBs蛋白肽段抑制肿瘤在小鼠体内生长
根据NUCBs蛋白具体负责结合LY49H的氨基酸序列，合成了如图5A-C所示的肽段序列及对照实验用肽段序列：
NUCB1肽段(45-81位氨基酸)：
Figure PCTCN2021143241-appb-000012
NUCB2肽段(49-85位氨基酸)：
Figure PCTCN2021143241-appb-000013
对照肽段：
Figure PCTCN2021143241-appb-000014
C57的野生型小鼠皮下都接种5×10 5的MC38细胞并分为3组，5天后开始腹腔注射多肽。分别注射对照肽段，NUCB1肽段和NUCB2肽段，每天早晚两次，每次剂量为100ug/只。从第5天开始测量肿瘤大小，每2天测量一次。
如图5D,E所示，10天后，注射NUCB1肽段或NUCB2肽段都能明显的减小肿瘤的大小。
人和鼠NUCBs蛋白进化上高度保守，比较功能肽段序列，人鼠NUCB1肽段相似性97.3％，NUCB2肽段相似性91.9％(图5F)。结果提示，人NUCB肽段也可能存在潜在的抑制肿瘤的作用。
实施例6.构建获得NUCB1与NUCB2单敲除小鼠和NUCB1与NUCB2同时敲除的双敲除小鼠
C57BL/6小鼠的NUCB1基因共有13个外显子，其中第2到13为编码外显子。用CRISPR-Cas9的方法，选择在2号外显子编码信号肽序列处设计sgRNA而达到敲除目的。测序确认了NUCB1基因2号外显子敲除了信号肽内的10个碱基，达到了移码的目的(图6A,B)。基因组PCR和mRNA水平的荧光定量PCR都确认了NUCB1的敲除(图6C,D)。
NUCB2基因共有14个外显子，其中外显子第3到14为编码外显子。用CRISPR-Cas9的方法，选择在3号外显子编码信号肽序列处设计2条sgRNA而达到一定长度敲除目的。测序确认NUCB2基因3号外显子敲除了包括部分信号肽编码序列在内的83个碱基，也达到了移码的目的(图6E,F)。分别在基因组水平和蛋白质水平都确定了NUCB2敲除小鼠构建成功(图6G,H)。分别拿到NUCB1和NUCB2单基因编辑小鼠后，两种小鼠杂交得到双基因编辑小鼠，进而得到NUCB1和NUCB2双基因纯合敲除小鼠(NUCB1&2)。
实施例7.NUCBs基因敲除小鼠促进了肿瘤的生长
NUCBs家族包括同源性很高的两个成员，即NUCB1和NUCB2。生化实验表明：NUCB1和NUCB2都能结合LY49H。
小鼠实验表明：细胞过表达NUCB1或NUCB2都有显著的抑制肿瘤的作用；外加NUCB1肽段或NUCB2肽段也能显著抑制肿瘤生长。利用获得的NUCBs敲除小鼠，分别皮下都接种5×10 5的MC38细胞于NUCB1和NUCB2单独敲除小鼠以及NUCB1&2双敲除小鼠。
结果显示：不管是雄鼠(图7A-C)还是雌鼠(图7D-F)，双敲除NUCB1&2都促进了肿瘤的生长，减短了生存期。但是单独敲除NUCB1或NUCB2没有效果，说明NUCBs家族的两个基因有代偿效应，沉默其中一个功能可能会被另外一个同源基因补偿。
综上实验结果，基因敲除小鼠更进一步地证明了NUCBs的抗肿瘤作用。
实施例8.NUCBs蛋白促进LY49H阳性NK细胞的扩增
图8A是流式细胞分析LY49H阳性细胞情况的圈门流程图。野生型C57雄鼠皮下接种MC38细胞和NUCB1/2过表达MC38细胞，第20天时过表达细胞肿瘤体积远小于野生型，收集脾脏流式分析发现LY49H阳性的NK细胞比例在过表达NUCB1/2的小鼠中显著增高(图8B)。同时，第15天时比较载瘤(MC38)的野生型雌鼠和NUCB1&2双敲除雌鼠，敲除小鼠的肿瘤重量显著高于野生型小鼠，流式分析显示敲除小鼠脾脏的LY49H阳性NK细胞比例显著降低(图8C)。C57野生型雄鼠皮下接种MC38细胞，从第2天开始，连续8天腹腔注射NUCB2肽段和肽段溶剂PBS，第14天时NUCB2多肽组肿瘤体积显著减小，脾脏的LY49H阳性NK细胞比例增加(图8D)。图8E为NUCBs促进LY49H阳性NK细胞增殖模式图。
结果表明，NUCB蛋白过表达减小了肿瘤大小，促进了小鼠脾脏LY49H阳性NK细胞的扩增(8B)；NUCB1&2基因敲除增加了肿瘤的重量，降低了小鼠脾脏LY49H阳性NK细胞的数量(8C)；腹腔注射NUCB2肽段减小肿瘤体积，增加LY49H阳性NK细胞的数量(8D)。
正反两方面的结果表明NUCBs蛋白结合LY49H后的一个重要的生理作用是促进了LY49H阳性的NK细胞的扩增，进而减小肿瘤。
实施例9.人中的NUCB1结合NK细胞表面受体KIR3DS1和KIR3DL1
图9A-C显示人中NUCB1蛋白结合KIR家族中的激活型受体KIR3DS1(B中KIR2DS1-5为KIR家族激活型受体，C中KIR2DL1-5，KIR3DL1-3为KIR家族中抑制性受体)。相比较于人NK细胞表面其他受体，如NKG2D,TIGIT,DNAM1,NKP30和NKP46，人NUCB1也高特异性的结合KIR3DS1(图9D)。根据人NUCB1的二级机构预测分片段显示NUCB1的45-138位是主要结合KIR3DS1的区段(图9E)。进一步细分核心氨基酸序列，显示人NUCB1的45-63位氨基酸是结合KIR3DS1和KIR3DL1的核心(图9F)。与前面小鼠中NUCBs的44-80位一致，而且更加的核心。
讨论
人中NUCBs对NK细胞的作用和受体
NUCBs在进化中人鼠高度保守，在小鼠中表现出显著的***的作用。后续一方面将通过人NK细胞系NK92和分离原代人血NK细胞，检测NUCBs蛋白对人NK细胞的作用和机制。由于人NK细胞中对应LY49受体家族的为KIR受体家族，包括8个抑制性受体:KIR2DL1-5，KIR3DL1-3，以及6个激活型受体， KIR2DS1-5，KIR3DS1，其中多个受体目前没有发现其配体。这些是最重要的潜在NUCBs的受体。后续第二方面本发明人将首要验证KIR受体家族及NKG2C/CD94受体，找到人NK细胞NUCBs受体。以上受体作为NK细胞的潜在靶点，对肿瘤的免疫治疗具有重要意义。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种核连蛋白(Nucleobindin，NUCB)或其促进剂的用途，其特征在于，用于制备药物或制剂，所述药物或制剂(a)用于促进NK细胞的扩增，(b)***；和/或(c)杀伤或抑制肿瘤细胞。
  2. 如权利要求1所述的用途，其特征在于，所述NUCB蛋白包括NUCB1蛋白和/或NUCB2蛋白。
  3. 如权利要求1所述的用途，其特征在于，所述NUCB1蛋白的氨基酸序列如SEQ ID NO.:4或SEQ ID NO.6所示或与SEQ ID NO.:4或SEQ ID NO.6的相同性≥85％(较佳地≥90％，更佳地≥95％或≥98％)。
  4. 如权利要求1所述的用途，其特征在于，所述NUCB2蛋白的氨基酸序列如SEQ ID NO.:5或SEQ ID NO.7所示或与SEQ ID NO.:5或SEQ ID NO.7的相同性≥85％(较佳地≥90％，更佳地≥95％或≥98％)。
  5. 如权利要求1所述的用途，其特征在于，所述NUCB蛋白包括全长的NUCB蛋白、成熟的NUCB蛋白、NUCB的与LY49H结合的关键区段、或含所述关键区段的NUCB活性片段。
  6. 如权利要求1所述的用途，其特征在于，所述NUCB蛋白特异性结合NK细胞表面受体LY49H。
  7. 一种体外促进NK细胞扩增的方法，其特征在于，包括步骤：
    (a)提供一NUCB蛋白或其促进剂；和
    (b)在所述NUCB蛋白或其促进剂的存在下，培养NK细胞，从而促进NK细胞扩增；。
  8. 如权利要求7所述的方法，其特征在于，所述NK细胞为表面受体LY49H阳性的NK细胞。
  9. 一种分离的复合物，其特征在于，所述复合物为NUCB蛋白与LY49H相结合所形成的二元复合物。
  10. 如权利要求9所述的复合物，其特征在于，所述NUCB蛋白的氨基酸序列选自下组：
    (a)具有SEQ ID NO.:4或5所示的氨基酸序列；
    (b)将如SEQ ID NO.:4或5所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述NUCB活性的由(a)衍生的多肽；或
    (c)氨基酸序列与SEQ ID NO.:4或5所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述NUCB活性的多肽。
  11. 一种如权利要求9所述的复合物的用途，其特征在于，用于筛选促进NK细胞扩增的药物或化合物。
  12. 一种筛选促进NK细胞扩增药物或化合物的方法，其特征在于，包括步骤：
    (a)在测试组中，在待测物质存在下，培养NK细胞，并且设置无待测物质的对照组；
    (b)检测测试组中所述复合物的含量H1并与对照组中的复合物含量H0进行比较，其中当H1显著高于H0，则表示所述测试物为促进NK细胞扩增的药物或化合物。
  13. 一种***的方法，其特征在于，向需要的对象施用治疗有效量的NUCB蛋白或其促进剂。
  14. 如权利要求13所述的方法，其特征在于，所述肿瘤选自下组：结肠癌、乳腺癌、肺癌、胃癌、肝癌、多发性骨髓瘤、肾癌、胰腺癌、黑色素瘤、淋巴瘤、甲状腺癌、或其组合。
  15. 一种LY49H蛋白或其活性片段的用途，其特征在于，用于制备一种治疗NUCB蛋白过高的相关疾病的药物组合物。
  16. 如权利要求15所述的方法，其特征在于，所述NUCB蛋白过高的相关疾病选自下组：多囊性卵巢综合征(Polycystic Ovary Syndrome)、高血压(Hypertention)、焦虑(Anxiety)、癫痫(Epilepsy)或其组合。
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