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WO2023016509A1 - 抑制肿瘤细胞转移的药物及其用途 - Google Patents

抑制肿瘤细胞转移的药物及其用途 Download PDF

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WO2023016509A1
WO2023016509A1 PCT/CN2022/111658 CN2022111658W WO2023016509A1 WO 2023016509 A1 WO2023016509 A1 WO 2023016509A1 CN 2022111658 W CN2022111658 W CN 2022111658W WO 2023016509 A1 WO2023016509 A1 WO 2023016509A1
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lgmn
integrin
protein
subunit
antibody
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陈剑峰
刘翠
王军磊
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Center for Excellence in Molecular Cell Science of CAS
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the invention relates to the field of tumor treatment, in particular to a drug for inhibiting tumor cell metastasis and its application.
  • Breast cancer is one of the most common malignant tumors in women worldwide, and distant metastasis of breast cancer is the main cause of high mortality. Metastasis of vital organs throughout the body such as bone, lung, liver, and brain is life-threatening. Tumor metastasis is a complex multi-stage process. Tumor cells invade lymphatic vessels, blood vessels, etc. from the primary site, and reach other tissue sites to continue to grow and form metastases. Although some achievements have been made in the treatment of breast cancer in recent years, there is no completely effective treatment for breast cancer metastasis, and its five-year survival rate is only 26%, which has become the main cause of death of breast cancer patients. Therefore, exploring effective targets for breast cancer metastasis is also a research hotspot and frontier topic in the field of life sciences. A better understanding of the mechanism of breast cancer metastasis can provide new ideas for the treatment of metastatic tumors.
  • Integrin is a kind of adhesion molecule (cell adhesion molecule, CAM) widely expressed on the surface of cell membrane, which is a heterodimer formed by two subunits of ⁇ and ⁇ through non-covalent bonds. Integrins can be roughly divided into two types according to their ligand-binding properties, one is binding to extracellular matrix proteins (ECM), and the other is binding to cell surface adhesion molecules.
  • ECM extracellular matrix proteins
  • Asparagine endopeptidase is a member of the C13 cysteine protease family, which can specifically cut the asparagine residue at the protein end.
  • the pro-LGMN synthesized in the cytoplasm is an inactive zymogen form.
  • the activation of LGMN needs to be cleaved under the strict acidic conditions of lysosomes.
  • LGMN protein can also be secreted to the outside of the cell as a component of the extracellular matrix. Studies have shown that LGMN is highly expressed in various tumor tissues compared with normal tissues.
  • Small molecule drugs in the prior art inhibit the growth of tumor in situ mainly by inhibiting the activity of LGMN enzymes.
  • In-depth study of the mechanism of action of LGMN in tumor cells will help to develop relevant therapeutic and diagnostic methods and products.
  • LGMN is related to cancer metastasis and prognosis, and it binds to integrin ⁇ subunit through RGD sequence, thereby regulating tumor metastasis.
  • the first aspect of the present invention provides the use of a reagent for inhibiting the interaction between LGMN protein and integrin or its ⁇ subunit in the preparation of a drug for inhibiting tumor metastasis or tumor cell metastasis.
  • the interaction is between the RGD sequence of the LGMN protein and integrin or its beta subunit.
  • the ⁇ subunit of the integrin is selected from one or more of the following: ⁇ 1 , ⁇ 3 , ⁇ 5 , ⁇ 6 and ⁇ 8 ; the integrin contains the ⁇ Integrin subunit.
  • the integrin is selected from one or more of the following: ⁇ v ⁇ 3, ⁇ v ⁇ 1 , ⁇ v ⁇ 5 , ⁇ v ⁇ 6 , ⁇ v ⁇ 8 , ⁇ 5 ⁇ 1 and ⁇ 8 ⁇ 1 .
  • the agent is selected from one or more of the following:
  • mutant LGMN proteins and/or their targeting vectors wherein, compared with the wild type, the RGD sequence of the mutant LGMN has a mutation that causes the interaction between LGMN and integrin or its ⁇ subunit to be weakened or disappeared;
  • mutant integrin or its ⁇ subunit and/or its targeting vector wherein, compared with the wild type, the mutant integrin or its ⁇ subunit has one or more in the ligand binding region interacting with LGMN protein Multiple mutations that lead to reduced or absent interaction with LGMN protein;
  • nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described in (3) or (4), or a nucleic acid construct comprising the nucleic acid molecule;
  • EDTA ethylenediaminetetraacetic acid
  • NTA aminotriacetic acid
  • CA citric acid
  • TA tartaric acid
  • GA gluconic acid
  • Reagents for knocking down or knocking down the expression of LGMN protein and/or integrin or its beta subunit such as ZFN and/or TALEN and/or CRISPR/Cas9 reagents and/or small interfering RNA;
  • the mutation is a RGD sequence mutation to RGE, RAD, RAE, KGD or KGE.
  • the integrin or its beta subunit has a mutation at the D120 and/or S121 position; preferably, the mutation is a substitution mutation; more preferably, the substitution The last amino acid residue is alanine or glutamic acid.
  • the small interfering RNA has the sequence shown in SEQ ID NO:5 or 6.
  • the tumor is selected from one or more of the following: breast cancer, lung adenocarcinoma, colon cancer, gastric adenocarcinoma, breast cancer, hepatocellular carcinoma, pancreatic cancer.
  • mutant LGMN protein, mutant integrin or ⁇ subunit thereof, anti-LGMN antibody or antigen-binding fragment thereof, and small interfering RNA are as described in any embodiment herein.
  • the present invention also provides a mutant LGMN protein.
  • the RGD sequence of the mutant LGMN protein has one or more mutations that lead to the weakening or disappearance of the interaction between LGMN and integrin or its ⁇ subunit.
  • the precursor is pro-LGMN protein.
  • the RGD sequence of the mutant LGMN protein is mutated to RGE, RAD, RAE, KGD or KGE compared to the wild type.
  • the present invention also provides a mutant integrin or its beta subunit, which has one or more mutations in the ligand-binding region interacting with the LGMN protein compared with the wild type, resulting in the weakening or disappearance of its interaction with the LGMN protein;
  • a mutation at D120 and/or S121 preferably, the mutation is a substitution mutation; more preferably, the amino acid residue after substitution is alanine or glutamic acid.
  • the ⁇ subunit of the integrin is selected from one or more of the following: ⁇ 1 , ⁇ 3 , ⁇ 5 , ⁇ 6 and ⁇ 8 ; the integrin contains the ⁇ Integrin subunit.
  • the integrin is selected from one or more of the following: ⁇ v ⁇ 3, ⁇ v ⁇ 1 , ⁇ v ⁇ 5 , ⁇ v ⁇ 6 , ⁇ v ⁇ 8 , ⁇ 5 ⁇ 1 and ⁇ 8 ⁇ 1 .
  • This article also provides the use of the LGMN protein and/or integrin or its ⁇ subunit as a target in screening drugs for inhibiting tumor metastasis or tumor cell metastasis, or as a molecular indicator for clinical diagnosis of tumor metastasis.
  • the tumor is selected from one or more of the following: breast cancer, lung adenocarcinoma, colon cancer, gastric adenocarcinoma, breast cancer, hepatocellular carcinoma, pancreatic cancer.
  • the medicament inhibits the interaction between LGMN protein and integrin or its beta subunit.
  • the drug attenuates the interaction of the RGD sequence of LGMN protein with integrin or its beta subunit.
  • the ⁇ subunit of the integrin is selected from one or more of the following: ⁇ 1 , ⁇ 3 , ⁇ 5 , ⁇ 6 and ⁇ 8 ;
  • the integrin is an integrin comprising the beta subunit.
  • the integrin is selected from one or more of the following: ⁇ v ⁇ 3, ⁇ v ⁇ 1 , ⁇ v ⁇ 5 , ⁇ v ⁇ 6 , ⁇ v ⁇ 8 , ⁇ 5 ⁇ 1 and ⁇ 8 ⁇ 1 .
  • the target site includes the RGD sequence of the LGMN protein, and/or, the ligand-binding region of integrin or its beta subunit that interacts with the LGMN protein.
  • the present invention also provides an anti-LGMN antibody or an antigen-binding fragment thereof, the CDRs of which are as follows: HCDR1 contains GFTFSSYA, HCDR2 contains IGNSGNYT, HCDR3 contains AKSSDSFNY, LCDR1 contains QSISSY, LCDR2 contains DAS, and LCDR3 contains QQAYANPDT.
  • the antibody inhibits the interaction between an LGMN protein and an integrin or a beta subunit thereof.
  • the FR region of the VH of the anti-LGMN antibody is the FR region of the VH shown in SEQ ID NO:1
  • the FR region of the VL is the FR region of the VL shown in SEQ ID NO:2.
  • the VH and VL of the antibody have the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 or variants having 90% sequence identity thereto.
  • the amino acid sequence of the heavy chain constant region of the antibody has the sequence shown in SEQ ID NO:3, and/or the amino acid sequence of the light chain constant region has the sequence shown in SEQ ID NO:4 .
  • the anti-LGMN antibody according to any embodiment of the present invention is a chimeric antibody or a fully human antibody; preferably a fully human antibody.
  • the present invention also provides a nucleic acid molecule or a nucleic acid construct comprising said nucleic acid molecule, said nucleic acid molecule comprising a sequence selected from: (1) a sequence encoding an anti-LGMN antibody or an antigen-binding fragment thereof described herein, or (2 ) the complementary sequence of (1).
  • the nucleic acid construct is a cloning vector, an integrating vector or an expression vector.
  • the invention also provides host cells that: (1) express an anti-LGMN antibody or antigen-binding fragment thereof described herein, or (2) comprise a nucleic acid molecule or nucleic acid construct described herein.
  • the present invention also provides the coding sequence of the mutant LGMN protein, the mutant integrin or its ⁇ subunit, or the anti-LGMN antibody or its antigen-binding fragment described herein or its complementary sequence, and the nucleic acid containing the coding sequence or its complementary sequence Use of the construct, and host cells expressing the mutant LGMN protein, mutant integrin or its ⁇ subunit, or anti-LGMN antibody in the preparation of drugs for inhibiting tumor metastasis or tumor cell metastasis.
  • the host cell contains the coding sequence or its complement or the nucleic acid construct.
  • the present invention also provides a pharmaceutical composition, which contains:
  • the present invention also provides a small interfering RNA for knocking down LGMN, which has the sequence shown in SEQ ID NO: 5 or 6.
  • the present invention also provides a small interfering RNA for knocking down integrin or its beta subunit, which has the sequence shown in SEQ ID NO:7.
  • the present invention also provides a method for inhibiting tumor cell metastasis in vitro, including the step of weakening the interaction between LGMN protein of tumor cells and integrin or its beta subunit.
  • the method includes weakening the interaction between the LGMN protein of tumor cells and integrin or its ⁇ subunit by any one or more of the following methods:
  • the interaction is between the RGD sequence of LGMN and an integrin or a beta subunit thereof.
  • the ⁇ subunit of the integrin is selected from one or more of the following: ⁇ 1 , ⁇ 3 , ⁇ 5 , ⁇ 6 and ⁇ 8 ; the integrin contains the ⁇ Integrin subunit.
  • the integrin is selected from one or more of the following: ⁇ v ⁇ 3, ⁇ v ⁇ 1 , ⁇ v ⁇ 5 , ⁇ v ⁇ 6 , ⁇ v ⁇ 8 , ⁇ 5 ⁇ 1 and ⁇ 8 ⁇ 1 .
  • the method has one or more features selected from:
  • the RGD sequence of the mutant LGMN protein has a mutation that causes the interaction with integrin or its ⁇ subunit to be weakened or disappeared;
  • the mutant integrin or its ⁇ subunit has one or more mutations in the ligand-binding region interacting with LGMN that lead to the weakening or disappearance of its interaction with LGMN;
  • the anti-LGMN antibody inhibits the interaction between LGMN and integrin or its beta subunit
  • the reagent that can reduce the concentration of divalent metal ions is EDTA;
  • the reagent for knocking down or knocking out the expression of LGMN and/or integrin or its beta subunit is ZFN and/or TALEN and/or CRISPR/Cas9 reagent and/or small interfering RNA.
  • mutant LGMN protein, mutant integrin or ⁇ subunit thereof, anti-LGMN antibody or antigen-binding fragment thereof, and small interfering RNA are as described in any embodiment herein.
  • the invention also provides the use of the reagent for detecting LGMN protein in the preparation of a kit for diagnosing tumor metastasis.
  • the reagent detects the expression, content or sequence of LGMN protein in tumor tissue.
  • the reagents include: antibodies against the LGMN protein, primers and/or probes that hybridize to the coding sequence of the LGMN protein.
  • the present invention also provides a method for screening agents that weaken or destroy the interaction between intracellular LGMN protein integrins or their ⁇ subunits.
  • the present invention also provides the use of LGMN in activating FAK, Src and RhoA.
  • the present invention also provides a method for constructing a mouse model of breast cancer bone metastasis, comprising the step of injecting breast cancer cells expressing markers into nude mice through the left ventricle.
  • the breast cancer cell is SCP2.
  • the marker is luciferase.
  • FIG. 1 Highly expressed LGMN is associated with breast cancer metastasis and prognosis.
  • A Venn diagram showing genes associated with breast cancer in three databases GSE45255, GSE65194, GSE22219.
  • B Gene Ontology analysis of 25 genes related to breast cancer metastasis.
  • C-E Kaplan-Meier analysis of the relationship between the expression level of LGMN and the metastasis and survival rate of breast cancer patients, the data are from GSE45255, GSE65194, GSE22219 databases, respectively.
  • F-G Cell migration and invasion abilities of normal breast epithelial cells MCF10A and breast cancer cell lines.
  • H-I Western blot assay to detect the expression level of LGMN in normal breast epithelial cells MCF10A and breast cancer cell lines (H) and the content of LGMN secreted into the culture medium (I).
  • Pro-LGMN is positively correlated with breast cancer metastasis.
  • A 25 genes associated with metastasis are listed.
  • B-C Kaplan-Meier survival analysis of the relationship between LGMN expression and metastasis-free survival (B) and overall survival (C) of breast cancer patients, data from http://kmplot.com/analysis/.
  • D-I The relationship between the content of pro-LGMN and the activated form of LGMN in whole cell lysate (WCL) and conditioned medium (CM) and breast cancer cell migration ability (D, F, H) and invasion ability (E, G , I) the correlation between.
  • LGMN mediates binding to ⁇ 3 integrin through its RGD sequence.
  • A Co-immunoprecipitation results of endogenous LGMN and ⁇ 3 integrin in Hs578T, MDA-MB-231 and SCP2 cells;
  • B Co-IP assay to detect the effect of LGMND120E on the binding of ⁇ 3 integrin to LGMN;
  • C Co-IP Experimental detection of the effect of EDTA on the binding of LGMN to ⁇ 3 integrin;
  • D Co-IP test to detect the effect of MIDAS mutation ⁇ 3S121A on the binding of ⁇ 3 integrin to LGMN;
  • E Co-IP test to detect the effect of LGMN enzyme activity mutation LGMNC189S on ⁇ 3 integration The effect of the binding of the protein to LGMN.
  • LGMN promotes tumor metastasis dependent on binding to integrins.
  • Hs578T, MDA-MB-231 and SCP2 cells stably and lowly expressing LGMN the control plasmid, wild-type LGMN, mutant LGMN D120E and LGMN C189S were respectively transfected to the three kinds of tumor cell wound healing experiments (A), Transwell migration (B ) and invasion (C) effects. Effects of purified LGMN wild-type protein and LGMN D120E protein on wound healing (D), Transwell migration (E) and invasion (F) of three tumor cells knocked down by LGMN. All data were averaged from more than three parallel experiments and error bars were drawn.
  • FIG. 7 the expression of integrin ⁇ v ⁇ 3.
  • Flow cytometry was used to detect the expression levels of integrin ⁇ v ⁇ 3 in Hs578T, MDA-MB-231 and SCP2 cells in different stable transfected cells expressing LGMN.
  • the P value was analyzed by one-way ANOVA statistical method.
  • FIG. 8 LGMN binding to integrins promotes bone metastasis of breast cancer in vivo.
  • the effects of control group, stable low-expression LGMN and stable expression of wild-type LGMN or point mutation LGMND120E on bone metastasis ability of breast cancer cell SCP2 were detected.
  • the P value was analyzed by one-way ANOVA statistical method, *P ⁇ 0.05, ***P ⁇ 0.001.
  • LGMN expression does not affect breast cancer growth in situ.
  • the growth of SCP2 breast cancer cells in situ breast cancer in the control group, stable low-expression LGMN and stable expression of wild-type LGMN or point mutation LGMND120E were detected respectively (n 5-6/group).
  • FIG. 10 Knockdown of LGMN or integrin ⁇ 3 can inhibit lung metastasis of orthotopic tumors in SCP2 breast cancer cells.
  • the SCP2 cells of the control group, LGMN knockdown group and ⁇ 3 knockdown group were injected into the mammary fat pad of B-NDG mice respectively, and the fluorescence representation (A) and fluorescence quantitative results (B) of orthotopic tumor lung metastasis are shown in the figure shown.
  • n 6/group.
  • P values were obtained using the statistical method of one-way ANOVA.
  • NS Not significant. **P ⁇ 0.005.
  • FIG. 13 Anti-LGMN antibodies identified by phage screening.
  • A,B ELISA assay to detect the binding of scFv to LGMN protein (A) and RGD polypeptide (B).
  • C The monoclonal antibody inhibited the binding of LGMN protein to SCP2 cells by flow cytometry.
  • C10 antibody inhibits bone metastasis of breast cancer.
  • A The binding between C10 antibody and LGMN protein was determined by Octet RED 96 instrument using the principle of biofilm interference. Biotin-conjugated LGMN was captured by streptavidin and immobilized on the chip to test its binding ability to C10 antibody with various concentration gradients. Binding kinetics was evaluated using a 1:1 Langmuir binding model and ForteBio Data Analysis 9.0 software.
  • B IC50 of C10 antibody inhibiting the binding of SCP2 cell surface integrin to FITC-labeled LGMN protein.
  • C10 antibody can specifically inhibit the interaction between LGMN and integrin ⁇ v ⁇ 3.
  • Pull-down experiments were used to detect the interaction between pro-LGMN-flag protein and integrin ⁇ v ⁇ 3, and to detect the effect of C10 antibody treatment on the binding of pro-LGMN-flag protein and integrin ⁇ v ⁇ 3.
  • LGMN is highly expressed in various tumor tissues.
  • the GEPIA database compares the expression of LGMN in various tumor tissues and their corresponding normal tissues.
  • C10 antibody can specifically inhibit the migration ability of T47D cells.
  • the effect of C10 antibody treatment on the migration of T47D cells co-expressed with LGMN and integrin ⁇ v ⁇ 3 was detected, and human IgG was used as the control group.
  • C10 antibody can specifically inhibit the migration ability of MNK-45 gastric cancer cells and A549 lung cancer cells.
  • the effect of C10 antibody treatment on the migration of MNK-45 gastric cancer cells and A549 lung cancer cells was detected, and human IgG was used as the control group.
  • A Western blot experiments showed that compared with normal breast epithelial cells MCF-10A, both MNK-45 gastric cancer cells and A549 lung cancer cells had higher expression of LGMN.
  • B The results of transwell experiments show that C10 antibody can inhibit the migration ability of MNK-45 gastric cancer cells and A549 lung cancer cells in vitro.
  • C10 antibody can specifically inhibit the migration ability of SW480 colon cancer cells.
  • the expression level of LGMN in SW480 colon cancer cells was detected by Western blot, and A549 lung cancer cells were used as positive control (A).
  • A A549 lung cancer cells
  • B human IgG was used as the control group.
  • FIG. 20 Binding of integrins ⁇ 1, ⁇ 5, ⁇ 6 and ⁇ 8 to LGMN protein.
  • Co-IP assay was used to detect the binding of integrins ⁇ 1, ⁇ 5, ⁇ 6 and ⁇ 8 to LGMN protein in Hs578T, MDA-MB-231 and SCP2 cells.
  • Inner FAK-Src-RhoA signaling pathway promotes migration and invasion of breast cancer cells.
  • the RGD sequence mutation can block the combination of LGMN and integrin, thereby blocking the migration and invasion of breast cancer cells mediated by LGMN and the metastasis of breast cancer tissues in mice. Therefore, blocking the combination of RGD domain of LGMN and integrin will be one of the effective ways to inhibit the metastasis of breast cancer.
  • breast cancer cells include but are not limited to MDA-MB-231, SCP2, Hs578T, MCF7, T47D breast cancer cells or other breast cancer cells expressing LGMN known to those skilled in the art.
  • the breast cancer described herein can be a breast cancer associated with or comprising any of these breast cancer cells.
  • lung adenocarcinoma includes lung adenocarcinoma associated with or comprising the cells of the LUAD line of lung adenocarcinoma cells; colon cancer includes colon cancer associated with or comprising the cells of the COAD line of colon cancer cells; gastric adenocarcinoma includes those associated with the STAD line of gastric adenocarcinoma cells Gastric adenocarcinoma related to cancer cells or containing the cells; hepatocellular carcinoma includes hepatocellular carcinoma related to or containing the cells of the LIHC line of hepatocellular carcinoma cells; pancreatic cancer includes pancreatic cancer related to or containing the cells of the PAAD line of pancreatic cancer cells.
  • LGMN is synthesized and secreted in the cytoplasm in the form of pro-LGMN. After pro-LGMN is cleaved, it will form 47KD, 46KD LGMN intermediates and mature active LGMN (active-LGMN, 26-289aa). See Structure and function of legumain in health and disease, Elfriede Dal et, al. Biochimie, 2016. Thus, "LGMN” or “LGMN protein” herein includes pro-LGMN, active LGMN, and LGMN intermediates. Similarly, "mutated LGMN” or “mutated LGMN protein” includes mutated pro-LGMN, mutated active LGMN, and mutated LGMN intermediates.
  • the invention provides a method for inhibiting tumor cell metastasis in vitro, including the step of weakening the interaction between LGMN protein of tumor cells and integrin or its beta subunit.
  • the interaction is between the RGD sequence of LGMN and integrin or its beta subunit.
  • the method weakens the interaction by any one or more of the following methods: (1) knocking out or knocking down the expression of LGMN protein in tumor cells; (2) knocking out or knocking down integrin or its ⁇ in tumor cells Subunit expression; (3) In wild-type or knockout LGMN protein tumor cells express mutant LGMN protein with weakened or disappeared interaction with integrin or its ⁇ subunit; (4) In wild-type or knockout integrin In the tumor cells expressing the mutant integrin or its ⁇ subunit whose interaction with LGMN protein is weakened or disappeared; protein contacting of an anti-LGMN antibody or antigen-binding fragment thereof; (6) contacting tumor cells with an agent that reduces the concentration of divalent metal ions.
  • the method includes, but is not limited to: using an expression vector and/or an integration vector of a mutant LGMN protein to treat tumor cells, and/or using an expression vector and/or an expression vector of a mutant integrin or its ⁇ subunit Integrate the vector to treat tumor cells, and/or use anti-LGMN antibody or its antigen-binding fragment to treat tumor cells, and/or use reagents that can reduce the concentration of divalent metal ions to treat tumor cells, and/or transfer gene knockout in tumor cells Remove the carrier to knock down the expression of LGMN protein and/or the expression of integrin or its ⁇ subunit in the tumor cells, and/or use technologies such as ZFN, TALEN or CRISPR/Cas9 to knock down the expression and/or integration of LGMN protein in tumor cells expression of LGMN or its ⁇ subunit, and/or knock down the expression of LGMN protein and/or the expression of integrin or its ⁇ subunit by interfering RNA-mediated gene silencing,
  • the expression frame of the mutant LGMN protein and/or The expression cassette of mutant integrin or its ⁇ subunit whose interaction with LGMN protein is weakened or disappeared is integrated into the genome of tumor cells, thereby weakening or destroying the interaction between intracellular LGMN protein integrin or its ⁇ subunit .
  • the invention provides a reagent for inhibiting the interaction between LGMN protein and integrin or its beta subunit and its use in the preparation of drugs for inhibiting tumor metastasis or tumor cell metastasis.
  • the "interaction" of an LGMN protein with an integrin as described herein is the interaction between the RGD sequence of the LGMN protein and the integrin beta subunit.
  • "Beta subunits" of integrins as described herein include, but are not limited to: ⁇ 1 , ⁇ 3 , ⁇ 5 , ⁇ 6 and ⁇ 8 .
  • the integrin is an integrin comprising the beta subunit , such as ⁇ v ⁇ 3 , ⁇ v ⁇ 1 , ⁇ v ⁇ 5 , ⁇ v ⁇ 6 , ⁇ v ⁇ 8 , ⁇ 5 ⁇ 1 and ⁇ 8 ⁇ 1 .
  • wild-type LGMN is shown in GenBank No.CAG33687.1, wherein RGD is located at position 118; wild-type pro-LGMN is shown in GenBank No.CAG33687.1; the amino acid sequence of wild-type ⁇ v is shown in GenBank No.AGC09592 .1; the amino acid sequence of wild-type ⁇ 3 is shown in GenBank No.AAI27668.1.
  • the agent for inhibiting the interaction between LGMN protein and integrin or its beta subunit can be mutant LGMN protein and/or its targeting vector, wherein, compared with wild type, the RGD sequence of mutant LGMN causes LGMN to interact with integrin Mutations that weaken or disappear the interaction of its ⁇ subunits.
  • the mutation is a mutation of the RGD sequence to RGE, RAD, RAE, KGD or KGE.
  • the agent for inhibiting the interaction between LGMN protein and integrin or its ⁇ subunit can also be a mutant integrin or its ⁇ subunit and/or its targeting vector, wherein, compared with the wild type, the mutant integrin or its
  • the ⁇ subunit has one or more mutations in the ligand-binding region that interacts with the LGMN protein, resulting in weakened or disappearance of its interaction with the LGMN protein.
  • the integrin or its ⁇ subunit has a mutation at the D120 and/or S121 position, such as a substitution mutation; preferably the substitution is alanine or glutamic acid.
  • the reagent that inhibits the interaction between LGMN protein and integrin or its ⁇ subunit can also be a reagent that can reduce the concentration of divalent metal ions, such as ethylenediaminetetraacetic acid (EDTA), aminotriacetic acid (NTA), citric acid ( CA), tartaric acid (TA) and gluconic acid (GA), etc.
  • EDTA ethylenediaminetetraacetic acid
  • NTA aminotriacetic acid
  • CA citric acid
  • TA tartaric acid
  • GA gluconic acid
  • the reagent for inhibiting the interaction between LGMN protein and integrin or its ⁇ subunit can also be a reagent for knocking down or knocking out the expression of LGMN protein and/or integrin or its ⁇ subunit, such as ZFN and/or TALEN and/or or CRISPR/Cas9 reagents and/or small interfering RNA.
  • a reagent for knocking down or knocking out the expression of LGMN protein and/or integrin or its ⁇ subunit such as ZFN and/or TALEN and/or or CRISPR/Cas9 reagents and/or small interfering RNA.
  • the inhibitory agent is LGMN or integrin-specific shRNA or its construct, wherein the shRNA specifically recognizes LGMN or integrin genes or transcripts thereof.
  • a "transcript” comprises a UTR region (eg 3' UTR) and a CDS region.
  • the small interfering RNA with a better effect of knocking out LGMN has the sequence shown in SEQ ID NO: 5 or 6; sequence shown.
  • ZFN, TALEN and CRISPR/Cas9 technologies suitable for use in the present invention are well known in the art. Each technology realizes the knockout of the target gene through the joint action of the DNA recognition domain and the endonuclease.
  • the agent that inhibits the interaction between LGMN protein and integrin or its beta subunit can also be an anti-LGMN antibody or an antigen-binding fragment thereof that inhibits the interaction between LGMN protein and integrin or its beta subunit.
  • the agent that inhibits the interaction between LGMN protein and integrin or its beta subunit can also be an antibody or antigen-binding fragment thereof of integrin or its beta subunit, or a receptor inhibitor of integrin or its beta subunit (e.g. Receptor inhibitors, including but not limited to ⁇ v ⁇ 3 and ⁇ v ⁇ 5 inhibitors Cilengitide, ⁇ IIb ⁇ 3 inhibitors Tirofiban and Epifibatide, ⁇ IIb ⁇ 3 antibody Abciximab, ⁇ v ⁇ 1 inhibitor PLN-1474 and antibody Volociximab, ⁇ v ⁇ 6 antibody 264RAD, ⁇ 4 ⁇ 1 antibody Natalizumab wait).
  • Receptor inhibitors including but not limited to ⁇ v ⁇ 3 and ⁇ v ⁇ 5 inhibitors Cilengitide, ⁇ IIb ⁇ 3 inhibitors Tirofiban and Epifibatide, ⁇ IIb ⁇ 3 antibody Abciximab, ⁇ v ⁇ 1 inhibitor PLN-1474 and antibody Voloc
  • Agents described herein that inhibit the interaction between LGMN proteins and integrins or their beta subunits can also be used as vaccine adjuvants.
  • the vaccine adjuvant can be used in combination with a vaccine (such as OVA) to form a vaccine composition, which can be used to prevent and/or treat tumor metastasis.
  • antibody includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), Diabodies and single chain molecules, as well as antibody fragments, especially antigen-binding fragments, eg, Fab, Fab', F(ab')2, Fv and scFv).
  • immunoglobulin Ig
  • antibody antibody
  • the antigen-binding fragment is composed of or comprises a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient
  • the affinity, for LGMN is preferably at least equal to 1/100, in a more preferred manner at least equal to 1/10, of the affinity of the antibody from which it originates.
  • Such antibody fragments will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which they are derived.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds depending on the heavy chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2 and CH3 for each of the ⁇ and ⁇ chains) and four (CH1, CH1, CH3 for the ⁇ and ⁇ isotypes).
  • VH variable domain
  • VL variable domain
  • CL constant domain
  • immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called alpha, delta, epsilon, gamma, and mu, respectively.
  • the gamma and alpha classes can be further divided into subclasses based on relatively minor differences in CH sequence and function, eg humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
  • VH and VL of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody.
  • the variable domains of the heavy and light chains can be referred to as "VH” and “VL”, respectively. These domains are usually the most variable part of the antibody (relative to other antibodies of the same type) and contain the antigen binding site.
  • the VH and VL of the antibodies described herein have the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 or variants having 90% sequence identity thereto.
  • variable refers to the fact that certain segments of the variable domains vary widely among antibody sequences.
  • the variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across all amino acids spanned by a variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light and heavy chain variable domains), namely HCDR1, HCDR2, HCDR3 and light LCDR1, LCDR2, and LCDR3 of the chain variable region.
  • HVRs hypervariable regions
  • FR framework regions
  • variable domains of native heavy and light chains each comprise four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a ⁇ -sheet conformation connected by the formation of loops and in some cases forming ⁇ -sheet structures Part of three HVR connections.
  • the HVRs in each chain are held together in close proximity by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institutes of Health Institute, Bethesda, MD, 1991).
  • the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.
  • the constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as the involvement of antibodies in antibody-dependent cell-mediated cytotoxicity.
  • the CDRs of the anti-LGMN antibody of the present invention are as follows: HCDR1 contains GFTFSSYA, HCDR2 contains IGNSGNYT, HCDR3 contains AKSSDSFNY, LCDR1 contains QSISSY, LCDR2 contains DAS, and LCDR3 contains QQAYANPDT.
  • the CDRs described herein were obtained using the IMGT antibody numbering system. After knowing the sequence of the antibody or antigen-binding fragment thereof, those skilled in the art can easily use other numbering systems to obtain the corresponding CDRs.
  • Fc region fragment crystallizable region
  • Fc domain Fc domain refers to the C-terminal region of an antibody heavy chain, which mediates the binding of the immunoglobulin to host tissues or factors, including those located in the immune system. Binding to Fc receptors on various cells (eg, effector cells), or to the first component (Clq) of the classical complement system.
  • the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc region of IgM and IgE is present in each polypeptide chain Contains three heavy chain constant domains (CH domains 2-4).
  • the human IgG heavy chain Fc region is generally defined as the stretch from the amino acid residue at positions C226 or P230 of the heavy chain to the carboxy-terminus, where this numbering is according to the EU index.
  • the Fc region can be a native sequence Fc or a variant Fc.
  • the sequence of the variant described herein may be at least 95%, 96%, 97%, 98% or 99% identical to its source sequence. Sequence identity according to the invention can be measured using sequence analysis software. For example the computer program BLAST, especially BLASTP or TBLASTN, using default parameters.
  • the anti-LGMN antibodies of the invention can be modified to affect function.
  • the invention includes anti-LGMN antibodies with modified glycosylation patterns, or may be modified to remove undesired glycosylation sites.
  • the anti-LGMN antibody of the present invention can be prepared by conventional methods in the art, such as hybridoma technology well known in the art.
  • the anti-LGMN antibodies of the invention may be expressed in cell lines other than hybridoma cell lines.
  • Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the invention. Transformation can be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used will depend on the host to be transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation , Encapsulation of polynucleotides in liposomes and microinjection of DNA directly into nuclei, etc.
  • Mammalian cell lines useful as hosts for expression are well known in the art and include, but are not limited to, various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO ) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial LGMN binding properties. Anti-LGMN antibodies can be fused with other polypeptides that need to be expressed.
  • ATCC American Type Culture Collection
  • CHO Chinese Hamster Ovary
  • HeLa cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells eg, HepG2
  • Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial LGMN binding properties.
  • Anti-LGMN antibodies can be fused with other
  • the invention includes nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof of the invention.
  • a nucleic acid molecule can be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • the present invention also includes degenerate variants of nucleic acid molecules encoding polypeptides or proteins, that is, nucleic acid molecules that encode the same amino acid sequence but differ in nucleotide sequence.
  • the coding sequence refers to the part of the nucleic acid sequence that directly determines the amino acid sequence of its protein product (such as an antibody or its antigen-binding fragment, etc.).
  • the boundaries of the coding sequence are usually determined by the ribosome binding site (for prokaryotes) immediately upstream of the 5' open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA.
  • a coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • the coding sequence of the antibody described herein can be in the same expression frame as the coding sequence of other polypeptides that need to be expressed in fusion.
  • the nucleic acid molecules described herein can generally be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequence disclosed herein, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as a template, related sequences were amplified. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.
  • the nucleic acid molecules described herein can also be directly synthesized.
  • nucleic acid constructs for expression by cells involve nucleic acid constructs.
  • the nucleic acid constructs herein comprise the nucleic acid molecules described herein, and one or more regulatory sequences operably linked to these sequences.
  • Regulatory sequences may be suitable promoter sequences, transcription terminator sequences, leader sequences, origins of replication functional in at least one organism, convenient restriction enzyme sites and one or more selectable markers, which are known in the art within the knowledge of the technician.
  • nucleic acid molecules of the invention can be manipulated in a variety of ways to ensure expression of the antibody or therapeutic protein. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the differences or requirements of the expression vector. Techniques for altering the sequence of nucleic acid molecules using recombinant DNA methods are known in the art.
  • the nucleic acid construct is a vector.
  • the vector can be a cloning vector, an expression vector, or a homologous recombination vector.
  • the nucleic acid molecules of the invention can be cloned into many types of vectors, eg, plasmids, phagemids, phage derivatives, animal viruses and cosmids.
  • Vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art.
  • expression vectors can be transferred into host cells by physical, chemical or biological means.
  • Physical methods for introducing nucleic acid molecules into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • Biological methods for introducing nucleic acid molecules of interest into host cells include the use of DNA and RNA vectors.
  • Chemical means of introducing nucleic acid molecules into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipid body.
  • Biological methods for introducing nucleic acid molecules into host cells include the use of viral vectors, such as vectors derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, especially retroviral vectors.
  • the selected gene can be inserted into a vector and packaged into a retroviral particle, such as a lentiviral particle, using techniques known in the art.
  • a host cell contains, expresses and/or secretes an antibody or antigen-binding fragment thereof and optionally a therapeutic polypeptide described herein.
  • a cell contains or contains, expresses, or secretes a molecule such as a polypeptide
  • "contains” means that the molecule is contained in or on the surface of the cell;
  • expression means that the cell produces the molecule "secretion” means that the cell secretes the expressed molecule out of the cell.
  • Host cells include not only cells that are ultimately used for protein expression, but also various engineering cells used in the process of producing the cells, such as E. coli cells, for example, providing the coding sequence of the protein of the present invention or providing the vectors described herein .
  • This article also provides the use of LGMN protein and/or integrin or its ⁇ subunit as a target in screening potential substances for inhibiting tumor metastasis or tumor cell metastasis, or as a molecular indicator for clinical diagnosis of tumor metastasis.
  • the method for screening the potential substances includes: treating and detecting (1) LGMN protein, and/or (2) integrin or its ⁇ subunit, and/or (3) LGMN protein and integrin or its ⁇ subunit The interaction system; and detecting the content of the corresponding protein in the system or the strength of the interaction.
  • the target specifically includes the RGD sequence of the LGMN protein, and/or, the ligand binding region of the integrin or its ⁇ subunit interacting with the LGMN protein.
  • the candidate substance down-regulates the LGMN protein or inhibits the interaction (such as the interaction between the RGD sequence of the LGMN protein and integrin or its ⁇ subunit), it indicates that the candidate substance is a potential substance for inhibiting tumor metastasis or tumor cell metastasis .
  • the system for detecting the interaction can be a system expressing LGMN protein and integrin, such as cells or cell culture.
  • the cells can be endogenously expressing LGMN protein and integrin; or can be recombinantly expressing LGMN protein and integrin.
  • the system for expressing LGMN protein and integrin can also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal, such as a mouse, rabbit, sheep, monkeys, etc.) etc.
  • the method for detecting the interaction between LGMN protein and integrin in the system is known in the art, detecting downstream signals (such as FAK-Src-RhoA signal) or cell migration.
  • the candidate substances are, for example, the antibodies described herein.
  • Inhibitory reagents of the invention can be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with LGMN, such as binding assays to detect and/or quantify LGMN expressed in tumor tissues or cells LGMN.
  • the present invention provides detection of the presence or level of LGMN in a sample using classical immunohistological methods known to those skilled in the art. Detection of LGMNs can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of LGMNs include ELISA, FACS, RIA, and the like.
  • anti-LGMN antibodies are typically labeled with a detectable labeling group.
  • Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides, fluorescent groups, enzymatic groups, chemiluminescent groups, biotinyl groups or recognition by secondary reporters The predetermined polypeptide epitope.
  • Various methods for labeling proteins are known in the art and can be used to carry out the present invention.
  • One aspect of the invention provides for the identification of cells expressing anti-LGMN antibodies.
  • the antibody is labeled with a labeling group and binding of the labeled antibody to LGMN is detected.
  • binding of the antibody to LGMN is detected in vivo.
  • the antibody-LGMN is isolated and measured using techniques known in the art.
  • Another aspect of the invention provides detection of the presence of a test molecule that competes with an antibody of the invention for binding to LGMN.
  • An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of LGMN in the presence or absence of a test molecule. An increase in the amount of free antibody (ie, antibody that does not bind LGMN) will indicate that the test molecule is able to compete with the antibody for binding to LGMN.
  • the antibody is labeled with a labeling group.
  • the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of one or more anti-LGMN antibodies of the present invention and a pharmaceutically acceptable carrier, which includes but not limited to diluents, carriers, solubilizers, emulsifiers , preservatives and/or adjuvants.
  • acceptable carriers and the like in pharmaceutical compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • a pharmaceutical composition may contain ingredients for improving, maintaining or retaining, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or The rate of release, absorption or penetration of such substances.
  • Such substances are known in the prior art, see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, edited by A.R. Genrmo, 1990, Mack Publishing Company.
  • the optimum pharmaceutical composition will be determined by the intended route of administration, mode of delivery and desired dosage.
  • compositions of the invention may be selected for parenteral delivery. Alternatively, compositions may be selected for inhalation or delivery through the alimentary tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • compositions will be apparent to those skilled in the art, including formulations comprising anti-LGMN antibodies in sustained or controlled release delivery formulations.
  • Techniques for formulating various other sustained or controlled delivery modes such as liposomal vehicles, bioerodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • compositions for in vivo administration are generally presented as sterile preparations. Sterilization is achieved by filtration through sterile filtration membranes. When the composition is lyophilized, this method can be used for sterilization either before or after lyophilization and reconstitution.
  • Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are usually presented in containers with sterile access ports, eg, intravenous solution strips or vials with a hypodermic needle-punctureable stopper.
  • kits for producing single dosage administration units may each contain a first container with a dry protein and a second container with an aqueous formulation.
  • kits containing single and multi-lumen prefilled syringes eg, liquid syringes and lyophilized syringes are provided.
  • the invention also provides methods of treating a patient by administering an anti-LGMN antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof according to any embodiment of the invention.
  • the terms "patient”, “individual”, and “subject” are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g. rat, mouse, dog, cat, rabbit, etc.) , and most preferably a human.
  • “Treatment” refers to the subject's use of the treatment regimens described herein to achieve at least one positive therapeutic effect (e.g., a decrease in cancer cell number, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth. ).
  • Therapeutic regimens that effectively treat a patient can vary depending on factors such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.
  • the therapeutically effective amount of a pharmaceutical composition comprising an anti-LGMN antibody or antigen-binding fragment thereof of the invention to be employed will depend, for example, on the extent and goal of the treatment. Those skilled in the art will appreciate that appropriate dosage levels for therapy will depend in part on the molecule being delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. conditions) vary. In certain embodiments, the clinician can titrate the dose and vary the route of administration to achieve optimal therapeutic effect.
  • the frequency of dosing will depend on the pharmacokinetic parameters of the particular anti-LGMN antibody in the formulation used.
  • the clinician typically administers the composition until a dosage is reached to achieve the desired effect.
  • the composition may thus be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter.
  • the route of administration of the pharmaceutical composition is according to known methods, such as oral, injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; Either by a sustained release system or by an implanted device.
  • Cell lines breast cancer cell lines MDA-MB-231, SCP2, Hs578T, MCF7, T47D, and breast epithelial cell line MCF10A.
  • the peptide sequence of Legumain antigen is: PQNFLAVLRGDAEAVKGIG (SEQ ID NO: 8), synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.
  • the phage library is Tomlinson I+J (purchased from Geneservice, I library capacity is 1.47 ⁇ 10 8 , J library capacity is 1.37 ⁇ 10 8 ).
  • the plasmid of packaging virus is transferred into 293T cells according to a certain ratio
  • Ultracentrifugation can be performed if necessary, the virus is concentrated, and centrifuged at 27,000 rpm at 4°C for 2 hours.
  • antibiotics such as puromycin, neomycin
  • Binding buffer Protein A purification binding buffer
  • NC nitrocellulose membrane
  • HRP HRP The coupled secondary antibody was incubated at room temperature for 1 hr; the NC membrane was washed twice with TBST, 5 min each time. Then ECL detection reagent for fluorescence development.
  • the shRNA sequences for knocking down human LGMN protein are: 5-GCCATGCCTACCAGATCATTC-3(shLGMN-1) and 5-GTATTGAGAAGGGTCATATTT-3(shLGMN-2).
  • the shRNA sequence for knocking down human ⁇ 3 integrin is: 5-GCTTAGCTTGAGGGTGACTAT-3.
  • control shRNA sequence is: 5-CCTAAGGTTAAGTCGCCCTCG-3.
  • FITC-labeled Legumain protein and serially diluted antibody were incubated in 50ul PBS (1mM Ca 2+ , 1mM Mg 2+ ) system for 30 minutes at room temperature, and then incubated with SCP2 breast cancer cells for 30 minutes at room temperature. Cells were washed twice and analyzed by flow cytometry. Humanized IgG was incubated with FITC-labeled legumain protein at the same concentration as a negative control.
  • the cells that migrated to the lower surface of the small chamber were fixed with 2% paraformaldehyde and stained with DAPI, and the upper surface was wiped with a cotton swab to remove cells that did not migrate there.
  • the number of migrated cells in 5 fields of view was recorded under a fluorescence microscope and averaged.
  • mice Inject 1 ⁇ 10 5 luciferase-labeled SCP2 breast cancer cells into the left ventricle of anesthetized 5-week-old nude mice, and inject C10 antibody (2 mg/kg) intraperitoneally every two days starting from two days before tumor cell injection and etc. A small amount of human IgG was used as a control until the end of the experiment. Weekly intraperitoneal injections of 75 mg/kg luciferase substrate were performed, and tumor growth in mice was detected by an in vivo imaging system. Finally, the mice treated for the sixth week were taken to take X-Ray and micro-CT to detect the bone damage of the mice.
  • RGD antigen polypeptide 100 ⁇ g/ml was coated on the immune tube, and the Tomlinson I+J phage library was subjected to three rounds of affinity enrichment screening of adsorption-elution-amplification in the immune tube. During the three rounds of enrichment screening, the intensity of non-specific washing was gradually increased to fully remove non-specifically bound phages. After each round of eluted bound phages were amplified, the next round of screening was performed.
  • the RGD polypeptide or LGMN protein was coated on a 96-well microtiter plate, and the same concentration of bovine serum albumin (BSA) was used as a negative control. After blocking with 2% skimmed milk, 50 ⁇ l of phage supernatant was added to each well and incubated at room temperature for 2 hours. After the plate was washed three times with PBST (0.1% Tween 20), HRP-anti-KM13 antibody (1:5000, 50 ⁇ l) was added to each well and incubated at room temperature for 1 h. After washing with PBST three times, add 100 ⁇ l TMB substrate to each well, and develop color in the dark. After the substrate turns blue, add 2 mol/L H 2 SO 4 to terminate the reaction, and finally read OD450 with a microplate reader.
  • BSA bovine serum albumin
  • BLI experiments were performed using an Octet Red 96 instrument (ForteBio, Inc.).
  • the principle is to immobilize the biotinylated LGMN protein on the streptavidin (SA) biosensor, and incubate with antibodies of different concentration gradients in the kinetic buffer, and observe the dynamic binding and dissociation of the two Condition.
  • SA streptavidin
  • the R2 value was ⁇ 0.95, and the KD value of the binding affinity of the C10 antibody to LGMN was calculated. Binding experiments were performed at 25°C. Data analysis was performed using Octet Data Analysis Software 9.0 (ForteBio, Menlo park, CA, USA).
  • LGMN The expression level of LGMN is related to the metastasis and prognosis of breast cancer
  • 2.4 LGMN promotes the migration and invasion of breast cancer cells by binding to integrin and activating the downstream signal FAK-Src-RhoA signal
  • the combination of integrins and ligands can change the reorganization of cytoskeletal proteins by activating downstream FAK, Src kinase and other molecules, and then affecting different guanosine triphosphate hydrolase (small GTPase) (mainly including: Rac1, RhoA and Cdc42). row, and finally realize the regulation of cell migration.
  • small GTPase small GTPase
  • the experimental results showed that LGMN can activate the classic downstream signal of integrin through FAK-Src, and activate RhoA to promote tumor cell metastasis (Fig. 6, GH).
  • Also adding LGMN wild-type protein treatment to the culture medium can also cause the activation of integrin downstream signals FAK, Src and RhoA, while LGMN D120E protein cannot activate the downstream signals ( Figure 6, IJ).
  • 2.11 C10 antibody can inhibit the migration ability of ER+ breast cancer cells, gastric cancer cells, lung cancer cells and colon cancer cells
  • C10 antibody can inhibit the migration and invasion ability of TNBC breast cancer cell SCP2 in vitro, and then we also tested the ability of C10 antibody to inhibit the migration of other tumor cells. Through transwell experiments, we found that C10 antibody can also inhibit the migration ability of ER+ breast cancer cells (T47D cells) overexpressing LGMN and integrin ⁇ v ⁇ 3 ( FIG. 17 ).
  • LGMN protein can bind to its ⁇ subunits ⁇ 1, ⁇ 5, ⁇ 6 and ⁇ 8 ( Figure 20).
  • the combination of LGMN and integrin depends on the conserved RGD sequence, so we think that the combination of LGMN and integrin of other RGD ligands in the present invention may have the ability to promote breast cancer metastasis.
  • LGMN is specifically highly expressed in breast cancer cells, while its expression is extremely low in normal tissues, and highly expressed LGMN has been proven to promote the development and metastasis of breast cancer. These characteristics make LGMN an ideal target for tumor therapy.
  • many studies have been conducted on the biological function of LGMN and its relationship with tumors, and it has become a new hotspot in the fields of tumor treatment, cellular immunity, disease diagnosis and prognosis.
  • Previous studies have developed small molecule inhibitors and tumor DNA vaccines for LGMN, and the mechanism of these small molecule drugs is mainly to inhibit the activity of LGMN enzymes to inhibit the growth of tumor in situ.

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Abstract

本文涉及抑制肿瘤细胞转移的药物及其用途。本发明提供抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂在制备抑制肿瘤或肿瘤细胞转移的药物中的用途。本发明所述试剂或组合物可以有效抑制肿瘤或肿瘤细胞转移。

Description

抑制肿瘤细胞转移的药物及其用途 技术领域
本发明涉及肿瘤治疗领域，具体涉及抑制肿瘤细胞转移的药物及其用途。
背景技术
乳腺癌是世界范围内女性最常见的恶性肿瘤之一，乳腺癌的远端转移是导致高致死率的主要原因。全身重要脏器的转移如骨，肺，肝脏和脑的转移都会危及生命。肿瘤的转移是一个复杂的多阶段过程，肿瘤细胞从原发部位侵入淋巴管，血管等，到达其他组织部位继续生长并形成转移灶。最近几年虽然在乳腺癌的治疗上取得了一定的成效，但是目前并没有完全有效地针对乳腺癌转移的治疗方法，其五年生存率只有26％，成为乳腺癌患者死亡的主要原因。因此探究有效的乳腺癌转移相关靶点研究，也是当前生命科学领域的研究热点和前沿性课题，更充分的了解乳腺癌转移机制可以为治疗转移性肿瘤供新的思路。
整合素(integrin)是一类广泛表达在细胞膜表面的黏附分子(cell adhesion molecule,CAM)，是由α和β两个亚基通过非共价键形成的异源二聚体。根据整合素的配体结合特性可大致分为两类，一类是与细胞外基质蛋白(extracellular matrix,ECM)结合，另一类与细胞表面黏附分子结合。
天冬酰胺内肽酶(Legumain,LGMN)是C13半胱氨酸蛋白酶家族成员之一，能特异性切割蛋白端的天冬酰胺残基，细胞质中合成的pro-LGMN是无活性的酶原形式，LGMN的活化需要在溶酶体严格的酸性条件下进行剪切完成，另外LGMN蛋白也可以被分泌至胞外作为细胞外基质的组分。研究表明，与正常组织中相比，LGMN在多种肿瘤组织中高表达。
现有技术中的小分子药物主要通过抑制LGMN酶的活性，来抑制原位瘤的生长。深入研究LGMN在肿瘤细胞中的作用机制有助于开发相关治疗和诊断方法和产品。
发明内容
发明人发现LGMN表达水平与癌症转移和预后相关，其通过RGD序列与整合素β亚基结合，进而调节肿瘤的转移。
因此，本发明第一方面提供抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂在制备抑制肿瘤转移或肿瘤细胞转移的药物中的用途。
在一个或多个实施方案中，所述相互作用是LGMN蛋白的RGD序列与整合素或其β亚基之间的相互作用。
在一个或多个实施方案中，所述整合素的β亚基选自以下一种或多种：β 1、β 3、β 5、β 6和β 8；所述整合素是含有所述β亚基的整合素。
在一个或多个实施方案中，所述整合素选自以下一种或多种：α vβ 3、α vβ 1、α vβ 5、α vβ 6、α vβ 8、α 5β 1和α 8β 1。
在一个或多个实施方案中，所述试剂选自以下的一种或多种：
(1)突变型LGMN蛋白和/或它们的打靶载体，其中，与野生型相比，突变型LGMN的RGD序列发生导致LGMN与整合素或其β亚基的相互作用减弱或消失的突变；
(2)突变型整合素或其β亚基和/或其打靶载体，其中，与野生型相比，突变型整合素或其β亚基在与LGMN蛋白相互作用的配体结合区域具有一个或多个导致其与LGMN蛋白相互作用减弱或消失的突变；
(3)抗LGMN抗体或其抗原结合片段，所述抗体抑制LGMN蛋白与整合素或其β亚基之间的相互作用；
(4)抗整合素或其β亚基的抗体或其抗原结合片段，所述抗体抑制LGMN蛋白与整合素或其β亚基之间的相互作用；
(5)编码(3)或(4)所述的抗体或其抗原结合片段的核酸分子或包含所述核酸分子的核酸构建物；
(6)表达(3)或(4)所述的抗体或其抗原结合片段的宿主细胞；
(7)整合素或其β亚基的抑制剂，所述抑制剂抑制LGMN蛋白与整合素或其β亚基之间的相互作用；
(8)能降低二价金属离子浓度的试剂，例如乙二胺四乙酸(EDTA)，氨基三乙酸(NTA)，柠檬酸(CA)、酒石酸(TA)和葡糖糖酸(GA)等；
(9)敲低或敲除LGMN蛋白和/或整合素或其β亚基的表达的试剂，例如ZFN和/或TALEN和/或CRISPR/Cas9试剂和/或小干扰RNA；
在一个或多个实施方案中，在(1)中，所述突变是RGD序列突变为RGE，RAD，RAE，KGD或KGE。
在一个或多个实施方案中，在(2)中，所述整合素或其β亚基在D120和/或S121位置上具有突变；优选地，所述突变为取代突变；更优选地，取代后的氨基酸残基为丙氨酸或谷氨酸。
在一个或多个实施方案中，所述小干扰RNA具有SEQ ID NO:5或6所示的序 列。
在一个或多个实施方案中，所述肿瘤选自以下的一种或多种：乳腺癌、肺腺癌、结肠癌、胃腺癌、乳腺癌、肝细胞癌、胰腺癌。
在一个或多个实施方案中，所述突变型LGMN蛋白、突变型整合素或其β亚基、抗LGMN抗体或其抗原结合片段、小干扰RNA如本文任一实施方案所述。
本发明还提供突变型LGMN蛋白，与野生型相比，突变型LGMN蛋白的RGD序列具有一个或多个导致LGMN与整合素或其β亚基的相互作用减弱或消失的突变。
在一个或多个实施方案中，所述前体是pro-LGMN蛋白。
在一个或多个实施方案中，与野生型相比，突变型LGMN蛋白的RGD序列突变为RGE，RAD，RAE，KGD或KGE。
本发明还提供突变型整合素或其β亚基，与野生型相比，其在与LGMN蛋白相互作用的配体结合区域具有一个或多个导致其与LGMN蛋白相互作用减弱或消失的突变；优选地，在D120和/或S121位置上具有突变；优选地，所述突变为取代突变；更优选地，取代后的氨基酸残基为丙氨酸或谷氨酸。
在一个或多个实施方案中，所述整合素的β亚基选自以下一种或多种：β 1、β 3、β 5、β 6和β 8；所述整合素是含有所述β亚基的整合素。
在一个或多个实施方案中，所述整合素选自以下一种或多种：α vβ 3、α vβ 1、α vβ 5、α vβ 6、α vβ 8、α 5β 1和α 8β 1。
本文还提供LGMN蛋白和/或整合素或其β亚基作为靶点在筛选抑制肿瘤转移或肿瘤细胞转移的药物中的用途，或用作分子指标在临床上用于诊断肿瘤转移。
在一个或多个实施方案中，所述肿瘤选自以下的一种或多种：乳腺癌、肺腺癌、结肠癌、胃腺癌、乳腺癌、肝细胞癌、胰腺癌。
在一个或多个实施方案中，所述药物抑制LGMN蛋白与整合素或其β亚基之间的相互作用。优选所述药物使LGMN蛋白的RGD序列与整合素或其β亚基的相互作用减弱。
在一个或多个实施方案中，在一个或多个实施方案中，所述整合素的β亚基选自以下一种或多种：β 1、β 3、β 5、β 6和β 8；所述整合素是含有所述β亚基的整合素。
在一个或多个实施方案中，所述整合素选自以下一种或多种：α vβ 3、α vβ 1、α vβ 5、α vβ 6、α vβ 8、α 5β 1和α 8β 1。
在一个或多个实施方案中，靶点包括LGMN蛋白的RGD序列，和/或，整合素或其β亚基的与LGMN蛋白相互作用的配体结合区域。
本发明还提供抗LGMN抗体或其抗原结合片段，其CDR如下所示：HCDR1包含GFTFSSYA，HCDR2包含IGNSGNYT，HCDR3包含AKSSDSFNY，LCDR1包含QSISSY，LCDR2包含DAS，LCDR3包含QQAYANPDT。
在一个或多个实施方案中，所述抗体抑制LGMN蛋白与整合素或其β亚基之间的相互作用。
在一个或多个实施方案中，所述抗LGMN抗体的VH的FR区为SEQ ID NO:1所示VH的FR区，和VL的FR区为SEQ ID NO:2所示VL的FR区。
在一个或多个实施方案中，所述抗体的VH和VL具有SEQ ID NO:1和SEQ ID NO:2所示的序列或与其具有90％序列相同性的变体。
在一些实施方案中，所述抗体的重链恒定区的氨基酸序列具有如SEQ ID NO:3所示的序列，和/或轻链恒定区的氨基酸序列具有如SEQ ID NO:4所示的序列。
在一个或多个实施方案中，本发明任一实施方案所述的抗LGMN抗体为嵌合抗体或完全人抗体；优选为完全人抗体。
本发明还提供核酸分子或包含所述核酸分子的核酸构建物，所述核酸分子包含选自以下的序列：(1)编码本文所述的抗LGMN抗体或其抗原结合片段的序列，或(2)(1)的互补序列。
在一个或多个实施方案中，所述核酸构建物是克隆载体、整合载体或表达载体。
本发明还提供宿主细胞，所述宿主细胞：(1)表达本文所述的抗LGMN抗体或其抗原结合片段，或(2)包含本文所述的核酸分子或核酸构建物。
本发明还提供本文所述的突变型LGMN蛋白、突变型整合素或其β亚基、或抗LGMN抗体或其抗原结合片段的编码序列或其互补序列，含有所述编码序列或互补序列的核酸构建体，以及表达所述突变型LGMN蛋白、突变型整合素或其β亚基、或抗LGMN抗体的宿主细胞在制备抑制肿瘤转移或肿瘤细胞转移的药物中的用途。
在一个或多个实施方案中，所述宿主细胞含有所述编码序列或其互补序列或所述核酸构建体。
本发明还提供一种药物组合物，所述药物组合物含有：
(1)药学上可接受的载体，和
(2)本发明任一实施方案所述的突变型LGMN蛋白、突变型整合素或其β亚基、或抗LGMN抗体或其抗原结合片段，和/或(3)含有编码(2)的核酸序列或其互补序列的核酸分子。
本发明还提供一种敲减LGMN的小干扰RNA，其具有SEQ ID NO:5或6所示的序列。
本发明还提供一种敲减整合素或其β亚基的小干扰RNA，其具有SEQ ID NO:7所示的序列。
本发明还提供一种体外抑制肿瘤细胞转移的方法，包括减弱肿瘤细胞的LGMN蛋白与整合素或其β亚基之间的相互作用的步骤。
在一个或多个实施方案中，所述方法包括通过以下任意一种或多种方式减弱肿瘤细胞的LGMN蛋白与整合素或其β亚基之间的相互作用：
(1)敲除或敲低肿瘤细胞的LGMN蛋白的表达；
(2)敲除或敲低肿瘤细胞中整合素或其β亚基表达；
(3)在野生型或敲除LGMN蛋白的肿瘤细胞中表达与整合素或其β亚基的相互作用减弱或消失的突变型LGMN蛋白；
(4)在野生型或者敲除整合素或其β亚基的肿瘤细胞中表达与LGMN蛋白的相互作用减弱或消失的突变型整合素或其β亚基；
(5)使肿瘤细胞与抗LGMN抗体或其抗原结合片段或含有所述抗LGMN抗体或其抗原结合片段的蛋白接触；
(6)使肿瘤细胞与降低二价金属离子浓度的试剂接触。
在一个或多个实施方案中，所述相互作用是LGMN的RGD序列与整合素或其β亚基之间的相互作用。
在一个或多个实施方案中，所述整合素的β亚基选自以下一种或多种：β 1、β 3、β 5、β 6和β 8；所述整合素是含有所述β亚基的整合素。
在一个或多个实施方案中，所述整合素选自以下一种或多种：α vβ 3、α vβ 1、α vβ 5、α vβ 6、α vβ 8、α 5β 1和α 8β 1。
在一个或多个实施方案中，所述方法具有选自以下的一个或多个特征：
所述突变型LGMN蛋白与野生型相比，其RGD序列发生导致与整合素或其β亚基的相互作用减弱或消失的突变；
所述突变型整合素或其β亚基与野生型相比，其在与LGMN相互作用的配体结合区域具有一个或多个导致其与LGMN相互作用减弱或消失的突变；
所述抗LGMN抗体抑制LGMN与整合素或其β亚基之间的相互作用；
所述能降低二价金属离子浓度的试剂是EDTA；
所述敲低或敲除LGMN和/或整合素或其β亚基表达的试剂是ZFN和/或TALEN和/或CRISPR/Cas9试剂和/或小干扰RNA。
在一个或多个实施方案中，
使用突变型LGMN蛋白的表达载体和/或整合载体处理肿瘤细胞，和/或
使用突变型整合素或其β亚基的表达载体和/或整合载体处理肿瘤细胞，和/或
使用抗LGMN抗体或其抗原结合片段处理肿瘤细胞，和/或
使用能降低二价金属离子浓度的试剂处理肿瘤细胞，和/或
在肿瘤细胞中转入基因敲除载体，以敲除所述肿瘤细胞中LGMN蛋白表达和/或整合素或其β亚基表达，和/或
采用ZFN、TALEN或CRISPR/Cas9等技术敲除肿瘤细胞LGMN蛋白表达和/或整合素或其β亚基的表达，和/或
通过干扰RNA介导的基因沉默敲低LGMN蛋白表达和/或整合素或其β亚基的表达，和/或
在肿瘤细胞中转入基因插入载体、以在敲除野生型LGMN蛋白和/或整合素或其β亚基的编码序列的同时将与整合素或其β亚基的相互作用减弱或消失的突变型LGMN蛋白的表达框和/或与LGMN蛋白的相互作用减弱或消失的突变型整合素或其β亚基的表达框整合到肿瘤细胞的基因组中，
从而减弱或破坏细胞内LGMN蛋白整合素或其β亚基之间的相互作用。
在一个或多个实施方案中，所述突变型LGMN蛋白、突变型整合素或其β亚基、抗LGMN抗体或其抗原结合片段、小干扰RNA如本文任一实施方案所述。
本发明还提供检测LGMN蛋白的试剂在制备诊断肿瘤转移的试剂盒中的用途。
在一个或多个实施方案中，所述试剂检测肿瘤组织中LGMN蛋白的表达、含量或序列。
在一个或多个实施方案中，所述试剂包括：针对LGMN蛋白的抗体、与LGMN 蛋白的编码序列杂交的引物和/或探针。
本发明还提供筛选减弱或破坏细胞内LGMN蛋白整合素或其β亚基之间的相互作用的试剂的方法。
本发明还提供LGMN在活化FAK、Src和RhoA中的用途。
本发明还提供一种构建乳腺癌骨转移小鼠模型的方法，包括将表达标记物的乳腺癌细胞通过左心室注射到裸鼠体内的步骤。
在一个或多个实施方案中，所述乳腺癌细胞是SCP2。
在一个或多个实施方案中，所述标记物是荧光素酶。
附图说明
图1，高表达的LGMN与乳腺癌的转移及预后相关。(A)韦恩图显示了三个数据库GSE45255、GSE65194、GSE22219中与乳腺癌相关的基因。(B)基因注释(Gene Ontology)分析25个与乳腺癌转移相关基因。(C-E)Kaplan-Meier分析LGMN的表达水平与乳腺癌患者的转移和生存率的关系，数据分别来自GSE45255、GSE65194、GSE22219数据库。(F-G)正常乳腺上皮细胞MCF10A与乳腺癌细胞系的细胞迁移和侵袭能力。(H-I)免疫印迹实验检测正常乳腺上皮细胞MCF10A与乳腺癌细胞系的LGMN的表达水平(H)和分泌到培养基中LGMN的含量(I)。
图2，Pro-LGMN与乳腺癌的转移呈正相关。(A)列举了25个与转移相关的基因。(B-C)Kaplan-Meier生存分析LGMN表达与乳腺癌病人的无转移发生生存率(B)及总生存率(C)的关系,数据来自http://kmplot.com/analysis/。(D-I)全细胞裂解液(WCL)及条件培养基(CM)中pro-LGMN和LGMN活化形式的含量与乳腺癌细胞迁移能力(D,F,H)之间及与侵袭能力(E,G,I)之间的相关性。
图3，敲低LGMN显著抑制乳腺癌细胞的转移能力。(A)在Hs578T、MDA-MB-231和SCP2细胞中，分别转染特异性针对LGMN的shLGMN(shLGMN-1和shLGMN-2)，其中扰乱的shRNA为阴性对照(Control)。WB检测LGMN的水平。敲低LGMN对三种肿瘤细胞伤口愈合实验(B),Transwell迁移(C)和在基质胶中侵袭(D)的影响。所有数据均为三次以上平行实验取平均值并作误差线。P值用t-text的统计方法计算，**P<0.01，***P<0.001。
图4，敲低LGMN不影响乳腺癌细胞的增殖能力。敲低LGMN对Hs578T、 MDA-MB-231和SCP2细胞CCK8增殖实验的影响(A)和克隆形成的影响(B)。所有数据均为三次以上平行实验取平均值并作误差线。P值用t-text的统计方法计算。
图5，LGMN通过其RGD序列介导与β3整合素结合。(A)Hs578T、MDA-MB-231和SCP2细胞内源LGMN和β3整合素免疫共沉淀结果；(B)Co-IP实验检测LGMND120E对β3整合素与LGMN结合的影响；(C)Co-IP实验检测EDTA对LGMN与β3整合素结合的影响；(D)Co-IP实验检测MIDAS突变β3S121A对β3整合素与LGMN结合的影响；(E)Co-IP实验检测LGMN酶活性突变LGMNC189S对β3整合素与LGMN结合的影响。
图6，LGMN促进肿瘤转移依赖与整合素的结合。在稳定低表达LGMN的Hs578T、MDA-MB-231和SCP2细胞当中分别转染对照质粒、野生型LGMN、突变型LGMN D120E和LGMN C189S对三种肿瘤细胞伤口愈合实验(A),Transwell迁移(B)和侵袭(C)的影响。纯化的LGMN野生型蛋白及LGMN D120E蛋白处理敲低LGMN的三种肿瘤细胞对于伤口愈合实验(D)，Transwell迁移(E)和侵袭(F)的影响。所有数据均为三次以上平行实验取平均值并作误差线。P值用t-text的统计方法计算，***P<0.01。(G)WB检测Hs578T、MDA-MB-231和SCP2细胞在敲低LGMN的基础上回补野生型LGMN及LGMN D120E突变的条件下，胞内整合素下游信号蛋白(FAK和Src)磷酸化水平的变化。(H)利用GTPase Pull-down技术检测胞内RhoA，Rac1和CDC42的活化水平。纯化的野生型LGMN蛋白及LGMN D120E蛋白处理LGMN敲低的Hs578T、MDA-MB-231和SCP2细胞，并检测FAK-Y397和Src-Y416磷酸化水平(I)及RhoA,Rac1和CDC42的活化水平(J)。
图7，整合素αvβ3的表达。流式细胞仪检测Hs578T、MDA-MB-231和SCP2细胞中不同的LGMN表达的稳转株细胞中整合素αvβ3的表达水平。P值用one-way ANOVA的统计方法分析得出。
图8，LGMN与整合素结合在体内促进乳腺癌骨转移。分别检测对照组及稳定低表达LGMN和稳定表达野生型LGMN或点突变LGMND120E对乳腺癌细胞SCP2骨转移能力的影响。(A,B)小动物活体成像、X-ray和Micro-CT检测骨转移部位的荧光水平的代表性图片(A)和统计图(B)(n＝5-7/组)。(C)ELISA检测小鼠血浆内人源LGMN的含量(n＝3-7/组)。分析小鼠CT扫描结果中骨体积分数(骨体积/材料体积)(D)，骨小梁数目(E),骨小梁分离度(F)的差异(n＝5/组)。P值用one-way ANOVA的统计方法分析得出，*P<0.05，***P<0.001。
图9，LGMN的表达不影响原位乳腺癌的生长。分别检测对照组及稳定低表达LGMN和稳定表达野生型LGMN或点突变LGMND120E对SCP2乳腺癌细胞原位乳 腺癌的生长(n＝5-6/组)。
图10，LGMN或整合素β3的敲低可以抑制SCP2乳腺癌细胞原位肿瘤的肺转移。对照组，LGMN敲低组与β3敲低组的SCP2细胞分别注射至B-NDG小鼠的乳房脂肪垫中，原位肿瘤的肺转移荧光代表图(A)和荧光定量结果(B)如图所示。n＝6只/组。P值用one-way ANOVA的统计方法得出。NS:没有显著性。**P<0.005。
图11，同时表达LGMN与整合素αvβ3可以增强T47D细胞在体外的迁移和侵袭力。A在T47D细胞中过表达LGMN与整合素αvβ3，并通过免疫印迹实验检测LGMN与整合素β3的表达水平。B-D伤口愈合实验，transwell细胞迁移实验和细胞侵袭实验检测T47D细胞的迁移和侵袭能力。结果显示为三次独立的重复实验。P值用one-way ANOVA的统计方法得出。NS:没有显著性。***P<0.001。
图12，同时表达LGMN与整合素αvβ3可以促进T47D细胞在小鼠体内的转移能力。T47D乳腺癌细胞原位乳腺癌模型(A)，小鼠肺部转移灶模式图(左)，肿瘤生物发光荧光统计结果(右)。T47D乳腺癌细胞左心室注射骨转移模型(B)，小鼠骨转移模式图(左)，骨转移肿瘤生物发光荧光统计结果(右)。P值用one-way ANOVA(A)或two-way ANOVA(B)的统计方法得出。NS:没有显著性。**P<0.05，***P<0.001。
图13，噬菌体筛选鉴定抗LGMN抗体。(A,B)ELISA实验检测scFv与LGMN蛋白(A)和RGD多肽(B)的结合。(C)流式细胞实验检测单克隆抗体抑制LGMN蛋白与SCP2细胞的结合。
图14，C10抗体抑制乳腺癌骨转移。(A)C10抗体与LGMN蛋白之间的结合利用生物膜干涉原理，用Octet RED 96仪器测定。生物素偶联的LGMN被链霉亲和素捕获并固定在芯片上，测试其与各种浓度梯度C10抗体的结合能力。结合动力学采用1:1的Langmuir结合模型，采用ForteBio Data Analysis 9.0软件进行评价。(B)C10抗体抑制SCP2细胞表面整合素与FITC标记的LGMN蛋白结合的IC50。伤口愈合实验(C)，Transwell迁移实验(D)和侵袭实验(E)检测C10抗体抑制SCP2细胞的迁移。(F)检测C10抗体处理SCP2敲低LGMN与对照组细胞后，胞内FAK-Y397和Src-Y416磷酸化水平及RhoA,Rac1和CDC42的活化水平。(G，H)小鼠活体成像，X-Ray和Micro-CT检测对照抗体组与C10抗体处理组中小鼠骨转移部位的荧光水平和骨损伤的代表性图片(G)和统计图(H)。(N＝6-7/组)。(I-K)分析C10抗体处理后小鼠CT扫描结果中骨体积/材料体积(I)，骨小梁数目(J),骨小梁分离度(K)的差异。(n＝5/组)。P值用t-text的统计方法(I-K)及two-way ANOVA的统计方式(C-F,H)分析得出，*p<0.05,***p<0.001。
图15，C10抗体可以特异性地抑制LGMN与整合素αvβ3的相互作用。Pull-down实验检测pro-LGMN-flag蛋白与整合素αvβ3的相互作用，并检测C10抗体处理对于pro-LGMN-flag蛋白与整合素αvβ3结合的影响。
图16，LGMN在多种肿瘤组织中高表达。GEPIA数据库比较多种肿瘤组织与其对应的正常组织中LGMN的表达。
图17，C10抗体可以特异性地抑制T47D细胞的迁移能力。在transwell细胞迁移实验中，检测C10抗体处理对于LGMN和整合素αvβ3共表达的T47D细胞迁移的影响，human IgG作为对照组。
图18，C10抗体可以特异性地抑制MNK-45胃癌细胞与A549肺癌细胞的迁移能力。在transwell细胞迁移实验中，检测C10抗体处理对于MNK-45胃癌细胞与A549肺癌细胞迁移的影响，human IgG作为对照组。(A)免疫印迹实验显示与正常乳腺上皮细胞MCF-10A相比，MNK-45胃癌细胞与A549肺癌细胞均含有较高的LGMN表达。(B)transwell实验结果表明C10抗体能抑制MNK-45胃癌细胞与A549肺癌细胞在体外的迁移能力。
图19，C10抗体可以特异性地抑制SW480结肠癌细胞的迁移能力。免疫印迹检测SW480结肠癌细胞的LGMN表达水平，以A549肺癌细胞为阳性对照(A)。伤口愈合实验中，检测C10抗体处理对SW480细胞迁移的影响(B)，以human IgG作为对照组。
图20，整合素β1、β5、β6和β8与LGMN蛋白的结合。Co-IP实验检测Hs578T、MDA-MB-231和SCP2细胞中整合素β1、β5、β6和β8与LGMN蛋白的结合。
具体实施方式
除非另有定义，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。这些技术在文献中有充分解释，诸如Molecular Cloning：A Laboratory Manual，第二版(Sambrook等，1989)；Oligonucleotide Synthesis(M.J.Gait编辑，1984)；Animal Cell Culture(R.I.Freshney编辑，1987)；Methods in Enzymology(Academic Press，Inc.)；Current Protocols in Molecular Biology(F.M.Ausubel等编辑，1987版及其定期更新版本)；PCR：The Polymerase Chain Reaction(Mullis等编辑，1994)；A Practical Guide to Molecular Cloning(Perbal Bernard V.，1988)；Phage Display：A Laboratory Manual(Barbas等，2001)。
本发明人发现高转移的乳腺癌细胞会大量分泌pro-LGMN至胞外，pro-LGMN 通过RGD序列与整合素(特别是αvβ3)结合，进而引发整合素的活化招募胞内信号分子，激活胞内FAK-Src-RhoA信号通路，促进乳腺癌细胞的迁移和侵袭。而将RGD序列突变能阻断LGMN与整合素结合，从而阻断LGMN介导的乳腺癌细胞的迁移和侵袭及乳腺癌组织在小鼠体内的转移。由此，阻断LGMN的RGD结构域与整合素的结合将是抑制乳腺癌转移的有效途径之一。此外，发明人通过噬菌体展示技术鉴定了一个可以阻断LGMN蛋白与整合素结合的人源化单克隆抗体C10，该抗体与LGMN结合后可以特异性地抑制其与整合素的相互作用，进而抑制了乳腺癌细胞的迁移和侵袭及乳腺癌细胞的骨转移。由此发明人发现，阻断LGMN的RGD位点与整合素的结合是抑制肿瘤转移的有效途径。此外，由于LGMN与整合素的结合促进细胞迁移运动所介导的胞内信号通路在体内是非常保守的，所以LGMN与整合素的相互作用促进肿瘤转移这一机制也适用于乳腺癌、肺腺癌、结肠癌、胃腺癌、乳腺癌、肝细胞癌、胰腺癌等其他LGMN与整合素高表达类型的肿瘤。
本文中，乳腺癌细胞包括但不限于MDA-MB-231、SCP2、Hs578T、MCF7、T47D系乳腺癌细胞或本领域技术人员已知的其他表达LGMN的乳腺癌细胞。本文所述的乳腺癌可以是与这些乳腺癌细胞中任意相关或包含这些乳腺癌细胞中任意的乳腺癌。示例性地，肺腺癌包括与LUAD系肺腺癌细胞相关或包含该细胞的肺腺癌；结肠癌包括与COAD系结肠癌细胞相关或包含该细胞的结肠癌；胃腺癌包括与STAD系胃腺癌细胞相关或包含该细胞的胃腺癌；肝细胞癌包括与LIHC系肝细胞癌细胞相关或包含该细胞的肝细胞癌；胰腺癌包括与PAAD系胰腺癌细胞相关或包含该细胞的胰腺癌。
LGMN在细胞质中合成并分泌的是pro-LGMN的形式。pro-LGMN经过剪切会形成47KD、46KD的LGMN中间体以及成熟的活性LGMN(active-LGMN，26-289aa)。参见Structure and function of legumain in health and disease,Elfriede Dall et,al.Biochimie,2016。因此，本文中的“LGMN”或“LGMN蛋白”包括pro-LGMN、活性LGMN以及LGMN中间体。类似地，“突变的LGMN”或“突变的LGMN蛋白”包括突变的pro-LGMN、突变的活性LGMN以及突变的LGMN中间体。
本发明提供体外抑制肿瘤细胞转移的方法，包括减弱肿瘤细胞的LGMN蛋白与整合素或其β亚基之间的相互作用的步骤。所述相互作用是LGMN的RGD序列与整合素或其β亚基之间的相互作用。所述方法通过以下任意一种或多种方式减弱所述相互作用：(1)敲除或敲低肿瘤细胞的LGMN蛋白的表达；(2)敲除或敲低肿瘤细胞中整合素或其β亚基表达；(3)在野生型或敲除LGMN蛋白的肿瘤细胞中表达与整合素或其β亚基的相互作用减弱或消失的突变型LGMN蛋白；(4)在野生型或者 敲除整合素或其β亚基的肿瘤细胞中表达与LGMN蛋白的相互作用减弱或消失的突变型整合素或其β亚基；(5)使肿瘤细胞与抗LGMN抗体或其抗原结合片段或含有所述抗LGMN抗体或其抗原结合片段的蛋白接触；(6)使肿瘤细胞与降低二价金属离子浓度的试剂接触。
在具体实施方案中，所述方法包括但不限于：使用突变型LGMN蛋白的表达载体和/或整合载体处理肿瘤细胞，和/或使用突变型整合素或其β亚基的表达载体和/或整合载体处理肿瘤细胞，和/或使用抗LGMN抗体或其抗原结合片段处理肿瘤细胞，和/或使用能降低二价金属离子浓度的试剂处理肿瘤细胞，和/或在肿瘤细胞中转入基因敲除载体，以敲除所述肿瘤细胞中LGMN蛋白表达和/或整合素或其β亚基表达，和/或采用ZFN、TALEN或CRISPR/Cas9等技术敲除肿瘤细胞LGMN蛋白表达和/或整合素或其β亚基的表达，和/或通过干扰RNA介导的基因沉默敲低LGMN蛋白表达和/或整合素或其β亚基的表达，和/或在肿瘤细胞中转入基因插入载体、以在敲除野生型LGMN蛋白和/或整合素或其β亚基的编码序列的同时将与整合素或其β亚基的相互作用减弱或消失的突变型LGMN蛋白的表达框和/或与LGMN蛋白的相互作用减弱或消失的突变型整合素或其β亚基的表达框整合到肿瘤细胞的基因组中，从而减弱或破坏细胞内LGMN蛋白整合素或其β亚基之间的相互作用。
试剂及其用途
本发明提供抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂及其在制备抑制肿瘤转移或肿瘤细胞转移的药物中的用途。本文所述LGMN蛋白与整合素的“相互作用”是LGMN蛋白的RGD序列与整合素β亚基之间的相互作用。本文所述整合素的“β亚基”包括但不限于：β 1、β 3、β 5、β 6和β 8。所述整合素是含有所述β亚基的整合素，例如α vβ 3、α vβ 1、α vβ 5、α vβ 6、α vβ 8、α 5β 1和α 8β 1。作为示例，野生型LGMN如GenBank No.CAG33687.1所示，其中RGD位于其118位；野生型pro-LGMN如GenBank No.CAG33687.1所示；野生型α v的氨基酸序列如GenBank No.AGC09592.1所示；野生型β 3的氨基酸序列如GenBank No.AAI27668.1所示。
抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂可以是突变型LGMN蛋白和/或其打靶载体，其中，与野生型相比，突变型LGMN的RGD序列发生导致LGMN与整合素或其β亚基的相互作用减弱或消失的突变。所述突变是RGD序列突变为RGE，RAD，RAE，KGD或KGE。
抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂还可以是突变型整合素或其β亚基和/或其打靶载体，其中，与野生型相比，突变型整合素或其β亚基在与LGMN蛋白相互作用的配体结合区域具有一个或多个导致其与LGMN蛋白相互作 用减弱或消失的突变。所述整合素或其β亚基在D120和/或S121位置上具有突变，例如取代突变；优选取代为丙氨酸或谷氨酸。
抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂还可以是能降低二价金属离子浓度的试剂，例如乙二胺四乙酸(EDTA)，氨基三乙酸(NTA)，柠檬酸(CA)、酒石酸(TA)和葡糖糖酸(GA)等。
抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂还可以是敲低或敲除LGMN蛋白和/或整合素或其β亚基的表达的试剂，例如ZFN和/或TALEN和/或CRISPR/Cas9试剂和/或小干扰RNA。在得知了靶序列后，制备敲低或敲除特定基因表达的干扰试剂的方法是本领域人员熟知的。针对蛋白及其编码序列构建所述敲低或敲除试剂的过程本领域周知。作为本发明的一种优选方式，所述抑制试剂是LGMN或整合素特异性的shRNA或其构建物，其中shRNA特异识别LGMN或整合素的基因或其转录本。本文中，“转录本”包含UTR区域(例如3’UTR)和CDS区。示例性地，敲除LGMN效果较佳的小干扰RNA具有SEQ ID NO:5或6所示的序列；敲除整合素或其β亚基效果较佳的小干扰RNA具有SEQ ID NO:7所示的序列。此外，适用于本发明的ZFN、TALEN和CRISPR/Cas9技术为本领域所周知。各技术各自通过DNA识别域与核酸内切酶的共同作用实现靶基因的敲除。
抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂还可以是抗LGMN抗体或其抗原结合片段，所述抗体抑制LGMN蛋白与整合素或其β亚基之间的相互作用。
抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂还可以是整合素或其β亚基的抗体或其抗原结合片段，或者整合素或其β亚基的受体抑制剂(例如受体抑制剂，包括但不限于αvβ3和αvβ5的抑制剂Cilengitide、αIIbβ3的抑制剂Tirofiban和Eptifibatide、αIIbβ3的抗体Abciximab、αvβ1的抑制剂PLN-1474和抗体Volociximab、αvβ6的抗体264RAD、α4β1的抗体Natalizumab等)。
本文所述的抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂也可用作疫苗佐剂。该疫苗佐剂可以和疫苗(例如OVA)联用，形成疫苗组合物，可用于预防和/或治疗肿瘤转移。
抗体
本文中，术语“抗体”包括单克隆抗体(包括全长抗体，其具有免疫球蛋白Fc区)，具有多表位特异性的抗体组合物，多特异性抗体(例如，双特异性抗体)，双抗体和单链分子，以及抗体片段，尤其是抗原结合片段，例如，Fab、Fab’、F(ab’)2、Fv和scFV)。本文中，术语“免疫球蛋白”(Ig)和“抗体”可互换地使用。优选地，所 述抗原结合片段，由其来源抗体的重链可变区或轻链可变区的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，对于LGMN，优选至少等于其来源抗体亲和力的1/100，在更优选方式中至少等于1/10。这种抗体片段将包含最少5个氨基酸，优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。
基本的4链抗体单元是由两条相同的轻链(L)和两条相同的重链(H)构成的异四聚体糖蛋白。每条轻链通过一个共价二硫键与重链相连，而两条重链通过一个或多个二硫键彼此相连，二硫键的数目取决于重链的同种型。每条重链和轻链还具有间隔规律的链内二硫桥。每条重链在N-末端具有可变结构域(VH)，接着是三个(对于每种α和γ链，CH1、CH2和CH3)和四个(对于μ和ε同种型，CH1、CH2、CH3和CH4)恒定结构域(CH)以及位于CH1结构域与CH2结构域之间的绞链区(Hinge)。每条轻链在N-末端具有可变结构域(VL)，接着是其另一端的恒定结构域(CL)。VL与VH排列在一起，而CL与重链的第一恒定结构域(CH1)排列在一起。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。成对的VH和VL一起形成一个抗原结合位点。关于不同类别抗体的结构和性质，参见如Basic and Clinical Immunology，第八版，Daniel P.Sties，Abba I.Terr和Tristram G.Parsolw编辑，Appleton&Lange，Norwalk，CT，1994，第71页和第6章。来自任何脊椎动物物种的轻链，根据其恒定结构域氨基酸序列，可归入两种称作κ和λ的截然不同型中的一种。根据其重链恒定结构域(CH)氨基酸序列，免疫球蛋白可归入不同的类或同种型。有五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，分别具有称作α、δ、ε、γ和μ的重链。根据CH序列和功能的相对较小差异，γ和α类可进一步分为亚类，例如人表达下列亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgA2。
抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基末端结构域。重链和轻链的可变结构域可分别称为“VH”和“VL”。这些结构域通常是抗体的最可变的部分(相对于相同类型的其它抗体)并含有抗原结合位点。在一个或多个实施方案中，本文所述抗体的VH和VL具有SEQ ID NO:1和SEQ ID NO:2所示的序列或与其具有90％序列相同性的变体。
术语“可变的”指可变结构域中的某些区段在抗体序列中差异广泛的情况。可变结构域介导抗原结合并限定特定抗体对其特定抗原的特异性。然而，变异性并非均匀分布于可变结构域跨越的全部氨基酸。相反，其集中在三个称为高变区(HVR)的区段(在轻链和重链可变结构域中均有)，即分别为重链可变区的HCDR1、HCDR2、HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3。可变结构域中更为高度保守 的部分称为构架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(FR1、FR2、FR3和FR4)，它们大多采取β-折叠构象，通过形成环状连接且在有些情况中形成β-折叠结构一部分的三个HVR连接。每条链中的HVR通过FR区非常接近的保持在一起，并与另一条链的HVR一起促成抗体的抗原结合位点的形成(参见Kabat等，Sequences of Immunological Interest，第五版，国立卫生研究所，Bethesda，MD，1991)。通常，轻链可变区的结构为FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4，重链可变区的结构为FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如在抗体依赖性细胞介导的细胞毒性中抗体的参与。在优选实施方案中，本发明的抗LGMN抗体的CDR如下所示：HCDR1包含GFTFSSYA，HCDR2包含IGNSGNYT，HCDR3包含AKSSDSFNY，LCDR1包含QSISSY，LCDR2包含DAS，LCDR3包含QQAYANPDT。本文所述CDR使用IMGT抗体编号系统获得。在知晓了抗体或其抗原结合片段的序列之后，本领域技术人员可以容易地使用其他编号系统获得相应CDR。
“Fc区”(可结晶片段区域)或“Fc结构域”或“Fc”是指抗体重链的C-末端区域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫系统的各种细胞(例如，效应细胞)上的Fc受体的结合，或者与经典补体系统的第一组分(C1q)的结合。在IgG，IgA和IgD抗体同种型中，Fc区由来自抗体两条重链的CH2结构域和CH3结构域的两个相同的蛋白片段构成；IgM和IgE的Fc区在每个多肽链中包含三个重链恒定结构域(CH结构域2-4)。虽然免疫球蛋白重链的Fc区的边界可以变化，但是人IgG重链Fc区通常定义为从重链位置C226或P230的氨基酸残基到羧基端的序列段，其中该编号是根据EU索引。如本文所使用的，Fc区可以是天然序列Fc或变体Fc。
在不实质性影响抗体活性的前提下，本领域技术人员可以对本发明的序列取代、添加和/或缺失一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得所述抗体或其功能性片段序列的变体。它们都被视为包括在本发明保护的范围内。如在可变区的FR和/或CDR区中将具有类似性质的氨基酸进行取代。取代优选是保守性取代；可进行保守性取代的氨基酸残基为本领域所周知。在一些实施方案中，本发明所述变体的序列可以与其来源序列有至少有95％、96％、97％、98％或99％的一致性。本发明所述的序列一致性可以使用序列分析软件测量。例如使用缺省参数的计算机程序BLAST，尤其是BLASTP或TBLASTN。
本发明的抗LGMN抗体可以被修饰以影响功能。本发明包括具有修饰的糖基化模式的抗LGMN抗体，或者可进行修饰以除去不期望的糖基化位点。
可采用本领域常规的方法制备本发明的抗LGMN抗体，如本领域熟知的杂交瘤技术。或者，本发明的抗LGMN抗体可在除杂交瘤细胞系以外的细胞系中表达。可用编码本发明抗体的序列转化合适的哺乳动物宿主细胞。转化可采用任何已知的方法进行，例如包括将多核苷酸包装在病毒(或病毒载体中)并用病毒(或载体)转导宿主细胞。所用的转化程序取决于将转化的宿主。用于将异源多核苷酸引入哺乳动物细胞中的方法为本领域所熟知，包括葡聚糖介导的转染、磷酸钙沉淀、聚凝胺介导的转染、原生质体融合、电穿孔、将多核苷酸囊封在脂质体中和将DNA直接微注射至核中等。可用作用于表达的宿主的哺乳动物细胞系为本领域所熟知，包括但不限于可从美国典型培养物保藏中心(ATCC)获得的多种永生化细胞系，包括但不限于中国仓鼠卵巢(CHO)细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，HepG2)等。尤其优选的细胞系通过确定哪些细胞系具有高表达水平并产生具有基本LGMN结合特性的抗体来进行选择。抗LGMN抗体可以与其他需要表达的多肽融合表达。
核酸分子
本发明包括编码本发明所述抗体或其抗原结合片段的核酸分子。核酸分子可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明也包括编码多肽或蛋白的核酸分子的简并变异体，即编码相同的氨基酸序列但核苷酸序列有所不同的核酸分子。
本文中，编码序列指核酸序列中直接确定其蛋白产物(例如抗体或其抗原结合片段等)的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括，但不限于DNA、cDNA和重组核酸序列。本文所述抗体的编码序列可以与其他需要融合表达的多肽的编码序列处于同一表达框。
本文所述的核酸分子通常可以用PCR扩增法获得。具体而言，可根据本文所公开的核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。或者，也可直接合成本文所述的核酸分子。
核酸构建物和细胞
构建表达载体而由细胞表达的方法涉及核酸构建物。本文的核酸构建物含有本文所述的核酸分子，以及与这些序列操作性连接的一个或多个调控序列。调控序列可 以是合适的启动子序列、转录终止子序列、前导序列、在至少一种有机体中起作用的复制起点、方便的限制酶位点和一个或多个可选择的标记，这些在本领域技术人员的知识范围内。
本发明所述的核酸分子可以多种方式被操作以保证所述抗体或治疗用蛋白的表达。在将核酸构建物插入载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变核酸分子序列的技术是本领域已知的。
在某些实施方案中，所述核酸构建物是载体。载体可以是克隆载体，也可以是表达载体，或者是同源重组载体。本发明的核酸分子可被克隆入许多类型的载体，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。将核酸分子引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。将感兴趣的核酸分子引入宿主细胞的生物学方法包括使用DNA和RNA载体。将核酸分子引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。将核酸分子引入宿主细胞的生物学方法包括使用病毒载体，例如源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒的载体，特别是逆转录病毒载体。可利用本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒，例如慢病毒颗粒。
本文中，宿主细胞含有、表达和/或分泌本文所述的抗体或其抗原结合片段和任选的治疗用多肽。本文中，当提及细胞含有或包含、表达、分泌某种分子如多肽时，“含有”指所述所述分子含于所述细胞内或表面上；“表达”指该细胞生产所述分子；“分泌”指该细胞将所表达的分子分泌出细胞外。宿主细胞既包括最终用于蛋白表达的细胞，也包括生产该细胞过程中使用到的各种工程细胞，如大肠杆菌细胞，以用于如提供本发明蛋白的编码序列或提供本文所述的载体。
药物筛选
本文还提供LGMN蛋白和/或整合素或其β亚基作为靶点在筛选抑制肿瘤转移或肿瘤细胞转移的潜在物质中的用途，或用作分子指标在临床上用于诊断肿瘤转移。筛选所述潜在物质的方法包括：用候选物质处理检测(1)LGMN蛋白，和/或(2)整合素或其β亚基，和/或(3)LGMN蛋白与整合素或其β亚基的相互作用的体系；和检测所述体系中相应蛋白的含量或所述相互作用的强弱。所述靶点具体包括LGMN蛋白的RGD序列，和/或，整合素或其β亚基的与LGMN蛋白相互作用的配体结合 区域。若所述候选物质下调LGMN蛋白或抑制所述相互作用(例如LGMN蛋白的RGD序列与整合素或其β亚基的相互作用)，则表明该候选物质是抑制肿瘤转移或肿瘤细胞转移的潜在物质。例如，所述检测相互作用的体系可以是表达LGMN蛋白和整合素的体系，例如细胞或细胞培养物。所述的细胞可以是内源性表达LGMN蛋白和整合素的细胞；或可以是重组表达LGMN蛋白和整合素的细胞。所述的表达LGMN蛋白和整合素的体系还可以是亚细胞体系、溶液体系、组织体系、器官体系或动物体系(如动物模型，优选非人哺乳动物的动物模型，如鼠、兔、羊、猴等)等。体系中LGMN蛋白和整合素的相互作用的检测方法本领域已知，检测下游信号(例如FAK-Src-RhoA信号)或细胞迁移。所述候选物质例如本文所述抗体。
诊断用途、测定和试剂盒
本发明的抑制试剂(例如抗LGMN抗体)可用于诊断目的，用来检测、诊断或监控与LGMN相关的疾病和/或病况，例如结合测定来检测和/或定量在肿瘤组织或细胞中表达的LGMN。本发明提供使用本领域技术人员已知的经典免疫组织学方法检测样本中LGMN的存在或水平。可以体内或体外进行LGMN的检测。适用于检测LGMN的存在的方法实例包括ELISA、FACS、RIA等。
对于诊断应用来说，通常用可检测的标记基团来标记抗LGMN抗体。合适的标记基团本领域周知，包括(但不限于)以下：放射性同位素或放射性核素、荧光基团、酶促基团、化学发光基团、生物素基基团或由二级报导体识别的预定多肽表位。用于标记蛋白质的各种方法在本领域中已知且可用来进行本发明。
本发明的一个方面提供识别表达抗LGMN抗体的细胞。在一个具体实施方案中，用标记基团标记抗体并检测经过标记的抗体与LGMN的结合。在另一个具体实施方案中，体内检测抗体与LGMN的结合。在另一个具体实施方案中，使用本领域中已知的技术来分离和测量抗体-LGMN。
本发明的另一方面提供检测与本发明的抗体竞争结合LGMN的测试分子的存在。一种所述测定的实例将涉及在存在或不存在测试分子的情形下检测含有一定量LGMN的溶液中的游离抗体的量。游离抗体(即，未结合LGMN的抗体)的量增加将表示测试分子能与该抗体竞争结合LGMN。在一个实施方案中，用标记基团标记抗体。或者，标记测试分子并在存在或不存在抗体的情形下监控游离测试分子的量。
药物组合物、施用途径
本发明提供药物组合物，其包含治疗有效量的一种或多种本发明的抗LGMN抗体以及药学上可接受的载体，所述载体包括但不限于稀释剂、载剂、增溶剂、乳化剂、防腐剂和/或佐剂。
在某些实施方案中，药物组合物中可接受的载体等优选地在所采用的剂量和浓度下对接受者无毒。在某些实施方案中，药物组合物可含有用于改善、维持或保留例如组合物的pH、渗透性、粘度、澄清度、颜色、等渗性、气味、无菌性、稳定性、溶解或释放速率、吸收或渗透的这类物质。这些物质为现有技术已知，例如可参见REMINGTON'S PHARMACEUTICAL SCIENCES，第18版，A.R.Genrmo编，1990，Mack Publishing Company。可视预期的施用途径、递送方式和所需的剂量来确定最佳的药物组合物。
可选择本发明的药物组合物用于肠胃外递送。或者，可选择组合物用于吸入或通过消化道(诸如经口)递送。所述药学上可接受的组合物的制备在本领域的技术内。
其它药物组合物将为本领域技术人员显而易见，包括在持续或控制释放递送配制物中包含抗LGMN抗体的配制物。用于配制多种其它持续或可控传递方式的技术(诸如脂质体载剂、生物易蚀微粒或多孔珠粒和积存注射)也为本领域技术人员所知。
用于体内施用的药物组合物通常以无菌制剂的形式提供。通过经无菌过滤膜过滤来实现灭菌。在组合物冻干时，可在冻干和复水之前或之后使用此方法进行灭菌。用于肠胃外施用的组合物可以冻干形式或在溶液中储存。肠胃外组合物通常放在具有无菌进入孔的容器中，例如具有皮下注射针可刺穿的塞子的静脉内溶液带或小瓶。
药物组合物一经配制，就以溶液、悬浮液、凝胶、乳液、固体、晶体或以脱水或冻干粉末的形式储存在无菌小瓶中。所述配制物可储存成即用形式或在施用前复水的形式(例如，冻干)。本发明还提供用于产生单剂量施用单位的试剂盒。本发明的试剂盒可各自含有具有干燥蛋白的第一容器和具有含水配制物的第二容器。在本发明的某些实施方案中，提供含有单腔和多腔预填充注射器(例如，液体注射器和冻干注射器)的试剂盒。
本发明也提供通过施用本发明任一实施方案所述的抗LGMN抗体或其抗原结合片段或其药物组合物来治疗患者的方法。
本文中，术语“患者”、“个体”、“对象”在本文中可互换使用，包括任何生物体，优选动物，更优选哺乳动物(例如大鼠、小鼠、狗、猫、兔等)，且最优选的是人。“治疗”指向对象采用本文所述治疗方案以达到至少一种阳性治疗效果(比如，癌症细胞数目减少、肿瘤体积减小、癌细胞浸润至周边器官的速率降低或肿瘤转移或肿瘤生长的速率降低)。有效治疗患者的治疗方案可根据多种因素(比如患者的疾病状态、年龄、体重及疗法激发对象的抗癌反应的能力)而变。
将采用的含有本发明抗LGMN抗体或其抗原结合片段的药物组合物的治疗有效量将取决于例如治疗程度和目标。本领域技术人员将了解，用于治疗的适当剂量水平 将部分取决于所递送的分子、适应症、施用途径和患者的大小(体重、体表或器官大小)和/或状况(年龄和一般健康状况)而变化。在某些实施方案中，临床医生可滴定剂量并改变施用途径来获得最佳的治疗效果。
给药频率将取决于所用配制物中特定抗LGMN抗体的药物动力学参数。临床医生典型地施用组合物直到达到实现所需效果的剂量。组合物因此可作为单次剂量施用，或随时间以作为两次或多次剂量(可含有或不含有相同量的所需分子)施用，或通过植入装置或导管以连续输液的方式施用。
药物组合物的施用途径是根据已知方法，例如经口、通过静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉或病灶内途径注射；通过持续释放系统或通过植入装置。
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非意图限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则均为本领域常规的材料和方法。
实施例
一、实验材料和方法
1.1材料
细胞系：乳腺癌细胞系MDA-MB-231、SCP2、Hs578T、MCF7、T47D，及乳腺上皮细胞系MCF10A。
Legumain抗原多肽序列为：PQNFLAVLRGDAEAVKGIG(SEQ ID NO:8)，由上海强耀生物科技有限公司合成。
噬菌体库为Tomlinson I+J(购自Geneservice，I库容量为1.47×10 8，J库容量为1.37×10 8)。
抗体
Figure PCTCN2022111658-appb-000001
Figure PCTCN2022111658-appb-000002
1.2方法
1.2.1磷酸钙法瞬时转染293T细胞
1.转染前一天分细胞，使细胞在转染时达到60～70％的密度；
2.转染前1hr，换成含有25μM氯喹的培养基，每10cm盘体积为10ml；
3.在15ml无菌离心管中，加入10μg DNA，用灭菌的MilliQ定溶1095μl，再加入155μl 2M CaCl2；
4.逐滴加入1,250μl 2×HBS，边加边轻柔混匀；
5.将该混合物直接逐滴加入细胞中，均匀地洒在整个平皿表面；
6.37℃培养7～11hr，漂洗一次，换成不含氯喹的培养基；
7.转染48～72hr后收集细胞。
1.2.2病毒包被、感染细胞
病毒包被
1.前一天分293T，第二天转染时密度达70％左右；
2.用磷酸钙转染的方法，将包装病毒的质粒按照一定比例转入293T细胞；
3. 48～72hr后收取培养液(含病毒)，用0.45μm针头滤器过滤后即可使用；
4.必要的话可进行超离，将病毒进行浓缩，27,000rpm，4℃离心2hr。
病毒感染细胞
1.前一天将要感染的细胞传代；
2.将细胞培养液，病毒和凝聚胺(polybrene，终浓度8μg/ml)混在一起，加入细胞中；
3. 18hr之后吸掉含病毒的培养液，加入新鲜的培养液；
4. 36～48hr后加入抗生素(如嘌呤霉素(puromycin)、新霉素(neomycin))进行筛选。
1.2.3 Protein A纯化LGMN蛋白
1.磷酸钙法转染293T细胞；
2.培养至培养液变黄，收取培养液(大约转染后3～4天)；
3.用等体积Protein A纯化结合缓冲液(Binding buffer)稀释培养液，稀释时，边加Binding buffer边搅拌，调pH至8.0；
4. 15,000rpm，4℃离心30min；
5.取上清用连通管加入Protein A柱子；
6.用Protein A纯化结合缓冲液(Binding buffer)洗柱子15个柱体积；
7.用Protein A纯化洗脱缓冲液(Elution buffer)洗脱7个柱体积，收集管中预先按1:10加入
8.Protein A纯化中和缓冲液(Neutralization buffer)；
9.收集洗脱液，浓缩，同时换buffer为HBS，蛋白分装后冻存于超低温冰箱。
1.2.4免疫共沉淀和免疫印迹
用预冷的TBS洗一遍细胞表面，用细胞刮将细胞刮下，溶于预冷的TBS中。3,000rpm离心3min后弃上清，用裂解缓冲液(Lysis buffer)重悬，冰上放置30min；12,000rpm离心15min，得到细胞裂解液。蛋白含量测定按照BCA法进行测定。在细胞裂解液中加入特定的抗体4度孵育过夜，以抗兔IgG作为对照，再加入protein G珠(bead)4度孵育2h。清洗beads三次后，制备样品。
同等量的蛋白样品用9％的SDS-PAGE电泳分离；电泳完毕后，利用湿式电转仪将蛋白转印到硝酸纤维素膜(NC)上，设定电流为280mA，时间80min；转印完毕后取出NC膜，含有转移蛋白的膜面向上，依次用封闭液(含5％脱脂奶粉的TBST)在室温孵育2hr；在特异抗体在4℃孵育过夜；TBST洗NC膜三次，每次5min；HRP耦联的二抗室温孵育1hr；TBST洗NC膜两次，每次5min。再ECL检测试剂进行荧光显影。
1.2.5 shRNA敲低基因
敲低人源LGMN蛋白的shRNA序列为：5-GCCATGCCTACCAGATCATTC-3(shLGMN-1)和5-GTATTGAGAAGGGTCATATTT-3(shLGMN-2).
敲低人源β 3整合素的shRNA序列为：5-GCTTAGCTTGAGGGTGACTAT-3.
对照shRNA序列为：5-CCTAAGGTTAAGTCGCCCTCG-3.
1.2.6流式细胞术
FITC标记的Legumain蛋白与梯度稀释的抗体于50ul PBS(1mM Ca 2+,1mM Mg 2+)体系中室温孵育30分钟，之后与SCP2乳腺癌细胞在室温下孵育30min。细胞洗涤2次后进行流式细胞术分析。人源化IgG与相同浓度FITC标记的legumain蛋白孵育组作为阴性对照。
1.2.7伤口愈合实验
将乳腺癌细胞铺至96孔板生长至80％-90％的密度后划线，细胞在含1％血清的培养基中生长16h，通过Incucyte FLR(Essen)自动成像仪记录16h内伤口愈合情况。伤口愈合的比例计算公式为，愈合比例＝[1-(16小时后伤口的宽度/起始伤口的宽度)]×100％。
1.2.8细胞迁移、侵袭实验
对于细胞迁移实验，将无血清培养基重悬的1×10 5SCP2细胞(100μl)铺至细胞迁移小室(Corning,8-μm滤膜)的上层，下层为600μl含10％血清的细胞培养基。细胞提前与抗体孵育1小时，并在迁移实验过一直维持相同浓度的抗体处理。对于细胞侵袭实验，在上述迁移实验中的迁移小室上层铺入基质胶，其他条件与迁移实验保持一致。细胞迁移或侵袭16小时后，将迁移至小室下底面的细胞用2％多聚甲醛固定并DAPI染色，上表面用棉签擦去未迁移过去的细胞。荧光显微镜下记录5个视野中的迁移细胞的数目并取平均值。
1.2.9乳腺癌骨转移实验
将1×10 5荧光素酶标记的SCP2乳腺癌细胞注射至麻醉的5周龄裸鼠的左心室，从肿瘤细胞注射前两天开始，每两天腹腔注射C10抗体(2mg/kg)及等量的人源IgG作为对照，直至实验结束。每周腹腔注射75mg/kg荧光素酶底物，并通过活体成像系统检测小鼠体内肿瘤的生长。最后取处理第六周的小鼠拍摄X-Ray及micro-CT检测小鼠骨损伤。
1.2.10噬菌体展示实验
将RGD抗原多肽100μg/ml包被免疫管，将Tomlinson I+J噬菌体库在免疫管中进行三轮吸附-洗脱-扩增的亲和富集筛选。在三轮富集筛选过程中逐步增加非特异性洗涤的强度，充分去除非特异性结合的噬菌体。每轮洗脱下来结合的噬菌体扩增后，进行下一轮的筛选。第四轮将纯化的LGMN蛋白20μg/ml作为抗原，进行第四轮吸附洗脱，最后将第四轮洗脱得到的噬菌体感染TG1(OD＝0.4-0.6)大肠杆菌菌株。从第四轮涂布的TYE平板上随机挑取单克隆至96孔细胞培养板中，分别制备单克隆的噬菌体上清进行噬菌体ELISA检测。
1.2.11 ELISA实验
包被RGD多肽或LGMN蛋白于96孔酶标板，以包被相同浓度的牛血清白蛋白(BSA)作为阴性对照。2％脱脂牛奶封闭后，每孔加入50μl噬菌体上清，室温孵育2h。PBST(0.1％土温20)洗板三次后，每孔加入HRP-抗-KM13抗体(1:5000，50μl)，室温孵育1h。PBST洗三次后，每孔加入100μl TMB底物，避光显色，待底物呈蓝色后，加入2mol/L H 2SO 4终止反应，最后酶标仪读取OD450。
1.2.12生物膜干涉实验(Bio-layer Interferometry，BLI)
BLI实验利用Octet Red 96仪器(ForteBio,Inc.)进行。原理为通过使生物素化的LGMN蛋白固定在链霉亲和素(streptavidin，SA)生物传感器上，并且与不同浓度梯度的抗体于动力学缓冲液中孵育，观察两者的动态结合与解离情况。根据LGMN与不同浓度梯度的抗体结合曲线整体拟合到1:1的Langmuir结合模型，R 2值≥0.95， 计算出C10抗体与LGMN结合亲和力的KD值。结合实验在25℃进行。数据分析采用Octet Data Analysis Software 9.0(ForteBio,Menlo park,CA,USA)。
二、实验结果
2.1 LGMN表达水平与乳腺癌转移和预后相关
为了探究与乳腺癌转移过程相关的基因，我们首先利用乳腺癌临床基因数据库(GSE45255,GSE65194和GSE22219)进行Univariate Cox proportional hazards regression分析。分析结果显示:在三个数据库中分别有795个基因，1402个基因和1896个基因与乳腺癌的转移呈正相关，且其中25个基因在三个数据库中都显示了可能与肿瘤转移相关(图1，A)，25个基因列表如图2，A所示。基因注释(Gene Ontology)分析揭示了这25个基因在细胞的基本定位和功能(图1，B)。其中LGMN蛋白作为一个蛋白酶主要分布在溶酶体中，参与蛋白的剪切和降解。Kaplan-Meier生存分析发现高表达LGMN的乳腺癌患者会更早出现转移(图1，C-E)，同时对于更大样本量的乳腺癌病人转移相关数据库的分析我们同样发现，高表达LGMN的乳腺癌病人转移率增加，生存期也更低(图2，B,C)。这些数据表明LGMN蛋白可能在乳腺癌细胞转移的过程中发挥了一定的功能。
为了进一步探究LGMN在乳腺癌转移中的功能，我们比较了正常乳腺上皮细胞MCF10A和几种常见的乳腺肿瘤细胞系MCF7、T47D、Hs578T、MDA-MB-231、SCP2中LGMN的表达水平及上述乳腺癌细胞的转移能力。细胞迁移实验和细胞侵袭实验发现与低转移的MCF7和T47D细胞相比，Hs578T、MDA-MB-231和SCP2细胞拥有更强的转移侵袭能力(图1，F-G)。免疫印迹实验表明，与正常乳腺上皮细胞MCF10A相比，几种乳腺癌肿瘤细胞均高表达LGMN，细胞裂解液中检测到56kDa的全长形式及36kDa活化形式的LGMN蛋白(图1，H)，且分析发现全长形式的LGMN在高转移的乳腺癌细胞Hs578T、MDA-MB-231和SCP2细胞中的表达更高。而在乳腺癌细胞分泌至细胞培养液中都是未成熟的全长形式的LGMN(图1，I)，同样分泌在胞外的pro-LGMN蛋白也是随着转移能力的增强而含量更高。且分析发现只有全长形式的pro-LGMN与乳腺癌细胞系的侵袭和转移能力呈正相关(图2，D-I)。由此表明乳腺癌细胞分泌至胞外的pro-LGMN蛋白在乳腺癌迁移和侵袭的过程中发挥着重要的作用。
2.2敲低LGMN显著抑制乳腺癌细胞的迁移和侵袭能力
为了进一步研究LGMN和乳腺癌细胞转移之间的关系，我们在Hs578T、MDA-MB-231和SCP2乳腺癌细胞通过shRNA介导的RNAi特异性敲低肿瘤细胞中的LGMN的表达水平，然后检测LGMN表达对于乳腺癌细胞迁移和侵袭能力的影响。 结果显示:两条针对LGMN的shRNA均可以有效地敲低LGMN在肿瘤细胞中的表达(图3，A)。且敲低LGMN显著地抑制了乳腺癌细胞的迁移和在基质胶中侵袭的能力(图3，B-D)。同时我们也验证了敲低LGMN并不影响乳腺癌细胞的生长和增殖能力(图4，A-B)。
2.3 LGMN通过RGD序列与整合素β3结合
进一步我们为了探究LGMN是否通过与整合素αvβ3结合调控乳腺癌细胞的迁移和侵袭，我们首先通过免疫共沉淀实验(Co-IP)检测了乳腺癌细胞中LGMN与整合素αvβ3的相互作用。RGD序列是整合素αvβ3与配体结合的重要位点。尽管全长形式和活化形式的LGMN蛋白都含有保守的RGD序列，但是我们在免疫共沉淀实验中发现只有分泌到胞外的全长形式的pro-LGMN可以与乳腺癌细胞表达的β3整合素结合(图5，A)。接下来我们探究RGD序列对于LGMN与整合素之间结合的影响。免疫共沉淀结果显示将LGMN蛋白中的RGD序列突变为RGE可以抑制LGMN和整合素β3的结合(图5，B)。整合素与配体的结合依赖于二价金属离子的调控。我们发现LGMN与整合素的相互作用同样也依赖于二价金属离子的存在，当用EDTA处理后，LGMN和整合素β3的结合也被显著抑制(图5，C)。另一方面，金属离子对整合素与配体结合的调控主要通过整合素上金属离子结合位点MIDAS发挥作用，我们同样发现β3整合素上的配体结合位点突变(β 3 S121A)同样不能与LGMN结合(图5，D)。LGMN作为蛋白酶其酶活性中心位点的突变(C189S)并不影响LGMN与整合素β3的结合(图5，E)。综合上述实验，我们发现pro-LGMN可以被乳腺癌细胞分泌到胞外，通过其RGD序列与细胞表面的整合素结合。
2.4 LGMN通过与整合素结合活化下游信号FAK-Src-RhoA信号促进乳腺癌细胞的迁移和侵袭
接下来我们想探究LGMN与整合素的结合在调控乳腺癌细胞的迁移和侵袭中所发挥的功能。为了研究这个问题，我们检测了稳定表达野生型、LGMN D120E和无酶活型LGMN C189S的稳转株Hs578T、MDA-MB-231和SCP2细胞的迁移和侵袭能力。结果发现，在细胞中重新表达野生型LGMN和酶活缺失的LGMN C189S后，肿瘤细胞的迁移和侵袭能力显著恢复(图6，A-C)。然而过表达突变型LGMN D120E，即破坏了LGMN与β3整合素的结合后，肿瘤细胞的转移能力受到抑制(图6，A-C)。以上结果直接证明与β3整合素的结合介导肿瘤细胞的转移进程，而LGMN的酶活性中心对于肿瘤细胞的转移并没有影响。由于整合素的表达水平也可能会调控乳腺癌细胞的转移,我们同时检测了不同的LGMN表达对于β3整合素的影响，流式细胞实验结果显示在不同的LGMN表达的稳转株中，β3的表达不受影响(图7)。为了进一步探究LGMN 促进乳腺癌细胞转移是否是通过自分泌pro-LGMN所介导的。我们在敲低LGMN的稳转株细胞培养基中加入LGMN野生型和点突变的LGMN D120E的纯化的蛋白，通过伤口愈合，细胞迁移和侵袭实验我们发现只有野生型pro-LGMN蛋白才能恢复乳腺癌细胞的迁移和侵袭能力(图6，D-F)。
整合素与配体的结合可以通过活化下游FAK，Src激酶等分子，然后通过影响不同的三磷酸鸟苷水解酶(small GTPase)(主要包括:Rac1、RhoA和Cdc42)从而改变细胞骨架蛋白的重排，最终实现对细胞迁移的调控。接下来我们利用免疫印迹相关实验检测了整合素下游相关信号活化情况，并确定经由哪一种GTPase而最终促进乳腺癌细胞转移。实验结果表明，LGMN可以活化整合素经典下游信号通过FAK-Src，并且活化RhoA进而促进肿瘤细胞转移(图6，G-H)。同样在培养液中加入LGMN野生型的蛋白处理同样可以引起整合素下游信号FAK、Src和RhoA的活化，而LGMN D120E蛋白则无法活化该下游信号(图6，I-J)。
2.5 LGMN与整合素结合在体内促进乳腺癌骨转移
通过上面的研究我们知道，LGMN可以被乳腺癌细胞分泌到细胞外作为整合素的配体活化整合素下游信号，进而促进乳腺癌细胞转移。为了进一步探究LGMN-整合素信号在体内对于乳腺癌转移的影响，我们建立了实验性乳腺癌骨转移模型。将稳定表达荧光素酶的SCP2乳腺癌细胞通过左心室注射到裸鼠体内，并于注射当天和实验6周内实时监测肿瘤生长情况。实验结果表明，表达LGMN野生型的SCP2细胞形成了更多的骨转移信号及骨组织的病灶(图8，A-B)。而LGMN D120E突变组及LGMN敲低组中，骨转移信号显著减少(图8，A-B)。监测小鼠血浆中人源LGMN的表达也发现，对照组和LGMN恢复组中血浆中LGMN的含量明显高于LGMN敲低和LGMN D120E恢复组(图8，C)。同时对骨的形态学测量分析发现回补野生型LGMN及LGMN C189S的小鼠的骨的受损程度高于LGMN敲低组及LGMN D120E回补组，表现为更低的骨含量(图8，D)，更少的骨小梁数(图8，E)，以及较高的骨小梁分离度(F)。为了进一步验证SCP2细胞在小鼠体内的增殖能力不受LGMN表达的影响，我们又构建了裸鼠原位乳腺癌模型，结果显示敲低或者回补LGMN的表达并不影响原位乳腺癌细胞的生长(图9)。
2.6敲低LGMN或整合素β3蛋白的表达可以抑制SCP2乳腺癌细胞原位肿瘤的肺转移
通过裸鼠骨转移模型，我们已经发现LGMN-整合素β3信号对于乳腺癌细胞在体内的转移具有重要的作用。接下来我们新构建了乳腺癌原位肿瘤模型，进一步探究LGMN-整合素αvβ3信号对于SCP2乳腺癌原位肿瘤转移的影响。我们将1×10 6SCP2 细胞与基质胶1:1混合后注射至B-NDG小鼠第四对乳腺脂肪垫中，原位肿瘤生长8周后，取小鼠的肺组织，通过生物发光成像检测肿瘤在肺中形成的转移灶。实验结果发现敲低LGMN或整合素β3的SCP2乳腺癌细胞原位肿瘤所形成的肺转移灶与对照组相比显著下调(图10)。
2.7 LGMN-整合素αvβ3信号也影响ER+乳腺癌T47D细胞的迁移与在小鼠体内的转移
为了进一步探究LGMN-整合素αvβ3信号是否也会影响ER+的乳腺癌细胞的迁移和侵袭能力。在之前的研究中我们检测了ER+乳腺癌细胞T47D低表达LGMN，同时我们检测发现T47D细胞也不表达整合素β3，所以我们构建了T47D细胞单独高表达LGMN或者整合素β3，以及同时表达LGMN和整合素β3的稳转株细胞，如图11，A所示。接下来我们通过细胞愈合实验、细胞迁移实验和细胞侵袭实验检测了其对于T47D细胞迁移和侵袭能力的影响。结果表明在T47D细胞中只有同时表达LGMN和整合素αvβ3才能促进细胞的迁移和侵袭，单独过表达LGMN或者整合素β3无法增强细胞的迁移和侵袭能力(图11，B-D)。
接下来我们进一步检测了LGMN-整合素αvβ3信号对于T47D细胞在小鼠体内的转移的影响。我们分别将T47D乳腺癌细胞通过原位注射至B-NDG小鼠乳腺脂肪垫或者通过左心室注射至裸鼠体内，分别构建原位肿瘤模型与乳腺癌骨转移模型，并分别统计原位肿瘤模型的肺转移灶形成与左心室模型的骨转移的发生。实验结果表明同时过表达LGMN与整合素αvβ3可以促进T47D细胞在体内的转移(图12)。
2.8筛选LGMN与整合素结合的抗体
上面的研究揭示了乳腺癌细胞可以通过分泌的pro-LGMN与细胞表面的整合素结合促进肿瘤转移。因此针对此机制，我们希望能筛选出抑制LGMN蛋白与整合素结合的抗体，从而特异性地抑制乳腺癌的转移。为了筛选封闭LGMN和整合素结合的抗体，我们采用了噬菌体展示技术进行了scFv的筛选和全长抗体的构建。首先，对Tomlinson I+J噬菌体库进行四轮的亲和富集筛选，前三轮以RGD多肽为抗原，最后一轮以全长LGMN蛋白为抗原。经过四轮的筛选，我们最后筛选得到了五个与LGMN蛋白及RGD多肽结合的scFv(图13，A-B)。进一步将轻重链的可变区序列构建至人源IgG1抗体骨架中，进行表达纯化。然后我们采用配体结合抑制实验对这些克隆进行功能鉴定。通过流式细胞仪检测在不同的抗体处理下，FITC标记的LGMN蛋白与SCP2细胞的结合我们发现C10抗体能抑制LGMN与SCP2细胞的结合(图13，C)。
为了进一步研究C10抗体的生理学功能，我们利用生物膜干涉原理通过小分子 间动力学实验检测了C10抗体与LGMN蛋白的亲和关系，最后计算得到C10抗体的解离常数为1.96nM(图14，A)。接下来我们用不同浓度的C10抗体处理FITC标记的LGMN，再将该混合物与SCP2细胞孵育，通过流式细胞仪检测C10抗体抑制LGMN与SCP2细胞结合的半抑制浓度。结果显示,MFI的半抑制浓度分别为22.1μg/ml(图14，B)。接下来我们探究了C10抗体对乳腺癌细胞迁移能力的影响。我们用在抑制SCP2细胞表面整合素与LGMN结合的最大抑制效果的最小浓度来处理SCP2敲低LGMN细胞及敲低对照组细胞，然后通过伤口愈合实验，Transwell实验和invasion实验发现，C10抗体能特异性地抑制SCP2细胞的迁移和侵袭(图14，C-E)。同时我们发现C10抗体能特异性地抑制LGMN介导的整合素下游信号FAK-Y397和Src-T416的磷酸化及RhoA的活化(图14，F)。
2.9 C10抗体在体内抑制乳腺癌细胞的转移
之前我们在分子水平和细胞水平验证了C10抗体的功能，接下来我们继续探究C10能否抑制LGMN表达的SCP2乳腺癌细胞在小鼠体内的转移。我们在裸鼠左心室注射SCP2乳腺癌细胞系构建的骨转移模型中，分别腹腔注射C10(2mg/kg)抗体及人源IgG对照抗体，每两天注射一次，持续6周。每周通过小鼠活体荧光成像系统实时检测乳腺癌的骨转移情况，如(图14，G-H)所示，在第六周，C10抗体组能降低小鼠乳腺癌细胞的骨转移灶的形成，X-Ray及Micro-CT结果也显示在第六周C10抗体组小鼠与对照组相对骨含量更高(图14，I)，骨小梁数更多(图14，J)，骨损伤更轻微(图14，K)。该结果表明C10抗体能抑制乳腺癌细胞在体内的转移。
2.10 C10抗体特异性抑制LGMN与整合素αvβ3结合
为了进一步探究C10抗体的功能，我们通过293T细胞体外纯化系统分别纯化得到了pro-LGMN-flag蛋白与整合素αvβ3保外段全长蛋白。接下里我们通过flag beads pull-down实验验证了pro-LGMN-flag蛋白与整合素αvβ3之间的结合(图15)。进一步在pull-down实验中，我们将pro-LGMN-flag提前与C10抗体预处理，我们发现C10抗体可以特异性地抑制pro-LGMN-flag蛋白与整合素αvβ3的相互作用(图15)。
2.11 C10抗体可以抑制ER+乳腺癌细胞、胃癌细胞、肺癌细胞与结肠癌细胞的迁移能力
除乳腺癌外，我们通过GEPIA数据库分析发现LGMN在肺腺癌，结肠癌，胃腺癌，乳腺癌，肝细胞癌，胰腺癌等癌症中表达增强(图16)。整合素αvβ3也在这些肿瘤中高表达。
之前我们已经验证了C10抗体可以抑制TNBC乳腺癌细胞SCP2在体外的迁移和侵袭能力，接下来我们也检测了C10抗体抑制其他肿瘤细胞迁移的能力。通过 transwell实验我们发现，C10抗体同样可以抑制过表达LGMN与整合素αvβ3的ER+乳腺癌细胞(T47D细胞)的迁移能力(图17)。
同时我们也检测了C10抗体对于其他肿瘤细胞迁移能力的影响。我们首先检测了MNK-45胃癌细胞与A549肺癌细胞LGMN的蛋白表达。免疫印迹实验显示与正常乳腺上皮细胞MCF-10A相比，MNK-45胃癌细胞与A549肺癌细胞均含有较高的LGMN表达(图18，A)。进一步transwell实验结果也表明C10抗体能抑制MNK-45胃癌细胞与A549肺癌细胞在体外的迁移能力(图18，B)。
此外，我们还检测了C10抗体对结肠癌细胞迁移能力的影响。免疫印迹结果显示，以A549肺癌细胞为阳性对照，SW480结肠癌细胞也含有较高水平的LGMN表达(图19，A)。并且伤口愈合实验结果表明C10抗体也能显著地抑制SW480结肠癌细胞的迁移能力(图19，B)。
三、结果讨论
由于LGMN与整合素的结合促进细胞迁移运动所介导的胞内信号通路在体内是非常保守的，所以LGMN与整合素的相互作用促进肿瘤转移这一机制也适用于其他LGMN与整合素αvβ3高表达类型的肿瘤。除整合素αvβ3以外，我们通过免疫共沉淀实验验证了其他RGD配体的整合素如αvβ1、αvβ5、αvβ6、αvβ8、α5β1和α8β1，LGMN蛋白可以与其β亚基β1、β5、β6和β8结合(图20)。LGMN与整合素的结合依赖于保守的RGD序列，由此我们认为本发明中LGMN与其他RGD配体的整合素的结合可能都具有促进乳腺癌转移的能力。
LGMN在乳腺癌细胞中特异性高表达，而在正常组织中表达极低，且高表达的LGMN已被证实能促进乳腺癌的发展及转移，这些特性让LGMN成为了肿瘤治疗的理想靶标。目前针对LGMN的生物学功能及其与肿瘤的关系进行了不少的研究，已成为肿瘤治疗，细胞免疫，疾病诊断和预后判断等领域的新热点。之前的研究针对LGMN研发了小分子抑制剂和肿瘤DNA疫苗，而这些小分子药物的机制主要是抑制LGMN酶的活性，来抑制原位瘤的生长。在本发明中我们发现肿瘤细胞自分泌的pro-LGMN与整合素结合，通过活化胞内FAK-Src-RhoA信号通路，促进肿瘤的转移和侵袭。由此我们认为LGMN介导的乳腺癌的转移主要是通过发挥其整合素配体的功能实现的而不依赖于LGMN的酶活功能，同时我们通过抗体特异性抑制LGMN与整合素的结合能特异性地阻断肿瘤的转移。因此抑制肿瘤细胞整合素与其自分泌的LGMN的相互作用是抑制肿瘤转移的治疗有效靶点。

Claims (14)

  1. 一种突变型LGMN蛋白，与野生型相比，突变型LGMN蛋白的RGD序列具有一个或多个导致LGMN与整合素或其β亚基的相互作用减弱或消失的突变，
    优选地，与野生型相比，突变型LGMN蛋白的RGD序列突变为RGE，RAD，RAE，KGD或KGE。
  2. 一种突变型整合素或其β亚基，与野生型相比，其在与LGMN蛋白相互作用的配体结合区域具有一个或多个导致其与LGMN蛋白相互作用减弱或消失的突变；
    优选地，
    所述突变型整合素或其β亚基在D120和/或S121位置上具有突变；和/或
    所述整合素的β亚基选自以下一种或多种：β 1、β 3、β 5、β 6和β 8，所述整合素是含有所述β亚基的整合素。
  3. 抗LGMN抗体或其抗原结合片段，其CDR如下所示：HCDR1包含GFTFSSYA，HCDR2包含IGNSGNYT，HCDR3包含AKSSDSFNY，LCDR1包含QSISSY，LCDR2包含DAS，LCDR3包含QQAYANPDT，
    优选地，所述抗体的VH和VL具有SEQ ID NO:1和SEQ ID NO:2所示的序列或与其具有90％序列相同性的变体，
    更优选地，所述抗体的重链恒定区的氨基酸序列具有如SEQ ID NO:3所示的序列，和/或轻链恒定区的氨基酸序列具有如SEQ ID NO:4所示的序列。
  4. 一种核酸分子，包含选自以下的序列：
    (1)编码权利要求3所述的抗LGMN抗体或其抗原结合片段的序列，或
    (2)(1)的互补序列。
  5. 包含权利要求4所述的核酸分子的核酸构建物，
    优选地，所述核酸构建物是克隆载体、整合载体或表达载体。
  6. 一种宿主细胞，所述宿主细胞：
    (1)表达权利要求3所述的抗LGMN抗体或其抗原结合片段，或
    (2)包含权利要求4所述的核酸分子或权利要求5所述的核酸构建物。
  7. LGMN蛋白和/或整合素或其β亚基作为靶点在筛选抑制肿瘤转移或肿瘤细胞转移的潜在物质中的用途，或用作分子指标在临床上用于诊断肿瘤转移的用途，
    优选地，
    所述肿瘤选自以下的一种或多种：乳腺癌、肺腺癌、结肠癌、胃腺癌、乳腺癌、肝细胞癌、胰腺癌；和/或
    所述潜在物质抑制LGMN蛋白与整合素或其β亚基之间的相互作用；和/或
    所述整合素的β亚基选自以下一种或多种：β 1、β 3、β 5、β 6和β 8，所述整合素是含有所述β亚基的整合素；和/或
    靶点包括：LGMN蛋白的RGD序列，和/或，整合素或其β亚基的与LGMN蛋白相互作用的配体结合区域。
  8. 一种药物组合物，所述药物组合物含有：
    (1)药学上可接受的载体，和
    (2)权利要求1所述的突变型LGMN蛋白、权利要求2所述的突变型整合素或其β亚基、或权利要求3所述的抗LGMN抗体或其抗原结合片段，和/或(3)含有编码(2)的核酸序列或其互补序列的核酸分子。
  9. 一种敲减LGMN的小干扰RNA，其具有SEQ ID NO:5或6所示的序列；或
    一种敲减整合素或其β亚基的小干扰RNA，其具有SEQ ID NO:7所示的序列。
  10. 抑制LGMN蛋白与整合素或其β亚基之间相互作用的试剂在制备抑制肿瘤转移或肿瘤细胞转移的药物中的用途，
    优选地，
    所述相互作用是LGMN蛋白的RGD序列与整合素或其β亚基之间的相互作用， 和/或
    所述整合素的β亚基选自以下一种或多种：β 1、β 3、β 5、β 6和β 8，所述整合素是含有所述β亚基的整合素；
    更优选地，所述试剂选自以下的一种或多种：
    (1)突变型LGMN蛋白和/或它们的打靶载体，其中，与野生型相比，突变型LGMN的RGD序列发生导致LGMN与整合素或其β亚基的相互作用减弱或消失的突变；优选地，所述突变是RGD序列突变为RGE，RAD，RAE，KGD或KGE；
    (2)突变型整合素或其β亚基和/或其打靶载体，其中，与野生型相比，突变型整合素或其β亚基在与LGMN蛋白相互作用的配体结合区域具有一个或多个导致其与LGMN蛋白相互作用减弱或消失的突变；优选地，所述整合素或其β亚基在D120和/或S121位置上具有突变；
    (3)抗LGMN抗体或其抗原结合片段，所述抗体抑制LGMN蛋白与整合素或其β亚基之间的相互作用；
    (4)抗整合素或其β亚基的抗体或其抗原结合片段，所述抗体抑制LGMN蛋白与整合素或其β亚基之间的相互作用；
    (5)编码(3)或(4)所述的抗体或其抗原结合片段的核酸分子或包含所述核酸分子的核酸构建物；
    (6)表达(3)或(4)所述的抗体或其抗原结合片段的宿主细胞；
    (7)整合素或其β亚基的抑制剂，所述抑制剂抑制LGMN蛋白与整合素或其β亚基之间的相互作用；
    (8)能降低二价金属离子浓度的试剂；
    (9)敲低或敲除LGMN蛋白和/或整合素或其β亚基的表达的试剂，例如ZFN和/或TALEN和/或CRISPR/Cas9试剂和/或小干扰RNA。
  11. 如权利要求10所述的用途，其特征在于，
    所述突变型LGMN蛋白如权利要求1所述，
    所述突变型整合素或其β亚基如权利要求2所述，
    所述抗LGMN抗体或其抗原结合片段如权利要求3所述，
    所述敲低或敲除LGMN蛋白和/或整合素或其β亚基的表达的试剂如权利要求9 所述。
  12. 如权利要求10或11所述的用途，其特征在于，所述肿瘤选自以下的一种或多种：乳腺癌、肺腺癌、结肠癌、胃腺癌、乳腺癌、肝细胞癌、胰腺癌。
  13. 一种体外抑制肿瘤细胞转移的方法，包括减弱肿瘤细胞的LGMN蛋白与整合素或其β亚基之间的相互作用的步骤，
    优选地，所述方法包括通过以下任意一种或多种方式减弱肿瘤细胞的LGMN蛋白与整合素或其β亚基之间的相互作用：
    (1)敲除或敲低肿瘤细胞的LGMN蛋白的表达；
    (2)敲除或敲低肿瘤细胞中整合素或其β亚基表达；
    (3)在野生型或敲除LGMN蛋白的肿瘤细胞中表达与整合素或其β亚基的相互作用减弱或消失的突变型LGMN蛋白；
    (4)在野生型或者敲除整合素或其β亚基的肿瘤细胞中表达与LGMN蛋白的相互作用减弱或消失的突变型整合素或其β亚基；
    (5)使肿瘤细胞与抗LGMN抗体或其抗原结合片段或含有所述抗LGMN抗体或其抗原结合片段的蛋白接触；
    (6)使肿瘤细胞与降低二价金属离子浓度的试剂接触，
    更优选地，所述方法具有选自以下的一个或多个特征：
    所述突变型LGMN蛋白与野生型相比，其RGD序列发生导致与整合素或其β亚基的相互作用减弱或消失的突变；
    所述突变型整合素或其β亚基与野生型相比，其在与LGMN相互作用的配体结合区域具有一个或多个导致其与LGMN相互作用减弱或消失的突变；
    所述抗LGMN抗体抑制LGMN与整合素或其β亚基之间的相互作用；
    所述能降低二价金属离子浓度的试剂是EDTA；
    所述敲低或敲除LGMN和/或整合素或其β亚基表达的试剂是ZFN和/或TALEN和/或CRISPR/Cas9试剂和/或小干扰RNA。
  14. 检测LGMN蛋白的试剂在制备诊断肿瘤转移的试剂盒中的用途，
    优选地，所述试剂检测肿瘤组织中LGMN蛋白的表达、含量或序列，
    更优选地，所述试剂选自以下一种或多种：针对LGMN蛋白的抗体、与LGMN蛋白的编码序列杂交的引物和/或探针。
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