WO2023016509A1

WO2023016509A1 - Drug for inhibiting tumor cell metastasis and use thereof

Info

Publication number: WO2023016509A1
Application number: PCT/CN2022/111658
Authority: WO
Inventors: 陈剑峰; 刘翠; 王军磊
Original assignee: 中国科学院分子细胞科学卓越创新中心
Priority date: 2021-08-11
Filing date: 2022-08-11
Publication date: 2023-02-16
Also published as: CN115704020A

Abstract

The text relates to a drug for inhibiting tumor cell metastasis and a use thereof. The present invention provides a use of a reagent for inhibiting the interaction between LGMN and an integrin or a β subunit thereof in the preparation of a drug for inhibiting tumor or tumor cell metastasis. The reagent or composition of the present invention can effectively inhibit tumor or tumor cell metastasis.

Description

Drugs for inhibiting tumor cell metastasis and uses thereof

technical field

The invention relates to the field of tumor treatment, in particular to a drug for inhibiting tumor cell metastasis and its application.

Background technique

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 α and β 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.

Asparagine endopeptidase (Legumain, LGMN) 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. In addition, 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.

Contents of the invention

The inventors found that the expression level of LGMN is related to cancer metastasis and prognosis, and it binds to integrin β subunit through RGD sequence, thereby regulating tumor metastasis.

Therefore, the first aspect of the present invention provides the use of a reagent for inhibiting the interaction between LGMN protein and integrin or its β subunit in the preparation of a drug for inhibiting tumor metastasis or tumor cell metastasis.

In one or more embodiments, the interaction is between the RGD sequence of the LGMN protein and integrin or its beta subunit.

In one or more embodiments, the β subunit of the integrin is selected from one or more of the following: β ₁ , β ₃ , β ₅ , β ₆ and β ₈ ; the integrin contains the β Integrin subunit.

In one or more embodiments, the integrin is selected from one or more of the following: α _v β _3, α _v β ₁ , α _v β ₅ , α _v β ₆ , α _v β ₈ , α ₅ β ₁ and α ₈ β ₁ .

In one or more embodiments, the agent is selected from one or more of the following:

(1) 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 β subunit to be weakened or disappeared;

(2) mutant integrin or its β subunit and/or its targeting vector, wherein, compared with the wild type, the mutant integrin or its β 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;

(3) an anti-LGMN antibody or an antigen-binding fragment thereof, which inhibits the interaction between LGMN protein and integrin or its beta subunit;

(4) an antibody against integrin or its β subunit or an antigen-binding fragment thereof, which inhibits the interaction between LGMN protein and integrin or its β subunit;

(5) a 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;

(6) A host cell expressing the antibody or antigen-binding fragment thereof described in (3) or (4);

(7) an inhibitor of integrin or its β subunit, which inhibits the interaction between LGMN protein and integrin or its β subunit;

(8) Reagents 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.;

(9) 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;

In one or more embodiments, in (1), the mutation is a RGD sequence mutation to RGE, RAD, RAE, KGD or KGE.

In one or more embodiments, in (2), 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.

In one or more embodiments, the small interfering RNA has the sequence shown in SEQ ID NO:5 or 6.

In one or more embodiments, 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.

In one or more embodiments, the mutant LGMN protein, mutant integrin or β 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. Compared with the wild type, 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 β subunit.

In one or more embodiments, the precursor is pro-LGMN protein.

In one or more embodiments, 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; Preferably, there is 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.

This article also provides the use of the LGMN protein and/or integrin or its β 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.

In one or more embodiments, the medicament inhibits the interaction between LGMN protein and integrin or its beta subunit. Preferably, the drug attenuates the interaction of the RGD sequence of LGMN protein with integrin or its beta subunit.

In one or more embodiments, the β subunit of the integrin is selected from one or more of the following: β ₁ , β ₃ , β ₅ , β ₆ and β ₈ ; The integrin is an integrin comprising the beta subunit.

In one or more embodiments, 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.

In one or more embodiments, the antibody inhibits the interaction between an LGMN protein and an integrin or a beta subunit thereof.

In one or more embodiments, the FR region of the VH of the anti-LGMN antibody is the FR region of the VH shown in SEQ ID NO:1, and the FR region of the VL is the FR region of the VL shown in SEQ ID NO:2.

In one or more embodiments, 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.

In some embodiments, 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 .

In one or more embodiments, 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).

In one or more embodiments, 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 β 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 β subunit, or anti-LGMN antibody in the preparation of drugs for inhibiting tumor metastasis or tumor cell metastasis.

In one or more embodiments, 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:

(1) a pharmaceutically acceptable carrier, and

(2) mutant LGMN protein, mutant integrin or its β subunit, or anti-LGMN antibody or antigen-binding fragment thereof according to any embodiment of the present invention, and/or (3) nucleic acid containing encoding (2) sequence or its complement.

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.

In one or more embodiments, the method includes weakening the interaction between the LGMN protein of tumor cells and integrin or its β subunit by any one or more of the following methods:

(1) Knocking out or knocking down the expression of LGMN protein of tumor cells;

(2) Knocking out or knocking down the expression of integrin or its β subunit in tumor cells;

(3) express a mutant LGMN protein whose interaction with integrin or its β subunit is weakened or disappeared in tumor cells of wild type or knockout LGMN protein;

(4) Expressing mutant integrin or its beta subunit with weakened or disappeared interaction with LGMN protein in wild-type or tumor cells knocked out of integrin or its beta subunit;

(5) contacting the tumor cells with an anti-LGMN antibody or antigen-binding fragment thereof or a protein comprising said anti-LGMN antibody or antigen-binding fragment thereof;

(6) Contacting tumor cells with an agent that reduces the concentration of divalent metal ions.

In one or more embodiments, the interaction is between the RGD sequence of LGMN and an integrin or a beta subunit thereof.

In one or more embodiments, the method has one or more features selected from:

Compared with the wild type, the RGD sequence of the mutant LGMN protein has a mutation that causes the interaction with integrin or its β subunit to be weakened or disappeared;

Compared with the wild type, the mutant integrin or its β 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.

In one or more embodiments,

Treatment of tumor cells with expression vectors and/or integration vectors of mutant LGMN proteins, and/or

Treatment of tumor cells with expression vectors and/or integration vectors for mutant integrins or their beta subunits, and/or

Treatment of tumor cells with an anti-LGMN antibody or antigen-binding fragment thereof, and/or

Treatment of tumor cells with agents that reduce the concentration of divalent metal ions, and/or

Transducing a gene knockout vector into the tumor cells to knock down the expression of LGMN protein and/or the expression of integrin or its β subunit in the tumor cells, and/or

Use ZFN, TALEN or CRISPR/Cas9 technology to knock down the expression of LGMN protein and/or the expression of integrin or its β subunit in tumor cells, and/or

Knockdown of LGMN protein expression and/or integrin or its beta subunit expression by interfering RNA-mediated gene silencing, and/or

Introducing a gene insertion vector in tumor cells to knock out the coding sequence of wild-type LGMN protein and/or integrin or its β subunit while weakening or disappearing the interaction with integrin or its β subunit The expression box of type LGMN protein and/or the expression box of mutant integrin or its β 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 β subunit.

The invention also provides the use of the reagent for detecting LGMN protein in the preparation of a kit for diagnosing tumor metastasis.

In one or more embodiments, the reagent detects the expression, content or sequence of LGMN protein in tumor tissue.

In one or more embodiments, 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 β 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.

In one or more embodiments, the breast cancer cell is SCP2.

In one or more embodiments, the marker is luciferase.

Description of drawings

Figure 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).

Figure 2. 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.

Figure 3. Knockdown of LGMN significantly inhibits the metastatic ability of breast cancer cells. (A) In Hs578T, MDA-MB-231 and SCP2 cells, shLGMN (shLGMN-1 and shLGMN-2) specific for LGMN were transfected, respectively, and the scrambled shRNA was used as a negative control (Control). WB detected the level of LGMN. Effects of knockdown of LGMN on wound healing (B), Transwell migration (C) and invasion in Matrigel (D) of three tumor cells. All data were averaged from more than three parallel experiments and error bars were drawn. P value was calculated by t-text statistical method, **P<0.01, ***P<0.001.

Figure 4. Knockdown of LGMN does not affect the proliferation ability of breast cancer cells. Effects of LGMN knockdown on CCK8 proliferation assay (A) and colony formation (B) of Hs578T, MDA-MB-231 and SCP2 cells. All data were averaged from more than three parallel experiments and error bars were drawn. P values were calculated using the t-text statistical method.

Figure 5, LGMN mediates binding to β3 integrin through its RGD sequence. (A) Co-immunoprecipitation results of endogenous LGMN and β3 integrin in Hs578T, MDA-MB-231 and SCP2 cells; (B) Co-IP assay to detect the effect of LGMND120E on the binding of β3 integrin to LGMN; (C) Co-IP Experimental detection of the effect of EDTA on the binding of LGMN to β3 integrin; (D) Co-IP test to detect the effect of MIDAS mutation β3S121A on the binding of β3 integrin to LGMN; (E) Co-IP test to detect the effect of LGMN enzyme activity mutation LGMNC189S on β3 integration The effect of the binding of the protein to LGMN.

Figure 6. LGMN promotes tumor metastasis dependent on binding to integrins. In 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. P value was calculated by t-text statistical method, ***P<0.01. (G) WB detection of phosphorylation levels of integrin downstream signaling proteins (FAK and Src) in Hs578T, MDA-MB-231 and SCP2 cells under the condition of knocking down LGMN and complementing wild-type LGMN and LGMN ^D120E mutation The change. (H) The activation levels of intracellular RhoA, Rac1 and CDC42 were detected by GTPase Pull-down technology. Purified wild-type LGMN protein and LGMN ^D120E protein treated LGMN knockdown Hs578T, MDA-MB-231 and SCP2 cells, and detected the phosphorylation levels of FAK-Y397 and Src-Y416 (I) and the activation levels of RhoA, Rac1 and CDC42 (J).

Figure 7, the expression of integrin αvβ3. Flow cytometry was used to detect the expression levels of integrin αvβ3 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.

Figure 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. (A, B) Representative pictures (A) and statistical graphs (B) of fluorescence levels of bone metastases detected by in vivo imaging, X-ray and Micro-CT in small animals (n=5-7/group). (C) The content of human LGMN in mouse plasma was detected by ELISA (n=3-7/group). The differences in bone volume fraction (bone volume/material volume) (D), trabecular number (E), and trabecular separation (F) in mouse CT scan results were analyzed (n=5/group). The P value was analyzed by one-way ANOVA statistical method, *P<0.05, ***P<0.001.

Figure 9, 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).

Figure 10. Knockdown of LGMN or integrin β3 can inhibit lung metastasis of orthotopic tumors in SCP2 breast cancer cells. The SCP2 cells of the control group, LGMN knockdown group and β3 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.

Figure 11, simultaneous expression of LGMN and integrin αvβ3 can enhance the migration and invasion of T47D cells in vitro. A Overexpress LGMN and integrin αvβ3 in T47D cells, and detect the expression levels of LGMN and integrin β3 by immunoblotting. B-D wound healing assay, transwell cell migration assay and cell invasion assay to detect the migration and invasion abilities of T47D cells. Results are shown as three independent replicate experiments. P values were obtained using the statistical method of one-way ANOVA. NS: Not significant. ***P<0.001.

Fig. 12, simultaneous expression of LGMN and integrin αvβ3 can promote the metastatic ability of T47D cells in mice. Orthotopic breast cancer model of T47D breast cancer cells (A), model of lung metastases in mice (left), and statistical results of tumor bioluminescence and fluorescence (right). T47D breast cancer cell left ventricle injection bone metastasis model (B), mouse bone metastasis pattern (left), bone metastasis tumor bioluminescent fluorescence statistical results (right). P value was obtained by one-way ANOVA (A) or two-way ANOVA (B) statistical method. NS: Not significant. **P<0.05, ***P<0.001.

Figure 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.

Figure 14, 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. Wound healing assay (C), Transwell migration assay (D) and invasion assay (E) detected that C10 antibody inhibited the migration of SCP2 cells. (F) Detection of intracellular phosphorylation levels of FAK-Y397 and Src-Y416 and activation levels of RhoA, Rac1 and CDC42 after treatment of SCP2-knockdown LGMN and control cells with C10 antibody. (G, H) In vivo imaging of mice, X-Ray and Micro-CT detection of fluorescence levels of bone metastases and bone damage in the control antibody group and C10 antibody-treated group Representative pictures (G) and statistical diagrams (H) . (N=6-7/group). (I-K) Analysis of differences in bone volume/material volume (I), trabecular number (J), and trabecular separation (K) in CT scan results of mice treated with C10 antibody. (n=5/group). The P value was analyzed by t-text statistical method (I-K) and two-way ANOVA statistical method (C-F, H), *p<0.05, ***p<0.001.

Figure 15, C10 antibody can specifically inhibit the interaction between LGMN and integrin αvβ3. Pull-down experiments were used to detect the interaction between pro-LGMN-flag protein and integrin αvβ3, and to detect the effect of C10 antibody treatment on the binding of pro-LGMN-flag protein and integrin αvβ3.

Figure 16, 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.

Figure 17, C10 antibody can specifically inhibit the migration ability of T47D cells. In the transwell cell migration assay, the effect of C10 antibody treatment on the migration of T47D cells co-expressed with LGMN and integrin αvβ3 was detected, and human IgG was used as the control group.

Figure 18, C10 antibody can specifically inhibit the migration ability of MNK-45 gastric cancer cells and A549 lung cancer cells. In the transwell cell migration assay, 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.

FIG. 19 , 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). In the wound healing experiment, the effect of C10 antibody treatment on the migration of SW480 cells was detected (B), and human IgG was used as the control group.

Figure 20, Binding of integrins β1, β5, β6 and β8 to LGMN protein. Co-IP assay was used to detect the binding of integrins β1, β5, β6 and β8 to LGMN protein in Hs578T, MDA-MB-231 and SCP2 cells.

Detailed ways

The practice of the present invention will employ, unless otherwise defined, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (ed. M.J. Gait, 1984); Animal Cell Culture (ed. R.I. Freshney, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (Editors F.M.Ausubel et al., 1987 edition and its regular updates); PCR: The Polymerase Chain Reaction (Editors Mullis et al., 1994); A Practical Guide to Molecular Cloning ( Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

The inventors found that breast cancer cells with high metastases secrete a large amount of pro-LGMN to the outside of the cell. 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. In addition, the inventor identified a humanized monoclonal antibody C10 that can block the binding of LGMN protein and integrin through phage display technology. The migration and invasion of breast cancer cells and the bone metastasis of breast cancer cells. Therefore, the inventors found that blocking the combination of RGD site of LGMN and integrin is an effective way to inhibit tumor metastasis. In addition, because the intracellular signaling pathway mediated by the combination of LGMN and integrin to promote cell migration is very conserved in vivo, the mechanism of interaction between LGMN and integrin to promote tumor metastasis is also applicable to breast cancer, lung adenocarcinoma, etc. Carcinoma, colon cancer, gastric adenocarcinoma, breast cancer, hepatocellular carcinoma, pancreatic cancer and other tumors with high expression of LGMN and integrins.

Herein, 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. Exemplarily, 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 β 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 β subunit; (4) In wild-type or knockout integrin In the tumor cells expressing the mutant integrin or its β 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.

In a specific embodiment, 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 β 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 β 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 β subunit, and/or knock down the expression of LGMN protein and/or the expression of integrin or its β subunit by interfering RNA-mediated gene silencing, and/or transfer the gene insertion vector into tumor cells , while knocking out the coding sequence of wild-type LGMN protein and/or integrin or its β subunit, the interaction with integrin or its β subunit will be weakened or disappeared. The expression frame of the mutant LGMN protein and/or The expression cassette of mutant integrin or its β 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 β subunit .

Reagents and their uses

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: β ₁ , β ₃ , β ₅ , β ₆ and β ₈ . The integrin is an integrin comprising _the beta _subunit , such _as _αvβ3 _, _αvβ1 _, _αvβ5 _, _αvβ6 _, _αvβ8 , _α5β1 and _α8β1 . As an example, 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 α _v is shown in GenBank No.AGC09592 .1; the amino acid sequence of wild-type _β3 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 β 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 β subunit can also be a mutant integrin or its β subunit and/or its targeting vector, wherein, compared with the wild type, the mutant integrin or its The β 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 β 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 β 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.

The reagent for inhibiting the interaction between LGMN protein and integrin or its β subunit can also be a reagent for knocking down or knocking out the expression of LGMN protein and/or integrin or its β subunit, such as ZFN and/or TALEN and/or or CRISPR/Cas9 reagents and/or small interfering RNA. After the target sequence is known, methods for preparing interfering reagents for knocking down or knocking out specific gene expression are well known to those skilled in the art. Procedures for constructing such knockdown or knockout reagents for proteins and their coding sequences are well known in the art. As a preferred mode of the present invention, the inhibitory agent is LGMN or integrin-specific shRNA or its construct, wherein the shRNA specifically recognizes LGMN or integrin genes or transcripts thereof. Herein, a "transcript" comprises a UTR region (eg 3' UTR) and a CDS region. Exemplarily, 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. In addition, 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 αvβ3 and αvβ5 inhibitors Cilengitide, αIIbβ3 inhibitors Tirofiban and Epifibatide, αIIbβ3 antibody Abciximab, αvβ1 inhibitor PLN-1474 and antibody Volociximab, αvβ6 antibody 264RAD, α4β1 antibody Natalizumab wait).

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

As used herein, the term "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). Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably. Preferably, 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 α and γ chains) and four (CH1, CH1, CH3 for the μ and ε isotypes). CH2, CH3 and CH4) constant domain (CH) and the hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at its N-terminus followed by a constant domain (CL) at its other end. VL is aligned with VH and CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. The paired VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see e.g. Basic and Clinical Immunology, Eighth Edition, edited by Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, pp. 71 and 6 chapter. Light chains from any vertebrate species can, based on their constant domain amino acid sequence, be assigned to one of two distinct types called kappa and lambda. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, 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.

"Variable region" or "variable domain" 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. In one or more embodiments, 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.

The term "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. However, the 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. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation connected by the formation of loops and in some cases forming β-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). Generally, 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. In a preferred embodiment, 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) or "Fc domain" or "Fc" 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. In IgG, IgA and IgD antibody isotypes, 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). Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, 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. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

Under the premise of not substantially affecting the activity of the antibody, those skilled in the art can substitute, add and/or delete one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequence thereof. They are all considered to be included in the protection scope of the present invention. Amino acids with similar properties are substituted eg in the FR and/or CDR regions of the variable region. Substitutions are preferably conservative substitutions; amino acid residues which may be conservatively substituted are well known in the art. In some embodiments, 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. Alternatively, 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 are well known in the art and 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.

nucleic acid molecule

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.

Herein, 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. Specifically, 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. Alternatively, the nucleic acid molecules described herein can also be directly synthesized.

Nucleic acid constructs and cells

Methods for constructing expression vectors 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.

The 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.

In certain embodiments, 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.

Methods of introducing genes into cells and expressing genes into cells are known in the art. Vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, 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.

Herein, a host cell contains, expresses and/or secretes an antibody or antigen-binding fragment thereof and optionally a therapeutic polypeptide described herein. Herein, when it is mentioned that 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 .

drug screening

This article also provides the use of LGMN protein and/or integrin or its β 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 β subunit, and/or (3) LGMN protein and integrin or its β 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 β subunit interacting with the LGMN protein. If 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 β subunit), it indicates that the candidate substance is a potential substance for inhibiting tumor metastasis or tumor cell metastasis . For example, 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.

Diagnostic uses, assays and kits

Inhibitory reagents of the invention (e.g., anti-LGMN antibodies) 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.

For diagnostic applications, anti-LGMN antibodies are typically labeled with a detectable labeling group. Suitable labeling groups are well known in the art and 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. In a specific embodiment, the antibody is labeled with a labeling group and binding of the labeled antibody to LGMN is detected. In another specific embodiment, binding of the antibody to LGMN is detected in vivo. In another specific embodiment, 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. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.

Pharmaceutical composition, route of administration

The present invention provides 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.

In certain embodiments, acceptable carriers and the like in pharmaceutical compositions are preferably nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, 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.

Pharmaceutical 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.

Other pharmaceutical 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.

Pharmaceutical 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.

Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals or as dehydrated or lyophilized powders. The formulations can be stored in a ready-to-use form or reconstituted (eg, lyophilized) before administration. The invention also provides kits for producing single dosage administration units. The kits of the invention may each contain a first container with a dry protein and a second container with an aqueous formulation. In certain embodiments of the invention, 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.

Herein, 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.

The present invention will be illustrated below in the form of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and materials used in the examples, unless otherwise stated, are conventional materials and methods in the art.

Example

1. Experimental materials and methods

1.1 Materials

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 ).

Antibody

1.2 Method

1.2.1 Transient transfection of 293T cells by calcium phosphate method

1. Separate the cells one day before transfection, so that the cells reach a density of 60-70% during transfection;

2. 1 hr before transfection, replace with medium containing 25 μM chloroquine, with a volume of 10 ml per 10 cm dish;

3. In a 15ml sterile centrifuge tube, add 10μg DNA, dissolve 1095μl with sterilized MilliQ, then add 155μl 2M CaCl2;

4. Add 1,250μl 2×HBS drop by drop, and mix gently while adding;

5. Add the mixture directly to the cells drop by drop, and evenly sprinkle it on the entire plate surface;

6. Incubate at 37°C for 7-11 hours, rinse once, and replace with chloroquine-free medium;

7. Collect cells 48-72 hours after transfection.

1.2.2 Virus coating and infection of cells

virus coating

1. Divide 293T the day before, and the density will reach about 70% when transfected the next day;

2. Using the method of calcium phosphate transfection, the plasmid of packaging virus is transferred into 293T cells according to a certain ratio;

3. After 48-72 hours, collect the culture medium (containing virus), filter it with a 0.45 μm syringe filter and use it;

4. Ultracentrifugation can be performed if necessary, the virus is concentrated, and centrifuged at 27,000 rpm at 4°C for 2 hours.

virus infected cells

1. Subculture the cells to be infected the day before;

2. Mix the cell culture medium, virus and polybrene (final concentration 8 μg/ml) together and add to the cells;

3. After 18 hours, suck off the virus-containing culture medium and add fresh culture medium;

4. Add antibiotics (such as puromycin, neomycin) after 36-48 hours for screening.

1.2.3 Protein A purification of LGMN protein

1. Transfection of 293T cells by calcium phosphate method;

2. Cultivate until the culture medium turns yellow, and collect the culture medium (about 3 to 4 days after transfection);

3. Dilute the culture medium with an equal volume of Protein A purification binding buffer (Binding buffer). When diluting, add Binding buffer while stirring to adjust the pH to 8.0;

4. Centrifuge at 15,000 rpm for 30 minutes at 4°C;

5. Take the supernatant and add it to the Protein A column with a connecting tube;

6. Wash the column with Protein A purification binding buffer (Binding buffer) for 15 column volumes;

7. Elute with Protein A purification elution buffer (Elution buffer) for 7 column volumes, and add to the collection tube in advance at a ratio of 1:10

8. Protein A purification neutralization buffer (Neutralization buffer);

9. Collect the eluate, concentrate, and change the buffer to HBS at the same time, and freeze the protein in an ultra-low temperature refrigerator after aliquoting.

1.2.4 Co-immunoprecipitation and Western blotting

Wash the cell surface with pre-cooled TBS, scrape off the cells with a cell scraper, and dissolve in pre-cooled TBS. After centrifugation at 3,000rpm for 3min, discard the supernatant, resuspend in lysis buffer (Lysis buffer), place on ice for 30min; centrifuge at 12,000rpm for 15min to obtain cell lysate. The protein content was determined according to the BCA method. Specific antibodies were added to the cell lysate and incubated overnight at 4°C, anti-rabbit IgG was used as a control, and protein G beads (beads) were added to incubate at 4°C for 2h. After washing the beads three times, the samples were prepared.

The same amount of protein samples were separated by 9% SDS-PAGE electrophoresis; after the electrophoresis was completed, the protein was transferred to the nitrocellulose membrane (NC) using a wet electrotransfer device, and the current was set at 280mA for 80min; after the transfer was completed Take out the NC membrane, with the membrane containing the transfer protein facing up, incubate with blocking solution (TBST containing 5% skimmed milk powder) at room temperature for 2 hours; incubate with the specific antibody at 4°C overnight; wash the NC membrane three times with TBST, each time for 5 minutes; 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.

1.2.5 shRNA knockdown gene

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 _β3 integrin is: 5-GCTTAGCTTGAGGGTGACTAT-3.

The control shRNA sequence is: 5-CCTAAGGTTAAGTCGCCCTCG-3.

1.2.6 Flow cytometry

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.

1.2.7 Wound healing experiments

Spread the breast cancer cells on a 96-well plate and grow to a density of 80%-90%, then streak the cells, grow them in a medium containing 1% serum for 16 hours, and record the wound healing within 16 hours by an Incucyte FLR (Essen) automatic imager . The formula for calculating the ratio of wound healing is: healing ratio=[1-(the width of the wound after 16 hours/the width of the initial wound)]×100%.

1.2.8 Cell migration and invasion experiments

For cell migration experiments, plate 1×10 ⁵ SCP2 cells (100 μl) resuspended in serum-free medium on the upper layer of a cell migration chamber (Corning, 8-μm filter membrane), and the lower layer on 600 μl of cell culture medium containing 10% serum . Cells were incubated with antibody for 1 hour in advance, and the antibody treatment was maintained at the same concentration throughout the migration experiment. For the cell invasion experiment, Matrigel was laid on the upper layer of the migration chamber in the above migration experiment, and other conditions were kept the same as the migration experiment. After 16 hours of cell migration or invasion, 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.

1.2.9 Breast cancer bone metastasis experiment

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.

1.2.10 Phage display experiment

RGD antigen polypeptide 100 μg/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. In the fourth round, 20 μg/ml of the purified LGMN protein was used as the antigen for the fourth round of adsorption and elution, and finally the phage obtained in the fourth round of elution was infected with TG1 (OD=0.4-0.6) Escherichia coli strain. Single clones were randomly picked from the TYE plates coated in the fourth round and transferred to 96-well cell culture plates, and the phage supernatants of the single clones were prepared for phage ELISA detection.

1.2.11 ELISA experiment

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 μl 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 μl) was added to each well and incubated at room temperature for 1 h. After washing with PBST three times, add 100 μl 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.

1.2.12 Bio-layer Interferometry (BLI)

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. According to the overall fitting of the binding curves of LGMN and antibodies with different concentration gradients to the 1:1 Langmuir binding model, 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).

2. Experimental results

2.1 The expression level of LGMN is related to the metastasis and prognosis of breast cancer

In order to explore the genes related to breast cancer metastasis, we firstly performed Univariate Cox proportional hazards regression analysis using breast cancer clinical gene database (GSE45255, GSE65194 and GSE22219). The results of the analysis showed that 795 genes, 1402 genes and 1896 genes were positively correlated with the metastasis of breast cancer in the three databases, and 25 of them showed that they might be associated with tumor metastasis in the three databases (Fig. 1, A), the list of 25 genes is shown in Figure 2, A. Gene Ontology analysis revealed the basic location and function of these 25 genes in cells (Fig. 1, B). Among them, LGMN protein is mainly distributed in lysosome as a protease, and participates in protein shearing and degradation. Kaplan-Meier survival analysis found that breast cancer patients with high expression of LGMN would have metastasis earlier (Figure 1, C-E). Patients had an increased rate of metastasis and a lower survival time (Fig. 2, B, C). These data suggest that LGMN protein may play a certain role in the process of breast cancer cell metastasis.

In order to further explore the function of LGMN in breast cancer metastasis, we compared the expression levels of LGMN in normal breast epithelial cells MCF10A and several common breast tumor cell lines MCF7, T47D, Hs578T, MDA-MB-231, SCP2 and the expression levels of LGMN in the above breast cancer cells. Metastatic ability of cancer cells. Cell migration experiments and cell invasion experiments found that compared with low-metastatic MCF7 and T47D cells, Hs578T, MDA-MB-231 and SCP2 cells had stronger metastatic and invasive abilities (Fig. 1, F-G). Western blot experiments showed that compared with normal breast epithelial cells MCF10A, several breast cancer tumor cells highly expressed LGMN, and the full-length form of 56kDa and the activated form of LGMN protein of 36kDa were detected in the cell lysate (Fig. 1, H). And the analysis found that the expression of the full-length form of LGMN was higher in the highly metastatic breast cancer cells Hs578T, MDA-MB-231 and SCP2 cells. However, breast cancer cells secrete immature full-length LGMN in the cell culture medium (Fig. 1, I), and the pro-LGMN protein secreted extracellularly also has a higher content as the transfer ability increases. And the analysis found that only the full-length form of pro-LGMN was positively correlated with the invasion and metastasis abilities of breast cancer cell lines (Fig. 2, D-I). This indicates that the extracellular pro-LGMN protein secreted by breast cancer cells plays an important role in the process of breast cancer migration and invasion.

2.2 Knockdown of LGMN significantly inhibits the migration and invasion of breast cancer cells

In order to further study the relationship between LGMN and breast cancer cell metastasis, we specifically knocked down the expression level of LGMN in tumor cells by shRNA-mediated RNAi in Hs578T, MDA-MB-231 and SCP2 breast cancer cells, and then detected the expression level of LGMN in tumor cells. The effect of expression on the migration and invasion ability of breast cancer cells. The results showed that both shRNAs targeting LGMN could effectively knock down the expression of LGMN in tumor cells (Fig. 3, A). And knockdown of LGMN significantly inhibited the ability of breast cancer cells to migrate and invade in Matrigel (Fig. 3, B-D). At the same time, we also verified that knocking down LGMN did not affect the growth and proliferation of breast cancer cells (Figure 4, A-B).

2.3 LGMN binds to integrin β3 through the RGD sequence

Further, in order to explore whether LGMN regulates the migration and invasion of breast cancer cells by binding to integrin αvβ3, we first detected the interaction between LGMN and integrin αvβ3 in breast cancer cells by co-immunoprecipitation (Co-IP). The RGD sequence is an important site for integrin αvβ3 to bind to its ligand. Although both full-length and activated LGMN proteins contain a conserved RGD sequence, we found that only the extracellularly secreted full-length pro-LGMN can bind to β3 integrin expressed by breast cancer cells in co-immunoprecipitation experiments (Fig. 5, A). Next, we explored the effect of RGD sequence on the binding between LGMN and integrin. Co-immunoprecipitation results showed that mutating the RGD sequence in the LGMN protein to RGE could inhibit the binding of LGMN and integrin β3 (Fig. 5, B). The binding of integrins to ligands depends on the regulation of divalent metal ions. We found that the interaction between LGMN and integrin was also dependent on the presence of divalent metal ions, and the binding of LGMN and integrin β3 was also significantly inhibited when treated with EDTA (Fig. 5, C). On the other hand, metal ions regulate the binding of integrin and ligand mainly through the metal ion binding site MIDAS on integrin. We also found that the ligand binding site mutation (β ₃ ^S121A ) on β3 integrin also cannot Binds to LGMN (Fig. 5, D). As a protease, the mutation (C189S) of the enzymatic active center of LGMN did not affect the binding of LGMN to integrin β3 (Fig. 5, E). Based on the above experiments, we found that pro-LGMN can be secreted extracellularly by breast cancer cells and bind to integrins on the cell surface through its RGD sequence.

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

Next, we wanted to explore the function of the combination of LGMN and integrins in regulating the migration and invasion of breast cancer cells. To investigate this question, we examined the migration and invasion abilities of Hs578T, MDA-MB-231, and SCP2 cells, stably expressing wild-type, LGMN ^D120E , and enzyme-free LGMN ^C189S . It was found that the migration and invasion abilities of tumor cells were significantly restored after re-expression of wild-type LGMN and enzyme-deleted LGMN ^C189S in cells (Fig. 6, AC). However, after overexpressing the mutant LGMN ^D120E , that is, destroying the combination of LGMN and β3 integrin, the metastatic ability of tumor cells was inhibited (Fig. 6, AC). The above results directly prove that the combination with β3 integrin mediates the metastasis process of tumor cells, while the enzyme active center of LGMN has no effect on the metastasis of tumor cells. Since the expression level of integrin may also regulate the metastasis of breast cancer cells, we also detected the effect of different LGMN expressions on β3 integrin. Expression was not affected (Figure 7). In order to further explore whether LGMN promotes breast cancer cell metastasis is mediated by autocrine pro-LGMN. We added the purified protein of LGMN wild-type and point-mutated LGMN ^D120E to the cell culture medium of stable transfection cells knocking down LGMN. Through wound healing, cell migration and invasion experiments, we found that only wild-type pro-LGMN protein can restore breast cancer Migration and invasion ability of cells (Fig. 6, DF).

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. Next, we used Western blot related experiments to detect the activation of integrin downstream related signals and determine which GTPase ultimately promotes the metastasis of breast cancer cells. 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).

2.5 Combination of LGMN and integrin promotes bone metastasis of breast cancer in vivo

Through the above studies, we know that LGMN can be secreted by breast cancer cells to the outside of the cell as an integrin ligand to activate integrin downstream signals, thereby promoting breast cancer cell metastasis. To further explore the effect of LGMN-integrin signaling on breast cancer metastasis in vivo, we established an experimental breast cancer bone metastasis model. SCP2 breast cancer cells stably expressing luciferase were injected into nude mice through the left ventricle, and the tumor growth was monitored in real time on the day of injection and within 6 weeks of the experiment. The experimental results showed that the SCP2 cells expressing LGMN wild type formed more bone metastasis signals and bone tissue lesions ( FIG. 8 , AB). However, in the LGMN ^D120E mutation group and the LGMN knockdown group, the signal of bone metastasis was significantly reduced (Fig. 8, AB). Monitoring the expression of human-derived LGMN in mouse plasma also found that the content of LGMN in plasma in the control group and the LGMN recovery group was significantly higher than that in the LGMN knockdown and LGMN ^D120E recovery groups (Fig. 8, C). At the same time, the morphological measurement analysis of the bone found that the bone damage of mice replenished with wild-type LGMN and LGMN ^C189S was higher than that of the LGMN knockdown group and the LGMN ^D120E replenishment group, showing lower bone content (Figure 8, D), fewer trabecular numbers (Fig. 8, E), and higher trabecular separation (F). In order to further verify that the proliferation ability of SCP2 cells in mice is not affected by the expression of LGMN, we constructed an orthotopic breast cancer model in nude mice, and the results showed that knocking down or replenishing the expression of LGMN did not affect the proliferation of orthotopic breast cancer cells growth (Figure 9).

2.6 Knocking down the expression of LGMN or integrin β3 protein can inhibit the lung metastasis of SCP2 breast cancer cells in situ

Using a nude mouse bone metastasis model, we have found that LGMN-integrin β3 signaling plays an important role in the metastasis of breast cancer cells in vivo. Next, we newly constructed a breast cancer orthotopic tumor model to further explore the effect of LGMN-integrin αvβ3 signaling on SCP2 breast cancer orthotopic tumor metastasis. We mixed 1×10 ⁶ SCP2 cells with matrigel 1:1 and injected them into the fourth pair of mammary fat pads of B-NDG mice. After 8 weeks of orthotopic tumor growth, the lung tissues of the mice were taken and imaged by bioluminescence To detect metastases formed by tumors in the lungs. The experimental results found that the lung metastases formed by orthotopic tumors of SCP2 breast cancer cells knocked down LGMN or integrin β3 were significantly down-regulated compared with the control group ( FIG. 10 ).

2.7 LGMN-integrin αvβ3 signaling also affects the migration and metastasis of ER+ breast cancer T47D cells in mice

In order to further explore whether LGMN-integrin αvβ3 signaling also affects the migration and invasion of ER+ breast cancer cells. In the previous study, we detected the low expression of LGMN in ER+ breast cancer cells T47D, and we found that T47D cells did not express integrin β3, so we constructed T47D cells that express LGMN or integrin β3 alone, and express both LGMN and The stably transfected cells of integrin β3 are shown in Figure 11, A. Next, we tested its effect on T47D cell migration and invasion through cell healing assay, cell migration assay and cell invasion assay. The results showed that only the simultaneous expression of LGMN and integrin αvβ3 in T47D cells could promote cell migration and invasion, and overexpression of LGMN or integrin β3 alone could not enhance cell migration and invasion (Figure 11, B-D).

Next, we further examined the effect of LGMN-integrin αvβ3 signaling on the migration of T47D cells in mice. We respectively injected T47D breast cancer cells into the mammary fat pad of B-NDG mice or injected them into nude mice through the left ventricle to construct an orthotopic tumor model and a breast cancer bone metastasis model, and respectively counted the orthotopic tumor models Occurrence of pulmonary metastases formation and bone metastases in a left ventricular model. The experimental results showed that the simultaneous overexpression of LGMN and integrin αvβ3 could promote the transfer of T47D cells in vivo ( FIG. 12 ).

2.8 Screening for antibodies binding to LGMN and integrins

The above studies reveal that breast cancer cells can promote tumor metastasis through the combination of secreted pro-LGMN and integrins on the cell surface. Therefore, aiming at this mechanism, we hope to screen out antibodies that inhibit the combination of LGMN protein and integrin, so as to specifically inhibit the metastasis of breast cancer. In order to screen antibodies that block LGMN and integrin binding, we used phage display technology to screen scFv and construct full-length antibodies. First, four rounds of affinity enrichment screening were performed on the Tomlinson I+J phage library. The first three rounds used the RGD polypeptide as the antigen, and the last round used the full-length LGMN protein as the antigen. After four rounds of screening, we finally screened and obtained five scFvs binding to LGMN protein and RGD polypeptide (Fig. 13, A-B). The variable region sequences of the light and heavy chains were further constructed into the human IgG1 antibody framework for expression and purification. We then performed functional characterization of these clones using ligand binding inhibition assays. The binding of FITC-labeled LGMN protein to SCP2 cells was detected by flow cytometry under different antibody treatments. We found that the C10 antibody could inhibit the binding of LGMN to SCP2 cells ( FIG. 13 , C).

In order to further study the physiological function of the C10 antibody, we used the principle of biomembrane interference to detect the affinity relationship between the C10 antibody and the LGMN protein through small molecule kinetic experiments, and finally calculated the dissociation constant of the C10 antibody to be 1.96nM (Figure 14, A). Next, we treated FITC-labeled LGMN with different concentrations of C10 antibody, then incubated the mixture with SCP2 cells, and detected the half-inhibitory concentration of C10 antibody inhibiting the binding of LGMN to SCP2 cells by flow cytometry. The results showed that the half-inhibitory concentration of MFI was 22.1 μg/ml (Fig. 14, B). We next explored the effect of C10 antibody on the migration ability of breast cancer cells. We treated SCP2 knockdown LGMN cells and knockdown control cells with the minimum concentration that inhibits the maximum inhibitory effect of SCP2 cell surface integrin binding to LGMN, and then found through wound healing experiments, Transwell experiments and invasion experiments that the C10 antibody can specifically Sexually inhibited the migration and invasion of SCP2 cells (Fig. 14, C-E). At the same time, we found that the C10 antibody could specifically inhibit the phosphorylation of FAK-Y397 and Src-T416 and the activation of RhoA mediated by LGMN-mediated downstream integrin signals (Fig. 14, F).

2.9 C10 antibody inhibits the metastasis of breast cancer cells in vivo

Previously, we verified the function of C10 antibody at the molecular and cellular levels. Next, we continued to explore whether C10 could inhibit the metastasis of LGMN-expressing SCP2 breast cancer cells in mice. In the bone metastasis model established by injecting SCP2 breast cancer cell line into the left ventricle of nude mice, we injected C10 (2 mg/kg) antibody and human IgG control antibody intraperitoneally, once every two days, for 6 weeks. The bone metastases of breast cancer were detected in real time by the mouse in vivo fluorescence imaging system every week. As shown in (Figure 14, G-H), in the sixth week, the C10 antibody group could reduce the formation of bone metastases of breast cancer cells in mice, The results of X-Ray and Micro-CT also showed that the mice in the C10 antibody group had higher bone content (Fig. 14, I), more bone trabeculae (Fig. 14, J) and more bone damage than the control group in the sixth week. Slight (Fig. 14, K). This result indicates that the C10 antibody can inhibit the metastasis of breast cancer cells in vivo.

2.10 C10 antibody specifically inhibits the binding of LGMN to integrin αvβ3

In order to further explore the function of C10 antibody, we purified the pro-LGMN-flag protein and the full-length protein of integrin αvβ3 by using the 293T cell in vitro purification system. Next, we verified the binding between pro-LGMN-flag protein and integrin αvβ3 by flag beads pull-down experiment (Figure 15). Further in the pull-down experiment, we pretreated pro-LGMN-flag with C10 antibody in advance, and we found that C10 antibody could specifically inhibit the interaction between pro-LGMN-flag protein and integrin αvβ3 (Figure 15).

2.11 C10 antibody can inhibit the migration ability of ER+ breast cancer cells, gastric cancer cells, lung cancer cells and colon cancer cells

In addition to breast cancer, we found that the expression of LGMN was enhanced in lung adenocarcinoma, colon cancer, gastric adenocarcinoma, breast cancer, hepatocellular carcinoma, pancreatic cancer and other cancers through GEPIA database analysis (Figure 16). Integrin αvβ3 is also highly expressed in these tumors.

We have previously verified that 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 αvβ3 ( FIG. 17 ).

At the same time, we also detected the effect of C10 antibody on the migration ability of other tumor cells. We first detected the protein expression of LGMN in MNK-45 gastric cancer cells and A549 lung cancer cells. Western blot experiments showed that compared with normal mammary epithelial cells MCF-10A, both MNK-45 gastric cancer cells and A549 lung cancer cells had higher expression of LGMN (Fig. 18, A). The results of further transwell experiments also showed that the C10 antibody could inhibit the migration ability of MNK-45 gastric cancer cells and A549 lung cancer cells in vitro ( FIG. 18 , B).

In addition, we also examined the effect of C10 antibody on the migration ability of colon cancer cells. The results of immunoblotting showed that, taking A549 lung cancer cells as a positive control, SW480 colon cancer cells also contained higher levels of LGMN expression ( FIG. 19 , A). And the results of wound healing experiments showed that C10 antibody can also significantly inhibit the migration ability of SW480 colon cancer cells ( FIG. 19 , B).

3. Discussion of results

Since the intracellular signaling pathway mediated by the combination of LGMN and integrin to promote cell migration is very conserved in vivo, the mechanism of interaction between LGMN and integrin to promote tumor metastasis is also applicable to other LGMN and integrin αvβ3 high expressing tumor types. In addition to integrin αvβ3, we verified that integrins of other RGD ligands such as αvβ1, αvβ5, αvβ6, αvβ8, α5β1 and α8β1 by co-immunoprecipitation experiments, LGMN protein can bind to its β subunits β1, β5, β6 and β8 ( 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. At present, 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. In the present invention, we found that autocrine pro-LGMN of tumor cells binds to integrin, and promotes tumor metastasis and invasion by activating intracellular FAK-Src-RhoA signaling pathway. Therefore, we believe that the metastasis of breast cancer mediated by LGMN is mainly realized through the function of its integrin ligand and does not depend on the enzymatic function of LGMN. to block tumor metastasis. Therefore, inhibiting the interaction between tumor cell integrins and their autocrine LGMN is a therapeutically effective target for inhibiting tumor metastasis.

Claims

A mutant LGMN protein, compared with the wild type, the RGD sequence of the mutant LGMN protein has one or more mutations that cause the interaction between LGMN and integrin or its beta subunit to be weakened or disappeared,

Preferably, the RGD sequence of the mutant LGMN protein is mutated to RGE, RAD, RAE, KGD or KGE compared to the wild type.
A mutant integrin or its beta subunit, which has one or more mutations in the ligand-binding region interacting with the LGMN protein that cause its interaction with the LGMN protein to weaken or disappear compared with the wild type;

Preferably,

The mutant integrin or its beta subunit has a mutation at the D120 and/or S121 position; and/or

The β subunit of the integrin is selected from one or more of the following: β 1 , β 3 , β 5 , β 6 and β 8 , and the integrin is an integrin containing the β subunit.
Anti-LGMN antibody or 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, LCDR3 contains QQAYANPDT,

Preferably, the VH and VL of the antibody have the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 or variants with 90% sequence identity thereto,

More preferably, 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.
A nucleic acid molecule comprising a sequence selected from:

(1) the sequence encoding the anti-LGMN antibody or antigen-binding fragment thereof of claim 3, or

(2) The complementary sequence of (1).
A nucleic acid construct comprising the nucleic acid molecule of claim 4,

Preferably, the nucleic acid construct is a cloning vector, an integrating vector or an expression vector.
A host cell, said host cell:

(1) expressing the anti-LGMN antibody or antigen-binding fragment thereof according to claim 3, or

(2) comprising the nucleic acid molecule of claim 4 or the nucleic acid construct of claim 5.
The use of LGMN protein and/or integrin or its β 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,

Preferably,

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; and/or

The potential substance inhibits the interaction between LGMN protein and integrin or its beta subunit; and/or

The β subunit of the integrin is selected from one or more of the following: β 1 , β 3 , β 5 , β 6 and β 8 , the integrin is an integrin containing the β subunit; and/or

Targets include: the RGD sequence of LGMN protein, and/or, the ligand-binding region of integrin or its β subunit interacting with LGMN protein.
A pharmaceutical composition, which contains:

(1) a pharmaceutically acceptable carrier, and

(2) the mutant LGMN protein according to claim 1, the mutant integrin or its β subunit according to claim 2, or the anti-LGMN antibody or antigen-binding fragment thereof according to claim 3, and/or ( 3) A nucleic acid molecule comprising the nucleic acid sequence encoding (2) or its complementary sequence.
A small interfering RNA knocking down LGMN, which has the sequence shown in SEQ ID NO: 5 or 6; or

A small interfering RNA for knocking down integrin or its beta subunit has the sequence shown in SEQ ID NO:7.
Use of a reagent for inhibiting the interaction between LGMN protein and integrin or its beta subunit in the preparation of a drug for inhibiting tumor metastasis or tumor cell metastasis,

Preferably,

The interaction is the interaction between the RGD sequence of the LGMN protein and the integrin or its beta subunit, and/or

The β subunit of the integrin is selected from one or more of the following: β 1 , β 3 , β 5 , β 6 and β 8 , and the integrin is an integrin containing the β subunit;

More preferably, the reagent is selected from one or more of the following:

(1) 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 β subunit to be weakened or disappeared; preferably Preferably, the mutation is a RGD sequence mutation to RGE, RAD, RAE, KGD or KGE;

(2) mutant integrin or its β subunit and/or its targeting vector, wherein, compared with the wild type, the mutant integrin or its β subunit has one or more in the ligand binding region interacting with LGMN protein A plurality of mutations leading to the weakening or disappearance of its interaction with the LGMN protein; preferably, the integrin or its β subunit has mutations at D120 and/or S121 positions;

(3) an anti-LGMN antibody or an antigen-binding fragment thereof, which inhibits the interaction between LGMN protein and integrin or its beta subunit;

(4) an antibody against integrin or its β subunit or an antigen-binding fragment thereof, which inhibits the interaction between LGMN protein and integrin or its β subunit;

(5) a 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;

(6) A host cell expressing the antibody or antigen-binding fragment thereof described in (3) or (4);

(7) an inhibitor of integrin or its β subunit, which inhibits the interaction between LGMN protein and integrin or its β subunit;

(8) Reagents that can reduce the concentration of divalent metal ions;

(9) A reagent for knocking down or knocking out the expression of LGMN protein and/or integrin or its beta subunit, such as ZFN and/or TALEN and/or CRISPR/Cas9 reagent and/or small interfering RNA.
purposes as claimed in claim 10, is characterized in that,

The mutant LGMN protein is as described in claim 1,

The mutant integrin or its β subunit is as described in claim 2,

The anti-LGMN antibody or its antigen-binding fragment is as described in claim 3,

The reagent for knocking down or knocking out the expression of LGMN protein and/or integrin or its β subunit is as described in claim 9 .
The use according to claim 10 or 11, wherein 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 .
A method for inhibiting tumor cell metastasis in vitro, comprising the step of weakening the interaction between LGMN protein of tumor cells and integrin or its beta subunit,

Preferably, the method includes weakening the interaction between the LGMN protein of tumor cells and integrin or its β subunit by any one or more of the following methods:

(1) Knocking out or knocking down the expression of LGMN protein of tumor cells;

(2) Knocking out or knocking down the expression of integrin or its β subunit in tumor cells;

(3) express a mutant LGMN protein whose interaction with integrin or its β subunit is weakened or disappeared in tumor cells of wild type or knockout LGMN protein;

(4) Expressing mutant integrin or its beta subunit with weakened or disappeared interaction with LGMN protein in wild-type or tumor cells knocked out of integrin or its beta subunit;

(5) contacting the tumor cells with an anti-LGMN antibody or antigen-binding fragment thereof or a protein comprising said anti-LGMN antibody or antigen-binding fragment thereof;

(6) contacting the tumor cells with an agent that reduces the concentration of divalent metal ions,

More preferably, the method has one or more features selected from:

Compared with the wild type, the RGD sequence of the mutant LGMN protein has a mutation that causes the interaction with integrin or its β subunit to be weakened or disappeared;

Compared with the wild type, the mutant integrin or its β 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.
Use of a reagent for detecting LGMN protein in the preparation of a kit for diagnosing tumor metastasis,

Preferably, the reagent detects the expression, content or sequence of LGMN protein in tumor tissue,

More preferably, the reagent is selected from one or more of the following: antibodies against LGMN protein, primers and/or probes that hybridize to the coding sequence of LGMN protein.