Anti-tumor angiogenesis drug synergist, anti-tumor drug composition and application

Abstract

This invention belongs to the pharmaceutical field, specifically relating to an anti-tumor angiogenesis drug potentiator, an anti-tumor drug composition, and its application. Firstly, this invention discovers that SHCBP1 inhibitors can enhance the therapeutic effect of anti-tumor angiogenesis drugs and can be used as potentiators for anti-tumor angiogenesis drugs. Secondly, when SHCBP1 inhibitors are used in combination with anti-tumor angiogenesis drugs, or in combination with anti-tumor angiogenesis drugs and PD-1 inhibitors, tumor angiogenesis can be normalized and its window period prolonged, significantly improving the efficacy of immunotherapy, anti-angiogenesis, and anti-tumor therapy, showing broad application prospects.

Description

[0001] This application claims priority to the earlier application filed on March 31, 2023, with application number CN2023103403499, entitled "An anti-tumor angiogenesis drug synergist, anti-tumor drug composition and application", the entire contents of which are set forth in this application. Technical Field

[0002] This invention belongs to the medical field, specifically relating to an anti-tumor angiogenesis drug synergist, an anti-tumor drug composition, and its application. Background Technology

[0003] Abnormal vascular systems and immunosuppression are two major characteristics of solid tumors. Due to the malignant proliferation of tumor cells, their vascular systems struggle to maintain a high-load metabolic oxygen supply. Consequently, tumors secrete excessive angiogenesis-promoting factors, which on the one hand promote the rapid proliferation of vascular endothelial cells, leading to their disordered accumulation; on the other hand, they damage the endothelial cell basement membrane and inhibit pericytes and smooth muscle cells from covering new blood vessels, resulting in abnormalities in the tumor vascular membrane and lumen structure. Furthermore, the uncontrolled growth and expansion of tumor cells are restricted by the surrounding host tissue, generating mechanical forces that compress intratumoral blood vessels, causing lumen collapse. Therefore, tumor vascularization macroscopically manifests as incomplete vessels, increased permeability, increased interstitial hydraulic pressure, and insufficient vascular perfusion. Insufficient oxygen transport and the inability to timely remove metabolic products further lead to the release of angiogenesis-promoting factors and inflammatory factors, not only causing more abnormal angiogenesis but also creating a high-pressure, low-pH, and immunosuppressive tumor microenvironment.

[0004] Numerous studies have found that, compared to simply inhibiting angiogenesis and causing vascular degeneration, the key therapeutic goals and mechanisms lie in effectively inducing tumor angiogenesis normalization, promoting immune cell infiltration, and facilitating chemotherapy drug delivery. Currently, the use of anti-angiogenic drugs to regulate vascular normalization, combined with immunotherapy, has shown excellent anti-tumor efficacy and has been approved by the US FDA for the treatment of lung cancer and liver cancer, among others. However, insufficient vascular normalization induced by anti-angiogenic drugs or a short vascular normalization window can lead to malignant inhibition of angiogenesis and drug resistance outside this window, causing new immunosuppression and resulting in vascular and immune circulation resistance, posing a significant challenge to anti-angiogenic combined immunotherapy.

[0005] Therefore, there is still a need in this field to develop new anti-angiogenic combined immunotherapy methods. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide an anti-tumor angiogenesis drug synergist, an anti-tumor drug composition, and its application, specifically including the following:

[0007] In a first aspect, the present invention provides the application of an SHCBP1 inhibitor in the preparation of an anti-tumor angiogenesis drug potentiator; wherein the SHCBP1 inhibitor interferes with / inhibits SHCBP1 expression.

[0008] Preferably, the SHCBP1 inhibitor includes an RNA interference reagent or a small molecule inhibitor.

[0009] Preferably, the SHCBP1 inhibitor is a small molecule inhibitor, specifically shRNA and the small molecule inhibitor MS1943; the sequence of the shRNA is shown in SEQ ID NO. 1-2; the structural formula of MS1943 is shown in formula (Ⅰ) below:

[0010]

[0011] Preferably, the anti-tumor angiogenesis drug is a VEGFR2 inhibitor.

[0012] Preferably, the VEGFR2 inhibitor comprises an anti-VEGFR2 antibody or a VEGFR2 tyrosine kinase inhibitor.

[0013] Preferably, the VEGFR2 inhibitor is apatinib.

[0014] In a second aspect, the present invention provides a pharmaceutical composition for anti-tumor purposes, the pharmaceutical composition comprising an SHCBP1 inhibitor and an anti-tumor angiogenesis drug.

[0015] Preferably, the SHCBP1 inhibitor includes an RNA interference reagent or a small molecule inhibitor.

[0016] Preferably, the SHCBP1 inhibitor is a small molecule inhibitor, specifically shRNA and the small molecule inhibitor MS1943; the sequence of the shRNA is shown in SEQ ID NO. 1-2; the structural formula of MS1943 is shown in formula (Ⅰ) below:

[0017]

[0018] Preferably, the anti-tumor angiogenesis drug is a VEGFR2 inhibitor.

[0019] Preferably, the VEGFR2 inhibitor comprises an anti-VEGFR2 antibody or a VEGFR2 tyrosine kinase inhibitor.

[0020] Preferably, the VEGFR2 inhibitor is apatinib.

[0021] Preferably, the pharmaceutical composition further includes an immunotherapeutic agent.

[0022] Preferably, the immunotherapy drug includes a PD-1 inhibitor.

[0023] Preferably, the PD-1 inhibitor is a PD-1 antibody.

[0024] Thirdly, the present invention provides the use of the pharmaceutical composition described in the second aspect above in the preparation of antitumor drugs.

[0025] Preferably, the cancers include stomach cancer and breast cancer.

[0026] The beneficial effects of this invention are:

[0027] This invention reveals that: ① SHCBP1 knockout significantly enhances the sensitivity of anti-tumor angiogenesis drugs (apatinib) to tumor angiogenesis by influencing the aggregation and adhesion of α-SMA, PDGFR2, and vascular adhesion factor VCAM-1 in new blood vessels, thus promoting vascular normalization. This indicates that inhibiting SHCBP1 expression can enhance the sensitivity of anti-tumor angiogenesis drugs, meaning that SHCBP1 inhibitors can act as synergists for anti-tumor angiogenesis drugs; ② SHCBP1 knockout combined with anti-tumor angiogenesis drugs (apatinib) can promote tumor angiogenesis normalization and improve the immune microenvironment, inhibit breast cancer growth, and prolong survival prognosis. In other words, SHCBP1 knockout combined with anti-tumor angiogenesis drugs (such as apatinib) can effectively inhibit tumor growth and improve... Local hypoxia and vascular perfusion in tumors promote the infiltration of immune cells; therefore, the combination of SHCBP1 inhibitors and anti-tumor angiogenesis drugs has significant anti-tumor activity. ③ Knockout of SHCBP1 combined with anti-tumor angiogenesis drugs (apatinib) and immunotherapy drugs (PD1 antibodies) can significantly inhibit tumor cell growth and improve the efficacy of anti-tumor angiogenesis therapy. ④ Furthermore, the combination of the SHCBP1 inhibitor MS1943 with anti-tumor angiogenesis drugs (apatinib) and immunotherapy drugs (PD1 antibodies) can significantly inhibit tumor cell growth and improve the efficacy of anti-tumor angiogenesis therapy, indicating that the combination of SHCBP1 inhibitors with anti-tumor angiogenesis drugs and immunotherapy drugs further enhances anti-tumor activity and has broad application value. Attached Figure Description

[0028] Figure 1 Results of the sensitizing effect of SHCBP1 on apatinib's inhibition of angiogenesis;

[0029] Figure 2 SHCBP1-KO combined with apatinib inhibits tumorigenesis (AC) and promotes tumor angiogenesis normalization (DF).

[0030] Figure 3 SHCBP1-KO combined with apatinib improves tumor vascular leakage (shown in A) and tumor hypoxia (shown in B);

[0031] Figure 4SHCBP1 knockout combined with apatinib induces tumor PD-L1 expression (shown in A) and promotes CD4 expression in immune cells. + and CD8 + T's infiltration (as shown in BF);

[0032] Figure 5 SHCBP1 knockout combined with apatinib and PD1 antibody inhibits the growth of breast cancer cells.

[0033] Figure 6 SHCBP1 inhibitor MS1943, in combination with apatinib and PD1 antibody, inhibits the growth of breast cancer cells. Detailed Implementation

[0034] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially. Unless otherwise specified, the present invention will be practiced using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology, as well as those skilled in the art, and fully explained in references such as: Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989), Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, 1998), Academic Press; Animal Cell Culture (edited by RIFreshney, 1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998), Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JB Griffiths, and DG Newell, 1993-1998), J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by DMWeir and CCBlackwell); Gene Transfer Vectors for Mammalian Cells (edited by JMMiller and MPCalos, 1987); Current Protocols in Molecular Biology (FMEdited by Ausubel et al., 1987); PCR: The Polymerase Chain Reaction, (Edited by Mullis et al., 1994); Current Protocols in Immunology (Edited by J.E. Coligan et al., 1991); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (Edited by D. Catty., IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (Edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (Edited by M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995).

[0035] The anti-tumor angiogenesis drug described in this application is apatinib (medical grade, Jiangsu Hengrui Medicine Co., Ltd.); fetal bovine serum and ECM culture medium (Sciencell), MTT cell proliferation assay kit (Promega), SHCBP1 antibody (Sigma), VEGFR2 antibody (Cell Signaling Technology), immunohistochemical staining kit (Wuhan Boster Biological), Alexa Fluor 488 fluorescent secondary antibody and Alexa Fluor 647 fluorescent secondary antibody (Abcam), Biacor CM5 chip (GE Healthcare), Flag antibody gel beads (Sigma), and Matrigel (Corning).

[0036] Cell line: Human umbilical vein endothelial cells (HUVECs) were extracted from the umbilical cord of newborns and have passed the ethical review of the Second Clinical Medical College of Lanzhou University.

[0037] Experimental animals: SHCBP1 knockout mice and MMTV-PyMT spontaneous breast cancer tumor model mice were purchased from Shanghai Southern Model Biotechnology Development Co., Ltd.

[0038] Human gastric cancer clinical tissue samples: All gastric cancer clinical tissue samples were provided by the Second Hospital of Lanzhou University. All patients signed informed consent forms, and the experimental process was approved by the Human Ethics Committee of the Second Hospital of Lanzhou University.

[0039] The research methodology is as follows:

[0040] (1) Establishment of a co-culture model of gastric cancer tumor organoids and vascular endothelial cells (HUEVCs): ① Isolation and culture of HUEVCs: A 15cm neonatal umbilical cord was taken, washed with sterile PBS, and infused with 0.1% type I collagenase. The incubation was carried out at 37℃ for 15 min. The digestive fluid was collected, centrifuged at 1000 rpm, and cultured in ECM complete medium; ② Construction of gastric cancer organoids: A 1cm... 3 Fresh gastric cancer tissue was minced and washed, then digested with type II collagenase at 37°C for 60 min. After filtration through a cell strainer and centrifugation at 1000 rpm, the cells were resuspended in a 1:1 mixture of DMEM / F12 medium and 40 μL of matrix gel and placed in a 24-well plate. After the matrix gel solidified, Intesti Cult™ organoid culture medium was added for further culture. ③ Co-culture of organs and endothelial cells: 5 × 10⁶ cells / well were cultured in a 24-well plate. 4 One organoid per pore was seeded into the upper chamber of the Transwell, and vascular endothelial cells were introduced at a rate of 2 × 10⁻⁶. 5 Organoids were seeded per well in the lower chamber and cultured in Intesti Cult™ organoid medium with 10 ng / mL LFGF-2 and 100 μg / mL Heparin. The culture medium was changed after 24 h and the wells were placed in an anoxic incubator.

[0041] Eight HUEVCs from different individuals were isolated, and tumor organoids from two gastric cancer patients were established. A hypoxic co-culture model of HUEVCs and organoids was also established.

[0042] (2) Construction of SHCBP1 knockdown HUEVCs cell line: ① Two shRNAs targeting SHCBP1 were designed and synthesized based on the human SHCBP1 gene (as shown in Table 1); ② Cell infection: HUVECs were infected at a rate of 2 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of / wells in 6-well plates. Two shRNAs were transfected into HUVEC cells using Lipofectamine™ 3000. After 48 hours, a portion of the cells were harvested and Western blotting was used to verify the knockdown efficiency. The remaining cells were used for subsequent experiments.

[0043] Table 1. Two shRNA sequences targeting SHCBP1

[0044]

[0045] (3) Establishment of the SHCBP1 vascular endothelial conditional knockout mouse model: The model was obtained by crossing SHCBP1flox mice with vascular endothelial Cdh5-2A-Cre mice. The full name of the SHCBP1flox mouse is C57BL / 6J-SHCBP1em1(flox)Smoc (Southern Model Animal). The SHCBP1 gene was edited using CRISPR / Cas9 technology. Homozygous SHCBP1flox mice were obtained through gRNA and donor vector construction, microinjection, F0 generation, F1 generation, homozygous breeding, and genotyping. The Cdh5-2A-Cre mouse, named C57BL / 6-Cdh5em1(2A-CreERT2-WPRE-polyA)Smoc (Jackson Laboratory), is a tool mouse that specifically expresses Cre in vascular endothelium. SHCBP1flox mice are mated with vascular endothelial Cdh5-2A-Cre mice to obtain SHCBP1 conditional knockout mice, Cdh5-Cre SHCBP1flox.

[0046] (4) Screening of SHCBP1 small molecule inhibitors: SHCBP1 was modeled using Alpha Fold 2 to obtain its three-dimensional protein structure. The Site Finder in MOE software was used to search for potential small molecule binding pockets. Next, the DELopen technology from WuXi AppTec, a protein drug fishing method, was used to initially screen from 400 million DNA-encoded small molecules to obtain potential small molecule inhibitors that can bind to SHCBP1. Then, the Dock module in MOE software was used to perform virtual docking screening of these potential small molecule inhibitors. Finally, combined with SPR affinity screening, SHCBP1 inhibitors were obtained. Currently, a small molecule binding pocket model of SHCBP1 has been obtained, and the DELopen kit has been used to fish for potential SHCBP1 inhibitors; the results are currently being analyzed.

[0047] (5) Establishment of a humanized tumor xenograft model (PDX) for gastric cancer: 1cm 3 Fresh gastric cancer tumor tissue was washed three times with sterile PBS and cut into pieces approximately 3mm in size. 3 The tissue block was mixed with a high concentration of matrix gel. 10-12 week old NSG mice were anesthetized, and a 7mm surgical incision was made under the armpit after disinfection. The matrix gel-coated tumor tissue was implanted into the mouse's armpit, sutured, and then placed in a cage for observation of tumor size. These mice were P1 generation. The tumor was observed when it reached a volume of 500-1000 mm². 3 At that time, the same method as described above is used for passage expansion, and the P3 generation can be used for subsequent research.

[0048] Example 1: SHCBP1 modulates the sensitivity of apatinib to inhibit angiogenesis.

[0049] The applicant isolated HUEVCs from 8 different donors and used a cyclization assay to detect their sensitivity to apatinib. The results showed that apatinib-resistant cells exhibited high expression of SHCBP1. Figure 1 As shown in Figure AB), and the phenomenon of increased nuclear translocation ( Figure 1 (as shown in CD); knockdown or removal of SHCBP1 can sensitize apatinib's inhibition of angiogenesis (as shown in CD); Figure 1 (As shown in EH); In vitro culture of SHCBP1 knockout mouse carotid arteries revealed that SHCBP1 knockout mouse arterial endothelial cells exhibited higher sensitivity to apatinib. Figure 1 (As shown in Figure I). The above results indicate that inhibiting SHCBP1 expression can enhance the sensitivity of vascular endothelial cells to anti-tumor angiogenesis drugs, meaning that SHCBP1 expression inhibitors can act as sensitizers for anti-tumor angiogenesis drugs, enhancing the sensitivity of vascular endothelial cells to these drugs.

[0050] Example 2: SHCBP1 knockout sensitization with apatinib improves tumor vascularization in mice.

[0051] A spontaneous breast cancer tumor model with SHCBP1 knockout was established by crossing SHCBP1 knockout mice with PyMT breast cancer spontaneous tumor model mice, and the mice were treated with 60 mg / kg apatinib. Figure 2 As shown in Figure A), the results showed that SHCBP1 knockout mice combined with apatinib significantly inhibited breast cancer growth and prolonged survival in mice. Figure 2 (As shown in B and C); In addition, immunofluorescence staining of mouse tissues showed that SHCBP1 knockout significantly sensitized apatinib to inhibit tumor angiogenesis, promoted vascular normalization, and affected the aggregation and adhesion of α-SMA, PDGFR2, and vascular adhesion factor VCAM-1 in neovascularization (as shown in B and C respectively). Figure 2 (As shown in the diagram). The above results indicate that inhibiting SHCBP1 expression can enhance the sensitivity of anti-tumor angiogenesis drugs and promote tumor angiogenesis normalization. That is, SHCBP1 expression inhibitors can act as sensitizers for anti-tumor angiogenesis drugs, and their combination with anti-tumor angiogenesis drugs can promote tumor angiogenesis normalization and enhance anti-tumor activity.

[0052] Example 3: SHCBP1 knockout improves tumor vascular leakage and tumor hypoxia

[0053] The applicant examined tumor vascular leakage and tumor hypoxia, and the results showed that SHCBP1 knockout combined with apatinib significantly improved tumor leakage. Figure 3 (as shown in A) and tumor hypoxia ( Figure 3 (As shown in Figure B). The above results indicate that inhibiting SHCBP1 expression can improve tumor vascular leakage and tumor hypoxia. That is, the combination of SHCBP1 inhibitors and anti-tumor angiogenesis drugs can further improve tumor vascular leakage and tumor hypoxia activity, and enhance anti-tumor activity.

[0054] Example 4: SHCBP1 knockout can sensitize apatinib to improve the tumor microenvironment in mice.

[0055] Subsequently, the applicant examined the expression of PD-L1 and the infiltration of immune cells in the tumor cells, and the results were as follows: Figure 4 As shown, SHCBP1 knockout significantly induced an increase in PD-L1 expression in tumors. Figure 4 As shown in Figure A), it promotes the growth of CD4 immune cells. + and CD8 + T cell infiltration ( Figure 4 (As shown in BF). The above results indicate that inhibiting SHCBP1 expression can significantly induce PD-L1 expression in tumors, meaning that SHCBP1 expression inhibitors can induce PD-L1 expression in tumors and improve the tumor microenvironment.

[0056] Example 5: SHCBP1 knockout combined with apatinib and PD1 antibody significantly inhibited tumor growth in mice.

[0057] The applicant used SHCBP1 knockout mice to cross with PyMT spontaneous breast cancer tumor model mice to establish an SHCBP1 knockout spontaneous breast cancer tumor model, and treated the model mice with apatinib (60 mg / kg, orally, once every 3 days) or PD-1 antibody (200 μg / mouse, once every 3 days). Figure 5 As shown in Figure A), the results showed that SHCBP1-KO combined with apatinib and PD1 antibody significantly prolonged the survival prognosis of mice and inhibited breast cancer growth. Figure 5 (CBC). The above results indicate that inhibiting SHCBP1 expression, combined with anti-tumor angiogenesis drugs and PD1 antibodies, can significantly inhibit tumor cell proliferation, suggesting that the combination of SHCBP1 inhibitors with anti-tumor angiogenesis drugs and immunotherapy drugs can significantly inhibit tumor cell proliferation and enhance anti-tumor activity.

[0058] Example 6: The SHCBP1 inhibitor MS1943 combined with apatinib and PD1 antibody significantly inhibited tumor growth in mice.

[0059] The applicant used micro-thermal surge screening to identify the small molecule compound MS1943, which can directly target SHCBP1, with an affinity of 128.32 nM. Figure 6 (As shown in Figure A). This inhibitor, in combination with apatinib, can significantly inhibit the cyclization ability of vascular endothelial cells (as shown in Figure A). Figure 6 (As shown in BC). Furthermore, using the PyMT spontaneous breast cancer tumor model mouse, mice were treated with MS1943 (50 mg / kg, intraperitoneal injection, every 2 days) or 60 mg / kg of apatinib or 200 μg / mouse of PD-1 antibody. The results showed that the SHCBP1 inhibitor MS1943 combined with apatinib and PD-1 antibody significantly inhibited breast cancer growth (…). Figure 6 (DG). The above results indicate that the combination of SHCBP1 inhibitors with anti-tumor angiogenesis drugs and immunotherapy drugs can significantly inhibit tumor cell proliferation and enhance anti-tumor activity.

Claims

1. Application of SHCBP1 inhibitor and apatinib in the preparation of anti-breast cancer drugs; wherein the SHCBP1 inhibitor is shRNA or small molecule inhibitor MS1943; the sequence of the shRNA is shown in SEQ ID NO. 1-2; the structural formula of MS1943 is shown in formula (Ⅰ) below: Equation (Ⅰ).

2. A pharmaceutical composition for treating breast cancer, characterized in that, The pharmaceutical composition comprises an SHCBP1 inhibitor and apatinib; wherein the SHCBP1 inhibitor is an shRNA or a small molecule inhibitor MS1943; the sequence of the shRNA is shown in SEQ ID NO. 1-2; the structural formula of the MS1943 is shown in formula (I) below: Equation (Ⅰ).

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition also includes a PD-1 antibody.

4. The use of the pharmaceutical composition as described in claim 2 or 3 in the preparation of an anti-breast cancer drug.

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