CN107149682B - CD 47-targeted immune checkpoint inhibitor pharmaceutical composition and preparation method thereof - Google Patents

Abstract

Translated fromChinese

本发明属生物技术领域，涉及一种靶向CD47的免疫检查点抑制剂药物组合物的制备及其用途。本发明由靶向CD47的免疫检查点抑制剂与细胞自噬抑制剂组成组合药物或者药物复方制剂。所述的细胞自噬抑制剂能增强靶向CD47的药物对于肿瘤细胞的杀伤效果，从而增强该类药物的抗肿瘤疗效；所述的细胞自噬抑制剂与靶向CD47的药物组合复方制剂，能增强药物的靶向效果、以及对于CD47位点的封闭效果及抗肿瘤效果，同时保证原靶向CD47免疫检查点抑制剂的稳定性和靶向性。所述制剂可用于各类肿瘤的诊断、检测和治疗。该制剂的制备工艺简单、适应性广，可用于大规模生产。The invention belongs to the field of biotechnology, and relates to the preparation and use of a pharmaceutical composition of an immune checkpoint inhibitor targeting CD47. The invention consists of a CD47-targeting immune checkpoint inhibitor and a cell autophagy inhibitor to form a combination drug or a drug compound preparation. The autophagy inhibitor can enhance the killing effect of the drug targeting CD47 on tumor cells, thereby enhancing the anti-tumor efficacy of the drug; the autophagy inhibitor combined with the drug targeting CD47 is a compound preparation, It can enhance the targeting effect of the drug, as well as the blocking effect on the CD47 site and the anti-tumor effect, while ensuring the stability and targeting of the original targeted CD47 immune checkpoint inhibitor. The preparation can be used for the diagnosis, detection and treatment of various tumors. The preparation has simple preparation process and wide adaptability, and can be used for large-scale production.

Description

CD 47-targeted immune checkpoint inhibitor pharmaceutical composition and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a novel CD 47-targeted immune checkpoint inhibitor pharmaceutical composition, a preparation method and application thereof, in particular to a novel compound drug or pharmaceutical composition formed by combining a CD 47-targeted immune checkpoint inhibitor and one or more cell autophagy inhibitors, and a preparation method and application thereof.

Background

The prior art discloses that CD47 (also known as integrin associated protein IAP) is a protein expressed on the surface of tumor cellsA highly expressed cellular transmembrane protein comprising five transmembrane domains and one Ig-like domain; in the immune cells of the body, the ligand of CD47 is mainly signal regulatory protein alpha (SIRP alpha, also called MyD-1); CD47 regulates cell migration, phagocytic activity and immune homeostasis through binding to its ligand; in the immune system of the body, CD47 plays a class of "eat me" signals that can interact with the NH of SIRPa via Ig-like domains₂The combination of the terminal domains can generate signals for inhibiting phagocytosis of macrophages, thereby generating inhibition effect on the body innate immune system. The current research on CD47 targeting drugs is mostly directed to blocking the binding of CD47 to its ligand, thereby removing the inhibitory effect of the binding of CD47 to its ligand on the innate immune system. The existing medicines comprise an anti-CD 47 monoclonal antibody, a SIRP alpha protein, a SIRP alpha/MyD-1/CD 172-Fc fusion protein and the like.

CD47 acts as a "eat me" signal, enabling tumor cells to escape phagocytosis thereof by the body's immune system. The SIRP alpha-Fc fusion protein targeting CD47 can block the binding of CD47 and a ligand thereof for a long time, and researches on different human leukemia cell nude mouse transplanted tumor models find that the monoclonal antibody targeting CD47 activates the clearance of transplanted leukemia cells by organisms by blocking the binding of CD47-SIRP alpha (Chao MP, Alizadeh AA, Tang C, Myklebust JH, Varghese B, et al. cell 2010142: 699-channel 713). The Weissman group studies with blood-derived and non-blood-derived cancer cells showed higher expression of CD47 in tumor stem cells, and further indicated that high expression of CD47 is a poor prognostic factor in tumor patients. Due to the over-expression of CD47 on the surface of tumor cells, the elimination of the tumor cells by macrophages is inhibited, and the survival rate of the tumor cells in vivo is improved. The research of the Van group on a homologous SIRP alpha mutant mouse transplanted tumor model finds that the SIRP alpha signal does not influence the metastasis and growth of tumors by itself, but the combination of CD47-SIRP alpha seriously weakens the therapeutic effect of monoclonal antibodies (Zhao XW, Van Beek EM, Schornagel K, Van der Maaden H, Van Houdt M, et al. Proc. Natl. Acad. Sci. USA 2011108: 18342-47); these results provide a theoretical basis for targeting CD47-SIRP alpha to treat malignant tumor or enhance the drug effect of monoclonal antibody.

Research shows that the CD47 can improve the drug delivery efficiency to the tumor microenvironment, and particularly, the Rodriguez group combines the recombinant CD47 protein to the nanoparticles, so that the half-life period of the nanoparticles in circulation and the drug delivery efficiency of cytotoxic drugs to tumors are improved in vivo; nanoparticle-bound CD47 inhibits nanoparticle clearance by the body via Fc γ R, consistent with the aforementioned CD 47-sirpa binding impairing antibody-mediated tumor therapeutic effects (Rodriguez PL, Harada T, Christian DA, pantono DA, Tsai RK, discor de. science 2013339: 971-75); in another study, the nanoparticle is coated by a red blood cell membrane, and the inhibition of the phagocytosis of the nanoparticle by the body is found to be CD47 dependent (Hu C-MJ, Fang RH, Luk BT, Chen KN, Carpenter C, et al. Nanoscale 20135: 2664-68); these results suggest that targeting CD47 may be an effective means to improve the efficiency of drug delivery.

Autophagy (autophagy), also known as type ii programmed cell death (type ii programmed cell death), is an important process responsible for the turnover of intracellular substances evolved and conserved in eukaryotes, and can decompose damaged or redundant organelles and aged proteins in cells to generate nucleotides, amino acids and other small molecular substances for the cells to synthesize new proteins and maintain the stability of microenvironment in the cells. The research finds that the autophagy is closely related to the occurrence and development of various diseases, particularly tumors. Depending on the mode of intracellular substrate transport into the lysosomal cavity, autophagy in mammalian cells can be divided into three modes: large autophagy (macroautophagy), small autophagy (microautophagy) and chaperone-mediated autophagy (CMA). Among them, macroautophagy has mainly the following features: (1) the development process is rapid; (2) inducibility of autophagy; (3) degrading in batches; (4) no selective phagocytosis; (5) conservation of autophagy; the other two autophagies differ from the major autophagy mainly in that: small autophagy is the deformation of the lysosomal membrane itself, which then encapsulates the substrate in the phagocyte cytoplasm; chaperone-mediated autophagy, in turn, selectively degrades proteins.

Research discloses that autophagy can generate stress response to cell external environment change and various stimuli, and cell autophagy with lower level can occur under growth condition, also called basic autophagy, however, once receiving external stimuli such as hunger, hypoxia, high temperature, high cell density or growth factor deprivation, the level of autophagy of cell will be quickly up-regulated, for example, under the condition of nutrient deficiency, cell autophagy can decompose in vivo necrotic organelles to generate amino acid for cell synthesis new protein, and maintain the survival of cell [ Piacentini M, D' Eletto M, Falasca L, et al.2011; 197: 246; ② Cook KL, Shajahan AN, Clarke R.2011; 1283-94 parts in 11 (8); ③ Wirawan E, VandenBerghe T, Lippens S, et al.2012; 22(1):43-61.]. However, as one of programmed cell Death, autophagy can lead to cell Death directly or indirectly through multiple pathways, and this killing may occur by inducing mechanisms such as apoptosis, necrosis, aging, etc., or by causing cells to undergo autophagic Death directly [ denton d, Nicolson S, Kumar s.cell Death diff.2012; 19(1):87-95.].

The current fusion protein preparation targeting CD47 is prepared by the traditional preparation process, so that off-target conditions exist and the treatment effect needs to be improved. To date, no report has been made on the formation of a novel formulation of an autophagy inhibitor and a fusion protein targeting CD 47.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an immune checkpoint inhibitor pharmaceutical composition targeting CD47, and a preparation method and application thereof. The key technical problem solved by the invention is to prepare an immune checkpoint inhibitor pharmaceutical composition which targets CD47 and can obviously kill tumor cells, and simultaneously ensure the stability and targeting of the original targeted CD47 immune checkpoint inhibitor.

The scheme for solving the technical problem is that a combined medicine or a medicinal compound preparation is formed by an immune checkpoint inhibitor targeting CD47 and an autophagy inhibitor.

In the present invention, the autophagy inhibitor includes, but is not limited to, Chloroquine (CQ), 3-MA, womancillin, bavacomycin A-1, ammonium chloride and LY 294002; chloroquine is preferred in embodiments of the present invention;

in the present invention, immune checkpoint inhibitor drugs targeting CD47 include, but are not limited to: anti-CD 47 monoclonal antibody, SIRP alpha protein, SIRP alpha/MyD-1/CD 172-Fc fusion protein, etc.; preferred in embodiments of the invention are SIRP α -Fc (MyD-1-Fc) fusion proteins, especially recombinant human SIRP α/CD172-Fc (MyD-1/CD172-Fc) fusion proteins;

in the invention, the autophagy inhibitor can enhance the killing effect of a medicament targeting CD47 on tumor cells, thereby enhancing the anti-tumor curative effect of the medicaments; the combination of the autophagy inhibitor and the CD47 targeting drug is a compound preparation, which can enhance the targeting effect of the drug, and the blocking effect and the anti-tumor effect on the CD47 locus.

In the invention, the autophagy inhibitor can be prepared into a pharmaceutical composition with a CD 47-targeted drug, and the anti-tumor efficacy of the CD 47-targeted drug is enhanced by adopting a sequential use mode.

The composition or compound preparation of the invention consisting of the immune checkpoint inhibitor targeting CD47 and the autophagy inhibitor can be used for treating tumors including but not limited to: non-small cell lung cancer, lymphoma, chronic myelogenous leukemia, acute lymphoid myelogenous leukemia, breast cancer, melanoma; the invention further carries out an influence test of the fusion protein targeting CD47 on the autophagy level of the non-small cell lung cancer cells, an in vitro phagocytosis promotion comparison test of the fusion protein preparation targeting CD47 and the fusion protein targeting CD47, an in vivo treatment effect comparison test of the fusion protein preparation targeting CD47 and the fusion protein targeting CD47, and an in vivo treatment effect comparison test of the fusion protein preparation targeting CD47 and the common protein targeting CD 47; the results confirmed that the fusion protein can obviously cause the lung cancer cells to generate autophagy (shown in figures 1A and B) and autophagy flow (shown in figure 1C); the fusion protein preparation has stronger phagocytosis promotion effect on macrophages, has obviously improved phagocytosis promotion effect compared with the fusion protein (MyD-1-Fc) which is independently used, proves the anti-tumor effect of the novel fusion protein targeting CD47 in vivo, and can obviously improve the anti-tumor effect when being prepared into a novel preparation together with an autophagy inhibitor; the fusion protein preparation has stronger anti-tumor effect, and compared with the common fusion protein targeting CD47, the anti-tumor promotion effect is obviously improved (as shown in figures 4A and B).

The composition or compound preparation of the invention, which consists of the immune checkpoint inhibitor targeting CD47 and the autophagy inhibitor, can be used for diagnosing and detecting various tumors.

The invention provides a method for preparing a composition or compound preparation consisting of an immune checkpoint inhibitor targeting CD47 and an autophagy inhibitor, wherein the method comprises the following steps of 1) preparing an autophagy inhibitor medicament; 2) preparing a targeting CD47 drug mother solution; 3) preparation of a pharmaceutical composition of an immune checkpoint inhibitor targeting CD 47. The preparation process is simple, has wide adaptability, and can be used for large-scale production.

Drawings

FIG. 1 shows that a fusion protein targeting CD47 causes autophagy in non-small cell lung cancer cells,

wherein, A: laser confocal fluorescence microscope observation of autophagosome generation, B: electron microscopy to observe autophagosome generation and distribution, C: laser confocal fluorescence microscopy examined the entire course of autophagic flow.

FIG. 2 is a fusion protein preparation targeting CD47 promoting phagocytosis of non-small cell lung cancer cells by macrophages.

FIG. 3 shows the inhibition of the growth of nude mouse transplanted tumor by the fusion protein targeting CD47 and its preparation,

wherein, A: effect of fusion proteins targeting CD47 and their formulations on tumor volume growth, B: effect of fusion proteins targeting CD47 and their formulations on tumor weight.

FIG. 4 inhibition of growth of nude mouse transplantable tumors by fusion protein formulations targeting CD47 and by proteins targeting CD47 in general,

wherein, A: effect of fusion protein formulation targeting CD47 and protein targeting CD47 in general on tumor volume growth, B: effect of fusion protein formulations targeting CD47 and proteins targeting CD47 in general on tumor weight.

Detailed Description

Example 1

1) Preparation of autophagy inhibitor drug

(1) Preparing a chloroquine solution: dissolving appropriate amount of chloroquine in 0.01M PBS (pH 7.4) to prepare 10mmol/L stock solution, filtering and sterilizing with 0.1 μ M filter, storing at 4 deg.C, diluting 500 times and 1000 times in vitro laboratory for inhibiting autophagy;

(2) preparing an ammonium chloride solution: dissolving appropriate amount of ammonium chloride in 0.01M PBS (pH 7.4) to obtain 0.4mol/L stock solution, filtering with 0.1 μ M filter, sterilizing, storing at 4 deg.C, and diluting 50-80 times during in vitro experiment for inhibiting autophagy of cells;

(3) preparation of 3-MA solution: dissolving appropriate amount of 3-MA in 0.01M PBS (pH 7.4) to obtain 200mM stock solution, filtering with 0.1 μ M filter, sterilizing, storing at-20 deg.C, and diluting to 2mM for inhibiting autophagy; (4) preparation of LY294002 solution: dissolving an appropriate amount of LY294002 in 0.01M PBS (pH 7.4) to prepare 20mM stock solution, filtering and sterilizing the stock solution by using a 0.1 mu M filter, preserving the stock solution at-20 ℃, and diluting the stock solution to 20 mu M for inhibiting autophagy in an in vitro experiment;

2) preparation of targeting CD47 drug mother liquor

The CD47 targeting drug is obtained from R & D company, weighing 10mg targeting CD47 protein or fusion protein lyophilized powder, dissolving in 1mL PBS solution with 0.02M pH 7.4, stirring thoroughly, filtering with 0.1 μ M sterile filter, the concentration is 10mg/mL, storing at 4 deg.C, diluting to 10 μ g/mL during in vitro test;

3) preparation of novel CD 47-targeted immune checkpoint inhibitor pharmaceutical composition

The CD 47-targeted immune checkpoint inhibitor and the autophagy inhibitor were prepared as a novel pharmaceutical composition that would target CD 47: and mixing the drug mother liquor targeting CD47 with chloroquine solution according to a ratio of 1:1, so that the concentration of the fusion protein targeting CD47 is 1mg/mL, and the concentration of chloroquine is 1 mmol/L. Dilution 100-fold during in vitro experiments:

4) effect of fusion proteins targeting CD47 on levels of autophagy in non-Small cell Lung cancer cells

The fusion protein (MyD-1-Fc) targeting CD47 is added into a culture medium according to the volume ratio of 1:100, and the influence of the fusion protein on the autophagy level of the non-small cell lung cancer cell is respectively observed and detected by adopting a laser confocal fluorescence microscope, an electron microscope, protein imprinting and the like, and an experimental result shows that the fusion protein can obviously cause the lung cancer cell to generate autophagy (figures 1A and B) and autophagy flow (figure 1C).

5) Comparison of in vitro phagocytosis promoting effects of the fusion protein preparation targeting CD47 and the fusion protein targeting CD47 non-small cell lung cancer cells were labeled with CFSE, macrophages and non-small cell lung cancer cells were co-cultured, the above fusion protein preparation targeting CD47 (MyD-1-Fc + CQ) was added to the culture medium at a volume of 1:100, and the effect of the fusion protein preparation on phagocytosis of non-small cell lung cancer cells by macrophages was observed. The observation result of the laser confocal fluorescence microscope is shown in figure 2, and the experimental result shows that the fusion protein preparation has stronger phagocytosis promoting effect on macrophages, and the phagocytosis promoting effect is obviously improved compared with the single use of the fusion protein (MyD-1-Fc).

6) Comparison of in vivo therapeutic effects of the fusion protein preparation targeting CD47 and the fusion protein targeting CD47 the fusion protein preparation targeting CD47 (MyD-1-Fc + CQ) and the fusion protein targeting CD47 (MyD-1-Fc) were intraperitoneally injected into mice at a dose of 100 μ L per mouse, 2 times per week, and 4 weeks after continuous treatment, the broken line graph of tumor growth volume is shown in fig. 2, and the experimental results show that the fusion protein preparation has a strong anti-tumor effect (fig. 3A, B), and compared with the fusion protein alone, the anti-tumor effect is significantly improved (fig. 3A, B). The anti-tumor effect of the novel fusion protein targeting CD47 in vivo is proved, and the anti-tumor effect can be obviously improved by preparing the novel fusion protein and an autophagy inhibitor into a novel preparation.

7) Comparison of in vivo therapeutic effects of the fusion protein preparation targeting CD47 and the fusion protein preparation targeting CD47, the fusion protein preparation targeting CD47 (MyD-1-Fc + CQ) and the fusion protein preparation targeting CD47 (B6H12) were injected intraperitoneally into mice at a dose of 100 μ L per mouse, 2 times per week, and 4 weeks after continuous treatment, the tumor growth volume line graph is shown in fig. 2, and the experimental results show that the fusion protein preparation has a strong anti-tumor effect (fig. 4A), and the anti-tumor promotion effect is significantly improved compared with the fusion protein targeting CD47 (fig. 4A, B).

Claims (6)

1. A synergistic medicine composition of an immune checkpoint inhibitor targeting CD47 is characterized in that a synergistic combination medicine or a synergistic medicine compound preparation is composed of the immune checkpoint inhibitor targeting CD47 and an autophagy inhibitor;

the cell autophagy inhibitor is selected from chloroquine or ammonium chloride;

the immune checkpoint inhibitor drug targeting the CD47 is as follows: an anti-CD 47 monoclonal antibody, a SIRPa protein, or a SIRPa/MyD-1/CD 172-Fc fusion protein.

2. The synergistic pharmaceutical composition of CD 47-targeted immune checkpoint inhibitors according to claim 1, wherein the CD 47-targeted immune checkpoint inhibitor drug is a sirpa-Fc fusion protein.

3. The synergistic pharmaceutical composition of CD 47-targeted immune checkpoint inhibitors according to claim 2, wherein the sirpa-Fc fusion protein is a MyD-1-Fc fusion protein.

4. The synergistic pharmaceutical composition of CD 47-targeted immune checkpoint inhibitors according to claim 3, wherein the MyD-1-Fc fusion protein is a recombinant human MyD-1/CD172-Fc fusion protein.

5. Use of the synergistic pharmaceutical composition of CD 47-targeted immune checkpoint inhibitors of claim 1 for the preparation of a medicament for enhancing the targeting effect of tumor-treating drugs and the blocking effect on the CD47 site as well as the anti-tumor effect.

6. The use according to claim 5, wherein said neoplasm is non-small cell lung cancer, lymphoma, chronic myelogenous leukemia, acute lymphoblastic leukemia, breast cancer or melanoma.

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