CN110960543A

CN110960543A - anti-VEGF-anti-PD 1 combined bispecific antibody for preparing anti-cancer drug

Info

Publication number: CN110960543A
Application number: CN201911261207.3A
Authority: CN
Inventors: 汪国兴; 袁红; 武婷; 樊丽
Original assignee: Anhui Rubiox Vision Biotechnology Co ltd
Current assignee: Anhui Rubiox Vision Biotechnology Co ltd
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2020-04-07

Abstract

The invention relates to the technical field of anti-cancer drugs, and particularly discloses an anti-cancer drug of ouabain combined with an anti-VEGF-anti-PD 1 bispecific antibody, wherein the anti-cancer drug comprises ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, the content of the anti-VEGF-anti-PD 1 bispecific antibody in 0.1ml of the anti-cancer drug is 0.01mg, and the content of ouabain in 0.1ml of the anti-cancer drug is 0.02 mg-0.06 mg; the CDR-H1 of the heavy chain variable region of the anti-VEGF-anti-PD 1 bispecific antibody is an amino acid sequence shown in SEQ ID NO. 1, the CDR-H2 is an amino acid sequence shown in SEQ ID NO. 2, and the CDR-H3 is an amino acid sequence shown in SEQ ID NO. 3; and the CDR-L of the light chain variable region of the anti-VEGF-anti-PD 1 bispecific antibody is an amino acid sequence shown in SEQ ID NO. 4. The invention overcomes the defects of the prior art, and the anticancer drug utilizes the combined action of ouabain and bispecific antibody to improve the anticancer performance of the drug.

Description

anti-VEGF-anti-PD 1 combined bispecific antibody for preparing anti-cancer drug

Technical Field

The invention relates to the technical field of anti-cancer drugs, and particularly belongs to an anti-cancer drug of ouabain combined with an anti-VEGF-anti-PD 1 bispecific antibody.

Background

Vascular Endothelial Growth Factor (VEGF), also known as Vascular Permeability Factor (VPF), is a highly specific vascular endothelial cell growth factor, and has the effects of promoting vascular permeability increase, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and angiogenesis. Vascular endothelial growth factor is a family, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and Placental Growth Factor (PGF). VEGF is commonly known as VEGF-A. VEGF-A promotes neovascularization and increases vascular permeability, VEGF-B plays a role in non-neovascularization tumors, VEGF-C and VEGF-D play a role in the formation of neovascularization and neolymphangiogenesis in cancer tissues, VEGF-E is also a potential neovascularization factor, PGF promotes neovascularization, increases vascular permeability, and PGF expression in experimental choroidal neovascularization is significantly increased. The high affinity receptor that specifically binds to vascular endothelial growth factor is called Vascular Endothelial Growth Factor Receptor (VEGFR), and is mainly classified into 3 types VEGFR-1, VEGFR-2, and VEGFR-3. VEGFR-1 and VEGFR-2 are mainly distributed on the surface of tumor vascular endothelium and regulate the generation of tumor blood vessels; VEGFR-3 is distributed mainly on the surface of lymphatic endothelium and regulates the generation of tumor lymphatic vessels. VEGF is a highly conserved homodimeric glycoprotein. Two single chains each having a molecular weight of 24kDa form a dimer with disulfide bonds. VEGF-decomposing monomers are inactive and removal of the N2 glycosyl has no effect on biological effects, but may play a role in cellular secretion. At least 5 protein forms of VEGF121, VEGF145, VEGF165, VEGF185, VEGF206 and the like are produced due to different shearing modes of mRNA, wherein VEGF121, VEGF145 and VEGF165 are secreted soluble proteins and can directly act on vascular endothelial cells to promote the proliferation of the vascular endothelial cells and increase the vascular permeability. In 1990, Philippine Folkman, university of Harvard, USA, proposed a well-known Folkman theory, that is, tumor tissue growth, which must rely on neovascularization to provide sufficient oxygen and nutrients to sustain. Is considered to be the basis for the clinical application of VEGF. The monoclonal antibody of anti-VEGF and VEGFR can inhibit vascular endothelial growth factor, and can be used for treating various metastatic cancers.

Programmed death receptor 1 (PD-1), an important immunosuppressive molecule, is an immunoglobulin superfamily, is a membrane protein of 268 amino acid residues. It was originally cloned from the apoptotic mouse T cell hybridoma 2B 4.11. The immunoregulation taking PD-1 as a target point has important significance for resisting tumor, infection, autoimmune disease, organ transplantation survival and the like. The ligand PD-L1 can also be used as a target, and the corresponding antibody can also play the same role. PD-1 and PD-L1 bind to initiate programmed death of T cells, allowing tumor cells to gain immune escape. PD-1 has at least two ligands, one is PD-L1 and one is PD-L2; PD-L1 has at least two ligands, one is PD-1 and one is CD 80; PD-L2 has at least two ligands, one PD-1 and one RGMB. PD-L1/L2 is expressed in antigen presenting cells, and PD-L1 is also expressed in various tissues. Binding of PD-1 to PD-L1 mediates a co-inhibitory signal of T cell activation, regulating T cell activation and proliferation, and acting like a negative regulator of CTLA-4. The chinese scientist shoji laboratory first found that PD-L1 is highly expressed in tumor tissue and modulates the function of tumor-infiltrating CD8T cells. Therefore, the immunoregulation taking PD-1/PD-L1 as a target has important significance for resisting tumors. In recent years, a plurality of anti-PD-1/PD-L1 antibodies have been rapidly developed in clinical research of tumor immunotherapy. Pembrolizumab and Nivolumab are currently FDA approved for advanced melanoma, and Nivolumab has recently also been FDA approved in the United states for treatment of advanced squamous non-small cell lung cancer. In addition, MPDL3280A (anti-PD-L1 mab), Avelumab (anti-PD-L1 mab), and the like have also entered several advanced clinical studies covering multiple tumor species such as non-small cell cancer, melanoma, bladder cancer, and the like. Due to the broad anti-tumor prospects and surprising potency of PD-1 antibodies, it is widely accepted in the industry that antibodies directed against the PD-1 pathway will bring about breakthrough advances in the treatment of a variety of tumors: can be used for treating non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma, leukemia, anemia, etc. After the clinical efficacy data on PD-1 antibody drugs revealed in the american cancer association (AACR) annual meeting in 2012 and 2013 and in the american clinical oncology Association (ASCO) annual meeting, PD-1 antibody became the most popular in-research antibody drug in the pharmaceutical industry worldwide.

A diabody is a bispecific antibody, which is a non-natural antibody whose two arms that bind to an antigen have different specificities. The construction of bifunctional antibodies usually employs biological methods and chemical cross-linking methods, and with the development of antibody engineering and molecular biology techniques, a new method for constructing bifunctional antibodies, i.e., genetic engineering method, has been developed in recent years. The genetic engineering method can construct multifunctional and multipurpose bifunctional antibody and humanized bifunctional antibody. The bifunctional antibody has potential application value in clinical treatment as a novel secondary targeting system. The bispecific antibody Blincyto (Blinatumomab) developed by FDA approval Anojin in U.S. at 03 th 12 th 2014 is marketed and used for treating acute lymphocytic leukemia. Blinatumomab is a CD19, CD3 bispecific antibody, and Blinatcyto (Blinatumomab) is the first bispecific antibody approved by the FDA in the United states. At present, more than 40 bifunctional antibody formats have been demonstrated to exist, but the development of bispecific antibodies has been difficult due to problems such as low production efficiency and poor pharmacokinetic properties.

The Chinese patent application No. CN201910734971.1 and the patent name 'anti-VEGF-anti-PD 1 bispecific antibody with a novel structure' provide a bifunctional antibody for resisting VEGF-anti-PD-1, which is formed by combining a PD1 antibody as a framework and a VEGF antibody as a single chain. On the basis of the bifunctional antibody, the invention actually explores how to prepare the medicine with good anti-cancer effect by using the bifunctional antibody.

Disclosure of Invention

The invention aims to provide an anticancer drug combining ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, overcomes the defects of the prior art, and improves the anticancer performance of the drug by combining the ouabain and the bispecific antibody.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

an anti-cancer drug of ouabain combined with an anti-VEGF-anti-PD 1 bispecific antibody, wherein the anti-cancer drug comprises ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, the content of the anti-VEGF-anti-PD 1 bispecific antibody in 0.1ml of the anti-cancer drug is 0.01mg, and the content of ouabain in 0.1ml of the anti-cancer drug is 0.02 mg-0.06 mg; the CDR-H1 of the heavy chain variable region of the anti-VEGF-anti-PD 1 bispecific antibody is an amino acid sequence shown in SEQ ID NO. 1, the CDR-H2 is an amino acid sequence shown in SEQ ID NO. 2, and the CDR-H3 is an amino acid sequence shown in SEQ ID NO. 3; and the CDR-L of the light chain variable region of the anti-VEGF-anti-PD 1 bispecific antibody is an amino acid sequence shown in SEQ ID NO. 4.

Further, the anti-cancer medicine comprises ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, the content of the anti-VEGF-anti-PD 1 bispecific antibody in 0.1ml of the anti-cancer medicine is 0.01mg, and the content of the ouabain in 0.1ml of the anti-cancer medicine is 0.03 mg-0.05 mg.

Further, the anti-cancer medicine comprises ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, the content of the anti-VEGF-anti-PD 1 bispecific antibody in 0.1ml of the anti-cancer medicine is 0.01mg, and the content of the ouabain in 0.1ml of the anti-cancer medicine is 0.04 mg.

Further, the heavy chain amino acid sequence of the anti-VEGF-anti-PD 1 bispecific antibody is shown as SEQ ID NO. 5, and the light chain amino acid sequence of the anti-VEGF-anti-PD 1 bispecific antibody is shown as SEQ ID NO. 6.

Further, the preparation method of the anti-VEGF-anti-PD 1 bispecific antibody comprises the following steps: constructing an expression vector containing the encoding genes of the single-chain antibody and the monoclonal antibody; introducing the expression vector into a host cell to obtain a host cell for stably expressing the bispecific antibody; culturing the host cell, and separating and purifying to obtain the bispecific antibody.

Furthermore, the anti-cancer drug is applied to drugs for treating pancreatic cancer, lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, nephroma, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal cancer, brain tumor, cervical cancer, leukemia, bone cancer, lymph cancer or other cancers.

Compared with the prior art, the invention has the following implementation effects:

according to the anti-cancer medicine of ouabain combined with the anti-VEGF-anti-PD 1 bispecific antibody, ouabain can inhibit the growth of various tumor cells, and the effect of the medicine on inhibiting the tumor cells and the coverage are improved by the combined action of the ouabain and the anti-VEGF-anti-PD 1 bispecific antibody.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples, and any modification is within the scope of the present invention without departing from the spirit of the present invention.

Example 1

The anti-cancer medicine comprises ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, the content of the anti-VEGF-anti-PD 1 bispecific antibody in 0.1ml of the anti-cancer medicine is 0.01mg, and the content of the ouabain in 0.1ml of the anti-cancer medicine is 0.02 mg; the CDR-H1 of the heavy chain variable region of the anti-VEGF-anti-PD 1 bispecific antibody is an amino acid sequence shown in SEQ ID NO. 1, the CDR-H2 is an amino acid sequence shown in SEQ ID NO. 2, and the CDR-H3 is an amino acid sequence shown in SEQ ID NO. 3; and the CDR-L of the light chain variable region of the anti-VEGF-anti-PD 1 bispecific antibody is an amino acid sequence shown in SEQ ID NO. 4.

The heavy chain amino acid sequence of the anti-VEGF-anti-PD 1 bispecific antibody is shown as SEQ ID NO. 5, and the light chain amino acid sequence of the anti-VEGF-anti-PD 1 bispecific antibody is shown as SEQ ID NO. 6.

The preparation method of the anti-VEGF-anti-PD 1 bispecific antibody comprises the following steps: constructing an expression vector containing the encoding genes of the single-chain antibody and the monoclonal antibody; introducing the expression vector into a host cell to obtain a host cell for stably expressing the bispecific antibody; culturing the host cell, and separating and purifying to obtain the bispecific antibody.

The anticancer drug is applied to the drugs for treating pancreatic cancer, lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, nephroma, ovarian cancer, prostatic cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymph cancer or other cancers.

Example 2

Based on example 1, 0.04mg of ouabain in 0.1ml of the anticancer drug, and the other components and the proportion are the same as example 1.

Example 3

Based on example 1, 0.06mg of ouabain in 0.1ml of the anticancer agent, and the other components and the proportion are the same as example 1.

Experimental example 1:

materials used for the experiments: lung cancer cell line 95-D; dissolving ouabain in 10% FBS + 90% DMEM medium, packaging, and storing at 4 deg.C; dissolving the anti-VEGF-anti-PD 1 bispecific antibody in 10% FBS + 90% DMEM medium, subpackaging, and storing at 4 ℃; detecting the proliferation and activity of CCK8 cells; 10% FBS + 90% DMEM medium.

Cell experiment process:

(1) taking the well conditioned 95-D cells, adjusting the density to 2.0X 10⁴mL, inoculate cell suspension in 96-well plates (0.1 mL/well) (set up blank experiment, add medium only);

(2) adherent growth was carried out for 48 hours, and the original medium was discarded. Adding culture medium (0.1mL) into control group, adding anti-VEGF-anti-PD 1 bispecific antibody (0.1mL anti-VEGF-anti-PD 1 bispecific antibody content 0.01mg) into experimental group, adding ouabain and anti-VEGF-anti-PD 1 bispecific antibody (0.1mL ouabain content 0.02mg, 0.04mg, 0.06mg and anti-VEGF-anti-PD 1 bispecific antibody content 0.01mg) into experimental group, and replacing culture medium from blank experimental group, and culturing for 48 hr;

(3) all wells of the medium were discarded, and CCK8 solution (10uL) was added to the wells and the culture was continued for 2 hours;

(4) measuring the absorbance of the sample at the wavelength of 450nm by using a microplate reader;

(5) calculating the cell viability:

cell viability ═ (a experiment-a blank)/(a control-a blank)

The results are shown in Table 1:

TABLE 195-D Lung cancer cell line survival rate statistical table

And (4) analyzing results:

the survival rate of 95-D lung cancer cell strains after being processed by a culture medium is 100 percent, the survival rate after being processed by the anti-VEGF-anti-PD 1 bispecific antibody is 56 percent, and the survival rate after being combined by ouabain (0.02mg, 0.04mg and 0.06mg) and the anti-VEGF-anti-PD 1 bispecific antibody is 50 percent, 42 percent and 46 percent. Compared with the survival rate of 59% of the single anti-VEGF-anti-PD 1 bispecific antibody, the combined use of ouabain (0.02mg, 0.04mg and 0.06mg) and the anti-VEGF-anti-PD 1 bispecific antibody has no statistical significance (P is more than 0.05), and the combined use of ouabain (0.04mg) and the anti-VEGF-anti-PD 1 bispecific antibody has statistical significance (P is less than 0.05); according to the experimental results of the separate treatment of ouabain with different dosages and the combined use of the anti-VEGF-anti-PD 1 bispecific antibody, the mass ratio of 4:1 can be selected as the ratio used in the subsequent experiments.

Experimental example 2:

materials used for the experiments: lung cancer cell strains 95-D, H596 and A549; dissolving ouabain in 10% FBS + 90% DMEM medium, packaging, and storing at 4 deg.C; dissolving the anti-VEGF-anti-PD 1 bispecific antibody in 10% FBS + 90% DMEM medium, subpackaging, and storing at 4 ℃; detecting the proliferation and activity of CCK8 cells; 10% FBS + 90% DMEM medium. The reagents used in the experiment are all common reagents in the market.

Cell experiment process:

(1) taking out the cells in good state, adjusting the density to 2.0 × 10⁴mL, inoculate cell suspension in 96-well plates (0.1 mL/well) (set up blank experiment, add medium only);

(2) adherent growth was carried out for 48 hours, and the original medium was discarded. Grouping and then adding: culture medium (0.1 mL/well) only in control group, anti-VEGF-anti-PD 1 bispecific antibody (0.1mL PD-1 content 0.01mg) in experimental group, ouabain and anti-VEGF-anti-PD 1 bispecific antibody combined use (0.1mL ouabain content 0.04mg and PD-1 content 0.01mg) in experimental group, and culture medium (0.1 mL/well) in blank experimental group for 48 hr;

(3) all wells were discarded and CCK8 solution (10ul) was added to the wells and incubation continued for 2 hours;

(5) calculating the cell viability;

cell viability ═ (a experiment-a blank)/(a control-a blank)

The results are shown in Table 2:

TABLE 2 statistics table for survival rate of different lung cancer cell strains

And (4) analyzing results:

the results of the experiments on the different cell strains in the 3 groups show that compared with the 100% cell viability of the control group, the ouabain and the anti-VEGF-anti-PD 1 bispecific antibody combined group have significant statistical difference (P is less than 0.01), and the results of the experiments on the anti-VEGF-anti-PD 1 bispecific antibody alone and the control group have smaller difference than those of the combined group and have statistical difference (P is less than 0.05). The fact that the combined use of ouabain and the anti-VEGF-anti-PD 1 bispecific antibody has better cancer inhibition effect than the use of the anti-VEGF-anti-PD 1 bispecific antibody alone.

Experimental example 3:

mouse experimental procedures:

taking a plurality of normal nude mice of 6 weeks old, wherein each mouse is male and has the weight of about 20 g; a549 cell line; dissolving ouabain in sterile PBS solution, packaging, and storing at 4 deg.C; dissolving the anti-VEGF-anti-PD 1 bispecific antibody in sterile PBS solution, subpackaging, and storing at 4 ℃;

a549 cells with the cell density of 1.0 multiplied by 107/mL are taken to disinfect the skin of a part to be injected of a mouse, 0.1mL of cell suspension is injected into the tail vein of each mouse, the lung tumor mass of the mouse is observed after 5 weeks, and when the size is 2-3mm, the lung cancer model of the mouse is successfully prepared.

Mice successfully modeled were randomly divided into 4 groups, PBS control group, ouabain experimental group, PD1 antibody + CD40 antibody experimental group, ouabain and PD1 antibody + CD40 antibody combined use experimental group. Sterile PBS (intraperitoneal injection 0.1mL) is administered to a control group, ouabain (intraperitoneal injection 0.1mL10mg/kg/d), PD1 antibody + CD40 antibody (intraperitoneal injection 0.1mL 2mg/kg/d), ouabain is used in combination with PD1 antibody + CD40 antibody (intraperitoneal injection 0.1mL ouabain 10mg/kg/d and PD1 antibody + CD40 antibody 2mg/kg/d), and the control group and the experimental group are continuously administered for 3 weeks.

The mice were sacrificed after 24 hours of weighing, the tumor mass was stripped off, the transfer was observed, weighed and stored. Tumor inhibition was calculated as mean tumor weight. Tumor inhibition rate ═ [ (control tumor volume-experimental tumor volume)/control tumor volume ] × 100%.

The results of the mouse experiments are shown in table 3:

TABLE 3 statistical table of mouse tumor inhibition rates

The results show that the tumor inhibition rate of the experimental group combining the anti-VEGF-anti-PD 1 bispecific antibody and ouabain is obviously higher than that of the PBS control group, the difference is statistically significant (P is less than 0.05), and the tumor inhibition rate of the experimental group combining the anti-VEGF-anti-PD 1 bispecific antibody and ouabain is higher than that of the experimental group combining ouabain and the anti-VEGF-PD 1 bispecific antibody (P is less than 0.05).

The foregoing is merely exemplary and illustrative of the present inventive concept and various modifications, additions and substitutions of similar embodiments may be made to the specific embodiments described by those skilled in the art without departing from the inventive concept or exceeding the scope of the claims as defined in the accompanying claims.

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Claims

1. An anti-cancer drug of ouabain combined with an anti-VEGF-anti-PD 1 bispecific antibody, which is characterized in that: the anti-cancer medicine comprises ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, the content of the anti-VEGF-anti-PD 1 bispecific antibody in 0.1ml of the anti-cancer medicine is 0.01mg, and the content of the ouabain in 0.1ml of the anti-cancer medicine is 0.02 mg-0.06 mg; the CDR-H1 of the heavy chain variable region of the anti-VEGF-anti-PD 1 bispecific antibody is an amino acid sequence shown in SEQ ID NO. 1, the CDR-H2 is an amino acid sequence shown in SEQ ID NO. 2, and the CDR-H3 is an amino acid sequence shown in SEQ ID NO. 3; and the CDR-L of the light chain variable region of the anti-VEGF-anti-PD 1 bispecific antibody is an amino acid sequence shown in SEQ ID NO. 4.

2. The anti-cancer drug of oubain in combination with anti-VEGF-anti-PD 1 bispecific antibody according to claim 1, wherein: the anti-cancer medicine comprises ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, the content of the anti-VEGF-anti-PD 1 bispecific antibody in 0.1ml of the anti-cancer medicine is 0.01mg, and the content of the ouabain in 0.1ml of the anti-cancer medicine is 0.03 mg-0.05 mg.

3. The anti-cancer drug of oubain in combination with anti-VEGF-anti-PD 1 bispecific antibody according to claim 1, wherein: the anti-cancer medicine comprises ouabain and an anti-VEGF-anti-PD 1 bispecific antibody, the content of the anti-VEGF-anti-PD 1 bispecific antibody in 0.1ml of the anti-cancer medicine is 0.01mg, and the content of the ouabain in 0.1ml of the anti-cancer medicine is 0.04 mg.

4. The anti-cancer drug of oubain in combination with anti-VEGF-anti-PD 1 bispecific antibody according to claim 1, wherein: the heavy chain amino acid sequence of the anti-VEGF-anti-PD 1 bispecific antibody is shown as SEQ ID NO. 5, and the light chain amino acid sequence of the anti-VEGF-anti-PD 1 bispecific antibody is shown as SEQ ID NO. 6.

5. The anti-cancer drug of oubain in combination with anti-VEGF-anti-PD 1 bispecific antibody according to claim 1, wherein: the preparation method of the anti-VEGF-anti-PD 1 bispecific antibody comprises the following steps: constructing an expression vector containing the encoding genes of the single-chain antibody and the monoclonal antibody; introducing the expression vector into a host cell to obtain a host cell for stably expressing the bispecific antibody; culturing the host cell, and separating and purifying to obtain the bispecific antibody.

6. The anti-cancer drug of oubain in combination with anti-VEGF-anti-PD 1 bispecific antibody according to claim 1, wherein: the anticancer drug is applied to the drugs for treating pancreatic cancer, lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, nephroma, ovarian cancer, prostatic cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, lymph cancer or other cancers.