WO2016115953A1 - Preparation and use of antibody fusion protein targeting vegfr2 - Google Patents

Abstract

The present invention belongs to the technical field of genetically engineered antibodies, and provides a preparation method for and a use of a fusion protein of tumor VEGFR2 (KDR3) antibody and MHC class I polypeptide-related A (MICA). In particular, the fusion protein is expressed and prepared by linking a full-length antibody of VEGFR2 with MICA through a flexible peptide. The protein can inhibit or destroy tumor angiogenesis, increase the content of MICA on the surface of the tumor cell, reestablish the immunological surveillance of a body activated by NKG2D pathway, and enhance ADCC effect mediated by the Fc region of the antibody.

Description

Preparation and application of an antibody fusion protein targeting VEGFR2 Technical field

The invention belongs to the field of bioengineering, and particularly relates to a high-affinity human-derived fusion protein capable of specifically binding to tumor vascular endothelial growth factor receptor (VEGFR/KDR) and NK cell activated receptor (NKG2D), which can inhibit Activation of vascular endothelial growth factor (KDR) receptor, which inhibits the growth of HUVECs that highly express vascular endothelial growth factor receptor human vascular endothelial cells, and enhances antibody-dependent cell-mediated cells of NK cells to human breast cancer cells Toxic effect (ADCC) is a highly specific genetically engineered fusion protein with anti-tumor and angiogenic activities targeting VEGFR and NKG2D.

Background technique

How to kill tumor cells without harming patients is a difficult problem we have been facing. At present, the key to the design of anti-tumor drugs is not only to kill tumor cells, but also to restore the body's immune surveillance, allowing the immune system to kill tumor cells. The interaction between tumor and immune system can be divided into three stages: early immune-mediated tumor "clearing", "equilibrium" and tumor "escape". An important mechanism of immune system immune monitoring is the expression of MHC class I-related antigen molecules A and B (MICA/B) on the surface of tumor cells. MICA/ B is expressed in natural killing. Ligand of the activated receptor NKG2D of cells (Natural killer cells, NK cells). NK cells are close to and bind to tumor cells by the interaction of MICA/B with NKG2D, thereby inducing the lysis of tumor cells by releasing cytokines and the like, thereby killing tumor cells. However, the study found that serum soluble MICA (sMIC) levels were significantly increased, leading to NK cell function defects, and further pointed out that soluble MICA is produced by the detachment of tumor cell surface MIC-A, and immune escape occurs when tumor cells escape immunity. Accordingly, some researchers have designed soluble peptides and anti-MICA antibodies to prevent MICA from falling off, which may develop into a new anti-tumor treatment.

A conventional drug that activates and rebuilds tumor immunity is antibodies, such as antibodies that have been approved for marketing by blocking CTLA4. Anti-tumor antibodies In addition to the well-known neutralization or inhibition of cell proliferation induced by certain factors to inhibit tumor growth, the main mechanism of its immune effector function is the passage of antibodies through its Fc segment to Fc receptors on the surface of NK or macrophages ( Fc gamma receptor, FcγR), FcγRIIIa binds, immune cells are close to antibody-bound tumor cells, and immune cells are activated by antibody-dependent cell-mediated cytotoxicity (ADCC).

Anti-vascular endothelial growth factor receptor antibody (VEGFR2 antibody)

Human VEGFR2 is called KDR (kinase inserted domain containing receptor), and vascular endothelial growth factor (VEGF) is a major receptor that stimulates proliferation and migration of endothelial cells and promotes neovascularization. The formation of tumor neovascularization is an essential condition for the tumor to transform into a malignant tumor. In addition, VEGFR-2 is also highly expressed in various tumor cells such as breast cancer, colon cancer, non-small cell lung cancer, and leukemia. Therefore, an inhibitor of VEGF or VEGFR2 can inhibit the neonatal angiogenesis, block the nutrient uptake of the tumor by "starvation therapy", and inhibit the proliferation of tumor cells or promote apoptosis.

The currently used VEGF antibody, bevacizumab, is a monoclonal antibody that inhibits the VEGF pathway. In April 2014, the FDA approved the Ramucirumab (IMC-1121B) antibody targeting VEGFR2 for the treatment of gastric cancer and gastroesophageal junction adenocarcinoma. Compared with bevacizumab, which is associated with clinical side effects such as hypertension and hemorrhage, anti-VEGFR2 antibody has better safety.

MHC-I related antigen molecule A (MICA) on the surface of tumor cells

The major histocompatibility complex (MHC) is involved in antigen recognition during the immune response and is closely related to the immune system. These include the MIC (MHC class cainrelated Gene) family, which plays an important role in immunity. The MIC family, also known as PerB11, is located in the MHC class and is highly polymorphic with multiple alleles. It includes 7 members of MICA, MICB, MICC, MICD, MICE, MICF and MICG. Among them, only MICA and MICB have the functions of encoding, expressing and transcribing proteins, and the rest are pseudogenes. Among them, MICA is located about 46 kb upstream of the HLA-B site, and the full length of l722 bp encodes a l382 bp transcript.

MICA is concentrated in the intestinal epithelium in human body, and is not expressed in other tissues such as brain, heart, lung, etc., but MICA can be expressed in most epithelial tumor cells (breast cancer, lung cancer, kidney cancer and ovarian cancer). It is considered to be a tumor-associated antigen. Therefore, tumor cells positively expressed by MIC-A are difficult to escape in the immune mechanism of the body, and the shedding of MICA makes the above-mentioned tumor cells positive for MICA, but can still escape immune surveillance. Moreover, in most of the above-mentioned MICA-positive epithelial tumor cells (breast cancer, lung cancer, colon cancer, etc.), vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed, so VEGFR-2 antibody It can be used as an effective carrier to link MIC-A protein and activate the NKG2D pathway to restore NK cell immune surveillance.

Summary of the invention

Purpose of the invention

The present invention provides an antibody fusion protein targeting VEGFR2 with potential medical and pharmaceutical value. The fusion protein of the present invention is characterized in that it specifically binds to the extracellular region 3 of human KDR and NKG2D, and can inhibit the growth of human breast cancer cell MDA-MB-231 and human vascular endothelial cell HUVEC in vitro, and can enhance NK cell to tumor cell. The ADCC effect, which induces an ADCC effect that is superior to that induced by the VEGFR2 antibody.

Technical solutions

An antibody fusion protein targeting VEGFR2, which is based on a full-length anti-VEGFR2 full-length antibody and a ligand MICA of NKG2D, and is constructed by fusion into a fusion protein, and the two peptides are linked by a flexible peptide. .

One of the strands consists of the VEGFR2 full-length antibody heavy chain and the MICA protein, and its amino acid sequence is SEQ NO. 1; the other strand is the VEGFR2 full-length antibody light chain, and its amino acid sequence is SEQ NO.

An expression purification method for isolating and purifying the above fusion protein.

An isolated nucleic acid, which encodes the VEGFR2-targeting antibody fusion protein of claim 1.

A set of expression vectors comprising the nucleic acid of claim 4.

A recombinant host cell comprising the expression vector of claim 5.

The use of a protein or protein fragment of any of the above, characterized in that binding to VEGFR2/NKG2D selectively or inhibiting the binding of VEGFR2/NKG2D to a VEGFR2 ligand/NKG2D ligand blocks the transduction of its signal.

A conjugate of a protein or protein fragment of any of the above.

The use of antibody fusion proteins targeting VEGFR2 in the treatment of tumors.

Further explanation of the invention:

The antibody fusion protein targeting VEGFR2 in the present invention consists of two strands, one of which is linked by a full length antibody and a MICA protein via a flexible peptide (GGGGS) and the other is a light chain of a full length antibody.

Expression vectors and host cell lines containing the VEGFR2 antibody/MICA fusion protein gene of the present invention are all within the scope of the present invention.

Another object of the present invention is to provide a method for expressing and purifying the above fusion protein.

The invention utilizes the PCR technology to clone and recombine the MVEGF protein obtained by screening and the full-length VEGFR2 antibody constructed by the patent single-chain antibody (patent number: ZL200910264180.3), and construct a recombinant vector of VEGFR2 full-length antibody/MICA fusion protein, and electricity. Transfer into CHO cells, integrate the fusion protein gene into the chromosome, and screen the stable cell line with high expression of the fusion antibody by using neomycin G418; expand the culture of the stable cell line, centrifuge the supernatant at low temperature, and clear the supernatant through Protein A. The column was isolated and purified; the antibody was isolated and purified by Western Blot; the affinity of the antibody to the antigen was analyzed by SPR assay; the growth inhibitory effect of the fusion protein on human vascular endothelial cell HUVEC and human breast cancer cell MDA-MB-231 was detected by MTT; ADCC experiments confirmed that the fusion protein can enhance the ADCC effect of NK cells on tumor cells, and the ADCC effect induced by it is better than that induced by VEGFR2 antibody.

DRAWINGS

Figure 1 is a recombination analysis of the fusion protein gene. The recombinant gene includes a heavy chain gene of about 2303 bp and a light chain gene of about 762 bp.

Figure 2 is a schematic diagram showing the structure of a fusion protein consisting of two strands, wherein the Fc region of the VEGFR2 antibody and the MICA protein are linked by a flexible peptide (GGGGS), while the heavy and light chains and heavy chains are self-passing in the expression cells. Disulfide bonds are assembled together.

Figure 3 is a diagram showing the SDS-PAGE protein electropherogram and Western Blot identification of the fusion protein. Figure 3A depicts the results of separation and purification of the fermented expressed fusion protein by a nickel column, and Figure 3BC depicts the results of the Western blot analysis of the isolated fusion protein. Figure 3B is an incubation of anti-H+L and Figure 3C is an incubation of anti-MICA. Lane 1 is the full-length antibody mAb04 of VEGFR2, lane 2 is the ligand MICA of NKG2D, and lane 3 is the isolated fusion protein mAb04-MICA.

Figure 4 is a graph showing the affinity of antigen-antibody affinity, showing the fusion protein mAb04-MICA and antigen VEGFR2 (Fig. 4A: ka(1/Ms): 6.18E+05, kd(1/s): 8.00E-04, KD ( M): 1.29E-09) or the binding test of ligand NKG2D (Fig. 4B: ka (1/Ms): 2.65E+08, kd (1/s): 188.2, KD (M): 7.10E-07) Experiment (Biacore).

Figure 5 is a flow-through assay showing the binding of the fusion protein mAb04-MICA to VEGFG2 or U937 cell surface NKG2D on the surface of HUVEC cells (HEK293 cells without normal expression of VEGFR2 and NKG2D as negative controls). Figure 5A, HUVEC cells; Figure 5B, U937 cells; Figure 5C, HEK293 cells.

Figure 6 is a graph depicting the growth inhibitory effect of the fusion protein mAb04-MICA on HUVEC cells (Fig. 6A, full-length antibody mAb04 with VEGFR2 as a positive control, ligand MICA with NKG2D as a negative control) or on human breast cancer cells Growth inhibition of MDA-MB-231 (Fig. 6B, the full-length antibody mAb04 of VEGFR2 was used as a positive control, and the ligand MICA of NKG2D was used as a negative control)

Figure 7 is a flow cytometric map depicting the apoptotic effect of the fusion protein mAb04-MICA on MDA-MB-231 cells.

Figure 8 is a bar graph of ADCC results, depicting the ADCC effect of the fusion protein mAb04-MICA on NK cell killing of human breast cancer cell line MDA-MB-231 (using the VEGFR2 full-length antibody mAb04 and NKG2D ligand MICA as a negative control) .

detailed description

Example 1 Construction of fusion protein mAb04-MICA

The mAb04 heavy light chain gene, MICA gene and pCA puro and pMH3 plasmids were used as templates to design and synthesize primers for PCR amplification. The construction of the fusion protein mAb04-MICA was based on the mAb04 heavy chain gene H', followed by overlapping PCR extension amplification to obtain complete H'-MICA gene. The PCR product was detected by 1.0% agarose gel electrophoresis, and the agarose gel recovery kit was used to recover the gene of interest. The final product of PCR amplification and the plasmids pCA puro and pMH3 were digested with restriction endonucleases, and the digested products were subjected to gelatinization recovery, and then ligated with T4 ligase at 16 ° C overnight. After ligation, E. coli HB2151 was competent, and the plate was coated. The next day, the monoclonal double digestion and sequencing were performed.

Example 2 Expression, purification and identification of the fusion protein mAb04-MICA

The recombinant plasmids H'-MICA-pCA puro, H'-MICA-pMH3, L-pCA puro, and L-pMH3 were electroporated into CHOs cell lines, and the high-yielding cell lines stably expressing the fusion protein were obtained by picking up the monoclonal cells in three rounds. The high-yielding cell line was expanded, and the supernatant was taken, centrifuged at 8000 rpm for 15 min, and filtered through a 0.22 μm filter. The sample was purified by a Protein A column and eluted with an eluate to obtain a purified protein. A.8% (12%) SDS-PAGE analysis collected samples and selected high purity proteins for identification. B. High purity protein samples were identified by Western Blot. Transfer the protein to PVDF membrane (purchased from Millipore) at 4 ° C, 250 mA constant current transfer for 1.5 h; at the end of transfer, the membrane was blocked in 5% skim milk at room temperature for 2 h; PBS was washed 3 times at a ratio of 1:2000. Anti-H+L, anti-Fc, anti-MICA antibody was added, incubated at 37 °C for 1 h, washed with PBS (TPBS) containing 0.05% Tween for 3 times, and then added with HRP-conjugated goat anti-mouse at a ratio of 1:5000. The IgG polyclonal antibody was incubated at 37 ° C for 1 h, TBS was washed 3 times, ECL luminescence solution was added dropwise, and the gel imager was exposed for photographing. The final purified and identified samples were dialyzed or ultrafiltered into PBS, snap frozen in liquid nitrogen, and stored at -70 °C.

Example 3 SPR experiment of fusion protein mAb04-MICA

In this experiment, the fusion protein mAb04-MICA was combined with the respective antigens to detect the interaction between the fusion protein and VEGFR2 or NKG2D using Biacore X100 as an SPR-dependent biosensor: A. Using a CM5 chip to ligate a recombinant fusion with an Fc fragment Protein molecule. VEGFR2 antigens with concentrations of 0.84375, 1.6875, 3.375, 6.25, 12.5, 25, 50, 100, 200 nmol/L were detected, and binding and dissociation curves were obtained. Ka(1/Ms) was analyzed by BIA evaluation software to be 6.18E+05. , kd (1/s) is 8.00E-04 calculated to obtain an equilibrium dissociation constant KD (M) value of 1.29E-09. B. Using a CM5 chip, a recombinant fusion protein molecule bearing an Fc fragment was ligated. NKG2D was detected at 3.906, 7.8125, 15.625, 31.25, 62.5, 125, 250 nmol/L, respectively, and the binding and dissociation curves were obtained. The BIA evaluation software was used to analyze ka(1/Ms) as 2.65E+08, kd(1/s). The equilibrium dissociation constant KD(M) value obtained for 188.2 is 7.1E-07.

Example 4 Flow Binding Experiment of Fusion Protein mAb04-MICA

In this experiment, the fusion protein mAb04-MICA was detected by flow cytometry with HUVEC cells expressing high VEGFR2 or U937 cells highly expressing NKG2D, and was used to detect the binding of the fusion protein to the antigen VEGFR2 and the ligand NKG2D to analyze the fusion protein. Binding ability. A.HUVCE cells were trypsinized and suspended in PBS-0.5% BSA, and the cells were incubated with the antibody for 30 min. After the antibody binds to the natural VEGFR2 on the cell surface, the anti-IgG antibody was added to the anti-IgG antibody for 60 min, and after PBS washing for 3 times, the antigen-antibody binding was analyzed by FACs Calibur flow cytometry to analyze the binding ability of the fusion protein to the antigen. B. U937 cells were suspended in PBS-0.5% BSA after centrifugation, and the cells were incubated with the antibody for 30 min. After the antibody binds to the natural VEGFR2 on the cell surface, the anti-IgG antibody was added to the anti-IgG antibody for 60 min, and after PBS washing for 3 times, the antigen-antibody binding was analyzed by FACs Calibur flow cytometry to analyze the binding ability of the fusion protein to the antigen.

Example 5 Growth inhibitory effect of fusion protein mAb04-MICA on human breast cancer cell line MDA-MB-231/vascular endothelial cell HUVEC

In this experiment, the fusion protein mAb04-MICA was treated with HUVEC cells highly expressing VEGFR2 or MDA-MB-231 cells highly expressing VEGFR2, and the experimental data were analyzed by SPSS software to evaluate the anti-angiogenesis and anti-tumor of the protein. active. A. 3×10 ³ to 5×10 ³ HUVEC cells were seeded into a 96-well cell culture plate, 100 μl/well, and cultured in a 37° C. 5% CO ₂ incubator. The sample group, the positive control mAb04, and the negative control MICA were each diluted to 10 different concentration gradients (0, 2.5, 5, 10, 20, 40, 80, 160, 200 nmol/) using 1% ECM medium containing 20 ng/ml VEGF. L), after 24 h, add 100 μl of the gradient-diluted antibody, set three parallel wells for each concentration, and continue to culture for 72 h. B. 1 × 10 ⁴ to 2 × 10 ⁴ A431 cells were seeded into a 96-well cell culture plate, 100 μl/well, and cultured in a 37 ° C 5% CO ₂ incubator. Sample group Db, positive control E10, and negative control AK404 were diluted to 10 different concentration gradients (0, 2.5, 5, 10, 20, 40, 80, 160, 200 nmol/L) with 1% fetal bovine serum medium. After 24 h, 100 μl of the gradient-diluted antibody was added, three parallel wells were set for each concentration, and incubation was continued for 72 h.

After observing the cell growth condition, 11 μL of MTT was added to each well, and incubation was continued for 4 hours at 37 ° C. The supernatant was carefully decanted, 150 μL of DMSO was added to each well, and the light absorption value (OD value) was measured at a wavelength of 570 nm/630 nm on the microplate reader to calculate cell proliferation. . The biological activity of the fusion protein was evaluated by the inhibition rate of cell growth, and the inhibition rate = (1 - experimental group OD value / control group OD value) × 100%.

Example 6 Apoptosis of Human Breast Cancer Cell Line MDA-MB-231 by Fusion Protein mAb04-MICA

Apoptosis experiments of the fusion protein mAb04-MICA on MDA-MB-231 demonstrated that the inhibition of cell proliferation by cells is through programmed cell death rather than cytotoxic death. 4×10 ⁵ cells of logarithmic growth phase MDA-MB-231 cells were seeded in 6-well cell culture plates, 1 ml/well, and supplemented with 1 ml/well complete medium, and cultured in a 37 ° C 5% CO ₂ incubator. The sample group fusion protein, the positive control mAb04, and the negative control MICA were diluted to 4 different concentration gradients (0, 10, 50, 250 nmol/L), and after 1.5 hours, 1.5 ml of the antibody was diluted and the culture was continued for 36 hours. .

After observing the cell growth condition, the medium was aspirated, washed twice with PBS, 0.25% trypsin-digested the plate cells, and the cells were gently washed twice with cold PBS; the cells were resuspended in 195 μL of 1×Binding Buffer to make the cell density 2~. 5×10 ⁵ /ml; add 5 μL of Annexin V-FITC to 195 μL of cell suspension, mix well, protect from light, incubate for 10 minutes at room temperature; wash cells once with 200 μL of 1× Binding Buffer, then resuspend the cells at 190 μL 1×Binding Buffer; 10 μL Propidium Iodie was added and analyzed by flow cytometry. The results showed that the apoptotic rate was positively correlated with the antibody concentration, demonstrating that the antibody can induce apoptosis of vascular endothelial cells and epidermal cancer tumor cells.

Example 7 ADCC experiment of fusion protein mAb04-MICA on human breast cancer cell line MDA-MB-231

In this experiment, the fusion protein mAb04-MICA was mixed with MDA-MB-231 cells and NK92 cells highly expressing VEGFR2 for 4 hours, and then detected by a cytotoxicity test kit. The experimental data were analyzed using SPSS software to evaluate the ADCC effect produced by the stimulation of the protein. MDA-MB-231 cells and NK92 cells were added to 96-well plates in proportion, and the fusion protein or positive control mAb04 or negative control MICA was added to the well plate for 4 hours at 100 nM, and the plate was centrifuged at 250 x g for 4 minutes, 50 μl. Transfer the supernatant to the enzyme assay plate, add 50 μl/well of the prepared substrate to the enzyme assay plate, cover the plate, incubate for 30 minutes at room temperature, protect from light, add 50 μl of stop solution to each well, and record absorbance at 490 nm. value. press

The formula calculates cytotoxicity and analyzes the results using software.

Claims (9)

An antibody fusion protein targeting VEGFR2, characterized in that the antibody is based on a full-length anti-VEGFR2 full-length antibody and a ligand MICA of NKG2D, and is constructed into a fusion protein by genetic recombination, and the two segments are made by using a flexible peptide. The proteins are linked together. The antibody fusion protein targeting VEGFR2 according to claim 1, wherein one strand consists of a VEGFR2 full-length antibody heavy chain and a MICA protein, and the amino acid sequence thereof is SEQ NO. 1; the other chain is The VEGFR2 full-length antibody light chain has an amino acid sequence of SEQ NO. An expression purification method for isolating and purifying the above fusion protein. An isolated nucleic acid, which encodes the VEGFR2-targeting antibody fusion protein of claim 1. A set of expression vectors comprising the nucleic acid of claim 4. A recombinant host cell comprising the expression vector of claim 5. Use of a protein or protein fragment according to any one of claims 1 or 2, characterized in that the selective binding to VEGFR2/NKG2D or inhibition of VEGFR2/NKG2D binding to a VEGFR2 ligand/NKG2D ligand blocks the transduction of its signal. A conjugate of a protein or protein fragment of any of claims 1 or 2. Use of the VEGFR2-targeting antibody fusion protein of claim 1 for the treatment of a tumor.

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