EA042220B1 - IMPROVED ANTI-VEGR-2 MONOCLONAL ANTIBODIES - Google Patents

Description

Technical field

The disclosed invention relates to the technical field of biopharmaceuticals and, in particular, to an improved monoclonal antibody against vascular endothelial growth factor receptor 2 (VEGFR-2).

Prior Art

The situation when tumor growth depends on the formation of new blood vessels is already being actively studied in tumor biology. Oxygen, nutrients, growth factors, hormones and proteolytic enzymes can be provided through angiogenesis, after which cells can be stimulated, diffuse and travel long distances, and tumor growth and progression can be accelerated. Angiogenesis is a very complex dynamic process and is regulated by many promoter/antiangiogenesis molecules. Turning on/off angiogenesis is considered as a malignant marker that promotes the prevalence of angiogenesis over antiangiogenesis. The vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/VEGFR) axis triggers several signaling networks and thus epithelial cell survival, mitosis, translocation and differentiation, vascular permeability, VEGF and their receptors play a central role in normal and pathological angiogenesis. In numerous human tumors, additional tumor angiogenesis and expression of a tumor-promoting angiogenesis factor has been shown to be associated with tumor classification and malignancy.

Vascular endothelial growth factor (VEGF), also called vascular permeability factor, is a specific cause of endothelial cell mitosis and is also an effective factor in inducing angiogenesis and permeability, the relevant receptors identified are VEGFR-1 (Flt-1, FMS-like tyrosine 1) , VEGFR-2 (also called KDR/Flk-1; kinase insert domain-containing receptor; fetal liver kinase-1), VEGFR-3(Flt4), neurofilament protein-1 (neuropilin-1), neurofilament protein-2. VEGFR-2 is the main VEGF receptor on vascular endothelial cells and is a glycoprotein, the extracellular region of this receptor has seven immunoglobulin-like regions (including ligand-binding domains and structural domains of receptor dimerization), the structural domain of tyrosine kinase catalysis is intracellular, and the receptor is mainly expressed on endothelial cells. cells and other cells such as megakaryocytes, retinal progenitor cells, mesenchymal stem cells and tumor cells such as melanoma cells, brain tumors and leukemia cells. As key molecules in the signal transduction pathway through specific vascular endothelial cell factor, VEGF and VEGFR-2 receptors are involved in the formation of new tumor vessels, the main biological functions of VEGF are carried out through VEGFR-2, VEGFR-2 and VEGF become dimers after binding, and intracellular tyrosine residues in VEGFR-2 undergo self-phosphorylation, so that cytomembrane/cytoplasmic kinase cascade signals are activated and transmitted to the cell nucleus, a number of endothelial cell changes can be obtained, including vascular endothelial cell proliferation, survival, cytoskeletal reorganization, cell migration, gene expression and so on. and ultimately cause hyperplasia of the blood vessels.

Due to the key function of the VEGF/VEGFR2 signaling pathway in the onset and development of tumors, there are a large number of drugs for the VEGF/VEGFR2 signaling pathway, such as the anti-VEGF antibody bevacumab and the anti-VEGFR-2 antibody ramusirumab.

Antibodies are high-tech medicines and the biggest challenges in biomedicine and since 2012, six of the top ten single-component medicines in global sales are antibody-based medicines, so antibody-based medicines have great potential to develop in the market . Antibody phage library technology is the most common technology in antibody-based medical screening, antibody phage library technology is a new technology developed on the basis of phage display technology in antibody genetic engineering, the gene pool with all genes of various kinds of antibody variable regions can be converted into a protein a library exposed on the surface of the phage, not only is it convenient, fast and efficient to obtain a monoclonal antibody in vitro, but also a new method for humanizing monoclonal antibodies has been developed, and the development of human monoclonal antibody production has been stimulated. In patent CN103333247 B of the Applicant's company, a series of VEGFR-2 antibodies were screened and obtained using computer-aided design and phage antibody technology, thus making the basis for obtaining drugs based on anti-VEGFR-2 antibodies, and further research by the Applicant's company showed, that affinity, biological activity, etc. antibodies presented in the patent can be further improved, so that on this basis, the antibodies are improved and optimized on the original basis.

Brief summary of the invention

The invention provides an improved monoclonal antibody against vasculoendothelial growth factor receptor 2 (VEGFR-2); According to the disclosure, two high-affinity antibodies in CN103333247B are used as templates for computer modeling, a new antibody phage library has been developed, and a new monoclonal anti-VEGFR-2 antibody has been obtained by multiple screening, with both affinity and bioactivity better than parent patented antibodies.

To achieve the above object, the invention provides an improved anti-VEGR-2 monoclonal antibody comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain variable region comprises any of SEQ NO: 1, SEQ NO: 2, or SEQ NO: 3; the amino acid sequence of the heavy chain variable region includes SEQ NO: 4.

The invention also provides an antibody, polypeptide or protein containing a light chain variable region or a heavy chain variable region.

The invention also provides a polynucleotide sequence or combination comprising the amino acid sequences of the light chain variable region or the heavy chain variable region.

The invention also provides an expression vector based on a recombinant deoxyribonucleic acid (DNA) containing the specified polynucleotide sequence or combination; The DNA sequence of the recombinant DNA expression vector includes nucleotide sequences for encoding the amino acid sequences of the heavy chain variable region, heavy chain constant region, light chain variable region, and light chain constant region of a monoclonal anti-VEGFR-2 antibody.

The invention also provides a host cell for transfection of a recombinant DNA expression vector, and the host cell includes a mammalian cell, an insect cell, an Escherichia coli cell, or a yeast cell, preferably a mammalian cell, and more preferably a HEK293E cell, a Chinese hamster ovary (CHO ) or cell NS0.

The heavy chain constant region of the invention is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4, or from the group consisting of mouse IgG1, IgG2a and IgG2b.

The invention also provides a complete antibody comprising a light chain variable region or a heavy chain variable region and a monoclonal anti-T1h antibody fragment, and the fragment includes, but is not limited to, Fab, Fab', F(ab') ₂ , Fv or ScFv.

The invention also provides a single chain antibody, a single domain antibody, a bispecific antibody, an antibody drug conjugate, and an immunotherapeutic agent using chimeric antigen receptor T cells comprising said light chain variable region or heavy chain variable region amino acid sequence.

The invention also provides a monoclonal antibody, artificial vector, drug or pharmaceutical composition containing said light chain variable region or heavy chain variable region.

The invention also provides a detection reagent or detection kit containing said light chain variable region or heavy chain variable region.

The antibody can be used to treat diseases caused by angiogenesis, and these diseases include, but are not limited to, tumors and macular degeneration.

When the ScFv is a single chain variable fragment; HEK293E cells are 293E human embryonic kidney cells; CHO cells are Chinese hamster ovary cells; NS0 cells are mouse NS0 thymoma cells.

Compared with the prior art, the present invention has the following advantages.

A monoclonal anti-VEGFR-2 antibody which has a higher affinity, is able to inhibit the binding of VEGFR-2 and vascular endothelial growth factor (VEGF) ligand in vitro well, has good in vitro biological activity, and has a broad development prospect.

The anti-VEGFR-2 monoclonal antibody provided herein is used to treat diseases caused by tumor angiogenesis, including, but not limited to, cancers such as non-small cell lung cancer, metastatic non-small cell lung cancer, gliomas, colorectal cancer, hepatocellular carcinoma , metastatic hepatocellular carcinoma, metastatic human epidermal growth factor receptor (HER2) negative breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma and metastatic renal cell carcinoma, and diseases such as macular degeneration.

Brief description of graphic materials

In FIG. 1 shows a graphical representation of the plasmid pScFvDisb-S;

in fig. 2 shows the relative affinity of a single chain anti-VEGFR-2 antibody when compared in a gradient ELISA (enzyme-linked immunosorbent assay);

in fig. 3 shows the protein expressed by the pTSE plasmid;

in fig. 4 shows a comparison of the binding abilities of monoclonal anti-VEGFR-2 antibodies with KDR;

- 2 042220 in FIG. 5 shows the binding of antibodies of the invention to VEGFR-2 on the cell surface;

in fig. 6 shows the effect of proliferation inhibition by the antibodies of the invention on human umbilical vein endothelial cells (HUVEC).

Detailed Description of Embodiments

Detailed methods for carrying out the present invention are shown in the embodiments, and all methods and reagents in the embodiments are conventional testing methods and conventional reagents unless otherwise indicated. The embodiments are used only to describe and interpret the disclosure, but are not inappropriate limitations of the invention.

The invention provides an improved monoclonal antibody against vascular endothelial growth factor receptor-2 (VEGFR-2) comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain variable region comprises any of SEQ NO: 1, SEQ NO: 2, or SEQ NO: 3; the amino acid sequence of the heavy chain variable region includes SEQ NO: 4.

SEQ NO.1 (VEGFR-2 light chain variable region sequence 1):

DIQMTQSPSSVSASIGDRVTITCRASQAIDNWLGWYQQKPGKAPKLLIYEGSNLNTGVPSRFSGSGSGTDFT LTISSLQAEDFAVYFCQQAKSFPPTFGGGTKVDIK;

SEQ NO.2 (VEGFR-2 light chain variable region sequence 2):

SEQ NO.3 (VEGFR-2 light chain variable region sequence 3):

SEQ NO.4 (VEGFR2 heavy chain variable region sequence 1):

Preferably, the invention also provides an antibody, polypeptide or protein comprising a light chain variable region or a heavy chain variable region.

More preferably, the invention also provides a polynucleotide sequence or combination comprising the amino acid sequences of the light chain variable region or the heavy chain variable region.

More preferably, the invention also provides a recombinant deoxyribonucleic acid (DNA) expression vector containing said polynucleotide sequence or combination; The DNA sequence of the recombinant DNA expression vector includes nucleotide sequences for encoding a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region of a monoclonal anti-VEGFR-2 antibody.

More preferably, the invention also provides a host cell for transfection of the recombinant expression DNA vector, and the host cell includes a mammalian cell, an insect cell, an Escherichia coli or yeast cell, preferably a mammalian cell, and even more preferably a HEK293E cell, a Chinese hamster ovary cell ( CHO) or cell NS0.

More preferably, the heavy chain constant region of the invention is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4, or from the group consisting of mouse IgG1, IgG2a and IgG2b.

More preferably, the invention also provides a complete antibody comprising a light chain variable region or a heavy chain variable region and a monoclonal anti-T1h antibody fragment, and this fragment includes, but is not limited to, Fab, Fab', F(ab') ₂ , Fv or ScFv .

More preferably, the invention also provides a single chain antibody, a single domain antibody, a bispecific antibody, an antibody drug conjugate, and a chimeric antigen receptor T cell immunotherapeutic agent comprising said light chain variable region or heavy chain variable region amino acid sequence.

More preferably, the invention also provides a monoclonal antibody, artificial vector, drug or pharmaceutical composition comprising said light chain variable region or heavy chain variable region.

More preferably, the monoclonal antibody is a fully human antibody.

More preferably, the invention also provides a detection reagent or detection kit comprising said light chain variable region or heavy chain variable region.

More preferably, the antibody can be used to treat diseases caused by angiogenesis, including, but not limited to, cancers such as non-small cell lung cancer, metastatic non-small cell lung cancer, gliomas, colorectal cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, metastatic breast cancer with negative human epidermal growth factor receptor (HER2), metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma and metastatic renal cell carcinoma, and diseases such as macular degeneration.

Detailed Description of Embodiments

The invention is described in detail with the help of drawings and embodiments.

Embodiment 1 Biopanning of single chain anti-VEGFR-2 antibodies.

The pCom3 vector (purchased from Biovector Science Lab, Inc.) has been modified with a number of gene cloning techniques and thus the vector can be used to generate and express a single chain antibody phage library. The modified vector is named pScFvDisb-S, a graphic representation of the vector plasmid is shown in FIG. 1, and based on this vector, a new fully synthetic antibody phage library was constructed based on the antibody sequences in patent CN10333247B.

The immunoassay tube is coated with the extracellular portion of VEGFR-2 as antigen, the amount of coating antigen is 2 μg/500 μl/tube, and the coating is performed overnight at 4°C. Accordingly, block the immunoassay tube and complete synthetic antibody phage library with 4% defatted milk powder/tween phosphate-buffered saline (PBST) for 1 hour at room temperature. The blocked antibody phage library is placed in an immunoassay tube for antigen-antibody binding, the amount of phage is about 109-10 ¹² , and the reaction is carried out for 1 hour at room temperature. Wash several times with PBST-phosphate buffer (PBST-PBS) to remove unbound phages, elute with 0.1 M glycine pH 2.2 and neutralize the eluted phage antibody solution with 1.5 M Tris-HCl pH 8.8 to pH about 7.0.

Infect neutralized phage with 10 ml of TG1 bacterial solution growing to logarithmic phase, leave for 30 min in an incubator at 37°C, remove part of the bacterial solution for gradient dilution, and cover the plate with 2YTAG to calculate the phage yield. Centrifuge the remaining bacterial solution to remove the supernatant, resuspend the bacteria in a small amount of medium, aspirate and cover a large 2YTAG plate for the next round of screening.

Scrap the bacteria that are infected and plate from the large plate, inoculate with 2YTAG liquid medium, shake to logarithmic phase, add M13 helper phage for superinfection, culture overnight at 28°C to amplify the phage, pellet and purify the phage with PEG6000- NaCl for the next round of screening, a total of phage library enriched and screened three times.

Embodiment 2 Identification of a positive cloning of an anti-VEGFR-2 phage single chain antibody.

After three rounds of screening, monocolonies with good separation are selected, inoculated into a deep 96-well plate with 2YTAG liquid medium, cultured at 37°C and 220 rpm to logarithmic phase, approximately 10 ¹⁰ M13K07 helper phage are added to each well, and left to stand and become infected within 30 minutes at 37°C. Centrifuge at 4000 rpm for 15 min at 4°C, remove the supernatant, resuspend and precipitate the bacteria with 2YTAK, and culture overnight at 28°C at 220 rpm. The phage supernatant is aspirated for identification by enzyme-linked immunosorbent assay (ELISA), the phage containing the biological antibody sequence (the heavy chain amino acid sequence is sequence No. 3 in CN10333247B and the light chain amino acid sequence is sequence No. 9 in CN10333247B) with the same vector, and the same construction method from CN10333247B is used as a positive control (named as 0-1), screened for high affinity monoclonal antibodies N-1, N-2 and N-3, and sequenced gene to confirm that the antibodies are different from the sequences specified in the patent CN10333247B.

Embodiment 3 Phage gradient ELISA to compare the affinity of single chain anti-VEGFR-2 antibodies.

The monocolonies obtained in embodiment 2 are isolated and purified and a phage gradient dilution ELISA is performed to test and identify affinity.

Coat the extracellular region of VEGFR-2 with carbonate buffer pH 9.6, coating overnight at 20 ng/well/100 μl at 4°C. Wash three times with PBST and block for 1 h with 4% milk-PBST at 37°C. Dilute the purified phage with a 5-fold gradient of 4% milk-PBST, place 100 µl of the diluted sample in each well and leave to stand for 1 hour at room temperature. Wash the ELISA plate with PBST buffer, place the HRP-anti-M13 monoclonal antibody diluted with 4% milk-PBST mixture in the ELISA plate and leave to stand for 1 hour at room temperature. Carry out staining with tetramethylbenzidine (TMB) staining kit for 10 min at room temperature. The reaction is stopped with 2M H ₂ SO ₄ and readings are taken at 450/630 nm. The results are shown in FIG. 2, and it is shown that all three selected different single chain antibodies can specifically bind to VEGFR-2, and all of them have a higher binding capacity than a conventional antibody.

0-1.

Embodiment 4 Preparation of complete anti-KDR antibodies.

Accordingly, the VH heavy chain and light chain genes of the three antibodies N-1, N-2 and N-3 are cloned into the pTSE vector (as shown in Fig. 3) containing the heavy and light chain constant region genes, and the pTSE vector for encoding the constant region human IgG1 (see SEQ: 5) and K chain constant region (see SEQ: 6) (the pTSE vector is shown in Fig. 3, and the production process is presented in paragraph [0019] on page 3 of the description CN103525868A).

SEQNO.5 (последовательность константной области человеческого IgGl): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. SEQ NO.6 (chain constant region sequence k):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Transient transfection was performed in HEK293E cells to express the full antibody, purification was performed using an AKTA protein A affinity column to obtain a complete antibody protein, and protein concentrations were determined using the Bicinchoninic Acid Disodium (BCA) kit (bicinchoninic acid disodium salt).

Embodiment 5 Full antibody and VEGFR2 extracellular region binding experiments.

Cover the extracellular region of the VEGFR with carbonate buffer solution pH 9.6, the coating is carried out in the amount of 20 ng/well/100 μl overnight at 4°C. Wash three times with 300 µl/well/PBST and then block for 1 h with 4% milk-PBST at 37°C and add biotin-labeled total antibodies at various dilutions. Human IgG (hIgG) is used as a homotypic control, the highest concentration of various total antibodies is 100 ng/ml, 8 gradients are made with 5-fold dilution and incubated for 2-3 hours at 37°C. Wash five times with 300 μl/well PBST and then incubate for 1 hour with streptavidin diluted 1:10,000 with 4% milk-PBST at 37°C. Wash eight times with 300 μl/well PBST, stain with TMB staining kit at 100 μl/well for 10 min at room temperature and stop the reaction with 2 M H ₂ SO ₄ . Read readings at 450 nm/630 nm. The results are shown in FIG. 4, all antibodies can bind well to KDR molecules on the cell surface, N3 has the highest affinity, and the affinity of N-1, N-2 and N-3 is higher than that of conventional 0-1 antibodies.

Embodiment 6: Cell surface binding specificity assay of total antibodies and VEGFR-2.

According to the invention, cell surface binding of different bispecific antibodies to EGFR is determined using CHO cells overexpressing VEGFR-2, and human IgG (hIgG) is used as a homotypic control. Digest with 0.25% pancreatin and centrifuge to collect cells. At the same time, various antibodies are diluted with the highest concentration of 100 nM in a 4-fold gradient. Wash the harvested cells with PBS + 1% BSA three times, then add PBS + 1% BSA to resuspend the cells, place the cells in a 96-well plate, 1 x 10 ⁵ cells per well, add 100 µl of diluted bispecific antibodies and incubate for 1 h at room temperature; centrifuge to remove supernatant, wash three times with PBS, then resuspend cells with diluted anti-human IgG FC antibody labeled with Alexa488, then incubate for 1 h without light, at room temperature, wash three times with PBS, resuspend in 100 μl PBS and determine fluorescence intensity by flow cytometry. The results are analyzed using Graphpad Prism. The results show that N3 is better able to bind to VEGFR-2 expressed by the cells and the binding abilities of all of N1, N2 and N3 are better than 0-1 (see Fig. 5).

Embodiment 7 Human umbilical vein endothelial cell (HUVEC) proliferation inhibition experiment with total antibodies.

Human umbilical vein endothelial cell (HUVEC) has already been widely used to investigate vascular endothelial cell proliferation, cell signaling pathways and multiple mechanisms of tumorigenesis, and the description investigated the inhibition of HUVEC proliferation by various antiVEGFR-2 antibodies. When HUVEC cells have grown to a density of 80%, the medium is replaced with fresh EGM medium with 5% FBS, digested with pancreatin after 6 hours, then the digested cells are washed 4-5 times with serum-free medium, if possible completely removing the medium after each centrifugation, resuspended cells are counted , inoculate 5000 cells/well into 96-well plates, avoiding the marginal wells of 96-well plates, place 100 μl of serum-free medium into each well, and starve the cells overnight. Aspirate the supernatant after 14-16 hours, add 50 µl of VEGF/ECM (200 ng/ml) to a concentration of 100 ng/ml, mix evenly with different concentrations of different antibodies and culture for 24 hours.

- 5 042220 determine cell proliferation. The results show that the IC ₅₀ (ng/l) for N1, N2 and N3 are 12.76; 17.32 and 10.53, respectively, and the IC ₅₀ (ng/l) for 0-1 is 18.59; the ability of all of N1, N2 and N3 to inhibit the proliferation of human umbilical vein endothelial cells is higher than that of 0-1, and N3 has a better ability to inhibit proliferation (see Fig. 6). Embodiments 5-7 show that both the molecular and cytological results for N1, N2 and N3 are better than for the 0-1 optimal antibody of the original patent CN103525868A, which means that the candidate molecules of this patent have good prospects for development and applications.

The antibody can be used to treat diseases caused by tumor angiogenesis, including, but not limited to, cancers such as non-small cell lung cancer, metastatic non-small cell lung cancer, gliomas, colorectal cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER2 negative metastatic breast cancer , metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma and metastatic renal epithelial cell carcinoma, and diseases such as macular degeneration.

For those skilled in the art, these embodiments only illustratively describe the invention, and it is obvious that specific implementations of the invention are not limited to the above options. Any minor improvements based on the ideas and technical diagrams of the invention or application of the ideas and technical diagrams of the invention without improvement in relation to other situations fall within the protection scope of this invention.

Claims (10)

CLAIM 1. Monoclonal anti-VEGFR-2 antibody (antibody to vascular endothelial growth factor receptor 2) comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region comprises any of SEQ ID NO: 1, SEQ ID NO : 2 or SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4. 2. The anti-VEGFR-2 monoclonal antibody of claim 1, which further comprises a heavy chain constant region, and the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 or from the group consisting of IgG1, IgG2a and mouse IgG2b. 3. A polynucleotide sequence encoding the amino acid sequence of the light chain variable region of the monoclonal anti-VEGFR-2 antibody of claim 1. 4. Expression vector based on recombinant deoxyribonucleic acid (DNA) containing the polynucleotide sequence according to claim 3. 5. A host cell for transfecting the recombinant DNA expression vector of claim 4, wherein the host cell includes a mammalian cell, an insect cell, an Escherichia coli cell, or a yeast cell. 6. The host cell according to claim 5, which is a mammalian cell. 7. A host cell according to claim 6, which is a HEK293E, a CHO (Chinese Hamster Ovary) cell, or an NS0 cell. 8. Pharmaceutical composition containing a monoclonal anti-VEGFR-2 antibody according to claim 1. 9. Reagent for detection of VEGFR-2 antigen, containing a monoclonal anti-VEGFR-2 antibody according to claim 1. 10. The use of the anti-VEGFR-2 monoclonal antibody according to claim 1 as a drug for the treatment of diseases caused by angiogenesis, and the diseases include tumors or macular degeneration.