WO2018161798A1 - 一种改进的抗vegfr-2单克隆抗体 - Google Patents

一种改进的抗vegfr-2单克隆抗体 Download PDF

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WO2018161798A1
WO2018161798A1 PCT/CN2018/076847 CN2018076847W WO2018161798A1 WO 2018161798 A1 WO2018161798 A1 WO 2018161798A1 CN 2018076847 W CN2018076847 W CN 2018076847W WO 2018161798 A1 WO2018161798 A1 WO 2018161798A1
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variable region
chain variable
antibody
heavy chain
light chain
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周海平
谷香果
白义
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北京东方百泰生物科技有限公司
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Priority to KR1020197025428A priority Critical patent/KR102276489B1/ko
Priority to JP2019546906A priority patent/JP6843262B2/ja
Priority to EP18764068.5A priority patent/EP3590964A4/en
Priority to CA3054615A priority patent/CA3054615C/en
Priority to AU2018229915A priority patent/AU2018229915B2/en
Priority to EA201991788A priority patent/EA201991788A1/ru
Priority to US16/487,833 priority patent/US11370834B2/en
Publication of WO2018161798A1 publication Critical patent/WO2018161798A1/zh

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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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Definitions

  • the present invention relates to the field of biopharmaceutical technology, and in particular to the present invention relates to an improved anti-VEGFR-2 monoclonal antibody.
  • Angiogenesis provides oxygen, nutrients, growth factors, hormones and proteolytic enzymes to promote the spread and metastasis of tumor cells to distant areas, accelerating tumor growth and deterioration.
  • Angiogenesis is a highly complex dynamic process regulated by many pro-/anti-angiogenic molecules.
  • the angiogenic switch is thought to be a malignant marker of pro-angiogenesis over anti-angiogenesis.
  • the VEGF/VEGFR axis triggers multiple signaling networks leading to epithelial cell survival, mitosis, metastasis and differentiation, and vascular permeability, and VEGF and its receptors play a central role in normal and pathological angiogenesis.
  • increased tumor vascularization and expression of tumor proangiogenic factors have been shown to be associated with tumor grade and malignancy.
  • VEGF Vascular endothelial growth factor
  • vascular permeability factor is a specific mitotic source of endothelial cells and an effective angiogenesis and permeability-inducing factor.
  • the corresponding receptor has been identified as VEGFR-1. (Flt-1, FMS-like tyrosine kinase 1), VEGFR-2 (also known as KDR/Flk-1, kinase insertion chimeric receptor, fetal liver kinase-1), VEGFR-3 (Flt4), nerve fibers Protein-1 (neuropilin-1), neurofibrin-2.
  • VEGFR-2 is a major VEGF receptor on vascular endothelial cells, a glycoprotein with seven immunoglobulin-like regions (including ligand binding domain and receptor dimerization domain) in the extracellular region.
  • the catalytic domain of tyrosine kinase is inserted, mainly expressed on endothelial cells, such as megakaryocytes, distant progenitor cells, mesenchymal stem cells, tumor cells such as melanoma cells, brain tumors and certain leukemia cells.
  • VEGF and VEGFR-2 receptors are involved in the angiogenesis of tumors as a key vascular endothelial cell-specific factor signaling pathway.
  • VEGFR-2 The main biological functions of VEGF are achieved by VEGFR-2, and VEGFR-2 and VEGF are combined.
  • Polymerization, and phosphorylation of tyrosine residues in the VEGFR-2 cell which activates and transmits a signal to the nucleus of the cell membrane/cytoplasmic kinase cascade, triggers a series of changes in endothelial cells, including vascular endothelial cell proliferation. Survival, cytoskeletal rearrangement, cell migration, and gene expression, and eventually cause vascular proliferation.
  • VEGF/VEGFR2 Because of the key role of the VEGF/VEGFR2 signaling pathway in the development of tumors, there are many drugs that target the signaling pathway of VEGF/VEGFR2, such as the anti-VEGF antibody bevacizumab and the anti-VEGFR-2 antibody. .
  • Antibodies are the most technically and most difficult class of drugs in biomedicine. Since 2012, six of the top ten single drugs in global sales have been antibody-based drugs. Therefore, the development potential of the antibody-based drug market is huge.
  • the most commonly used technique for antibody drug screening is the phage antibody library technology, a new technology for genetic antibody engineering developed by phage display technology, which can convert all antibody variable region genes containing different species.
  • the translation of the gene bank into a protein library displayed on the surface of the phage not only makes the production of monoclonal antibodies more convenient, rapid and efficient in vitro, but also opens up a new way of humanizing monoclonal antibodies and promotes the production of human monoclonal antibodies. development of. Our patent No.
  • CN103333247 B is a series of anti-VEGFR-2 antibodies screened by computer-aided design and phage antibody library technology, which lays a foundation for obtaining anti-VEGFR-2 antibody drugs.
  • Our company is further researching. It has been found that the antibodies involved in the above patents have further improvement in affinity and biological activity. Based on this, we have improved the above-mentioned antibodies based on the original research.
  • the invention provides an improved anti-VEGFR-2 monoclonal antibody; the invention adopts the highest affinity of the CN103333247B patent as a template, performs computer-aided simulation design, designs a new phage antibody library, and performs multiple rounds of screening. A new anti-VEGFR-2 monoclonal antibody with affinity and biological activity higher than the original patent antibody was obtained.
  • an improved anti-VEGFR-2 monoclonal antibody comprising:
  • a light chain and a heavy chain comprising any one of SEQ NO. 1, SEQ NO. 2 or SEQ NO. 3; the amino acid sequence of the heavy chain variable region comprising SEQ NO .4.
  • the present invention also provides an antibody, polypeptide or protein comprising the above-described light chain variable region or the above heavy chain variable region.
  • the present invention also provides a polynucleotide sequence or combination comprising the above-described light chain variable region or the above-described heavy chain variable region amino acid sequence.
  • the present invention also provides a recombinant DNA expression vector comprising the above polynucleotide sequence or combination; the DNA sequence of the recombinant DNA expression vector comprises the above heavy chain variable region, heavy chain encoding an anti-VEGFR-2 antibody Amino acid sequences of the constant region, the light chain variable region, and the light chain constant region.
  • the present invention also provides a host cell transfected with the above recombinant DNA expression vector, the host cell comprising mammalian cells, insect cells, Escherichia coli or yeast; preferably mammalian cells; further preferably HEK293E cells, CHO cells or NSO cells.
  • heavy chain constant region of the present invention is selected from human IgG1, IgG2, IgG3, IgG4 or murine IgG1, IgG2a, IgG2b.
  • the present invention also provides a fragment of the full length antibody and the anti-T1h monoclonal antibody comprising the above light chain variable region or the above heavy chain variable region, the fragment including but not limited to Fab, Fab', F ( Ab') 2 , Fv or ScFv.
  • the present invention also provides a single chain antibody, a single domain antibody, a bispecific antibody, an antibody drug conjugate and a chimeric antigen receptor comprising the amino acid sequence of the above light chain variable region or the above heavy chain variable region T cell immunotherapy.
  • the present invention provides a monoclonal antibody, an artificial vector, a pharmaceutical or a pharmaceutical composition comprising the above-described light chain variable region or the above heavy chain variable region.
  • the present invention also provides a detection reagent or kit comprising the above-described light chain variable region or the above heavy chain variable region.
  • the antibody is useful for treating diseases caused by neovascularization, including but not limited to tumors and macular degeneration.
  • the ScFv is a single-chain fragment variable
  • the HEK293E cell is a human embryonic kidney 293E cell
  • the CHO cell is a Chinese hamster ovary cell
  • the NSO cell For mouse NSO thymoma cells.
  • the anti-VEGFR-2 monoclonal antibody provided by the invention has high affinity and can inhibit the binding of VEGFR-2 and its ligand VEGF well in vitro, and has good biological activity in vitro; and has broad development prospects.
  • the anti-VEGFR-2 monoclonal antibody provided by the invention is used for treating diseases caused by neovascularization of tumors, including but not limited to the following diseases: non-small cell lung cancer, metastatic non-small cell lung cancer, glioma, colorectal cancer Hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER2-negative metastatic breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma, metastatic renal cell carcinoma and other diseases such as macular degeneration.
  • the present invention provides an improved anti-VEGFR-2 monoclonal antibody comprising:
  • a light chain and a heavy chain comprising any one of SEQ NO. 1, SEQ NO. 2 or SEQ NO. 3; the amino acid sequence of the heavy chain variable region comprising SEQ NO .4;
  • VFGFR-2 light chain variable region sequence 1 DIQMTQSPSSVSASIGDRVTITCRASQAIDNWLGWYQQKPGKAPKLLIYEGSNLNTGVPSRFSGSGSGTDFTLTISSLQAEDFAVYFCQQAKSFPPTFGGGTKVDIK;
  • SEQ NO. 2 (VEGFR-2 light chain variable region sequence 2): DIQMTQSPSSVSASIGDRVTITCRASDAIDQWLGWYQQKPGKAPKLLIYEASNLDTGVPSRFSGSGSGTDFTLTISSLQANQFAVYFCQQAKSFPPTFGGGTKVDIK;
  • SEQ NO. 3 (VEGFR-2 light chain variable region sequence 3): DIQMTQSPSSVSASIGDRVTITCRASQGIDQWLGWYQQKPGKAPKLLIYEGSNLNTGVPSRFSGSGSGTDFTLTISSLQANQFAVYFCQQAKSFPPTFGGGTKVDIK;
  • VFGFR-2 heavy chain variable region sequence 1 QVQLVESGGGLVKPGGSLRLSCAASAFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDLWGQGTMVTVSS.
  • the present invention also provides an antibody, polypeptide or protein comprising the above-described light chain variable region or the above heavy chain variable region.
  • the present invention also provides a polynucleotide sequence or combination comprising the above-described light chain variable region or the above-described heavy chain variable region amino acid sequence.
  • the present invention also provides a recombinant DNA expression vector comprising the above polynucleotide sequence or combination; the DNA sequence of the recombinant DNA expression vector comprising the above heavy chain variable region encoding an anti-VEGFR-2 antibody, Amino acid sequences of the heavy chain constant region, the light chain variable region, and the light chain constant region.
  • the present invention also provides a host cell transfected with the above recombinant DNA expression vector, the host cell comprising a mammalian cell, an insect cell, Escherichia coli or yeast; preferably a mammalian cell; further preferably HEK293E cell, CHO Cells or NSO cells.
  • the heavy chain constant region of the invention is selected from the group consisting of human IgGl, IgG2, IgG3, IgG4 or murine IgGl, IgG2a, IgG2b.
  • the present invention also provides a fragment of the full length antibody and the anti-T1h monoclonal antibody comprising the above-described light chain variable region or the above heavy chain variable region, including but not limited to Fab, Fab', F(ab') 2 , Fv or ScFv.
  • the present invention also provides a single chain antibody, a single domain antibody, a bispecific antibody, an antibody drug conjugate, and a chimeric antigen comprising the amino acid sequence of the above light chain variable region or the above heavy chain variable region.
  • Receptor T cell immunotherapy a single chain antibody, a single domain antibody, a bispecific antibody, an antibody drug conjugate, and a chimeric antigen comprising the amino acid sequence of the above light chain variable region or the above heavy chain variable region.
  • the present invention also provides a monoclonal antibody, artificial vector, drug or pharmaceutical composition comprising the above-described light chain variable region or the above heavy chain variable region.
  • the monoclonal antibody is of all human origin.
  • the present invention also provides a detection reagent or kit comprising the above-described light chain variable region or the above heavy chain variable region.
  • the antibody can be used to treat diseases caused by neovascularization, including but not limited to non-small cell lung cancer, metastatic non-small cell lung cancer, glioma, colorectal cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma , HER2-negative metastatic breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma, metastatic renal cell carcinoma and other diseases such as macular degeneration.
  • diseases caused by neovascularization including but not limited to non-small cell lung cancer, metastatic non-small cell lung cancer, glioma, colorectal cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma , HER2-negative metastatic breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma, metastatic renal cell carcinoma and other diseases such as macular degeneration.
  • the vector pCom3 vector (purchased from the Chinese plasmid vector strain cell strain gene collection) was modified by a series of gene cloning methods for use in the construction and expression of a phage single-chain antibody library.
  • the engineered vector was named pScFvDisb-S, and its plasmid map is shown in Figure 1. Based on this vector, a new fully synthetic phage antibody library based on the antibody sequence in the patent CN10333247B was constructed.
  • the extracellular domain of VEGFR-2 was used as an antigen to coat the immunotube, and the antigen coating amount was 2 ug/500 ul/tube, and coated at 4 ° C overnight.
  • the immunotubes and the fully synthetic phage antibody library were blocked with 4% skim milk powder/PBST, respectively, and blocked at room temperature for 1 hour.
  • the blocked phage antibody library was added to the immunotube for antigen-antibody binding, and the phage input amount was about 10 9 -10 12 , and the reaction was carried out at room temperature for 1 hour.
  • the PBST-PBS was washed several times to remove unbound phage, eluted with 0.1 M PH2.2 glycine, and the eluted phage antibody solution was neutralized to about pH 7.0 with 1.5 M PH8.8 Tris-HCl.
  • the above-mentioned neutralized phage was infected with 10 ml of TG1 bacterial solution grown to log phase, and allowed to stand in a 37 ° C incubator for 30 min, and some of the bacterial liquid was taken out for gradient dilution and applied to 2YTAG plates for calculation of phage yield. .
  • the remaining bacterial liquid was centrifuged to discard the supernatant, and the bacterial pellet was resuspended in a small amount of medium, and then aspirated and applied to a 2YTAG large plate to prepare for the next round of screening.
  • the above-mentioned infected plate was scraped from the large plate, and the cells were inoculated to 2YTAG liquid medium. After the logarithmic phase, M13 helper phage super infection was added, and the phage was expanded overnight at 28 ° C, and PEG6000-NaCl was purified. Phage were used for the next round of screening. A total of three rounds of phage library enrichment screening were performed.
  • the well-separated monoclonal colonies were picked and inoculated into 96-well deep-well plates supplemented with 2YTAG liquid medium, and cultured at 37 ° C, 220 rpm to their logarithmic growth phase, adding about 10 10 per well.
  • the phage M13KO7 was inactivated for 30 minutes at 37 °C. After centrifugation at 4000 rpm for 15 minutes at 4 ° C, the supernatant was discarded, the cells were resuspended in 2YTAK, and cultured overnight at 28 ° C, 220 rpm. The phage-containing supernatant was aspirated and identified by ELISA.
  • the phage of the biological antibody sequence of CN10333247B (the heavy chain is CN10333247B sequence No.3, the light chain is CN10333247B sequence No.9) was used as a positive control (named as O-1).
  • the monoclonal antibodies N-1, N-2 and N-3 with higher affinity were screened, and the sequencing of the gene was determined to be different from the sequence in the original patent CN10333247B.
  • Example 2 The clone obtained in Example 2 was subjected to display and purification of monoclonal phage, and phage gradient dilution ELISA assay was performed to identify affinity.
  • the extracellular domain of VEGFR-2 was coated with a pH 9.6 carbonate buffer, 20 ng/well/100 ul, and coated overnight at 4 °C.
  • the cells were washed three times with PBST and blocked with 4% milk-PBST at 37 ° C for 1 hour h.
  • the purified phage was diluted with a 5 fold gradient of 4% milk-PBST, 100 ul of the diluted sample was added to each well, and allowed to stand at room temperature for 1 hour.
  • the ELISA plate was washed with PBST, and the 4% milk-PBST diluted HRP-anti-M13 monoclonal antibody was added to the ELISA plate and allowed to stand at room temperature for 1 h.
  • the TMB color development kit was developed and developed at room temperature for 10 minutes. After termination with 2M H 2 SO 4 , the reading was taken at 450 nm / 630 nm. As shown in Fig. 2, the three different single-chain antibodies screened were able to specifically bind to VEGFR-2, and the binding ability was higher than that of O-1.
  • the heavy chain VH and light chain k genes of the above three antibodies N-1, N-2 and N-3 were cloned into the vector pTSE containing the heavy and light chain constant region genes, respectively (see Figure 3), encoding human IgG1.
  • the constant region (see SEQ. 5) and the pTSE vector of the constant region of the k chain (see SEQ. 6) (pTSE vector is shown in Figure 3, and the preparation process is described in paragraph [0019] on page 3 of the CN103525868A specification).
  • SEQ NO. 6 (constant region sequence of k-strand): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;
  • HEK293E cells were transiently transfected for full antibody expression.
  • Whole antibody protein was obtained using an AKTA instrument protein A affinity column purification. Protein concentration determination was performed using a BCA kit.
  • the extracellular domain of VEGFR was coated with a pH 9.6 carbonate buffer, 20 ng/well/100 ul, overnight coating at 4 °C. Wash three times with 300 ul/well PBST, and then add 4% milk-PBST for 1 h at 37 °C. Different dilutions of biotinylated whole antibodies were added. Human IgG (hIgG) was used as an isotype control, the highest concentration of various whole antibodies was 100 ng/ml, 8 gradients were made to 8 gradients, and incubated at 37 °C for 2-3 hours. Wash five times with 300 ul/well PBST, and then add streptavidin diluted 1 : 10000 with 4% milk-PBST for 1 h at 37 °C.
  • the cells were washed eight times with 300 ul/well PBST, developed by TMB colorimetric kit, 100 ul/well, developed at room temperature for 10 min, and then terminated with 2M H 2 SO 4 . 450 nm / 630 nm reading.
  • the experimental results are shown in Figure 4. All the antibodies bind well to the KDR molecules on the cell surface.
  • the affinity of N3 is relatively high, and N-1, N-2 and N-3 are higher than the original O. -1.
  • CHO cells overexpressing VEGFR-2 are used to detect the binding of different bispecific antibodies to cell surface EGFR, and human IgG (hIgG) is used as an isotype control.
  • the cells were harvested by centrifugation with 0.25% trypsin. Diluted various antibodies at the same time, the highest concentration was 100 nM, 4 times gradient dilution. The collected cells were washed three times with PBS+1% BSA, and the cells were resuspended with PBS+1% BSA, and then the cells were plated in 96-well plates at 1 ⁇ 10 5 cells per well, and 100 ⁇ l of diluted dispecific was added.
  • the antibody was incubated for 1 hour at room temperature; the supernatant was removed by centrifugation, the cells were washed three times with PBS, and the cells were resuspended with diluted Alexa488-labeled anti-human IgG FC antibody, incubated at room temperature for 1 hour in the dark, washed three times with PBS, and then washed again.
  • the cells were resuspended in 100 ul PBS and the fluorescence intensity was measured by flow cytometry.
  • the results were analyzed using Graphpad Prism. The results showed that N3 could better bind to VEGFR-2 expressed by cells, and the binding ability of N1, N2 and N3 was stronger than that of O-1 (see Figure 5).
  • Example 7 Total antibody inhibits proliferation of HUVEC cells
  • HUVEC Human umbilical vein endothelial cells
  • HUVEC Human umbilical vein endothelial cells
  • the present inventors have studied the inhibitory effects of different anti-VEGFR-2 antibodies on HUVEC cell proliferation. Wait for HUVEC cells to grow to 80% abundance, replace with fresh EPM medium containing 5% FBS, trypsin digest after 6 hours, then wash the digested cells with serum-free medium for 4-5 times. Pour the medium, resuspend the cell count, connect 5,000 cells per well to the 96-well plate, discard the 96-well plate edge, 100 ul of serum-free medium per well, and starve overnight.
  • the above antibodies of the invention can be used for treating diseases caused by neovascularization, including but not limited to non-small cell lung cancer, metastatic non-small cell lung cancer, glioma, colorectal cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER2 negative Metastatic breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma, metastatic renal cell carcinoma and other diseases as well as macular degeneration.
  • diseases caused by neovascularization including but not limited to non-small cell lung cancer, metastatic non-small cell lung cancer, glioma, colorectal cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER2 negative Metastatic breast cancer, metastatic gastric adenocarcinoma, metastatic colorectal cancer, metastatic melanoma, metastatic renal cell carcinoma and other diseases as well as macular degeneration.

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Abstract

本发明属于生物医药技术领域，提供了一种改进的抗VEGFR-2单克隆抗体及其应用。本发明通过计算机辅助模拟设计，设计了新的噬菌体抗体库，经多轮筛选得到了改进的抗VEGFR-2单克隆抗体，所述抗体的亲和力和生物学活性均高于原抗，在体外能够很好地抑制VEGFR-2和其配体VEGF的结合，可用于治疗肿瘤和黄斑变性等新生血管引起的疾病。

Description

一种改进的抗VEGFR-2单克隆抗体 技术领域
本发明涉及生物制药技术领域，具体涉及本发明涉及一种改进的抗VEGFR-2单克隆抗体。
背景技术
肿瘤生长依赖新生血管的形成已经在肿瘤生物学上研究的比较透彻。血管生成可提供氧气、营养物质、生长因子、荷尔蒙及蛋白水解酶，从而促进肿瘤细胞向远处扩散和转移，加速肿瘤的生长和恶化。血管生成是一个高度复杂的动态过程，由许多促/抗血管生成分子调节。血管生成的开关被认为是促血管生成超过抗血管生成的一个恶性标志。VEGF/VEGFR轴触发多重信号网络，导致上皮细胞存活、有丝分裂、转移和分化、血管渗透，VEGF及其受体在正常和病理血管生成中发挥着中枢作用。在人类许多种癌症中，增加的肿瘤血管化作用和肿瘤促血管生成因子的表达被证实与肿瘤的分级和恶性与否有关。
血管内皮细胞生长因子(VEGF)又称血管通透因子，它是内皮细胞的特异性有丝分裂源，也是一种有效的血管形成和通透性诱导因子，已被鉴定的相应受体为VEGFR-1(Flt-1，FMS样酪氨酸激酶1)，VEGFR-2(又称为KDR/Flk-1，激酶插入嵌合受体、胎肝激酶-1)，VEGFR-3(Flt4)，神经纤维蛋白-1(neuropilin-1)，神经纤维蛋白-2。VEGFR-2是血管内皮细胞上的主要VEGF受体，是一种糖蛋白，其胞外区含有七个免疫球蛋白样区(含配体结合域和受体二聚化结构域)，胞内插入了酪氨酸激酶催化结构域，主要在内皮细胞上表达，其他如巨核细胞、视网膜远祖细胞、间充质干细胞，肿瘤细胞如黑色素瘤细胞、脑瘤和某些白血病细胞等。VEGF和VEGFR-2受体作为关键的血管内皮细胞特异因子信号传导途径的分子参与肿瘤新生血管的生成，VEGF的主要生物学功能都是通过VEGFR-2实现，VEGFR-2和VEGF结合后发生二聚体化，并且VEGFR-2胞内的酪氨酸残基自身磷酸化，从而激活并将细胞膜/细胞质激酶级联反应信号传递到细胞核，可引发内皮细胞的一系列变化，包括血管内皮细胞增殖、存活、细胞骨架重排、细胞迁移以及基因表达等，并最终引起血管增生。
由于VEGF/VEGFR2信号通路在肿瘤的发生发展中的关键作用，因此有不少针对VEGF/VEGFR2的信号通路的药物，如抗VEGF抗体贝伐单抗和抗VEGFR-2的抗体雷莫芦单抗。
抗体是生物医药中技术含量最高、难度最大的一类药物，从2012年起，全球销售排名前十名的单药中有六个是抗体类药物，因此，抗体类药物市场的发展潜力巨大。抗体药物筛选最普遍应用的技术是噬菌体抗体库技术，噬菌体抗体库技术是继噬菌体展示技术发展而来的一项基因抗体工程的新技术，它可将含不同物种的全部抗体可变区基因的基因库转化成展示在噬菌体表面的蛋白库，不仅使单克隆抗体的生产更方便、快速、高效地在体外进行，还开辟了单克隆抗体人源化的新途径，促进了人类单克隆抗体生产的发展。我公司专利号为 CN103333247 B的专利就是通过计算机辅助设计和噬菌体抗体库技术筛选得到的一系列抗VEGFR-2的抗体，为获得抗VEGFR-2的抗体药物奠定了基础，我公司在进一步地研究中发现，上述专利中涉及的抗体在亲和力和生物学活性等方面还存在进一步的提升可能，基于此，我们在原有的研究基础上，对上述抗体进行了改进性优化。
发明内容
本发明提供了一种改进的抗VEGFR-2单克隆抗体；本发明以CN103333247B专利中亲和力最高的两个为模板，进行计算机辅助模拟设计，设计了新的噬菌体抗体库，并进行了多轮筛选，得到了一株亲和力和生物学活性均高于原专利抗体的新的抗VEGFR-2单克隆抗体。
为实现上述目的，本发明提供了一种改进的抗VEGFR-2单克隆抗体，包括：
轻链和重链；所述轻链可变区的氨基酸序列包含SEQ NO.1、SEQ NO.2或SEQ NO.3中的任一种；所述重链可变区的氨基酸序列包含SEQ NO.4。
其中，本发明还提供了一种包含上述轻链可变区或上述重链可变区的抗体、多肽或蛋白。
其中，本发明还提供了一种包含上述轻链可变区或上述重链可变区氨基酸序列的多核苷酸序列或组合。
其中，本发明还提供了一种包含上述多核苷酸序列或组合的重组DNA表达载体；所述重组DNA表达载体的DNA序列中包含编码抗VEGFR-2抗体的上述重链可变区、重链恒定区、轻链可变区和轻链恒定区的氨基酸序列。
其中，本发明还提供了一种转染上述重组DNA表达载体的宿主细胞，所述宿主细胞包含哺乳动物细胞、昆虫细胞、大肠杆菌或酵母；优选哺乳动物细胞；进一步优选HEK293E细胞、CHO细胞或NSO细胞。
其中，本发明的重链恒定区选自人的IgG1、IgG2、IgG3、IgG4或鼠的IgG1、IgG2a、IgG2b。
其中，本发明还提供了一种包含上述轻链可变区或上述重链可变区的全长抗体和抗T1h单克隆抗体的片段，所述片段包含但不限于Fab、Fab’、F(ab’) 2、Fv或ScFv。
其中，本发明还提供了一种包含上述轻链可变区或上述重链可变区的氨基酸序列的单链抗体、单域抗体、双特异抗体、抗体药物偶联物和嵌合抗原受体T细胞免疫疗法。
其中，本发明还提供了一种含有上述轻链可变区或上述重链可变区的单克隆抗体、人工载体、药物或药物组合物。
其中，本发明还提供了一种含有上述轻链可变区或上述重链可变区的检测试剂或试剂盒。
其中，所述抗体可用于治疗新生血管引起的疾病，所述疾病包括但不限于肿瘤和黄斑变性。
其中，所述ScFv为单链抗体(single-chain fragment variable)；所述HEK293E细胞为人胚肾293E细胞(human embryonic kidney 293E cell)；CHO细胞为中国仓鼠卵巢细胞(chinese hamster ovary cell)；NSO细胞为小鼠NSO胸腺瘤细胞。
本发明与现有技术相比具有以下优点：
本发明提供的抗VEGFR-2单克隆抗体具有较高的亲和力，在体外能够很好的抑制VEGFR-2和其配体VEGF的结合，在体外具有良好的生物学活性；开发前景广阔。
本发明提供的抗VEGFR-2单克隆抗体用于治疗由肿瘤新生血管生成引起的疾病，包括但不限于下列疾病：非小细胞肺癌、转移性非小细胞肺癌、神经胶质瘤、结直肠癌、肝细胞癌、转移性肝细胞癌、HER2阴性的转移性乳腺癌、转移性胃腺癌、转移性结直肠癌、转移性黑色素瘤、转移性肾细胞癌等癌症以及黄斑变性等疾病。
说明书附图
图1、pScFvDisb-S质粒图谱；
图2、梯度ELISA比较抗VEGFR-2单链抗体的相对亲和力；
图3、蛋白质表达pTSE质粒图谱；
图4、抗VEGFR-2单克隆抗体与KDR结合能力比较；
图5、本发明抗体与细胞表面的VEGFR-2结合情况；
图6、本发明抗体对人脐静脉内皮细胞HUVEC增殖抑制作用。
具体实施方式
本发明详细的实施方法参见实施例，实施例中所述的实验方法和试剂，若无特殊说明均为常规实验方法和试剂。以下实施例仅用于说明和解释本发明，而不是以任何方式限制本发明。
本发明提供了一种改进的抗VEGFR-2单克隆抗体，包括：
轻链和重链；所述轻链可变区的氨基酸序列包含SEQ NO.1、SEQ NO.2或SEQ NO.3中的任一种；所述重链可变区的氨基酸序列包含SEQ NO.4；
SEQNO.1(VEGFR-2轻链可变区序列1)：DIQMTQSPSSVSASIGDRVTITCRASQAIDNWLGWYQQKPGKAPKLLIYEGSNLNTGVPSRFSGSGSGTDFTLTISSLQAEDFAVYFCQQAKSFPPTFGGGTKVDIK；
SEQ NO.2(VEGFR-2轻链可变区序列2)：DIQMTQSPSSVSASIGDRVTITCRASDAIDQWLGWYQQKPGKAPKLLIYEASNLDTGVPSRFSGSGSGTDFTLTISSLQANQFAVYFCQQAKSFPPTFGGGTKVDIK；
SEQ NO.3(VEGFR-2轻链可变区序列3)：DIQMTQSPSSVSASIGDRVTITCRASQGIDQWLGWYQQKPGKAPKLLIYEGSNLNTGVPSRFSGSGSGTDFTLTISSLQANQFAVYFCQQAKSFPPTFGGGTKVDIK；
SEQNO.4(VEGFR-2重链可变区序列1)：QVQLVESGGGLVKPGGSLRLSCAASAFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDLWGQGTMVTVSS。
优选地，本发明还提供了一种包含上述轻链可变区或上述重链可变区的抗体、多肽或蛋白。
更加优选地，本发明还提供了一种包含上述轻链可变区或上述重链可变区氨基酸序列的多核苷酸序列或组合。
更加优选地，本发明还提供了一种包含上述多核苷酸序列或组合的重组DNA表达载体；所述重组DNA表达载体的DNA序列中包含编码抗VEGFR-2抗体的上述重链可变区、重链恒定区、轻链可变区和轻链恒定区的氨基酸序列。
更加优选地，本发明还提供了一种转染上述重组DNA表达载体的宿主细胞，所述宿主细胞包含哺乳动物细胞、昆虫细胞、大肠杆菌或酵母；优选哺乳动物细胞；进一步优选HEK293E细胞、CHO细胞或NSO细胞。
更加优选地，本发明的重链恒定区选自人的IgG1、IgG2、IgG3、IgG4或鼠的IgG1、IgG2a、IgG2b。
更加优选地，本发明还提供了一种包含上述轻链可变区或上述重链可变区的全长抗体和抗T1h单克隆抗体的片段，所述片段包含但不限于Fab、Fab’、F(ab’) 2、Fv或ScFv。
更加优选地，本发明还提供了一种包含上述轻链可变区或上述重链可变区的氨基酸序列的单链抗体、单域抗体、双特异抗体、抗体药物偶联物和嵌合抗原受体T细胞免疫疗法。
更加优选地，本发明还提供了一种含有上述轻链可变区或上述重链可变区的单克隆抗体、人工载体、药物或药物组合物。
更加优选地，所述单克隆抗体是全人源的。
更加优选地，本发明还提供了一种含有上述轻链可变区或上述重链可变区的检测试剂或试剂盒。
更加优选地，所述抗体可用于治疗新生血管引起的疾病，包括但不限于非小细胞肺癌、转移性非小细胞肺癌、神经胶质瘤、结直肠癌、肝细胞癌、转移性肝细胞癌、HER2阴性的转移性乳腺癌、转移性胃腺癌、转移性结直肠癌、转移性黑色素瘤、转移性肾细胞癌等癌症以及黄斑变性等疾病。
具体实施例
以下结合附图和实施例详述本发明。
实施例1、抗VEGFR-2的单链抗体的生物淘选
采用一系列基因克隆的方法对载体pCom3载体(购自中国质粒载体菌株细胞株基因保藏中心)进行改造，使之用于噬菌体单链抗体库的构建和表达。改造后的载体命名为pScFvDisb-S，其质粒图谱如图1所示，并以此载体为基础，构建基于专利CN10333247B中抗 体序列的新的全合成噬菌体抗体库。
以VEGFR-2胞外区为抗原包被免疫管，抗原包被量为2ug/500ul/管，4℃包被过夜。再用4％脱脂奶粉/PBST分别封闭免疫管和全合成噬菌体抗体库，室温封闭1小时。封闭后的噬菌体抗体库加入免疫管中进行抗原抗体结合，噬菌体投入量约为10 9-10 12个，室温反应1小时。PBST-PBS清洗多次以除去未结合的噬菌体，0.1M PH2.2的甘氨酸洗脱，用1.5M PH8.8的Tris-HCl中和洗脱下来的噬菌体抗体溶液至PH 7.0左右。
将上述中和后的噬菌体感染10ml生长至对数期的TG1菌液，37℃培养箱中静置30min，取出部分菌液进行梯度稀释，涂布于2YTAG平板上，用于计算噬菌体产出量。剩余的菌液离心弃上清，将菌体沉淀重悬于少量培养基，吸出后涂布于2YTAG大平板，为下一轮筛选做准备。
将上述感染后涂板的菌体从大平板上刮下，接菌至2YTAG液体培养基，摇至对数期后加入M13辅助噬菌体超感染，28℃培养过夜扩增噬菌体，PEG6000-NaCl沉降纯化噬菌体用于下一轮筛选。共进行三轮噬菌体库富集筛选。
实施例2、抗VEGFR2噬菌体单链抗体阳性克隆的鉴定
经过三轮筛选后，挑取分隔良好的单克隆菌落，接种于加有2YTAG液体培养基的96孔深孔板，37℃，220rpm培养至其对数生长期，每孔加入约10 10的辅助噬菌体M13KO7，37℃静止感染30分钟。4000rpm，4℃离心15分钟，弃去上清，菌体用2YTAK重悬沉淀，28℃，220rpm培养过夜。吸取含噬菌体上清进行ELISA鉴定，用相同载体同样构建方法的CN10333247B的生物学抗体序列(重链为CN10333247B序列的No.3，轻链为CN10333247B序列No.9)的噬菌体作为阳性对照(命名为O-1)。筛选得到亲和力较高的单克隆抗体N-1、N-2和N-3，基因测序确定为不同于原专利CN10333247B中序列。
实施例3、梯度稀释噬菌体ELISA比较抗VEGFR2单链抗体的亲和力
将实施例2中获得的克隆进行单克隆噬菌体的展示和纯化，进行噬菌体梯度稀释ELISA实验鉴定亲和力。
用pH 9.6的碳酸盐缓冲液包被VEGFR-2胞外区，20ng/孔/100ul，4℃包被过夜。PBST洗涤三次，4％milk-PBST 37℃封闭1小时h。将纯化后的噬菌体用4％milk-PBST五倍梯度稀释，每孔加入100ul稀释后的样品，室温静置1小时。用PBST洗涤ELISA板，将4％milk-PBST稀释后的HRP-anti-M13单克隆抗体加入ELISA板中，室温放置1h。TMB显色试剂盒显色，室温显色10分钟。用2M H 2SO 4终止后，450nm/630nm读数。结果如图2所示，筛选出的3株不同的单链抗体均能与VEGFR-2进行特异性结合，且结合能力均高于的O-1。
实施例4、抗KDR全抗体的制备
将上述3个抗体N-1、N-2和N-3的重链VH和轻链k基因分别克隆至装有重链和轻链恒 定区基因的载体pTSE(见图3)，编码人IgG1的恒定区(见SEQ.5)和k链的恒定区(见SEQ.6)的pTSE载体中(pTSE载体如图3所示，制备过程参见CN103525868A说明书第3页第[0019]段)。
SEQNO.5(人IgG1的恒定区序列)：ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG；
SEQ NO.6(k链的恒定区序列)：RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
瞬时转染HEK293E细胞，进行全抗体表达。使用AKTA仪器protein A亲和柱纯化获得全抗体蛋白。使用BCA试剂盒进行蛋白浓度测定。
实施例5、全抗体与VEGFR2胞外区的结合实验
用pH9.6的碳酸盐缓冲液包被VEGFR胞外区，20ng/孔/100ul，4℃过夜包被。用300ul/孔PBST洗三次，再加入4％milk-PBST在37℃封闭1h。加入不同稀释度的生物素标记的全抗体。用人的IgG(hIgG)作为同型对照，各种全抗体最高浓度是100ng/ml，5倍稀释做8个梯度，37℃孵育2-3h。用300ul/孔PBST洗五次，再加入用4％mi lk-PBST 1∶10000稀释的链霉亲和素37℃孵育1h。用300ul/孔PBST洗八次，TMB显色试剂盒显色，100ul/孔，室温显色10min，然后用2M H 2SO 4终止。450nm/630nm读数。实验结果如图4所示，所有抗体均能很好的与细胞表面的KDR分子结合，其中N3的亲和力相对较高，且N-1、N-2、N-3均高于原有的O-1。
实施例6、全抗体与细胞表面VEGFR-2的结合特异性分析
本发明采用过表达VEGFR-2的CHO细胞来检测不同的双特异抗体与细胞表面EGFR的结合情况，用人的IgG(hIgG)作为同型对照。用0.25％胰酶消化、离心收集细胞。同时稀释各种抗体，最高浓度为100nM，4倍梯度稀释。将收集的细胞用PBS+1％BSA洗三遍，再加PBS+1％BSA重悬细胞，然后铺细胞于96孔板中，每孔1×10 5个细胞，加入100ul稀释好的双特异性抗体，室温孵育1小时；离心去上清，用PBS洗细胞三遍，再用稀释好的Alexa488标记的抗人IgG FC抗体重悬细胞，室温避光孵育1小时，PBS洗三遍，再用100ul PBS重悬，用流式细胞仪检测荧光强度。结果用Graphpad Prism分析。结果显示，N3能够更好的结合细胞表达的VEGFR-2，N1、N2、N3的结合能力均强于O-1(见图5)。
实施例7、全抗体抑制HUVEC细胞增殖实验
人脐静脉内皮细胞(HUVEC)，已被广泛用于血管内皮细胞增殖、细胞信号途径和多种肿 瘤发生机制的研究，本发明研究了不同抗VEGFR-2抗体对HUVEC细胞增殖抑制作用。等HUVEC细胞长到80％丰度，换新鲜含5％FBS的EGM培养基，6小时后用胰酶消化，再用无血清培养基洗消化下来的细胞4-5遍，每次离心后尽量倒净培养基，重悬细胞计数，5000个细胞每孔接入96孔板，放弃96孔板边缘孔，100ul无血清培养基每孔，饥饿过夜。14-16小时后吸去上清，加入50ul VEGF/ECM(200ng/ml)，至终浓度为100ng/ml，和不同浓度的不同抗体混匀后培养24小时。然后加入CCK-8，检测细胞增殖情况。结果显示，N1、N2和N3的IC 50(ng/L)分别为12.76、17.32、和10.53，O-1的IC 50(ng/L)为18.59；N1、N2、N3抑制血管内皮细胞增殖能力均强于O-1，其中N3最好(见图6)。综合实施例5-7，可以看出N1、N2、N3均无论在分子还是在细胞学水平的结果均优于原专利CN103525868A的最佳抗体O-1，这也意味着本专利中的候选分子更具有开发和应用前景。
本发明上述抗体可用于治疗新生血管引起的疾病，包括但不限于非小细胞肺癌、转移性非小细胞肺癌、神经胶质瘤、结直肠癌、肝细胞癌、转移性肝细胞癌、HER2阴性的转移性乳腺癌、转移性胃腺癌、转移性结直肠癌、转移性黑色素瘤、转移性肾细胞癌等癌症以及黄斑变性等疾病。
对于本领域的普通技术人员而言，具体实施例只是对本发明进行了示例性描述，显然本发明具体实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种非实质性的改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围之内。

Claims (12)

  1. 一种抗VEGFR-2单克隆抗体，其特征在于，包括：轻链和重链；所述轻链可变区的氨基酸序列包含SEQ NO.1、SEQ NO.2或SEQ NO.3中的任一种；所述重链可变区的氨基酸序列包含SEQ NO.4。
  2. 一种包含上述权利要求1所述的轻链可变区或重链可变区的抗体、多肽或蛋白。
  3. 一种包含权利要求1所述的轻链可变区或重链可变区氨基酸序列的多核苷酸序列或组合。
  4. 一种包含如权利要求3所述的多核苷酸序列或组合的重组DNA表达载体；所述重组DNA表达载体的DNA序列中包含编码抗VEGFR-2抗体的所述重链可变区、重链恒定区、所述轻链可变区和轻链恒定区的氨基酸序列。
  5. 一种转染如权利要求4所述的重组DNA表达载体的宿主细胞，所述宿主细胞包含哺乳动物细胞、昆虫细胞、大肠杆菌或酵母；优选哺乳动物细胞；进一步优选HEK293E细胞、CHO细胞或NSO细胞。
  6. 根据权利要求1所述的单克隆抗体，其特征在于，重链恒定区选自人的IgG1、IgG2、IgG3、IgG4或鼠的IgG1、IgG2a、IgG2b。
  7. 一种包含权利要求1所述的轻链可变区或重链可变区的全长抗体和抗T1h单克隆抗体的片段，所述片段包含但不限于Fab、Fab’、F(ab’)2、Fv或ScFv。
  8. 一种包含如权利要求1所述的轻链可变区或重链可变区的氨基酸序列的单链抗体、单域抗体、双特异抗体、抗体药物偶联物和嵌合抗原受体T细胞免疫疗法。
  9. 一种包含权利要求1所述的轻链可变区或重链可变区的单克隆抗体、人工载体、药物或药物组合物。
  10. 根据权利要求1所述的单克隆抗体，其特征在于，所述单克隆抗体是全人源的。
  11. 一种包含权利要求1所述的轻链可变区或重链可变区的检测试剂或试剂盒。
  12. 一种包含权利要求1所述的轻链可变区或重链可变区的抗体用于治疗新生血管引起的疾病，所述疾病包含肿瘤和黄斑变性。
PCT/CN2018/076847 2017-03-07 2018-02-14 一种改进的抗vegfr-2单克隆抗体 WO2018161798A1 (zh)

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JP2019546906A JP6843262B2 (ja) 2017-03-07 2018-02-14 改良型抗ｖｅｇｆｒ−２モノクローナル抗体
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AU2018229915A AU2018229915B2 (en) 2017-03-07 2018-02-14 Improved anti-VEGFR-2 monoclonal antibody
EA201991788A EA201991788A1 (ru) 2017-03-07 2018-02-14 Улучшенные моноклональные анти-vegr-2 антитела
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CN106674349B (zh) 2017-03-07 2018-03-13 北京东方百泰生物科技有限公司 一种改进的抗vegfr‑2单克隆抗体
WO2021013061A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 一种人源化抗vegfr2抗体及其应用
CN111024949A (zh) * 2019-12-31 2020-04-17 上海博威生物医药有限公司 一种重组抗vegfr2单克隆抗体的生物活性分析方法及其应用
CN112094351B (zh) * 2020-09-11 2021-04-20 北京东方百泰生物科技股份有限公司 一种抗vegfr-2单克隆抗体的检测性抗体、应用及检测方法
CN115724968B (zh) * 2021-08-27 2023-08-08 三优生物医药(上海)有限公司 Vegf结合分子及其用途
CN114081945A (zh) * 2021-11-19 2022-02-25 北京东方百泰生物科技股份有限公司 一种治疗肿瘤疾病的联合用药组合物及应用
CN115957319B (zh) * 2022-10-14 2023-06-30 北京东方百泰生物科技股份有限公司 一种抗nkg2a单克隆抗体的注射制剂
CN116254315A (zh) * 2022-12-23 2023-06-13 北京东方百泰生物科技股份有限公司 一种vegf/vegfr抑制剂生物学活性的检测方法

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