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WO2016115953A1 - Préparation et utilisation d'une protéine de fusion de type anticorps ciblant vegfr2 - Google Patents

Préparation et utilisation d'une protéine de fusion de type anticorps ciblant vegfr2 Download PDF

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WO2016115953A1
WO2016115953A1 PCT/CN2015/097367 CN2015097367W WO2016115953A1 WO 2016115953 A1 WO2016115953 A1 WO 2016115953A1 CN 2015097367 W CN2015097367 W CN 2015097367W WO 2016115953 A1 WO2016115953 A1 WO 2016115953A1
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vegfr2
fusion protein
antibody
mica
protein
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Chinese (zh)
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张娟
王旻
王佑富
解伟
柳芳
任学艳
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China Pharmaceutical University
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China Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • 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 is a highly specific genetically engineered fusion protein with anti-tumor and angiogenic activities targeting VEGFR and NKG2D.
  • MHC class I-related antigen molecules A and B 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.
  • serum soluble MICA sMIC
  • 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, Fc ⁇ R), Fc ⁇ RIIIa binds, immune cells are close to antibody-bound tumor cells, and immune cells are activated by antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • VAGFR2 antibody Anti-vascular endothelial growth factor receptor antibody
  • VEGFR2 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.
  • VEGF vascular endothelial growth factor
  • the formation of tumor neovascularization is an essential condition for the tumor to transform into a malignant tumor.
  • 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.
  • VEGF antibody is a monoclonal antibody that inhibits the VEGF pathway.
  • the FDA approved the Ramucirumab (IMC-1121B) antibody targeting VEGFR2 for the treatment of gastric cancer and gastroesophageal junction adenocarcinoma.
  • Ramucirumab IMC-1121B
  • anti-VEGFR2 antibody has better safety.
  • MHC-I related antigen molecule A (MICA) on the surface of tumor cells
  • MHC The major histocompatibility complex
  • MIC MHC class cainrelated Gene
  • PerB11 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.
  • VEGFR2 vascular endothelial growth factor receptor 2
  • 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.
  • 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 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.
  • a conjugate of a protein or protein fragment of any of the above is a conjugate of a protein or protein fragment of any of the above.
  • 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.
  • GGGGS flexible peptide
  • 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.
  • 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.
  • GGGGS flexible peptide
  • 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
  • Figure 3BC depicts the results of the Western blot analysis of the isolated fusion protein.
  • Figure 3B is an incubation of anti-H+L
  • 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
  • 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) .
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • PVDF membrane purchased from Millipore
  • 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.
  • 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.
  • M equilibrium dissociation constant
  • 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.
  • 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.
  • 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.
  • FACs Calibur flow cytometry to analyze the binding ability of the fusion protein to the antigen.
  • 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
  • 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.
  • 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 ⁇ l 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 ⁇ l/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 ⁇ l of the gradient-diluted antibody was added, three parallel wells were set for each concentration, and incubation was continued for 72 h.
  • 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. .
  • 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 ⁇ L of 1 ⁇ Binding Buffer to make the cell density 2 ⁇ .
  • Example 7 ADCC experiment of fusion protein mAb04-MICA on human breast cancer cell line MDA-MB-231
  • 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 ⁇ l.
  • the formula calculates cytotoxicity and analyzes the results using software.

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Abstract

La présente invention appartient au domaine technique des anticorps produits par génie génétique, et concerne une méthode de préparation et une utilisation d'une protéine de fusion constituée d'un anticorps (KDR3) contre le VEGFR2 tumoral et de la protéine MICA (MHC class I polypeptide-related sequence A). En particulier, la protéine de fusion est exprimée et préparée en liant à MICA un anticorps de pleine longueur contre VEGFR2, via un peptide flexible. La protéine peut inhiber ou détruire l'angiogenèse tumorale, augmenter la teneur en MICA en surface de la cellule tumorale, rétablir la surveillance immunologique d'un corps activé par la voie NKG2D, et améliorer l'effet de cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC, pour "antibody-dependant cell-mediated cytotoxicity") médiée par la région Fc de l'anticorps.
PCT/CN2015/097367 2015-01-21 2015-12-15 Préparation et utilisation d'une protéine de fusion de type anticorps ciblant vegfr2 Ceased WO2016115953A1 (fr)

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