CN112552410A - Antibody fusion protein, preparation method thereof and application thereof in anti-tumor - Google Patents

Antibody fusion protein, preparation method thereof and application thereof in anti-tumor Download PDF

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CN112552410A
CN112552410A CN201910916586.9A CN201910916586A CN112552410A CN 112552410 A CN112552410 A CN 112552410A CN 201910916586 A CN201910916586 A CN 201910916586A CN 112552410 A CN112552410 A CN 112552410A
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fusion protein
antibody fusion
seq
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amino acid
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朱祯平
黄浩旻
顾昌玲
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Priority to CN202080058822.9A priority patent/CN114667297B/en
Priority to PCT/CN2020/113556 priority patent/WO2021057451A1/en
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The invention belongs to the field of tumor treatment, and discloses an antibody fusion protein, a preparation method thereof and application thereof in tumor resistance. More specifically, it comprises the anti-HER 2 monoclonal antibody IgG and the D2 domain of VEGFR1, the D2 domain of VEGFR1 being linked to the C-terminus of the IgG heavy chain by a peptide linker L. The antibody fusion protein can block HER2 and VEGFR2 signal channels at the same time, has the effect of inhibiting tumor proliferation better than that of a monoclonal antibody, provides a candidate drug with better treatment effect for anti-tumor treatment, and has wide application prospect in the treatment of tumor diseases.

Description

Antibody fusion protein, preparation method thereof and application thereof in anti-tumor
Technical Field
The invention belongs to the technical field of tumor treatment and biology, and relates to an antibody fusion protein composed of anti-HER 2 monoclonal antibody IgG and D2 structural domain of VEGFR1, and a preparation method and application thereof.
Background
HER2(human epidermal growth factor receptor 2), which has receptor tyrosine protein kinase activity, is one of the human epidermal growth factor receptor family members and is expressed only at low levels in a small number of normal tissues of adults. Studies have shown that HER2 is overexpressed in various tumors, such as in about 30% of breast cancer patients and 16% of gastric cancer patients, and that HER2 overexpression in tumors can significantly promote tumor growth and enhance tumor invasion and metastasis ability, which is an important indicator of poor prognosis in such patients. Thus, as early as 1998, the first monoclonal antibody drug, Herceptin, targeting HER2 was FDA approved for marketing and use in the treatment of HER2 overexpressed breast and gastric cancers.
There are two phases of tumor growth, from the slow avascular growth phase to the rapid angiogenic proliferation phase. If no blood vessels are formed inside the tumor, the primary tumor grows slowly and metastasis cannot be achieved. Inhibition of tumor angiogenesis is therefore considered to be one of the currently promising approaches to tumor therapy. Among the Vascular Endothelial Growth Factor (VEGFs) family, VEGF-A165 (hereinafter referred to as VEGF) is the most abundant active subtype. VEGF, by binding to the type II receptor VEGFR2, activates a signaling pathway to undergo a cascade of reactions that promote neovascularization and maintain its integrity. However, the type I receptor VEGFR1 binds VEGF much more strongly than VEGFR2 and acts mainly at the extracellular domain D2 of VEGFR 1. VEGFR1-D2 blocks the binding of VEGFR2 and VEGF by competing with the binding of VEGF, thereby blocking the signal pathway, inhibiting the proliferation and angiogenesis of endothelial cells, and thus inhibiting the rapid proliferation and metastasis of tumors.
The invention introduces an antibody fusion protein capable of blocking HER2 and VEGFR2 signal pathways at the same time, and our research shows that the antibody fusion protein can be combined with HER2 antigen on the surface of a tumor cell to inhibit tumor proliferation; on the other hand, it can compete with VEGF binding and inhibit endothelial cell proliferation and angiogenesis. The action mechanism occurs in a tumor microenvironment, and can effectively inhibit the generation of blood vessels in the tumor, thereby inhibiting the growth of the tumor. The antibody fusion protein has the effect of inhibiting tumor proliferation better than HER2 monoclonal antibody and HER2 monoclonal antibody + FcD2, and has wide application prospect in the treatment of tumor diseases.
Disclosure of Invention
The invention aims to provide an antibody fusion protein capable of blocking HER2 and VEGFR2 signal pathways at the same time, and provides a nucleotide molecule for coding the antibody fusion protein; providing an expression vector comprising said nucleotide molecule; a host cell providing the expression vector; providing a method for preparing the antibody fusion protein; providing a pharmaceutical composition comprising the antibody fusion protein; provides the application of the antibody fusion protein in preparing medicaments.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an antibody fusion protein capable of blocking signal paths of HER2 and VEGFR2 at the same time, which comprises anti-HER 2 monoclonal antibody IgG and a D2 domain of VEGFR1, wherein the D2 domain of VEGFR1 is connected to the C terminal of an IgG heavy chain through a peptide linker L.
An "antibody fusion protein" of the invention is a recombinantly produced antigen binding molecule in which an antibody or antibody fragment is linked to another protein or peptide. It comprises the anti-HER 2 monoclonal antibody IgG and the D2 domain of VEGFR1, the D2 domain of VEGFR1 linked to the C-terminus of the IgG heavy chain by a peptide linker L.
The "anti-HER 2 monoclonal antibody IgG" of the invention is an approximately 150kDa molecule consisting of four peptide chains, containing two identical gamma heavy chains of approximately 50kDa and two identical light chains of approximately 25kDa, thus having a tetrameric quaternary structure. The two heavy chains are interconnected by disulfide bonds and are each linked to one light chain. The resulting tetramer has two identical halves that form a fork or Y-like shape, with each end of the fork containing an identical antigen binding site. IgG antibodies can be divided into subclasses (e.g., IgG1, 2, 3, 4) based on minor differences in amino acid sequences in the constant region of the heavy chain.
Preferably, the heavy chain of the IgG comprises the complementarity determining region HCDR1-3, wherein the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown as SEQ ID NO:2, the amino acid sequence of HCDR3 is shown as SEQ ID NO: 3 is shown in the specification;
the light chain of the IgG comprises the complementarity determining region LCDR1-3, wherein the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown as SEQ ID NO: 5, the amino acid sequence of LCDR3 is shown in SEQ ID NO: and 6.
In the art, the binding region of an antibody typically comprises a light chain variable region and a heavy chain variable region, each variable region comprising three domains of 3 CDRs. The CDR domains of the heavy and light chains of an antibody are referred to as HCDR and LCDR, respectively. Thus, a conventional antibody antigen-binding site comprises six CDRs, including sets of CDRs from heavy and light chain V regions, respectively.
Preferably, the amino acid sequence of the heavy chain variable region of the IgG is as shown in SEQ ID NO: 7, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.
Preferably, the amino acid sequence of the peptide linker L is as shown in SEQ ID NO: shown at 9.
Preferably, the heavy chain amino acid sequence of the antibody fusion protein is as shown in SEQ ID NO: 10, and the light chain amino acid sequence is shown as SEQ ID NO: shown at 11.
In constructing the antibody fusion proteins of the present invention, problems associated with the chemical and physical stability of the antibody fusion proteins are also solved, such as expression of physically stable molecules, increased heat and salt dependent stability, reduced aggregation, increased solubility at high concentrations, and maintenance of affinity for HER2 and VEGF, respectively.
In another aspect, the invention provides a nucleotide molecule encoding an antibody fusion protein as described in any one of the above.
Preferably, the nucleotide sequence of the heavy chain of the antibody fusion protein encoded by the nucleotide molecule is as shown in SEQ ID NO: 12, and the nucleotide sequence encoding the light chain is shown as SEQ ID NO: shown at 13.
The preparation method of the nucleotide molecule is a conventional preparation method in the field, and preferably comprises the following preparation methods: the nucleotide molecule encoding the above antibody fusion protein is obtained by a gene cloning technique such as a PCR method or the like, or obtained by a method of artificial complete sequence synthesis.
Those skilled in the art know that the nucleotide sequence encoding the amino acid sequence of the above-described antibody fusion protein may be appropriately substituted, deleted, altered, inserted or added to provide a polynucleotide homolog. The polynucleotide homologue of the present invention may be prepared by substituting, deleting or adding one or more bases of a gene encoding the antibody fusion protein within a range in which the activity of the antibody is maintained.
In another aspect, the present invention provides an expression vector comprising any one of the nucleotide molecules described above.
Wherein the expression vector is conventional in the art, refers to an expression vector comprising appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate sequences. The expression vector may be a virus or a plasmid, such as a suitable phage or phagemid, for more technical details see for example Sambrook et al, Molecular Cloning: a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and Protocols for nucleic acid manipulation are described in Current Protocols in Molecular Biology, second edition, Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.4(+), pDFFR or pTT 5.
The invention further provides a host cell containing the expression vector.
The host cell of the present invention is any host cell that is conventional in the art, provided that the recombinant expression vector is stably self-replicating and the nucleotide is efficiently expressed. Wherein the host cell comprises prokaryotic expression cells and eukaryotic expression cells, the expression vector preferably comprises: COS, CHO (Chinese hamster Ovary), NS0, sf9, sf21, DH5 α, BL21(DE3) or TG1, more preferably e.coli TG1, BL21(DE3) cells (expressing single chain or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies). The recombinant expression transformant of the present invention can be obtained by transforming the expression vector into a host cell. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.
Preferably, the host cell is a eukaryotic cell, preferably selected from the group consisting of CHO cells and 293E cells.
In another aspect, the present invention provides a method for preparing the above antibody fusion protein, the method comprising the steps of:
a) culturing a host cell as described in any of the above under expression conditions to express an antibody fusion protein that blocks both HER2 and VEGFR2 signaling pathways;
b) isolating and purifying the antibody fusion protein of step a).
The culture method of the host cell and the separation and purification method of the antibody are conventional methods in the field, and for the specific operation method, reference is made to the corresponding cell culture technical manual and antibody separation and purification technical manual. Using the above method, the recombinant protein can be purified as a substantially homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis.
The antibody fusion protein disclosed in the present invention can be isolated and purified by affinity chromatography, and the antibody fusion protein bound to the affinity column can be eluted by a conventional method such as high salt buffer, PH change, etc., depending on the characteristics of the affinity column used. The inventor of the invention carries out detection experiments on the obtained antibody fusion protein, and the experimental results show that the antibody fusion protein can be well combined with target cells and antigens and has higher affinity.
In another aspect of the invention, a composition is provided comprising an antibody fusion protein as described above and one or more pharmaceutically acceptable carriers, diluents, or excipients.
The antibody fusion protein provided by the invention can form a pharmaceutical preparation composition together with a pharmaceutically acceptable carrier so as to exert a curative effect more stably, and the preparations can ensure the conformation integrity of the amino acid core sequence of the antibody fusion protein disclosed by the invention and simultaneously protect the multifunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation). In general, it is generally stable for at least one year at 2 ℃ to 8 ℃ for liquid formulations and at least six months at 30 ℃ for lyophilized formulations. The antibody fusion protein preparation can be suspension, hydro-acupuncture, freeze-drying and other preparations commonly used in the pharmaceutical field.
For hydro-acupuncture or lyophilized formulations of the antibody fusion proteins disclosed herein, pharmaceutically acceptable carriers preferably include, but are not limited to: one or a combination of a surfactant, a solution stabilizer, an isotonicity adjusting agent, and a buffer. Wherein the surfactant preferably includes, but is not limited to: nonionic surfactants such as polyoxyethylene sorbitol fatty acid esters (tween 20 or 80); poloxamer (such as poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; tetradecyl, oleyl, or octadecyl sarcosine; pluronics; MONAQUATTM, etc., in an amount to minimize the tendency of the antibody fusion protein to granulate. Solution stabilizers preferably include, but are not limited to, one or a combination of the following list: saccharides, for example, reducing sugars and non-reducing sugars; amino acids, such as monosodium glutamate or histidine; alcohols, for example: trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol, and the like, the solution stabilizer should be added in an amount such that the resulting formulation remains stable for a period of time deemed stable by one skilled in the art. The isotonicity adjusting agent preferably includes, but is not limited to, one of sodium chloride, mannitol, or a combination thereof. Buffers preferably include, but are not limited to: tris, histidine buffer, phosphate buffer, or a combination thereof.
The invention also provides the application of the antibody fusion protein or the pharmaceutical composition in preparing a medicament for treating tumors.
The medicament for treating the tumor is a medicament for inhibiting and/or treating the tumor, and can comprise delay of development of symptoms related to the tumor and/or reduction of severity of the symptoms, further relieve of existing symptoms related to the tumor and prevent other symptoms, and reduce or prevent metastasis of the tumor and the like.
The tumors targeted by the drugs of the present invention preferably include, but are not limited to: breast cancer, lung cancer, bone cancer, stomach cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, rectal cancer, colon cancer, cancer of the anal region, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, cancer of the penis, prostate cancer, pancreatic cancer, cancer of the brain, cancer of the testis, cancer of the lymph, transitional cell cancer, cancer of the bladder, kidney or ureter, cancer of the renal cell, carcinoma of the renal pelvis, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, solid tumor of childhood, lymphocytic lymphoma, Central Nervous System (CNS) tumor, primary central nervous system lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, melanoma, Kaposi's sarcoma, and, Epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic or acute leukemia and combinations of said carcinomas.
When the antibody fusion protein and the composition thereof of the present invention are administered to animals including human, the administration dose varies depending on the age and body weight of the patient, the nature and severity of the disease, and the administration route, and the results of animal experiments and various cases can be referred to, and the total administration dose cannot exceed a certain range. The dosage of intravenous injection is 1-1800 mg/day.
The antibody fusion protein and the composition thereof disclosed by the invention can also be combined with other anti-tumor drugs for administration so as to achieve the purpose of more effectively treating tumors, and the anti-tumor drugs comprise but are not limited to: 1. cytotoxic drugs: 1) drugs that act on the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosoureas, methyl sulfonates; platinum compounds such as Cisplatin (cissplatin), Carboplatin (Carboplatin), and Oxaliplatin (Oxaliplatin); antibiotics such as Adriamycin (Adriamycin/Doxorubicin), actinomycin D (dactinomycin D), Daunorubicin (Daunorubicin), Epirubicin (Epirubicin), Mithramycin (Mithramycin), etc.; 2) drugs that affect nucleic acid metabolism: dihydrofolate reductase inhibitors such as Methotrexate (MTX) and Pemetrexed (Pemetrexed); thymidine synthase inhibitors such as fluorouracils (5-fluorouracil, capecitabine), etc.; purine nucleoside synthase inhibitors such as 6-mercaptopurine and the like; ribonucleotide reductase inhibitors such as hydroxyurea (hydroxyarbamide) and the like; DNA polymerase inhibitors such as cytarabine (cytisine arabinoside) and Gemcitabine (Gemcitabine), etc.; 3) tubulin-acting drugs: docetaxel (Docetaxel), vinblastine (vinchristine), Vinorelbine (Vinorelbine), podophylline, homoharringtonine, etc.; 2. hormone drugs: antiestrogens such as Tamoxifen (Tamoxifen), Droloxifene (Droloxifene), Exemestane (Exemestane), etc.; aromatase inhibitors such as Aminoglutethimide (Aminoglutethimide), Formestane (Formestane), letrozole (letrozole), Anastrozole (Anastrozole), etc.; anti-androgens: flutamide RH-LH agonists/antagonists: norrad, etalone, and the like; 3. biological response modifier drugs: the medicine has anti-tumor effect mainly by regulating body immunity function, such as Interferon (Interferon); interleukin-2 (Interleukin-2); thymosin peptides (Thymosins), and the like; 4. monoclonal antibody drugs: trastuzumab (Trastuzumab), Rituximab (Rituximab), Cetuximab (Cetuximab), Bevacizumab (Bevacizumab), and the like; 5. other classes of anti-tumor drugs: including some drugs whose current mechanism is not clear and which are to be further studied, etc. The antibody fusion protein and the composition thereof disclosed by the invention can be combined with one of the anti-tumor drugs or the combination thereof.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
firstly, the antibody fusion protein provided by the invention can block signal paths of HER2 and VEGFR2 at the same time. The in vitro activity detection result shows that: on a molecular level, the affinity of the antibody fusion protein HD2 to HER2 antigen is equivalent to that of a monoclonal antibody; HD2 has an affinity for VEGF comparable to that of Fc-D2; on a cellular level, HD2 can inhibit the proliferation of HUVEC (human umbilical vein endothelial cells), and the biological activity is equivalent to that of Fc-D2; HD2 can inhibit the proliferation of HER2 positive tumor cells, wherein the inhibition effect on NCI-N87, SK-OV3 and SK-BR3 is better than that on HER2 monoclonal antibody and HER2 monoclonal antibody + FcD 2.
Secondly, animal experiments show that the tumor inhibition effect of the antibody fusion protein HD2 is obviously superior to that of the anti-HER 2 monoclonal antibody with the same molar concentration, and the VEGFR1-D2 structural domain of HD2 plays a synergistic anti-tumor role.
Thirdly, the antibody fusion protein provided by the invention has strong stability, provides a candidate drug with better treatment effect for anti-tumor treatment, and has great application prospect in the treatment of tumor diseases.
Drawings
FIG. 1: schematic structural diagram of antibody fusion protein HD 2.
FIG. 2A: HPLC-SEC detection profile of antibody fusion protein HD 2.
FIG. 2B: polyacrylamide gel electrophoresis image of antibody fusion protein HD 2.
FIG. 3A: ELISA detected binding of antibody fusion protein HD2 to HER 2.
FIG. 3B: ELISA detected binding of antibody fusion protein HD2 to VEGF.
FIG. 4: ELISA detects that antibody fusion protein HD2 blocks binding of VEGF to VEGFR 2.
FIG. 5: FACS detects binding of the antibody fusion protein HD2 to BT474 cells.
FIG. 6A: proliferation inhibition curve of antibody fusion protein HD2 on BT474 tumor cells.
FIG. 6B: the proliferation inhibition curve of the antibody fusion protein HD2 on SK-BR3 tumor cells.
FIG. 6C: the proliferation inhibition curve of the antibody fusion protein HD2 on SK-OV3 tumor cells.
FIG. 6D: inhibition of proliferation of NCI-N87 tumor cells by antibody fusion protein HD 2.
FIG. 7: proliferation inhibition curves of antibody fusion protein HD2 against HUVEC.
FIG. 8A: results of pharmacokinetic parameters of antibody fusion protein HD2 in rats calculated by HER2 coating ELISA.
FIG. 8B: results of pharmacokinetic parameters of the antibody fusion protein HD2 in rats calculated by proteinA coating ELISA.
FIG. 9: curve of antibody fusion protein HD2 inhibiting tumor proliferation in mouse tumor model.
FIG. 10A: DSC profile of antibody fusion protein HD 2.
FIG. 10B: thermostability HPLC-SEC of antibody fusion protein HD 2.
Detailed Description
The following examples and experimental examples are intended to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cellsndedition,Cold spring Harbor Laboratory Press.
The experimental materials and sources used in the following examples and the methods of formulating the experimental reagents are specifically described below.
Experimental materials:
293E cells: from the NRC biotechnology Research Institute.
Human umbilical vein endothelial cells HUVEC: purchased from Sciencell.
Human breast cancer cells BT 474: from the cell bank of the Chinese academy of sciences.
Human ovarian cancer cell SK-OV 3: from the cell bank of the Chinese academy of sciences.
Human breast cancer cell SK-BR 3: from the cell bank of the Chinese academy of sciences.
Human gastric cancer cell line NCI-N87: purchased from the American Type Culture Collection (ATCC).
Protein A chip: label No. 29139131-AA; and lot is 10261132.
SD rat: is purchased from Zhejiang vitamin Tonglihua laboratory animal technology Co., Ltd, and produces a license SCXK (Zhe) 2018-.
BALB/c nude mice: purchased from Shanghai Ling Biotech, Inc.
Experimental reagent:
VEGF-A165: designated herein as VEGF, purchased from R & D, cat # 293-VE-010.
VEGFR 2: constructed from R & D, cat # 357-KD.
Biotinylated VEGF antibody: from R & D, cat # BAF 293.
HRP-labeled murine anti-human Fab antibodies: purchased from sigma, cat # a 0293.
Streptavidin HRP: purchased from BD Biosciences, cat # 554066.
Goat anti-human IgG-FITC: purchased from sigma, cat # F4143.
PBS: purchased from Biotechnology (Shanghai) Inc., cat # B548117.
PBST：PBS+0.05％Tween 20。
BSA: purchased from Biotechnology (Shanghai) Inc., cat # A60332.
TMB: purchased from BD corporation under item number 555214.
FBS: purchased from Gibco, cat # 10099.
HBS-EP working solution: available from Life science, BR-1006-69.
An experimental instrument:
HiTrap MabSelectSuRe column: purchased from GE company.
Beckman Coulter CytoFLEX flow cytometer: purchased from Beckman corporation.
SpectraMax i3x microplate reader: purchased from Molecular Devices, Inc.
Spectra maxm5 microplate reader: purchased from Molecular Devices, Inc.
The HER2 monoclonal antibodies in the embodiment of the invention are human-mouse chimeric monoclonal antibodies obtained by a cell culture production process which is expressed and independently developed by a CHO cell expression system according to the amino acid sequence of Herceptin in the three kingdoms healthcare industry.
Example 1 molecular construction of antibody fusion protein HD2
The invention adopts a mode of connecting anti-HER 2 monoclonal antibody IgG and D2 structural domain of VEGFR1 in series to construct antibody fusion protein HD 2. The D2 domain of VEGFR1 (SEQ ID NO: 14) and the heavy chain of the anti-HER 2 monoclonal antibody (SEQ ID NO: 7) were linked by peptide Linker (SEQ ID NO: 9) to give the heavy chain of the fusion protein (SEQ ID NO: 10). The light chain of HER2 mab (SEQ ID NO: 11) remained unchanged. To increase the expression efficiency of this molecule in 293E cells, Kingchi corporation was entrusted with codon optimization of the nucleic acid sequence of HD2 molecule. Optimization mainly considers factors such as codon preference, GC content, mRNA secondary structure, repetitive sequence and the like, and then entrusts the Jinzhi company to synthesize. The nucleic acid sequence of the spliced HD2 heavy chain is SEQ ID NO: 12, the light chain nucleic acid sequence is SEQ ID NO: 13. see appendix for sequences. The structure of HD2 is shown in figure 1.
Example 2 expression and purification of antibody fusion protein HD2
The DNA fragments of the heavy chain and the light chain of HD2 were cloned into pTT5 vector, respectively, and recombinant plasmids were extracted to co-transfect CHO cells and/or 293E cells. After culturing the cells for 5 to 7 days, the culture solution was subjected to high-speed centrifugation, vacuum filtration through a microfiltration membrane, and then applied to a HiTrap MabSelectSuRe column, and the proteins were eluted in one step with an eluent containing 100mM citric acid and having a pH of 3.5, and then the target sample was recovered and dialyzed into PBS having a pH of 7.4. The purified protein is detected by HPLC, and the HPLC-SEC detection spectra of HD2 are respectively shown in FIG. 2A, the antibody molecular state is uniform, and the monomer purity is more than 98%.
And (3) adding the purified antibody fusion protein HD2 into a non-reducing electrophoresis buffer solution respectively, carrying out SDS-polyacrylamide gel electrophoresis detection, adding the purified antibody fusion protein HD2 into a reducing electrophoresis buffer solution respectively, boiling, carrying out SDS-polyacrylamide gel electrophoresis detection, and obtaining an electrophoresis chart shown in figure 2B, wherein the theoretical molecular weight of the antibody fusion protein HD2 is 169 KD.
Example 3 enzyme-linked immunosorbent assay (ELISA) for determining the affinity of HD2 for HER2 antigen and VEGF
To examine the affinity of the HD2 antibody fusion protein to HER2 antigen, Sansheng Jian's HER2-ECD-His protein was diluted to 250ng/ml with PBS buffer, pH7.4, and then 100. mu.l/well was added to the ELISA plate and incubated overnight at 4 ℃. The next day, plates were washed twice with PBST, blocked by adding PBST + 1% BSA per well, blocked for 1h at 37 deg.C, and plates were washed twice with PBST. Then adding the antibody fusion protein HD2 to be detected diluted by PBS + 1% BSA gradient, using anti-HER 2 monoclonal antibody as positive control, the initial concentration is 100nM, and gradually diluting by 3 times for 12 gradients. Incubate at 37 ℃ for 1h, wash the plate with PBSTTwo times, adding HRP-labeled mouse anti-human Fab antibody, incubating at 37 deg.C for another 40min, washing the plate with PBST for three times, patting to dry, adding 100. mu.l of TMB to each well, standing at room temperature (20 + -5 deg.C) for 5 min in the dark, and adding 50. mu.l of 2M H to each well2SO4The substrate reaction is stopped, and the OD value is read at 450nm of the microplate reader. GraphPad Prism7 data analysis, mapping and EC calculation50The experimental results are shown in fig. 3A, HD2 and the positive control HER2 mab, EC binding to HER2500.2622 and 0.1942 respectively, and the affinity of the two is equivalent.
To test the binding capacity of HD2 to VEGF, VEGF was diluted to 500ng/ml with PBS pH7.4, 100. mu.l/well was added to the microplate, and coated overnight at 4 ℃. The plates were washed 2 times with PBST, blocked by adding PBS + 2% BSA to 200. mu.l/well, and washed 1 time with PBST after standing at 37 ℃ for 1 hour. The antibody fusion protein HD2 to be detected, which was diluted with a PBS + 1% BSA gradient, and Fc-D2 as a positive control, was then added at an initial concentration of 200nM, and the 12 gradients were diluted 3-fold in stages. Adding the blocked ELISA plate, placing at 100. mu.l/well for 1 hour at 37 ℃, washing the plate for 2 times by PBST, adding the mouse anti-human Fc antibody marked by HRP, placing at 37 ℃ for 30 minutes, washing the plate for 3 times by PBST, patting the residual liquid drops on absorbent paper as dry as possible, adding 100. mu.l of TMB into each well, placing at room temperature (20 +/-5 ℃) for 5 minutes in the dark, adding 50. mu.l of 2M H into each well2SO4The stop solution stops the substrate reaction, and the OD value is read at 450nm of the microplate reader. GraphPad Prism7 data analysis, mapping and EC calculation50. The results of the experiment are shown in FIG. 3B, and the EC of the antibody fusion protein HD2 and the positive control Fc-D2 binding to VEGF500.05403 and 0.04474, respectively, with comparable affinities.
Example 4 enzyme-linked immunosorbent assay (ELISA) assay HD2 blocks VEGF binding to VEGFR2
Since VEGF is a key step in regulating the proliferation and migration of vascular endothelial cells by binding to VEGFR2, VEGF binds more strongly to VEGFR1 than to VEGFR 2. This experiment therefore examined the ability of HD2 to block VEGF binding to VEGFR 2.
VEGFR2 was diluted to 400ng/mL in PBS pH7.4, 100. mu.L/well was added to the microplate, and coated overnight at 4 ℃. PBST washing plate 2 times, 200 u l/hole adding PBS + 2% BSA for blocking, 37 degrees C after 1 hours PBSThe plates were washed 2 times for use, VEGF diluted to 4nM with 1% BSA in PBS, and the samples to be tested were diluted again with 4nM, 1% BSA in PBS, starting at 200nM and diluted three-fold steps of 12 gradients. Add 100 u l/hole blocked ELISA plate, 37 degrees C placed 1 hours, PBST washing plate 2 times, with PBS + 1% BSA diluted biotinylated VEGF antibody to 0.2 u g/mL, 100 u l/hole adding ELISA plate, 37 degrees C placed 1 hours PBST washing plate 2 times. Adding HRP labeled Streptavidin (SA), incubating at 37 deg.C for 30min, washing the plate with PBST three times, patting to dry, adding 100. mu.l TMB to each well, standing at room temperature (20 + -5 deg.C) in the dark for 5 min, adding 50. mu.l 2M H to each well2SO4The substrate reaction is stopped, and the OD value is read at 450nm of the microplate reader. GraphPad Prism7 data analysis, mapping and IC calculation50The experimental results are shown in FIG. 4, and the IC of the antibody fusion protein HD2 and the positive control Fc-D2 blocking the binding of VEGF to VEGFR2501.587 and 1.466, respectively, which have equivalent blocking ability.
Example 5 detection of binding of HD2 to target cell BT474
In the experiment, human breast cancer cells BT474 with high cell surface HER2 expression are used as target cells, washed three times by PBS containing 0.5% BSA, centrifuged for 5 minutes at 300g each time, supernatant is discarded, the cells are resuspended by PBS containing 0.5% BSA, and the cell concentration is 1X 106Adding 100 mu L of cell/mL into a 96-well plate, diluting an antibody fusion protein HD2 and a positive control HER2 monoclonal antibody to 400nM, gradually diluting the diluted cells by 11 gradients, adding 100 mu L of cell/well into the 96-well plate, uniformly mixing BT474 cells, incubating the mixed cells at 4 ℃ for 1h, washing the cells twice by PBS to remove unbound antibody to be detected, incubating the cells with 100 mu L of 1g/mL goat anti-human IgG-FITC at 4 ℃ for 30 minutes, centrifuging the cells for 5 minutes at 300g, washing the cells twice by PBS to remove unbound secondary antibody, finally suspending the cells in 200 mu L of PBS, and determining the binding affinity of HD2 to the cells by a Beckman Coulter Cytoflex flow cytometer. The obtained data are analyzed by GraphPad Prism7 software fitting, the experimental result is shown in FIG. 5, the EC of the antibody fusion protein HD2 and the positive control HER2 monoclonal antibody combined with BT474 cells501.238 and 1.054, respectively, HD2 and the positive control HER2 mab were comparable in affinity to BT 474.
Example 6 inhibition of proliferation of HD2 on HER2 Positive tumor cells in vitro
The surfaces of NCI-N87, SK-OV3, SK-BR3 and BT474 tumor cells all have HER2 antigen expression; the supernatants of the three-day cell culture were subjected to ELISA detection, and these tumor cells were found to secrete a small amount of VEGF, with the supernatant being about 1-1.5 ng/mL.
Cells NCI-N87, SK-OV3, SK-BR3 and BT474 in logarithmic growth phase cultured by adherence are digested by pancreatin, counted after being resuspended, the cell density is adjusted by using a culture medium containing 1% FBS, and a 96-well cell culture plate is paved at 100 mu L/well. Wherein NCI-N87 is 10000/hole, BT474, SK-OV3 and SK-BR3 are 5000/hole respectively. Adding 200 μ L/well of culture medium or PBS, sealing, standing at 37 deg.C, and adding 5% CO2The culture was carried out overnight in an incubator. The next day the antibody to be detected is added. Three groups of samples, namely HD2, HER2 monoclonal antibody and HER2 monoclonal antibody + FcD2, are diluted by a culture medium containing 1% FBS to prepare a 300nM solution, and then the solution is diluted by 3 times step by step to obtain 10 gradients in total. A panel of FcD2 inhibition experiments at 1500nM starting concentration was also set as a control. The diluted sample, 100. mu.L/well, was added to the corresponding 96-well plate cells, and the cells were incubated at 37 ℃ with 5% CO2The culture was continued for 6 days. Incubating the cell culture plate for 6 days, adding 10 μ L/well of CCK-8 for color development, and adding CO2And (4) continuously incubating for 2-5 h in the incubator, and measuring the OD value by using an enzyme-labeling instrument with 650nm as a reference wavelength and 450 nm. The data obtained were analyzed by GraphPad Prism7 software and the results are shown in fig. 6A, 6B, 6C, 6D.
The result shows that the proliferation inhibition effect of HD2 on SK-BR3, SK-OV3 and NCI-N87 is better than that of HER2 monoclonal antibody and HER2 monoclonal antibody + FcD2, and FcD2 at the initial concentration of 1500nM has no inhibition effect on tumor cells. This shows that HD2 fusion protein may exert a synergistic effect, while the HER2 antibody of HD2 binds to tumor cells, the VEGF secreted by the tumor cells is neutralized by the VEGFR1-D2 domain, and the VEGF in the tumor cell microenvironment may have a certain promotion effect on the proliferation.
Example 7 inhibition of cell HUVEC proliferation in vitro by HD2
VEGF can stimulate the proliferation of HUVEC (human epithelial vehicle endothial cell). HD2 inhibits HUVEC proliferation in vitro by binding to VEGF.
Digesting cultured HUVEC with pancreatin, re-suspending, counting cells with cell viability of above 95%, washing with sterile PBS once, and re-suspending to 3 × 10 with 0.5% FBS-containing ECM basal medium4cells/mL, 100. mu.L/well were added to the middle 60 wells of a 96-well cell culture plate, the remainder was filled with medium, left at 37 ℃ with 5% CO2The incubator was incubated overnight. The next day, VEGF was diluted to 60ng/mL using 0.5% FBS-containing ECM basal medium, and the sample to be tested, HD2, Fc-D2, negative control IgG1 starting concentration 400nM, was diluted three times step by step with 10 gradients, added to a 96 well plate with HUVEC in the middle, 37 ℃, 5% CO2After three days of culture in the incubator, the supernatant was aspirated, 10. mu.L/well of CCK-8 was added for color development, and the incubation was continued for 4 to 8 hours, and the OD value was measured at 450nm with 650nm as the reference wavelength using an microplate reader. The data obtained were analyzed by GraphPad Prism7 software, and the results are shown in FIG. 7, IC of HD2 and positive control Fc-D2500.2982 and 0.2429 respectively have equivalent inhibition rates, and the negative control IgG1 antibody has no inhibition effect on the proliferation of HUVEC.
Example 8 Octet determination of the affinity dissociation constant KD of HD2 for antigen
The kinetic parameters of the dissociation of the binding between HD2 and the antigen HER2-ECD-his were determined by the Protein A capture method, HD2 was bound to the Protein A chip at a concentration of 5. mu.g/ml, the antigen HER2-ECD-his was diluted with 1 XHBS working solution, and the antibody was bound to the antigen by 6 concentration gradients and dissociated in HBS working solution.
The kinetic parameters of the dissociation of the binding of HD2 and antigen VEGF-A165 were determined by the proteInA capture method, HD2 was bound to the Protein A chip at a concentration of 5. mu.g/ml, the antigen VEGF was diluted with 1 XHBS working solution, and the antibody was bound to the antigen by 6 concentration gradients and dissociated in HBS working solution.
The affinity dissociation constants of HD2 and the two groups of antigens are shown in the following table, and the results show that HD2 has good affinity with the antigens HER2 and VEGF.
TABLE 1
Figure BDA0002216270190000131
Figure BDA0002216270190000141
KD is the affinity constant; kon is the binding rate constant; kdis the dissociation rate constant.
Example 9 pharmacokinetic Studies of antibody fusion protein HD2
4 SD rats weighing about 200g were injected with 2mg of antibody fusion protein HD2 per rat via the tail vein. Blood was collected from the orbit at intervals after the administration, and serum was collected by centrifugation at 8000rpm/min after the blood had naturally coagulated. The drug concentration in the serum of HD2 was determined by the following method:
1) HER2-His coated ELISA plates, 50 ng/well, 4 ℃ coated overnight, next day PBST plate washing twice, then with PBS + 2% BSA at 37 ℃ blocked for 2 hours. The starting HD2 standard at 1000ng/mL was diluted two-fold in stages for 12 gradients. Serum samples were diluted 2000-fold, added to the blocked ELISA plates, incubated for one hour at 37 deg.C, then washed twice with PBST, added with HRP-labeled murine anti-human Fab antibody, diluted 1:3000, 100. mu.L/well. Incubate at 37 ℃ for 40 min. PBST was washed 4 times, patted dry, 100. mu.l of TMB was added to each well, and left at room temperature (20. + -. 5 ℃) in the dark for 5 minutes, 50. mu.l of 2M H was added to each well2SO4The stop solution stops the substrate reaction, and the OD value is read at 450nm of the microplate reader.
2) The ELISA plate was coated with protein A and the antibody Fab fragment was detected, with the amount of protein A coating being 100 ng/well, overnight at 4 deg.C, washed twice with the next day of PBST, and blocked with PBS + 2% BSA at 37 deg.C for 2 hours. The plates were washed twice with PBST and the HD2 standard was diluted two-fold stepwise starting from 1000ng/mL for 12 gradients. Diluting the rat serum sample by 2000 times, adding the two groups of samples into a sealed ELISA plate, incubating for 1 hour, washing the plate twice by PBST, adding a mouse anti-human Fab antibody marked by HRP, placing the plate for 30 minutes at 37 ℃, after washing the plate for 3 times by PBST, patting the residual liquid drops on absorbent paper as dry as possible, adding 100 mu l of TMB into each hole, placing the plate for 5 minutes at room temperature (20 +/-5 ℃) in a dark place, and adding 50 mu l of 2M H into each hole2SO4The stop solution stops the substrate reaction, and the OD value is read at 450nm of the microplate reader.
The half-life period of the antibody drug in the rat body is calculated by using Phoenix software, pharmacokinetic parameters are shown in the following table, experimental results are shown in figures 8A and 8B, the half-life period in the rat body detected by using the two ELISA methods is 183h and 203h respectively, the half-life period results calculated by the two ELISA methods are not very different, and the data are reliable.
The half-life of HD2 was calculated using HER2 test as shown in the table below.
TABLE 2
Figure BDA0002216270190000142
Figure BDA0002216270190000151
The half-life of HD2 was calculated using the proteinA assay as follows:
TABLE 3
Rat HL_Lambda_z(hr) Cmax(ug/mL)
1 159.76559 74
2 159.58479 73
3 261.44609 59
4 230.41973 53
Average 203h
Example 10 antitumor Effect of HD2 on NCI-N87 transplant tumor model
The human gastric cancer cell strain NCI-N87 expresses HER2 antigen on the cell surface, and HER2 antibody is combined with the cell strain NCI-N87, so that the cell signal path can be blocked, and the tumor proliferation can be inhibited. Collecting in vitro cultured human gastric cancer cell line NCI-N87 cells, adjusting cell concentration to 5 × 107Suspending the cells/mL in serum-free medium, inoculating 100 μ L of cell suspension under aseptic condition to the dorsal subcutaneous part of the nude mouse, measuring the length and width of the transplanted tumor with vernier caliper, calculating the tumor volume, and waiting for the tumor growth to 100-200mm3Animals were then randomized into groups. The dosage of the HD2 sample to be detected is divided into two groups, 17mg/kg and 1.7mg/kg, the dosage of the positive control drug HER2 monoclonal antibody is 15mg/kg, and the dosage is equal to the molar weight of HD 2. The control group was administered the same volume of PBS intraperitoneally at a volume of 0.2 mL/mouse (20g) twice weekly for three consecutive weeks, and 2 graft tumor volumes were measured weekly. The experimental result is shown in fig. 9, on the NCI-N87 nude mouse transplantation tumor model, the antibody fusion protein HD2 shows in vivo anti-tumor activity, and there is a dose-dependent relationship, and compared with HER2 monoclonal antibody of the same molar concentration, the HD2 fusion protein has better tumor-inhibiting effect than HER2 monoclonal antibody, which indicates that the second extracellular region D2 domain of VEGFR1 of HD2 exerts a synergistic anti-tumor effect.
Example 11 thermal stability Studies of HD2
The experiment used a MicroCal VP-Capillary DSC, which was filtered through a 0.22um filter with a sample and its buffer, and 400. mu.l of the sample and its matching buffer were placed in a 96-well plate and scanned at 25 ℃ to 100 ℃ at a scanning rate of 120 ℃ per hour, and HD2 was stored in PBS at pH 7.4. The Tm value of HD2 detected by DSC is shown in Table 4, and the map is shown in FIG. 10A, therefore, the antibody fusion protein HD2 is relatively stable, and the following 37 ℃ stability experiment result also verifies the point.
Table 4: DSC value of HD2
Sample number Tm Onset Tm1 Tm2
HD2 67.2 72.2 82.2
Fc-D2 63.77 71.3 82.1
Stability at 37 ℃ experiment: the HD2 fusion protein was dialyzed into Ph7.4 PBS buffer, adjusted to a concentration of 2mg/mL, placed in a 37 ℃ incubator at intervals, sampled and checked for purity by HPLC-SEC. As a result, the purity of HPLC-SEC was almost unchanged at 14 days, and was 97%, and the purity of HPLC-SEC was slightly decreased at 95% in the HPLC-SEC detection result at 28 days, indicating that the HD2 fusion protein was stable. The HPLC-SEC profile is shown in FIG. 10B.
As can be seen from the above experiments, the HD2 antibody fusion protein provided by the invention has the same affinity to antigen and target cells as the monoclonal antibody; meanwhile, the compound has good biological activity, can inhibit the proliferation of HER2 positive tumor cells, and has better inhibition effect than HER2 monoclonal antibody and HER2 monoclonal antibody + FcD2 on NCI-N87, SK-BR3 and SK-OV3 tumor cells; and can inhibit the proliferation of human umbilical vein endothelial cell HUVEC. The detection result on a mouse tumor model shows that HD2 with the same molar concentration has the effect of inhibiting tumor proliferation better than HER2 monoclonal antibody; and the antibody fusion protein has strong stability and wide application prospect.
Sequence listing
<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH
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ccttccgtgt tcctgttccc ccctaagccc aaggacaccc tgatgatttc caggacaccc 780
gaggtgacct gtgtggtggt ggacgtcagc cacgaggacc ccgaggtgaa attcaactgg 840
tacgtcgatg gcgtggaggt gcacaacgct aagaccaagc ccagggagga gcagtacaat 900
tccacctaca gggtggtgtc cgtgctgacc gtcctccatc aggactggct gaacggcaaa 960
gagtataagt gcaaggtgag caacaaggcc ctccctgctc ccatcgagaa gaccatcagc 1020
aaagccaagg gccagcccag ggaacctcaa gtctataccc tgcctcccag cagggaggag 1080
atgaccaaga accaagtgag cctcacatgc ctcgtcaagg gcttctatcc ttccgatatt 1140
gccgtcgagt gggagtccaa cggacagccc gagaacaact acaagacaac accccccgtg 1200
ctcgattccg atggcagctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 1260
caacaaggca acgtcttcag ttgcagcgtc atgcatgagg ccctccacaa ccactacacc 1320
cagaagagcc tctccctgag ccctggaaag ggcggtgggg gaagtggagg cggtgggagc 1380
gacaccggca ggcccttcgt ggagatgtac agcgaaatcc ccgaaatcat ccacatgacc 1440
gagggcaggg agctggtgat cccgtgcagg gtgaccagcc ccaacatcac cgtgaccctg 1500
aagaagttcc ccctggacac cctgattccc gacggcaaga ggatcatctg ggacagcagg 1560
aagggcttca tcatcagcaa cgccacctac aaggagatcg gcctgctgac ctgcgaggcc 1620
accgtcaacg gccacctgta caagaccaac tacctgaccc acaggcagac caataccatc 1680
<210> 13
<211> 642
<212> DNA
<213> Composite
<400> 13
gacatccaga tgacccagag ccctagctcc ctgagcgcga gcgtgggaga cagggtcacc 60
atcacatgca gggcctccca ggacgtgaac accgctgtcg cctggtacca gcagaagccc 120
ggcaaggccc ctaagctgct gatctacagc gccagcttcc tgtacagcgg cgtcccttcc 180
aggttctccg gaagcagatc cggcaccgac ttcaccctga ccatctccag cctccagccc 240
gaggacttcg ccacctacta ctgtcagcag cactacacca cccctcccac cttcggacag 300
ggcaccaagg tggagatcaa gagaaccgtc gccgctccca gcgtcttcat cttccccccc 360
agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480
gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600
ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642
<210> 14
<211> 101
<212> PRT
<213> Composite
<400> 14
Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
1 5 10 15
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
35 40 45
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
50 55 60
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
65 70 75 80
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95
Gln Thr Asn Thr Ile
100

Claims (14)

1. An antibody fusion protein capable of blocking both HER2 and VEGFR signal pathways, comprising the anti-HER 2 monoclonal antibody IgG and the D2 domain of VEGFR1, wherein the D2 domain of VEGFR1 is linked to the C-terminus of the IgG heavy chain via a peptide linker L.
2. The antibody fusion protein of claim 1, wherein the heavy chain of IgG comprises the complementarity determining region HCDR1-3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown as SEQ ID NO:2, the amino acid sequence of HCDR3 is shown as SEQ ID NO: 3 is shown in the specification;
the light chain of the IgG comprises the complementarity determining region LCDR1-3, wherein the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown as SEQ ID NO: 5, the amino acid sequence of LCDR3 is shown in SEQ ID NO: and 6.
3. The antibody fusion protein of claim 1, wherein the heavy chain variable region of the IgG has the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.
4. The antibody fusion protein of claim 1, wherein the amino acid sequence of the peptide linker L is as set forth in SEQ ID NO: shown at 9.
5. The antibody fusion protein of claim 1, wherein the heavy chain amino acid sequence of the antibody fusion protein is as set forth in SEQ ID NO: 10, and the light chain amino acid sequence is shown as SEQ ID NO: shown at 11.
6. A nucleotide molecule encoding the antibody fusion protein of any one of claims 1-5.
7. The nucleotide molecule of claim 6, wherein the nucleotide sequence encoding the heavy chain of the antibody fusion protein is as set forth in SEQ ID NO: 12, and the nucleotide sequence encoding the light chain is shown as SEQ ID NO: shown at 13.
8. An expression vector comprising the nucleotide molecule of any one of claims 6 or 7.
9. The expression vector of claim 8, wherein the expression vector is selected from the group consisting of pDR1, pcdna3.4(+), pDHFR and pTT 5.
10. A host cell comprising the expression vector of claim 8.
11. The host cell according to claim 10, wherein the host cell is a eukaryotic cell, preferably selected from the group consisting of CHO cells and 293E cells.
12. A method of producing an antibody fusion protein according to any one of claims 1 to 5, comprising the steps of:
a) culturing the host cell of any one of claims 10-11 under expression conditions such that an antibody fusion protein is expressed that blocks both HER2 and VEGFR signaling pathways;
b) isolating and purifying the antibody fusion protein of step a).
13. A composition comprising an antibody fusion protein of any one of claims 1-5 and one or more pharmaceutically acceptable carriers, diluents, or excipients.
14. Use of an antibody fusion protein according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 13, for the preparation of a medicament for the treatment of a tumor.
CN201910916586.9A 2019-09-26 2019-09-26 Antibody fusion protein, preparation method thereof and application thereof in anti-tumor Pending CN112552410A (en)

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