CN113698493B - Double-function protein aiming at VEGF and TGF-beta and application thereof - Google Patents

Double-function protein aiming at VEGF and TGF-beta and application thereof Download PDF

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CN113698493B
CN113698493B CN202110906277.0A CN202110906277A CN113698493B CN 113698493 B CN113698493 B CN 113698493B CN 202110906277 A CN202110906277 A CN 202110906277A CN 113698493 B CN113698493 B CN 113698493B
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CN113698493A (en
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白义
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Beijing Dongfang Baitai Biotechnology Co ltd
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Abstract

The invention relates to the field of biological medicine, and particularly provides a bifunctional protein aiming at VEGF and TGF-beta, which comprises an anti-VEGF monoclonal antibody, a connecting peptide linker and a TGF-beta RII extracellular domain polypeptide, wherein the anti-VEGF monoclonal antibody can be specifically combined with a VEGF antigen, and is connected with the TGF-beta RII extracellular domain polypeptide through the connecting peptide linker. The bifunctional protein aiming at VEGF and TGF-beta provided by the invention can target a tumor microenvironment with high expression of VEGF and reduce the side effect of a TGF-beta inhibitor, and the bifunctional protein can be used singly and can be combined with the existing immunotherapy drugs such as a PD-1/PD-L1 inhibitor to effectively improve the treatment effect of tumor diseases.

Description

Double-function protein aiming at VEGF and TGF-beta and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a double-function protein aiming at VEGF and TGF-beta and application thereof.
Background
With the continuous development of biological medicines, immunotherapy of the anti-PD-1/PD-L1 monoclonal antibody makes a major breakthrough in the field of cancer treatment, and more than 15 kinds of cancers, such as metastatic melanoma, bladder cancer, non-small cell lung cancer and the like, are treated more effectively. However, the anti-PD-1/PD-L1 monoclonal antibody is also effective in only 20-30% of cancer patients, and many patients develop resistance quickly even if they initially respond to treatment. At the same time, there is increasing evidence that many cancers can develop multiple strategies to evade treatment, and there is a need to develop new immunotherapies against cancer.
Research shows that the tumor microenvironment is a key factor of immunotherapy, the transforming growth factor TGF-beta is an important cell factor in the tumor microenvironment and is used as a multifunctional cell factor, and the TGF-beta plays an important role in physiological activities such as growth, differentiation and migration of cells and has multiple functions in the development of cancers. TGF-beta can inhibit the proliferation of tumor cells and promote their differentiation and apoptosis during the tumorigenic stage, and as tumors progress, the tumors become insensitive to TGF-beta due to loss of TGF-beta receptor expression or mutation of downstream signaling molecules, at which time the effect of TGF-beta translates into promoting tumor progression. It is documented that TGF- β is overexpressed in most cancers, and plays a key role in regulating the adaptive immune system as well as promoting Epidermal Mesenchymal Transition (EMT), infiltration, and metastasis of tumor cells. TGF-beta can not only inhibit the expression of IFN-gamma, limit the differentiation of TH1 cells, weaken the activation and cytotoxicity of CD8+ effector T cells and NK cells and inhibit the development of central memory T cells, but also can induce the differentiation of Tregs (an immunosuppressive CD4+ T cell which is an index of poor prognosis in various cancers) and is documented in the literature that high-level TGF-beta in serum is positively correlated with poor prognosis; the primary reason for the unresponsiveness to PD-1/PD-L1 inhibitors is the high level of TGF-. beta.in the tumor microenvironment.
Currently, drug development for TGF-beta targets is mainly divided into three categories: 1) blocking binding of TGF-beta to its receptor; 2) blocks TGF-beta signaling pathway; 3) interfering with the production of TGF-beta. However, the existing inhibitors targeting the TGF- β signaling pathway bring about cardiotoxicity by acting on TGF- β type I receptor kinase or inhibiting the activity of three isomers at the same time, and do not show clinical effectiveness at clinically acceptable doses, so antibody drugs targeting the TGF- β signaling pathway are still under constant research.
The vascular endothelial cell growth factor VEGF is a main regulator in the process of tumor angiogenesis, and is another protein molecule playing an important role in the tumor microenvironment, and has a plurality of common points with TGF-beta in the tumor microenvironment environment, such as promotion of angiogenesis, promotion of metastasis, inhibition of immune response, positive correlation between high-level expression and poor prognosis, and the like. The main VEGF-targeting drugs include bevacizumab (bevacizumab) and aflibercept (VEGF trap), of which bevacizumab was first approved by the FDA and widely used in clinical treatment of colorectal cancer, lung cancer and renal cancer. However, bevacizumab has fewer applicable symptoms, larger side effects, controversial curative effect and easy drug resistance, so that the bevacizumab has great improvement space. The research finds that: glioblastoma that is non-responsive to the VEGF-targeting antibody bevacizumab and breast cancer patients that are HER2 negative, TGF- β levels are higher in the tumor; in preclinical models and some clinical experiments, the combination of VEGF and TGF-beta inhibitor is better than single use.
In the tumor microenvironment, a variety of immune cells, including T cells and NK cells, as well as a variety of signaling factors, including VEGF and TGF- β, are involved in the response to tumors. The mechanism by which tumor cells utilize for escape is never unique, so treatments directed to a single target tend to be of limited effectiveness, while combinations of treatments directed to multiple targets generally yield better results. Both VEGF and TGF-beta have mechanistically effects in promoting tumor angiogenesis and in suppressing immune responses, while the TGF-beta signaling pathway is generally thought to promote VEGF expression; however, there have also been several studies showing that VEGF also regulates TGF-. beta.1 expression through the PI3K/Akt signaling pathway. Thus, TGF- β and VEGF can modulate each other through multiple pathways, and if both signaling factors can be blocked simultaneously, it may be more favorable to switch the tumor microenvironment in the tumor-suppressive direction. In China, a plurality of enterprises develop bifunctional proteins aiming at PD-L1 and TGF-beta and bifunctional proteins aiming at PD-1 and TGF-beta into clinical stages, but the information that bifunctional protein medicaments aiming at VEGF and TGF-beta enter the clinical stages is still not disclosed, so that the development of a bifunctional protein medicament aiming at VEGF and TGF-beta is urgently needed in order to meet the requirements of more cancer patients.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a bifunctional protein drug aiming at VEGF and TGF-beta, which targets the tumor microenvironment with high expression of VEGF through an anti-VEGF monoclonal antibody part, introduces a TGF-beta receptor part, neutralizes VEGF and TGF-beta in the tumor microenvironment, inhibits tumor angiogenesis, removes immune suppression and further realizes a better tumor suppression effect.
The specific technical scheme of the invention is as follows:
the invention provides a bifunctional protein aiming at VEGF and TGF-beta, which comprises an anti-VEGF monoclonal antibody, a connecting peptide linker and a TGF-beta RII extracellular domain polypeptide, wherein the anti-VEGF monoclonal antibody can be specifically combined with a VEGF antigen, and is connected with the TGF-beta RII extracellular domain polypeptide through the connecting peptide linker.
Further, the anti-VEGF monoclonal antibody comprises two heavy chains and two light chains, and the TGF-beta RII extracellular domain polypeptide is connected with the C terminal or the N terminal of the two heavy chains or the two light chains of the anti-VEGF monoclonal antibody through the connecting peptide linker.
Further, the amino acid sequence of the heavy chain is SEQ ID NO. 1, and the amino acid sequence of the light chain is SEQ ID NO. 2; the amino acid sequence of the TGF-beta RII extracellular domain polypeptide is SEQ ID NO 3.
Further, the general formula of the connecting peptide linker is (GGGGS) n;
wherein n is an integer of 1 to 6;
preferably, n is 4.
Further, the bifunctional protein is selected from any one of the following:
the protein EB-1 is formed by connecting the TGF-beta RII extracellular domain polypeptide with the C tail ends of two heavy chains of an anti-VEGF monoclonal antibody through a connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-1 is SEQ ID NO. 4, and the amino acid sequence of the light chain of the protein EB-1 is SEQ ID NO. 2;
the protein EB-2 is formed by connecting the TGF-beta RII extracellular domain polypeptide with the N tail ends of two heavy chains of an anti-VEGF monoclonal antibody through the connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-2 is SEQ ID NO. 5, and the amino acid sequence of the light chain of the protein EB-2 is SEQ ID NO. 2;
the protein EB-3 is formed by connecting the TGF-beta RII extracellular domain polypeptide with the C tail ends of two light chains of an anti-VEGF monoclonal antibody through the connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-3 is SEQ ID NO. 1, and the amino acid sequence of the light chain is SEQ ID NO. 6;
and the protein EB-4 is formed by connecting the TGF-beta RII extracellular domain polypeptide with the N tail ends of two light chains of the anti-VEGF monoclonal antibody through the connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-4 is SEQ ID NO. 1, and the amino acid sequence of the light chain is SEQ ID NO. 7.
The invention also provides a nucleotide molecule encoding a bifunctional protein targeting VEGF and TGF- β according to any one of claims 1 to 5.
The present invention also provides a recombinant DNA expression vector comprising the nucleotide molecule of claim 7.
The invention also provides a host cell transfected with the recombinant DNA expression vector, and the host cell comprises prokaryotic, yeast or mammalian cells.
The invention also provides the application of the bifunctional protein aiming at VEGF and TGF-beta in the preparation of drugs for treating tumor diseases or fibrotic diseases;
the tumor disease is selected from colorectal cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, renal cancer, gastric cancer, liver cancer, ovarian cancer, melanoma or glioma;
the fibrotic diseases include liver fibrosis and lung fibrosis.
The invention also provides the application of the bifunctional protein aiming at VEGF and TGF-beta and an immunoregulation medicament or an anti-inflammatory factor medicament in the preparation of medicaments for treating tumor diseases; the immunoregulatory drug is selected from an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA 4 monoclonal antibody, an anti-4-1 BB monoclonal antibody, an anti-OX-40 monoclonal antibody, an anti-PD-L2 monoclonal antibody, an anti-LAG-3 monoclonal antibody, an anti-TIGIIT monoclonal antibody, an anti-GITR monoclonal antibody, an anti-ICOS monoclonal antibody, an anti-PVR monoclonal antibody, an anti-PVRIG monoclonal antibody, an anti-VISTA monoclonal antibody or an anti-TIMS monoclonal antibody;
the anti-inflammatory agent is selected from: anti-TNFa monoclonal antibody, anti-IL-1 beta monoclonal antibody or IL-1 receptor antagonist, anti-IL-6R monoclonal antibody, anti-IL-8 monoclonal antibody, recombinant IL-15 or IL-15 agonist;
preferably, the neoplastic disease is selected from colorectal cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, renal cancer, gastric cancer, liver cancer, ovarian cancer, melanoma or glioma.
The invention has the following beneficial effects: the anti-VEGF and anti-TGF-beta bispecific antibody provided by the invention can target a VEGF high-expression tumor microenvironment and reduce the side effect of a TGF-beta inhibitor, the bispecific antibody can be used singly, and can be combined with the existing immunotherapy drugs such as a PD-1/PD-L1 inhibitor to effectively improve the treatment effect of tumor diseases, and the tumor diseases comprise but are not limited to the following: colorectal cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, ovarian cancer, melanoma or glioma and other solid tumors.
Drawings
FIG. 1 is a first diagram showing the structure of a bifunctional protein against VEGF and TGF-. beta.in example 2 of the present invention;
FIG. 2 is a diagram showing the structure of a bifunctional protein against VEGF and TGF- β according to example 2 of the present invention;
FIG. 3 is a diagram of the structure type III of a bifunctional protein against VEGF and TGF-beta in example 2 of the present invention;
FIG. 4 is a diagram of the structure type of a bifunctional protein against VEGF and TGF-beta in example 2 of the present invention;
FIG. 5 is a plasmid map of the pTSE vector in example 4 of the present invention;
FIG. 6 is a diagram showing the binding ability of the bifunctional protein molecules of example 5 of the present invention against VEGF and TGF- β to VEGF;
FIG. 7 is a diagram showing the binding ability of the bifunctional protein molecules against VEGF and TGF-beta to TGF-beta 1 in example 5 of the present invention;
FIG. 8 is a diagram showing the binding ability of the bifunctional protein molecules against VEGF and TGF-beta to TGF-beta 3 in example 5 of the present invention;
FIG. 9 is a diagram showing the binding ability of a bifunctional protein molecule against VEGF and TGF-beta to TGF-beta 2 in example 5 of the present invention;
FIG. 10 is a graph showing the inhibition of VEGF proliferation promotion by a bifunctional protein molecule against VEGF and TGF- β in example 6 of the present invention;
FIG. 11 is a graph showing the effect of VEGF on cell migration inhibition by bifunctional protein molecules directed against VEGF and TGF- β in example 7 of the present invention;
FIG. 12 is a graph showing that bifunctional protein molecules against VEGF and TGF- β of example 8 inhibit the release of cell surface TGF- β 1;
FIG. 13 is a graph showing the tumor suppression effect of bifunctional protein molecules against VEGF and TGF- β (administered alone) in a mouse colon cancer model in example 9 of the present invention;
FIG. 14 is a graph showing the tumor suppression effect of bifunctional protein molecules against VEGF and TGF- β (administered in combination with an anti-PD-1 antibody) in a mouse colon cancer model in example 10 of the present invention.
Detailed Description
In order that the invention may be more readily understood, before describing the embodiments, certain technical and scientific terms of the invention are set forth below:
as used herein, the term "TGF-beta (transforming growth factor-beta)", i.e., transforming growth factor-beta, belongs to a group of newly discovered TGF-beta superfamilies that regulate cell growth and differentiation.
The term "VEGF (vascular endothelial growth factor)", as used herein, is a vascular endothelial growth factor (vascular endothelial growth factor, also referred to earlier as Vascular Permeability Factor (VPF), which is a heparin-binding growth factor (vascular endothelial cell-specific factor) that induces angiogenesis in vivo. The factor can effectively promote the regeneration of blood vessels and has important effect on medical research.
The term "TGF-. beta.RII extracellular domain polypeptide" as used herein, is the extracellular domain of the type II receptor of TGF-. beta.s.
The term "antibody" as used herein encompasses whole antibodies and any antigen-binding fragment thereof, including murine, humanized, bispecific or chimeric antibodies, which may also be Fab, F (ab)2, Fv or ScFv (single chain antibody), which may be naturally occurring antibodies or antibodies which may be altered (e.g., mutated, deleted, substituted, etc.).
As used herein, the terms "variable region" and "constant region", i.e., the regions of the heavy and light chains of an antibody adjacent to the N-segment are variable regions (V regions), the remaining amino acid sequences adjacent to the C-segment are relatively stable, and are constant regions (C regions), the variable regions include 3 Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs), each of the light and heavy chain variable regions consists of 3 CDR regions and 4 FR regions, the 3 CDR regions of the heavy chain are represented by HCDR1, HCDR2 and HCDR3, respectively, and the 3 CDR regions of the light chain are represented by LCDR1, LCDR2 and LCDR3, respectively.
The term "CHO cell" is a chinese hamster ovary cell (chinese hamster ovary cell); the term "HEK 293 cells" was human embryonic kidney 293E cells (human embryo kidney 293E cells), and the term "NS 0 cells" was mouse NS0 thymoma cells.
The present invention will be described in further detail with reference to the following examples.
Example 1
The invention embodiment 1 constructs a bifunctional protein aiming at VEGF and TGF-beta, which comprises an anti-VEGF monoclonal antibody, a connecting peptide linker and TGF-beta RII extracellular domain polypeptide, wherein the anti-VEGF monoclonal antibody can be specifically combined with VEGF antigen, and is connected with the TGF-beta RII extracellular domain polypeptide through the connecting peptide linker. The anti-VEGF monoclonal antibody comprises two heavy chains and two light chains, and the TGF-beta RII extracellular domain polypeptide is connected with the C terminal or the N terminal of the two heavy chains or the two light chains of the anti-VEGF monoclonal antibody through a connecting peptide linker.
Example 2
In the embodiment 2 of the invention, on the basis of the embodiment 1, the anti-VEGF monoclonal antibody is further defined, the anti-VEGF monoclonal antibody defined herein is a monoclonal antibody taking VEGF as a target, the invention is preferably bevacizumab, the amino acid sequence of the heavy chain is SEQ ID NO. 1, and the amino acid sequence of the light chain is SEQ ID NO. 2; the amino acid sequence of the TGF-beta RII extracellular domain polypeptide is SEQ ID NO 3.
The linker of the connecting peptide has a general formula of (GGGGS) n; and n is 4.
The bispecific antibody is selected from any one of the following:
the protein EB-1 is formed by connecting TGF-beta RII extracellular domain polypeptide with the C tail ends of two heavy chains of an anti-VEGF monoclonal antibody through a connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-1 is SEQ ID NO. 4, the amino acid sequence of the light chain of the protein EB-1 is SEQ ID NO. 2, and the configuration is shown in figure 1;
the protein EB-2 is formed by connecting TGF-beta RII extracellular domain polypeptide with the N tail ends of two heavy chains of an anti-VEGF monoclonal antibody through a connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-2 is SEQ ID NO. 5, the amino acid sequence of the light chain of the protein EB-2 is SEQ ID NO. 2, and the configuration is shown in figure 2;
the protein EB-3 is formed by connecting TGF-beta RII extracellular domain polypeptide with the C tail ends of two light chains of an anti-VEGF monoclonal antibody through a connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-3 is SEQ ID NO. 1, the amino acid sequence of the light chain of the protein EB-3 is SEQ ID NO. 6, and the configuration is shown as figure 3;
the protein EB-4 is formed by connecting TGF-beta RII extracellular domain polypeptide with the N tail ends of two light chains of an anti-VEGF monoclonal antibody through a connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-4 is SEQ ID NO. 1, the amino acid sequence of the light chain is SEQ ID NO. 7, and the configuration is shown in figure 4.
The specific sequence is as follows:
1 (amino acid sequence of heavy chain of anti-VEGF monoclonal antibody)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
2 (amino acid sequence of light chain of anti-VEGF monoclonal antibody)
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
3 (amino acid sequence of TGF-beta RII extracellular domain polypeptide)
GIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD；
SEQ ID NO 4 (amino acid sequence of heavy chain of protein EB-1)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD；
SEQ ID NO:5 (amino acid sequence of heavy chain of protein EB-2)
GIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
SEQ ID NO 6 (amino acid sequence of light chain of protein EB-3)
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD；
SEQ ID NO. 7 (amino acid sequence of light chain of protein EB-4)
GIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
Example 3
The embodiment 3 of the invention provides a nucleotide molecule which encodes bifunctional proteins aiming at VEGF and TGF-beta.
The invention also provides a recombinant DNA expression vector which comprises the nucleotide molecule.
The invention also provides a host cell for transfecting the recombinant DNA expression vector, wherein the host cell comprises prokaryotic cells, yeast cells or mammalian cells.
The invention also provides the application of the bifunctional protein aiming at VEGF and TGF-beta in the preparation of the drugs for treating tumor diseases or fibrotic diseases;
the tumor disease is selected from colorectal cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, renal cancer, gastric cancer, liver cancer, ovarian cancer, melanoma or glioma;
fibrotic diseases include liver fibrosis and pulmonary fibrosis.
The invention also provides the application of the combination of the bifunctional protein aiming at VEGF and TGF-beta and an immunoregulation medicament or an anti-inflammatory factor medicament in preparing a medicament for treating tumor diseases; the immunomodulator is selected from anti-PD-1 monoclonal antibody, anti-PD-L1 monoclonal antibody, anti-CTLA 4 monoclonal antibody, anti-4-1 BB monoclonal antibody, anti-OX-40 monoclonal antibody, anti-PD-L2 monoclonal antibody, anti-LAG-3 monoclonal antibody, anti-TIGIT monoclonal antibody, anti-ICOS monoclonal antibody, anti-PVR monoclonal antibody, anti-PVRIG monoclonal antibody, anti-VISTA monoclonal antibody or anti-TIMS monoclonal antibody;
preferably, the immunomodulatory drug is an anti-PD-1 monoclonal antibody, and the anti-PD-1 monoclonal antibody is selected from Nivolumab, Pembrolizumab, Tereprinizumab, Cedilizumab, Terralizumab, Carrilizumab or DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 or DFPD1-13 disclosed in patent document with patent number ZL201510312910.8 and patent name of one anti-PD-1 monoclonal antibody and obtaining method thereof, and is not limited to the above definition of the anti-PD-1 monoclonal antibody, but also can be other anti-PD-1 monoclonal antibodies commercially used in experiments, as long as the target is PD-1 monoclonal antibody, and other anti-PD-1 monoclonal antibodies are not specifically limited herein.
In the present invention, the anti-PD-1 monoclonal antibody is preferably DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 or DFPD1-13 disclosed in patent No. ZL201510312910.8, a monoclonal antibody having a patent name of anti-PD-1 and a method for obtaining the same.
The anti-inflammatory agent is selected from: anti-TNFa monoclonal antibody, anti-IL-1 beta monoclonal antibody or IL-1 receptor antagonist, anti-IL-6R monoclonal antibody, anti-IL-8 monoclonal antibody, recombinant IL-15 or IL-15 agonist;
preferably, the neoplastic disease is selected from colorectal cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, renal cancer, gastric cancer, liver cancer, ovarian cancer, melanoma or glioma.
EXAMPLE 4 construction of bifunctional protein molecule expression vectors for VEGF and TGF-beta
(1) Corresponding gene sequences are designed according to example 1 with reference to the structural forms of the bifunctional proteins in FIGS. 1-4, for example, in example 2, pTSE is selected as an expression vector, genes encoding the heavy chain (SEQ ID NO:1) and the light chain (SEQ ID NO:2) amplified from the anti-VEGF monoclonal antibody engineered cell strain are cloned into the vector pTSE vector (the structure is shown in FIG. 5, the preparation process is shown in CN103525868A description page 3, paragraph [0019 ]), then the gene encoding the TGF-beta RII extracellular domain polypeptide (synthesized by Nanjing Kingsler Biotech limited) is cloned into one end of the heavy chain or the light chain of the anti-VEGF monoclonal antibody engineered cell strain by homologous recombination and a linker, transformed into TOP competence (Virginia, Cathaki, Cat. HT702-03), and after the sequencing is correct, 4 expression vectors capable of expression are obtained, the plasmids are respectively named as a carrier of protein EB-1, protein EB-2, protein EB-3 or protein EB-4, meanwhile, a coding gene of TGF-beta RII extracellular domain polypeptide is cloned to the C end of an expression carrier (pTSE is also selected as the expression carrier) filled with an Fc segment of IgG1 type by a homologous recombination mode and a connecting peptide linker, and a carrier capable of expressing a control molecule is obtained, wherein the sequence named as 052Trap and 052Trap is shown as SEQ ID NO: 8. The method comprises the steps of carrying out plasmid extraction by using an endotoxin-free large-extraction kit (CW 2104 purchased from Kangji century Biotechnology Co., Ltd.), operating according to the instruction provided by the kit in specific operation steps, finally determining the plasmid concentration, and storing the plasmids at the temperature of 20 ℃ below zero.
SEQ ID NO 8 (amino acid sequence of 052Trap)
GAASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD；
(2) HEK293E cells (purchased from the institute of basic medicine of Chinese academy of medical sciences, the product number is GNHu43) were transiently transfected according to the structural collocation in example 2 to perform bifunctional protein expression; two control molecules, bevacizumab and 052Trap, were expressed simultaneously. 4 bifunctional proteins (designated protein EB-1, protein EB-2, protein EB-3 and protein EB-4) and two control molecules (designated bevacizumab and 052Trap) were obtained by protein A affinity column purification using AKTA instrument, and protein concentration was determined using BCA kit (purchased from: Beijing Hui east science and technology Co., Ltd., Cat. No.: BCA0020), which showed very low yields (<1mg/L) of protein EB-3 and protein EB-4.
Example 5 binding experiments with VEGF or TGF-beta 1/2/3 against bifunctional protein molecules of VEGF and TGF-beta
VEGF, TGF-. beta.1, TGF-. beta.2 or TGF-. beta.3 was coated with a carbonate buffer pH9.6 at 100 ng/well/100. mu.l overnight at a temperature of 4 ℃. Washing with 300 μ l/well PBST five times, adding 1% BSA-PBST, blocking at room temperature for 1h, adding bifunctional protein of protein EB-1 or protein EB-2 diluted by 5 times gradient, adding control molecule 052Trap or bevacizumab as control, and incubating at room temperature for 1 h. The membrane was washed five times with 300. mu.l/well PBST, and Anti-human Fc-HRP diluted with 1% BSA-PBST 1:10000 was added and incubated for 1h at room temperature. After washing, the color was developed with TMB color development kit at 100. mu.l/well for 8min at room temperature, and then developed with 2MH2SO4The color development was terminated. Readings at 450nm/630nm were taken using GraphPad Prism 5.0 mapping software to generate antigen-antibody binding profiles, as shown in figures 6-8, and corresponding EC50 values were calculated as follows (no binding of all molecules to TGF- β 2, not shown):
Figure BDA0003201766070000091
to further examine the binding of bifunctional protein molecules to TGF-. beta.2, protein EB-1, protein EB-2, 052Trap or bevacizumab was coated with carbonate buffer pH9.6 at 1. mu.g/well/100. mu.l overnight at 4 ℃. The membrane was washed five times with 300. mu.l/well PBST, then blocked with 1% BSA-PBST for 1h at room temperature, and then incubated for 1h at room temperature with 5-fold gradient dilutions of TGF-. beta.2 molecules. The cells were washed five times with 300. mu.l/well PBST, and a biotin-labeled TGF-. beta.2 antibody diluted 1% BSA-PBST 1:1000 was added and incubated at room temperature for 1 h. The membrane was washed five times with 300. mu.l/well PBST, and Streptavidin-HRP diluted with 1% BSA-PBST 1:5000 was added and incubated at room temperature for 30 min. After washing, the color was developed with TMB color development kit at 100. mu.l/well for 8min at room temperature, and then developed with 2MH2SO4Color development is stopped. The antigen-antibody binding curves were made using GraphPad Prism 5.0 mapping software, reading at 450nm/630nm, as shown in fig. 9, and the corresponding EC50 values were calculated, as follows:
cloning 052Trap Bevacizumab Protein EB-1 Protein EB-2
EC50(ng/ml) 11.7 NA 21.1 26.0
Experimental results show that TGF- β RII extracellular domain polypeptides do not bind to TGF- β 2 coated ELISA plates, but bind to TGF- β 2 in solution. Protein EB-1 and protein EB-2 can be well combined with three TGF-beta, and the EC50 value is basically consistent with that of positive control molecule 052 Trap. Protein EB-1 and protein EB-2 also bound well to VEGF, with EC50 values of protein EB-1 and bevacizumab being substantially the same, and lower than EC50 value of protein EB-2. The position of TGF-beta RII extracellular domain polypeptide on the bifunctional protein molecule has little influence on the affinity of the same TGF-beta, but different positions have certain influence on the affinity of the same VEGF: when TGF-. beta.RII extracellular domain polypeptides are linked to the VEGF binding region at the N-terminus of the heavy chain of the bifunctional protein molecule, it is possible to affect the binding of the bifunctional protein molecule to VEGF, resulting in antibody EB-2 having a lower affinity than antibody EB-1 and bevacizumab.
Example 6 bifunctional protein molecules against VEGF and TGF-beta inhibit the effects of VEGF on cell proliferation
HUVEC cells were trypsinized, resuspended in basal Medium (ECM) and counted, adjusted to 2X 105cell/mL. VEGF was added to the cell suspension to a final concentration of 0.1. mu.g/mL, 50. mu.L/well in pre-coated 96-well plates. The initial action concentration of the protein EB-1, the protein EB-2, the negative control molecule 052Trap and the positive control antibody bevacizumab is 2 mug/mL, the preparation concentration is 4 mug/mL, the dilution is carried out by 2 times of gradient, the total is ten gradients, 50 mug/hole is added into a 96-hole plate, and the mixture is gently mixed. The 96-well plate is placed at 37 ℃ and 5% CO2Incubate in incubator for 72 h. After the incubation, adding CCK-8 color development solution 10 μ L/well, placing at 37 deg.C and 5% CO2And incubating for 2-4 h. The 96-well cell culture plate was removed and the absorbance of each well was read at a wavelength of 450nm using a microplate reader. Inhibition plots were made using GraphPad Prism 5.0 mapping software, as shown in fig. 10, and the corresponding IC50 values were calculated as follows:
cloning 052Trap Bevacizumab Protein EB-1 Protein EB-2
IC50(μg/ml) NA 3.454 3.151 5.160
The results show that both protein EB-1 and protein EB-2 can inhibit the effect of VEGF on HUVEC cell proliferation, and the inhibition effect of protein EB-2 is slightly weaker than that of protein EB-1.
Example 7 bifunctional protein molecules against VEGF and TGF-beta inhibit the effects of VEGF on cell migration
1) Treating a Cell Cultrue Inset kit with 0.2% gelatin before an experiment, wherein gelatin is required to be treated in advance outside a cup inside the cup, and the Cell Cultrue Inset kit is placed for more than 2 hours at 37 ℃; all samples were diluted with ECM medium. The loading of each group is shown in the following table:
experiment grouping Blank group Experimental group Control group
HUVEC + + +
VEGF + + +
Antibodies - + -
Control - - +
2) VEGF is diluted to the final concentration of 100ng/mL, and negative control molecules 052Trap, positive control molecules bevacizumab, protein EB-1 and protein EB-2 are respectively subjected to gradient dilution to ensure that the final concentrations are respectively as follows: 3000. mu.g/mL, 30. mu.g/mL and 3. mu.g/mL.
3) HUVEC cells were digested and collected, centrifuged to adjust cell density to 3X105And (4) adding 150 mu L of cell suspension into the kit, placing the kit into a 24-well culture plate, adding 300 mu of LECM culture medium to the outer side of the kit, placing the kit into a cell incubator, and culturing for 2 hours.
4) After the cells are attached to the wall, 750 mu L of diluted sample and VEGF diluent are added outside the kit according to groups and cultured in an incubator at 37 ℃ for 19 h.
5) After the culture is finished, the 24-hole plate is taken out of the incubator, the culture medium inside and outside the cup is completely absorbed, 500 mu L of 2ug/ml Calcein-AM diluent is added to the outer side of each cup, the cup is placed in the incubator at 37 ℃ for about 70min, the excitation light of the microplate reader is set to be 485nm, and the fluorescence value at 530nm is read.
6) And (6) data processing. The calculation of relative mobility was performed according to the following formula, and the histogram between cell mobility and drug concentration was plotted using graphpad.prism.v. 5.01 software.
Relative mobility (plug-in outside fluorescence intensity value under the action of antibody at different concentrations-blank plug-in outside fluorescence intensity value)/(plug-in outside fluorescence intensity value without antibody action-blank plug-in outside fluorescence intensity value)
Inhibition plots were made using GraphPad Prism 5.0 mapping software, as shown in fig. 11, and corresponding IC50 values were calculated as follows:
cloning 052Trap Bevacizumab Protein EB-1 Protein EB-2
IC50(μg/ml) NA 3.454 3.151 5.160
The results show that both protein EB-1 and protein EB-2 can inhibit the influence of VEGF on HUVEC cell migration, the inhibition effect of the protein EB-1 is stronger than that of the protein EB-2, and the dose effect is obvious.
Example 8 bifunctional protein molecules against VEGF and TGF-beta inhibit cell surface TGF-beta 1 release
One day ahead of time 50. mu.L (1X 10) of a cell line overexpressing TGF-. beta.1 was inoculated6Cells/ml) in a 96-well plate, and a series of concentration gradient dilutions of protein EB-1, protein EB-2, positive control molecule 052Trap, or negative control molecule bevacizumab were added, each 50 μ L incubated overnight, starting at 20 μ g/ml, followed by 3-fold concentration gradient dilutions, for a total of 7 concentration gradients per molecule. The next day 50. mu.L (1X 10) was added6Individual cells/ml) of stable PAI-LUC-expressing mv.1.lu mink lung epithelial cells (purchased from the institute of basic medicine, department of medicine, china, academy of science, cell resources center, cat #: 3111C0001CCC000195), luciferase activity was tested after 6 hours incubation in cell culture incubator. And a graph pad Prism 5.0 plotting software was used to make an inhibition curve of the antibody inhibiting the release of TGF β 1 as shown in fig. 12, and corresponding IC50 values were calculated as follows:
cloning 052Trap Bevacizumab Protein EB-1 Protein EB-2
IC50(ng/ml) 22.12 NA 61.55 76.79
The data and FIG. 12 show that both protein EB-1 and protein EB-2 can effectively inhibit the release of TGF beta 1 on the cell surface.
Example 9 in vivo tumor inhibition experiment
1. Experimental animals:
species lines: BALB/c, female, mouse;
the week age is as follows: 6-8 weeks;
experimental animal providers: beijing Huafukang Biotechnology GmbH.
2. The experimental method comprises the following steps:
each mouse was inoculated subcutaneously in the right dorsal part 3x105CT26 tumor cells (purchased from ATCC stock number: CRL-2638), the animals after tumorigenesis were randomly divided into several groups of 10, and 3 days later, were intraperitoneally injected with protein EB-1(10mg/kg) provided in the present invention, while several control groups were set up: the dosage of each protein is the same as that of the protein EB-1. Taking the medicine twice a week, measuring tumor volume three times a week and measuring body weight once a week, observing and recording tumor growth, wherein the tumor volume is more than 2000mm3The mice were sacrificed by cervical dislocation, and the tumors were carefully isolated with surgical scissors and weighed, and the results are shown in fig. 13.
As can be seen from fig. 13, tumor growth was not inhibited in the 052Trap group, compared to the isotype IgG control group, while tumor volume was significantly reduced in each of the other groups. When bevacizumab is combined with 052Trap, the tumor volume is smaller than that when bevacizumab is used alone; the use of protein EB-1 reduced tumor volume significantly compared to bevacizumab 052Trap, but was significantly reduced compared to bevacizumab alone. Therefore, the two targets of VEGF and TGF-beta are inhibited simultaneously, the inhibition on the growth of the CT26 tumor can be obviously improved, and the inhibition effect on the tumor by using the bifunctional protein EB-1 is better than that by combining bevacizumab with 052 Trap.
Example 10 in vivo tumor inhibition assay (combination anti-PD-1 antibody)
1. Experimental animals:
species lines: BALB/c, female, mouse;
the week age is as follows: 6-8 weeks;
experimental animal providers: beijing Huafukang Biotechnology GmbH.
2. Different administration groups are set, specifically as follows:
an anti-mouse PD-1 monoclonal antibody (purchased from Bio X Cell, cat # BE0146) single administration group (10mg/kg), a protein EB-1 combined anti-mouse PD-1 monoclonal antibody administration group (each 10mg/kg) and an isotype IgG control group (10 mg/kg);
each mouse was inoculated subcutaneously on the right dorsal side 5 x105The CT26 tumor cells are divided into several groups of 10 animals after tumor formation, and the administration is performed by intraperitoneal injectionGroup, dosing twice a week, measuring tumor volume three times a week and measuring body weight once a week, observing and recording tumor growth, and measuring tumor volume over 3000mm3The mice were sacrificed by cervical dislocation, and tumors were carefully isolated with surgical scissors and weighed. The results are shown in FIG. 14.
As can be seen from FIG. 14, the anti-mouse PD-1 monoclonal antibody alone exhibited substantially no anti-tumor effect as compared with the control group. Compared with the single administration group of the anti-mouse PD-1 monoclonal antibody and the single administration group of the protein EB-1, the administration group of the protein EB-1 combined anti-mouse PD-1 monoclonal antibody shows obvious tumor inhibition effect, the tumor of the mouse disappears completely at 18 days, and the superiority of the synergistic effect of the protein EB-1 combined anti-mouse PD-1 monoclonal antibody is reflected.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.
Sequence listing
<110> Beijing Oriental Baitai Biotechnology GmbH
<120> bifunctional protein for VEGF and TGF-beta and application thereof
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Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
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Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
450 455 460
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln
465 470 475 480
Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val
485 490 495
Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys
500 505 510
Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
515 520 525
Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu
530 535 540
Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His
545 550 555 560
Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu
565 570 575
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp
580 585 590
Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
595 600 605
Pro Asp
610
<210> 5
<211> 610
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
1 5 10 15
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
20 25 30
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
35 40 45
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
50 55 60
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
65 70 75 80
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
85 90 95
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
100 105 110
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
115 120 125
Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
145 150 155 160
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
165 170 175
Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn
180 185 190
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile
195 200 205
Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg
210 215 220
Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met
225 230 235 240
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr
245 250 255
Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val Trp Gly Gln
260 265 270
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
275 280 285
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
290 295 300
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
305 310 315 320
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
325 330 335
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
340 345 350
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
355 360 365
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
370 375 380
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
385 390 395 400
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
405 410 415
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
420 425 430
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
435 440 445
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
450 455 460
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
465 470 475 480
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
485 490 495
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
500 505 510
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
515 520 525
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
530 535 540
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
545 550 555 560
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
565 570 575
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
580 585 590
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
595 600 605
Gly Lys
610
<210> 6
<211> 371
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45
Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val
225 230 235 240
Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala
245 250 255
Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr
260 265 270
Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile
275 280 285
Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp
290 295 300
Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr
305 310 315 320
His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys
325 330 335
Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser
340 345 350
Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser
355 360 365
Asn Pro Asp
370
<210> 7
<211> 371
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
1 5 10 15
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
20 25 30
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
35 40 45
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
50 55 60
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
65 70 75 80
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
85 90 95
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
100 105 110
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
115 120 125
Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln
145 150 155 160
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
165 170 175
Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp
180 185 190
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr
195 200 205
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
210 215 220
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
225 230 235 240
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly
245 250 255
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
260 265 270
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
275 280 285
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
290 295 300
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
305 310 315 320
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
325 330 335
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
340 345 350
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
355 360 365
Gly Glu Cys
370
<210> 8
<211> 393
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
Gly Ala Ala Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
1 5 10 15
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
20 25 30
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
35 40 45
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
50 55 60
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
65 70 75 80
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
85 90 95
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
100 105 110
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
115 120 125
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
130 135 140
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
145 150 155 160
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
165 170 175
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
180 185 190
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
195 200 205
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
210 215 220
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
260 265 270
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
275 280 285
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
290 295 300
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
305 310 315 320
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
325 330 335
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
340 345 350
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
355 360 365
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
370 375 380
Glu Glu Tyr Asn Thr Ser Asn Pro Asp
385 390

Claims (8)

1. A bifunctional protein against VEGF and TGF- β, comprising an anti-VEGF monoclonal antibody capable of specifically binding to a VEGF antigen, a linker peptide linker and two TGF- β RII extracellular domain polypeptides, said anti-VEGF monoclonal antibody being linked to said two TGF- β RII extracellular domain polypeptides by said linker peptide;
the anti-VEGF monoclonal antibody comprises two heavy chains and two light chains, and the two TGF-beta RII extracellular domain polypeptides are respectively connected with the C terminal or the N terminal of the two heavy chains of the anti-VEGF monoclonal antibody through the connecting peptide linker;
the amino acid sequence of the heavy chain is SEQ ID NO. 1, and the amino acid sequence of the light chain is SEQ ID NO. 2; the amino acid sequence of the TGF-beta RII extracellular domain polypeptide is SEQ ID NO 3;
the general formula of the connecting peptide linker is (GGGGS)n；
Wherein n is 4.
2. The bifunctional protein of claim 1 directed to VEGF and TGF- β, wherein said bifunctional protein is selected from any one of:
the protein EB-1 is formed by respectively connecting two TGF-beta RII extracellular domain polypeptides with the C tail ends of two heavy chains of an anti-VEGF monoclonal antibody through a connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-1 is SEQ ID NO. 4, and the amino acid sequence of the light chain of the protein EB-1 is SEQ ID NO. 2;
and the protein EB-2 is formed by respectively connecting two TGF-beta RII extracellular domain polypeptides with the N tail ends of two heavy chains of the anti-VEGF monoclonal antibody through the connecting peptide linker, the amino acid sequence of the heavy chain of the protein EB-2 is SEQ ID NO. 5, and the amino acid sequence of the light chain of the protein EB-2 is SEQ ID NO. 2.
3. A nucleotide molecule encoding the bifunctional protein of claim 1 or 2 directed to VEGF and TGF- β.
4. A recombinant DNA expression vector comprising the nucleotide molecule of claim 3.
5. A host cell transfected with the recombinant DNA expression vector of claim 4, wherein said host cell comprises a prokaryotic, yeast, or mammalian cell.
6. Use of the bifunctional protein of claim 1 or 2 against VEGF and TGF- β in the manufacture of a medicament for the treatment of a neoplastic disease or fibrotic disease;
the tumor disease is selected from colorectal cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, renal cancer, gastric cancer, liver cancer, ovarian cancer, melanoma or glioma;
the fibrotic disease includes liver fibrosis and lung fibrosis.
7. Use of a bifunctional protein directed against VEGF and TGF- β according to claim 1 or 2 in combination with an immunomodulatory drug or an anti-inflammatory factor drug for the preparation of a medicament for the treatment of a neoplastic disease; the immunoregulatory drug is selected from an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA 4 monoclonal antibody, an anti-4-1 BB monoclonal antibody, an anti-OX-40 monoclonal antibody, an anti-PD-L2 monoclonal antibody, an anti-LAG-3 monoclonal antibody, an anti-TIGIIT monoclonal antibody, an anti-GITR monoclonal antibody, an anti-ICOS monoclonal antibody, an anti-PVR monoclonal antibody, an anti-PVRIG monoclonal antibody, an anti-VISTA monoclonal antibody or an anti-TIMS monoclonal antibody;
the anti-inflammatory agent is selected from: anti-TNFa monoclonal antibodies, anti-IL-1 beta monoclonal antibodies or IL-1 receptor antagonists, anti-IL-6R monoclonal antibodies, anti-IL-8 monoclonal antibodies, recombinant IL-15 or IL-15 agonists.
8. The use according to claim 7, wherein the neoplastic disease is selected from colorectal cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, renal cancer, gastric cancer, liver cancer, ovarian cancer, melanoma or glioma.
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