CN115707716A - TGF-beta/VEGF difunctional antibody fusion protein - Google Patents

TGF-beta/VEGF difunctional antibody fusion protein Download PDF

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CN115707716A
CN115707716A CN202110950470.4A CN202110950470A CN115707716A CN 115707716 A CN115707716 A CN 115707716A CN 202110950470 A CN202110950470 A CN 202110950470A CN 115707716 A CN115707716 A CN 115707716A
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黄浩旻
邓岚
朱祯平
刘利芬
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Dansheng Pharmaceutical Technology Shanghai Co ltd
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Priority to PCT/CN2022/113411 priority patent/WO2023020592A1/en
Priority to CN202280053404.XA priority patent/CN117858907A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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

Abstract

The invention provides a TGF-beta/VEGF bifunctional antibody fusion protein. The bifunctional antibody fusion proteins of the present invention are dimers formed from two monomers, each monomer comprising: a first binding domain Z1; and a second binding domain Z2; wherein the first binding domain specifically binds to the target molecule TGF-. Beta.s; the second binding domain specifically binds to the target molecule VEGF. The bifunctional antibody fusion protein can block VEGF and TGF-beta signal channels at the same time, and has important clinical significance for effectively treating ophthalmic diseases such as CNV, PVR, AMD and the like and treating tumors.

Description

TGF-beta/VEGF difunctional antibody fusion protein
Technical Field
The invention relates to the technical field of fusion proteins, in particular to a TGF-beta/VEGF bifunctional antibody fusion protein.
Background
Exudative aging-related macular degeneration (AMD), which results from Choroidal Neovascularization (CNV), is one of the leading causes of high-vision impairment in developed countries. Evidence is now available that Vascular Endothelial Growth Factors (VEGFs) play a central role in the development of CNV, and compounds that inhibit VEGF production or inhibit the VEGF signaling pathway can inhibit CNV. In recent years, anti-VEGF antibodies have shown greater therapeutic efficacy compared to traditional therapeutic approaches including photodynamic therapy. anti-VEGF drugs have become the primary choice for drug therapy for CNV, AMD, and the like. At present, the administration frequency of the main anti-VEGF drugs on the market is 2-4 weeks, and repeated intravitreal injection is carried out for many times, so that great psychological and physiological burden is brought to patients. Therefore, there is an urgent need in the art to develop a drug that targets VEGF for a longer duration and can be used for treating ophthalmic diseases such as CNV, AMD, and the like. In addition, VEGFs also play an important role in corneal neovascularization, and one strategy for treating corneal neovascular diseases (e.g., corneal alkali burns) is to inhibit VEGF activity.
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 the site of action is mainly the extracellular domain D2 of VEGFR 1. VEGFR1-D2 blocks the binding of VEGFR2 to VEGF by competing for binding to VEGF, thereby blocking the signaling pathway and inhibiting endothelial cell proliferation and angiogenesis.
Transforming growth factor-beta (TGF-beta) belongs to a TGF-beta superfamily which regulates the growth and differentiation of cells, and is a multifunctional cell factor. High levels of TGF- β have a broad effect on Retinal Pigment Epithelium (RPE) and play an important role in the pathogenesis of ophthalmic diseases such as Proliferative Vitreoretinopathy (PVR) and AMD. TGF-beta transmits signals by binding with a receptor complex thereof, induces phosphorylation of SMAD2 and SMAD3, activates downstream effector genes including Vascular Endothelial Growth Factor A (VEGFA), and thus plays a role in promoting angiogenesis. In addition, TGF- β promotes epithelial to mesenchymal transition, weakens the tightness of retinal barrier cell junctions, increases smooth muscle actin expression, enhances the contractile properties of fibrous RPE cells, leading to an increased risk of retinal detachment.
On the other hand, the VEGF signal channel is related to the migration, proliferation and survival of tumor neogenetic endothelial cells, plays an important role in the angiogenesis process around tumors, and has close relation with the morbidity of various common tumors and the metastasis of the tumors. TGF- β signaling can act on immune cells in the tumor microenvironment. On the one hand, secreted TGF- β inhibits cytotoxicity of effector T cells and natural killer cells (NK), which reduces the antitumor capacity of innate immune cells in the tumor microenvironment; in addition, the TGF-betSub>A expression is up-regulated, so that the secretion of factors such as VEGF-A and the like is promoted, and the angiogenesis is stimulated, thereby providing assistance for the growth and the metastasis of cancer cells. Recent studies have also shown that the TGF- β signalling pathway is also associated with resistance in tumour cells.
Therefore, the medicine for simultaneously blocking VEGF and TGF-beta signal channels has clinical potential in treating ophthalmic diseases such as CNV, PVR, AMD and the like and tumors. The development of a bifunctional drug targeting VEGF and TGF-beta simultaneously is urgently needed.
Disclosure of Invention
The invention aims to provide a TGF-beta/VEGF bifunctional antibody fusion protein.
In a first aspect of the present invention, there is provided a bifunctional fusion protein which is a dimer formed from two monomers, each monomer comprising:
a first binding domain Z1; and
a second binding domain Z2;
wherein the first binding domain specifically binds to the target molecule TGF-. Beta.s;
the second binding domain specifically binds to the target molecule VEGF.
In another preferred embodiment, Z1 is an antibody or antibody fragment that specifically binds TGF- β.
In another preferred embodiment, the antibody comprises: an antibody of animal origin (e.g., a murine antibody), a chimeric antibody, a humanized antibody.
In another preferred embodiment, the antibody fragment comprises a heavy chain variable region and a light chain variable region.
In another preferred embodiment, the antibody fragment comprises a single chain variable fragment (scFv) or a double chain variable fragment (dcFv).
In another preferred embodiment, Z2 is a polypeptide fragment that specifically binds VEGF.
In another preferred embodiment, the polypeptide fragment of VEGF is derived from a VEGF receptor.
In another preferred embodiment, Z2 is an extracellular region of a type I VEGF receptor (VEGFR 1).
In another preferred embodiment, said Z1 and said Z2 are linked by a linker or a peptide bond.
In another preferred example, the joint is a flexible joint.
In another preferred embodiment, the flexible linker comprises 5-30 amino acids, preferably 10-25 amino acids.
In another preferred embodiment, the flexible peptide linker comprises 1-6G 4S.
In another preferred embodiment, Z1 is an antibody and the linker is (G4S) n Wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6), preferably n =2 or 4.
In another preferred embodiment, Z1 is an anti-TGF- β monoclonal antibody, and Z2 is attached to Z1 at a position selected from the group consisting of: the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, the C-terminus of the light chain, or a combination thereof.
In another preferred embodiment, Z1 is an anti-TGF- β monoclonal antibody and Z2 is attached to the end of the heavy chain constant region of Z1 by a linker.
In another preferred embodiment, Z1 is an anti-TGF-. Beta.monoclonal antibody, and Z2 is attached to the end of the heavy chain variable region of Z1 via a linker.
In another preferred embodiment, Z1 is an anti-TGF- β monoclonal antibody and Z2 is attached to the end of the light chain constant region of Z1 by a linker.
In another preferred embodiment, each monomer in the bifunctional fusion protein has a structure from N-terminus to C-terminus as shown in formula I:
Figure BDA0003218436730000031
wherein the content of the first and second substances,
t1, T2, T3 are each independently absent or the extracellular region of a type I VEGF receptor (VEGFR 1), and at least one is not absent;
l1, L2, L3 are each independently a bond or linker element;
VL represents the light chain variable region of an anti-TGF-beta antibody;
CL represents the light chain constant region of an anti-TGF-. Beta.antibody;
VH represents the heavy chain variable region of an anti-TGF-beta antibody;
CH represents the heavy chain constant region of an anti-TGF-beta antibody;
"-" represents a disulfide bond or a covalent bond;
"-" represents a peptide bond;
wherein the bifunctional fusion protein has the activity of simultaneously binding VEGF and TGF-beta.
In another preferred embodiment, the term "to" refers to one or more interchain disulfide bonds between heavy chains or light chains.
In another preferred embodiment, L1, L2 and L3 are each independently (G4S) 2 、(G4S) 3 Or (G4S) 4 。
In another preferred embodiment, T2, T3, L2 and L3 are none.
In another preferred embodiment, T1 is the extracellular domain of a type I VEGF receptor.
In another preferred embodiment, the anti-TGF- β antibody is a monoclonal antibody.
In another preferred embodiment, the anti-TGF- β antibody is a humanized antibody.
In another preferred embodiment, the anti-TGF- β antibody is an IgG class antibody.
In another preferred embodiment, the heavy chain amino acid sequence of the anti-TGF- β antibody is as set forth in SEQ ID NO:13, the light chain amino acid sequence of the anti-TGF-beta antibody is shown as SEQ ID NO: shown at 9.
In another preferred embodiment, the fusion protein is selected from the group consisting of:
(1) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:1, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:3 is shown in the figure;
(2) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:5, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:3 is shown in the specification;
(3) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:7, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:9 is shown in the figure;
(4) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:11, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:9 is shown in the figure;
(5) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:13, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:14 is shown in the figure; or
(6) And (2) the polypeptide which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequences in the (1) to (5) and has the activity of simultaneously binding VEGF and TGF-beta.
In a second aspect of the invention there is provided a polynucleotide molecule encoding a fusion protein according to the first aspect of the invention.
In a third aspect of the invention there is provided an expression vector comprising a polynucleotide molecule according to the second aspect of the invention.
In a fourth aspect of the invention, there is provided a host cell comprising an expression vector according to the third aspect of the invention.
In a fifth aspect of the present invention, there is provided a method for producing the fusion protein according to the first aspect of the present invention, characterized in that the production method comprises the steps of:
a) Culturing the host cell according to the fourth aspect of the invention under expression conditions, thereby expressing the bifunctional fusion protein;
b) Isolating and purifying the fusion protein of step a).
In a sixth aspect of the invention, there is provided a pharmaceutical composition, characterized in that it comprises an effective amount of a fusion protein according to the first aspect of the invention and one or more pharmaceutically acceptable carriers, diluents or excipients.
In another preferred embodiment, the pharmaceutical composition is in unit dosage form.
In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating ophthalmic diseases.
In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a parenteral dosage form or a parenteral dosage form.
In another preferred embodiment, the parenteral dosage form comprises intravitreal injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.
In a seventh aspect of the invention there is provided a fusion protein according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, for use in the manufacture of a medicament for the treatment of an ophthalmic disorder.
In another preferred embodiment, the ophthalmic disease is selected from: exudative age-related macular degeneration (AMD), choroidal Neovascularization (CNV), proliferative Vitreoretinopathy (PVR), corneal neovascular diseases.
In another preferred embodiment, the ophthalmic disease is corneal alkali burn.
In an eighth aspect of the invention, there is provided a use of the fusion protein according to the first aspect of the invention, or the pharmaceutical composition according to the sixth aspect of the invention, in the manufacture of a medicament for the treatment of a tumour.
In another preferred embodiment, the tumor is pancreatic cancer.
In a ninth aspect of the invention, there is provided a method of treating an ophthalmic disease or tumour, the method comprising administering to a subject in need thereof a fusion protein according to the first aspect of the invention, or an immunoconjugate thereof, or a pharmaceutical composition according to the sixth aspect of the invention.
In a tenth aspect of the invention, there is provided an immunoconjugate comprising:
(a) A fusion protein according to the first aspect of the invention; and
(b) A coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
In another preferred embodiment, the conjugate moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.) capable of producing a detectable product.
In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate (ADC).
In another preferred embodiment, the immunoconjugate is for use in the preparation of a pharmaceutical composition for the treatment of an ophthalmic disorder.
In an eleventh aspect of the invention there is provided the use of an immunoconjugate according to the tenth aspect of the invention for the preparation of a pharmaceutical composition for the treatment of an ophthalmic disease or tumor.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows a schematic diagram of the structure of antibody fusion protein TV01, TV03 as in FIG. 1A, antibody fusion protein TV02, TV04 as in FIG. 1B, and antibody fusion protein TV05 as in FIG. 1C.
FIG. 2 shows HPLC detection of purified protein.
FIG. 3 shows ELISA detection of affinity of anti-TGF-. Beta./VEGF bifunctional fusion proteins to TGF-. Beta.1.
Figure 4 shows SMAD3 reporter gene suppression experiments.
FIG. 5 shows cellular experiments in which the TGF-. Beta./VEGF bifunctional fusion protein blocks the binding of VEGF to the receptor KDR.
FIG. 6 shows the anti-angiogenic effect of anti-TGF-. Beta./VEGF bifunctional fusion proteins in an alkali-burned corneal neovascular model.
FIG. 7 shows the anti-angiogenic effect of anti-TGF-. Beta./VEGF bifunctional fusion proteins in a matrigel angiogenesis model.
FIG. 8 shows the anti-tumor effect of anti-TGF-beta/VEGF bi-functional fusion protein on a human pancreatic cancer BxPC-3 nude mouse transplantation tumor model.
Detailed Description
The present inventors have extensively and intensively studied and, for the first time, constructed an anti-TGF-. Beta.VEGF bifunctional fusion protein comprising an anti-TGF-. Beta.antibody portion and an extracellular region of type I VEGF receptor (VEGFR 1). Experimental results show that the bifunctional fusion protein has good physical stability, can effectively block the combination of VEGF and a receptor KDR, can obviously inhibit the formation of corneal neovascularization in a mouse alkali burn corneal neovascularization model, and can obviously inhibit the formation of neovascularization in a mouse matrigel angiogenesis model. In addition, on a mouse tumor model of human pancreatic cancer, the bifunctional fusion protein can obviously inhibit tumor growth, and has important clinical application prospects. The present invention has been completed based on this finding.
Term(s) for
In the present invention, the term "fusion protein" refers to a novel polypeptide sequence obtained by fusing two or more identical or different polypeptide sequences. The term "fusion" refers to a linkage by peptide bonds either directly or by means of one or more linkers. The term "linker" refers to a short peptide, typically a peptide of 2-30 amino acids in length, that can link two polypeptide sequences.
In the present invention, the term "Antibody (Ab for short)" is an isotetraglycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has at one end a variable region (VH) followed by a constant region consisting of three domains, CH1, CH2, and CH 3. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region comprising a domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region of the heavy chain, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC) and the like. Heavy chain constant regions include IgG1, igG2, igG3, igG4 subtypes; light chain constant regions include Kappa (Kappa) or Lambda (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by interpoly disulfide bonds formed between the hinge regions.
In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically a mixture of different antibodies with epitopes against different antigens), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins.
In the present invention, the term "humanized" means that the CDRs are derived from an antibody of a non-human species (preferably a mouse), and the remaining part of the antibody molecule (including the framework region and the constant region) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity.
As used herein, the term "framework region" (FR) refers to a portion of an immunoglobulin variable region in which the amino acid composition and arrangement other than the hypervariable region are relatively less variable. The light and heavy chains of immunoglobulins each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can thus be referred to as (FR 1-L) - (CDR 1-L) - (FR 2-L) - (CDR 2-L) - (FR 3-L) - (CDR 3-L) - (FR 4-L) and the heavy chain variable domain can thus be represented as (FR 1-H) - (CDR 1-H) - (FR 2-H) - (CDR 2-H) - (FR 3-H) - (CDR 3-H) - (FR 4-H). Preferably, the FRs of the present invention are human antibody FRs or derivatives thereof that are substantially identical, i.e., 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, to the FRs of a naturally occurring human antibody.
Knowing the amino acid sequence of the CDR, one skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.
As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.
As used herein, the term "linker" or "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the domains of the light and heavy chains to fold into one or more amino acid residues that exchange the dual variable region immunoglobulin. In the present invention, preferred linkers refer to linkers L1, L2 and L3, wherein L1 connects the extracellular region of VEGFR1 to the N-terminus of the heavy chain of the anti-TGF- β antibody, L2 connects the extracellular region of VEGFR1 to the C-terminus of the heavy chain of the anti-TGF- β antibody, and L3 connects the extracellular region of VEGFR1 to the C-terminus of the light chain of the anti-TGF- β antibody.
Suitable linkers are flexible linkers, examples of which include monoglycine (Gly), or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker may vary with the type of secondary structural element that is desired to be achieved in the linker. In the present invention, the joint is (G4S) n Preferably (G4S) 2 、(G4S) 3 Or (G4S) 4 。
Bifunctional fusion proteins
The bifunctional fusion protein of the invention is an anti-VEGF and TGF-beta bifunctional fusion protein, comprising an anti-TGF-beta antibody part and an extracellular region of a type I VEGF receptor (VEGFR 1).
As used herein, "bifunctional fusion protein", "bifunctional antibody fusion protein of the invention", "TGF- β/VEGF bifunctional antibody fusion protein", "anti-VEGF and TGF- β bifunctional fusion protein" are used interchangeably and refer to a bifunctional fusion protein according to the first aspect of the invention comprising an anti-TGF- β antibody moiety and an extracellular domain of a type I VEGF receptor (VEGFR 1).
Preferably, the heavy chain amino acid sequence of the anti-TGF-beta antibody of the invention is as set forth in SEQ ID NO:13, the light chain amino acid sequence of the anti-TGF-beta antibody is shown as SEQ ID NO: shown at 9. One skilled in the art may also modify or engineer an anti-TGF- β antibody of the invention, e.g., by adding, deleting, and/or substituting one or more amino acid residues, by techniques well known in the art, to further increase the affinity or structural stability of the anti-TGF- β antibody, and obtain the modified or engineered results by conventional assay methods.
In the present invention, the bifunctional fusion protein of the present invention further comprises conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties as compared to the amino acid sequence of the bifunctional fusion protein of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
In the present invention, the terms "anti-and" binding "refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. Typically, the antibody is present in an amount less than about 10 -7 M, e.g. less than about 10 -8 M、10 -9 M、10 -10 M、10 -11 M or less binds the antigen with an equilibrium dissociation constant (KD). The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity of an antibody to an antigen is determined in a BIACORE instrument using Surface Plasmon Resonance (SPR for short) or the relative affinity of the binding of an antibody to an antigen is determined using ELISA.
The bifunctional fusion proteins of the present invention may be used alone, or in combination or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, or a combination of any of the above.
Coding nucleic acids and expression vectors
The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. In the present invention, the term "expression vector" refers to a vector, such as a plasmid, a viral vector (e.g., adenovirus, retrovirus), a phage, a yeast plasmid, or other vectors, carrying an expression cassette for expression of a particular protein of interest or other material. For example, expression vectors conventional in the art comprising appropriate regulatory sequences, such as promoters, terminators, enhancers, and the like, include, but are not limited to: viral vectors (e.g., adenovirus, retrovirus), plasmids, bacteriophages, yeast plasmids or other vectors. The expression vector preferably comprises pDR1, pcDNA3.4 (+), pDHFR or pTT5.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
In the present invention, the term "host cell" is a variety of host cells that are conventional in the art, as long as the vector can stably replicate autonomously and the polynucleotide molecule carried by the vector can be efficiently expressed. Wherein the host cell comprises prokaryotic expression cells and eukaryotic expression cells, preferably comprising: COS, CHO, NS0, sf9, sf21, DH5 α, BL21 (DE 3), TG1, BL21 (DE 3), 293F or 293E cells.
Pharmaceutical composition and use
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 4 to about 8, preferably from about 5 to about 7, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection.
In the present invention, the term "pharmaceutical composition" refers to a pharmaceutical composition of the bifunctional fusion protein of the present invention, which can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition for more stable therapeutic effect, and these pharmaceutical compositions can ensure the conformational integrity of the amino acid core sequence of the bifunctional fusion protein disclosed in the present invention, and at the same time protect the multifunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation).
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g. 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the above-mentioned bifunctional fusion protein of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the bifunctional fusion proteins of the present invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the bifunctional fusion protein or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
In the present invention, the term "effective amount" refers to an amount or dose that, upon administration of the pharmaceutical composition of the present invention to a subject, produces a desired effect in the treated subject, including improvement of the condition of the subject. The term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, rats and mice, and the like.
The main advantages of the invention include:
(1) The bifunctional fusion proteins of the present invention bind to VEGF and TGF-beta with high affinity.
(2) The bifunctional fusion protein has good physical stability.
(3) The difunctional fusion protein can effectively inhibit the Smad3 activation induced by TGF-beta 1.
(4) The bifunctional fusion protein can effectively block the combination of VEGF and a receptor KDR, and has better blocking function than a positive control Bevacizumab.
(5) The bifunctional fusion protein can obviously inhibit the formation of corneal neovascularization in an alkali burn cornea neovascularization model and can obviously inhibit the formation of neovascularization in a matrigel angiogenesis model.
(6) The bifunctional fusion protein can obviously inhibit the growth of tumors.
(7) The bifunctional fusion protein has the potential of effectively treating ophthalmic diseases such as CNV, PVR, AMD and the like and tumors. Particularly in the field of ophthalmic diseases, on one hand, the VEGF-induced ocular angiogenesis and leakage can be rapidly inhibited, on the other hand, the TGF-beta signal channel is blocked to inhibit VEGF-induced expression, so that the ocular region can maintain a low VEGF level for a long time, and the ophthalmic diseases such as CNV, PVR, AMD and the like can be more effectively treated.
The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
The sequence information referred to in the examples below is summarized in table 1.
TABLE 1 sequence information of antibodies of the invention
Figure BDA0003218436730000131
Figure BDA0003218436730000141
Wherein the TV02 light chain amino acid sequence is identical to the TV01 light chain;
the light chains of TV04, TV03 and 12.7 are identical;
the amino acid sequence of the TV05 heavy chain is the same as that of the 12.7 monoclonal antibody heavy chain.
Example 1 construction of antibody fusion protein molecules
The invention adopts a mode of respectively connecting the C end (figure 1A) or the N end (figure 1B) of an anti-TGF-beta monoclonal antibody IgG heavy chain and the C end (figure 1C) of a light chain in series with a D2 structural domain of VEGFR1 to construct an antibody fusion protein. The amino acid sequence of the fusion protein of the invention is shown in table 2 below:
table 2.
Figure BDA0003218436730000142
Figure BDA0003218436730000151
Figure BDA0003218436730000161
Figure BDA0003218436730000171
The structural schematic diagrams of the antibody fusion proteins TV01 and TV03 are shown in figure 1A, the structural schematic diagrams of the antibody fusion proteins TV02 and TV04 are shown in figure 1B, and the structural schematic diagram of the antibody fusion protein TV05 is shown in figure 1C. The specific connection method is shown in table 3.
Wherein, the sequence of the anti-TGF-beta monoclonal antibody in TV02 is derived from a humanized antibody 1D11-Hu (constructed by humanizing murine 1D11 in US20180244763A 1) constructed in PCT patent application PCT/CN2021/088153, and the heavy chain and the light chain are respectively 1D11-Hu-HC and 1D11-Hu-LC in example 2.1. The heavy chain of the anti-TGF- β monoclonal antibody in TV01 has mutated amino acid K to A at position 448 relative to TV 02. The anti-TGF- β monoclonal antibody sequences in TV04 and TV05 were derived from another antibody mAb127 constructed in PCT patent application PCT 202tcn 2021088153 (i.e. 12.7 mAb in this application whose heavy and light chain variable regions were derived from US20100136021 A1), heavy and light chains mAb127-HC and mAb127-LC, respectively, in example 2.1. The heavy chain of the anti-TGF- β monoclonal antibody in TV03 has mutated amino acid K to A at position 448 relative to TV 04.
Table 3.
Fusion proteins Connection mode Sources of antibodies
TV01 anti-TGF-beta antibody heavy chain C-terminal connection VEGFR-D2 1D11-Hu
TV02 anti-TGF-beta antibody heavy chain N-terminal connection VEGFR-D2 1D11-Hu
TV03 anti-TGF-beta antibody heavy chain C-terminal connection VEGFR-D2 mAb127
TV04 anti-TGF-beta antibody(iv) the N-terminal end of the body heavy chain is connected with VEGFR-D2 mAb127
TV05 anti-TGF-beta antibody light chain C-terminal connection VEGFR-D2 mAb127
Example 2 preparation of TGF-. Beta./VEGF bifunctional fusion proteins
The DNA fragments of the heavy chain and the light chain of the anti-TGF-beta/VEGF double-function fusion protein are respectively subcloned into a pcDNA3.4 vector (purchased from thermofisher, A14697), and recombinant plasmids are extracted to co-transfect CHO cells and/or 293F cells. After 7 days of cell culture, the culture fluid is subjected to high-speed centrifugation, vacuum filtration through a microfiltration membrane, then loaded on a HiTrap MabSelect SuRe column, protein is eluted by an eluent with 100mM citric acid and pH3.5 in one step, and a target sample is recovered and dialyzed to change the fluid to PBS. The purified protein is detected by HPLC, and the detection results show that the fusion protein has uniform molecular state and the monomer purity is more than 97 percent, as shown in FIGS. 2A-2E.
Example 3 determination of affinity of anti-TGF-. Beta./VEGF bifunctional fusion proteins for antigens by enzyme-Linked immunosorbent assay (ELISA)
3.1 ELISA (enzyme-Linked immuno sorbent assay) for detecting affinity of anti-TGF (transforming growth factor) -beta/VEGF (vascular endothelial growth factor) dual-function fusion protein and TGF-beta 1
TGF-. Beta.1 protein (purchased from microbiosystems, cat. # TG 1-H4212) was plated at 100 ng/well overnight at 4 ℃. The plates were washed 3 times with PBST, 200. Mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The antibody was diluted to 100nM with a 4-fold dilution to form 12 concentration gradients, and the blocked ELISA plates were added sequentially at 100. Mu.l/well and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and then HRP-labeled goat anti-human Fab antibody (purchased from abcam, cat. # ab 87422) was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); adding stop solution into each hole to stop the reaction of the substrate, reading OD value at 450nm of an enzyme-labeling instrument, and GraphData analysis by Pad Prism6, plotting and calculating EC 50 。
As shown in FIG. 3A, the anti-TGF-beta/VEGF bifunctional fusion proteins TV01, TV02, TV03, TV04, TV05 and the positive control 12.7 monoclonal antibodies all can effectively bind TGF-beta 1, and the EC50 (nM) values are 0.188,0.213,0.174,0.221,0.187 and 0.202 respectively, and the affinities are equivalent.
3.2 ELISA (enzyme-Linked immuno sorbent assay) for detecting affinity of anti-TGF-beta/VEGF (transforming growth factor-beta/VEGF) dual-functional fusion protein and VEGF
To test the binding capacity of the anti-TGF-. Beta./VEGF bifunctional fusion proteins to VEGF, VEGF protein (purchased from microbiosystems, cat. # VE 5-H4210) was plated at 100 ng/well overnight at 4 ℃. The plates were washed 3 times with PBST, 200. Mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. Diluting the antibody with a diluent to 200nM at a 4-fold ratio to form 12 concentration gradients, sequentially adding the sealed ELISA plates at 100 μ l/well, and standing at 37 deg.C for 1 hr. The plates were washed 3 times with PBST, and an HRP-labeled goat anti-human Fab antibody (purchased from abcam, cat. # ab 87422) was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); adding stop solution into each hole to stop the reaction of the substrate, reading OD value at 450nm of an enzyme-labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC 50 。
As shown in FIG. 3B, the anti-TGF-beta/VEGF dual-function fusion proteins TV01, TV02, TV03, TV04 and TV05 can effectively bind VEGF, the EC50 (nM) values are 0.478,0.394,0.340,0.381 and 0.393, and the affinities are equivalent. anti-TGF-. Beta.mAb 12.7 did not bind VEGF efficiently.
3.3 ELISA (enzyme-Linked immuno sorbent assay) for detecting capacity of anti-TGF-beta/VEGF (transforming growth factor-beta/VEGF) dual-function fusion protein for simultaneously combining TGF-beta 1 and VEGF
Steric hindrance may affect the ability of an anti-TGF-. Beta./VEGF bifunctional fusion protein to bind both antigens simultaneously. To test the ability of the anti-TGF-. Beta./VEGF bifunctional fusion proteins to bind both TGF-. Beta.1 and VEGF, TGF-. Beta.1 was plated at 100 ng/well overnight at 4 ℃. The plates were washed 3 times with PBST, 200. Mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. Diluting anti-TGF-beta/VEGF dual-function fusion protein by using diluentDiluting to initial concentration of 200nM at 2 times ratio to form 12 concentration gradients, sequentially adding the sealed ELISA plate at 100 μ l/well, and standing at 37 deg.C for 1 hr. PBST washing plate 3 times, according to 150 ng/hole adding Biotinylated VEGF (Yi Qiao Shen, cat. # 11066-H27H-B) antigen, 37 degrees C placed for 1 hours. After 3 PBST washes, HRP-labeled Streptavidin was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. Mu.l of 2M H per well 2 SO 4 Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC 50 。
As shown in FIG. 3C, the result of the experiment was that TV03 was able to bind both TGF-. Beta.1 and VEGF with an EC50 (nM) value of 5.855.
Example 4 determination of affinity dissociation constant KD of anti-TGF-beta/VEGF bifunctional fusion proteins for the antigens TGF-beta 1, TGF-beta 2
Kinetic parameters of the binding dissociation of the anti-TGF-. Beta./VEGF bifunctional fusion protein and the antigen TGF-. Beta.1/TGF-. Beta.2 were determined using a capture method using an octet molecular interaction analyzer, the AR2G probe was activated with 20mM EDC and 10mM s-NHS, the antigen TGF-. Beta.1/TGF-. Beta.2 was diluted to 5. Mu.g/ml with 10mM Sodium Acetate (Sodium Acetate, pH 6.0), bound to the activated AR2G probe, and the probe was blocked with 1M Ethanolamine (Ethanolamine, pH 8.5) solution, respectively. Fusion proteins were diluted with 1 × kinetic Buffer working solution and dissociated in 1 × kinetic Buffer working solution, setting 5 concentration gradients with a maximum concentration of 3.13nM/6.25nM, respectively.
The experimental results are shown in Table 4, the affinity dissociation constant of the anti-TGF-beta/VEGF double-function fusion proteins TV01 and TV03 is equivalent to that of a positive control anti-TGF-beta 1 monoclonal antibody 12.7 for TGF-beta 1; whereas for TGF-. Beta.2, TV03 has an affinity comparable to 12.7, TV01 has a slightly weaker affinity.
TABLE 4 affinity dissociation constants
Figure BDA0003218436730000201
Note: KD is the affinity constant; kon is the binding rate constant; kdis the dissociation rate constant.
Example 5 determination of affinity dissociation constant KD of anti-TGF-beta/VEGF bifunctional fusion proteins for antigen VEGF
Kinetic parameters of binding dissociation of anti-TGF-. Beta./VEGF bifunctional fusion protein and antigen VEGF-A165 were determined using a capture method using an octet molecular interaction analyzer, AR2G probe was activated with 20mM EDC and 10mM s-NHS, antigen VEGF-A165 was diluted to 5. Mu.g/ml with 10mM Sodium Acetate (Sodium Acetate, pH 6.0), bound to the activated AR2G probe, and the probe was blocked with 1M Ethanolamine (Ethanolamine, pH 8.5) solution. Fusion proteins were diluted with 1 × kinetic Buffer working solution and dissociated in 1 × kinetic Buffer working solution, setting 5 concentration gradients with a maximum concentration of 12.5 nM.
The experimental result is shown in Table 5, the affinity of the anti-TGF-beta/VEGF double-function fusion protein TV03 for VEGF-A165 is slightly better than that of a positive control anti-VEGF monoclonal antibody Bevacizumab.
TABLE 5 affinity dissociation constants
Figure BDA0003218436730000202
Figure BDA0003218436730000211
Note: KD is the affinity constant; kon is the binding rate constant; kdis the dissociation rate constant.
Example 6 SMAD3 reporter Gene inhibition assay
SBE Reporter HEK293 Cell (available from BPS bioscience, cat. # 60653) expresses Smad3 binding element (SEB) with a luciferase Reporter gene, and the in vitro activity of antibodies can be evaluated by studying the inhibitory effect of antibody proteins on TGF- β 1-induced Smad3 activation in this Cell. SBE293 cells grown in the logarithmic phase in adherent culture were taken, washed once with DPBS, digested with pancreatin, and neutralized with pancreatin. Trypan blue cells were counted and centrifuged at 300g for 5 min. Medium (all purchased from Gibico Co.) containing MEM (10% FBS, 1% non-essential amino acids, 1mM sodium pyruvate)#10095-098, 10091-148, 11140-050, 11360-070) after resuspension, counting the plates, adjusting the density to 35000/well, 100. Mu.l/well, placing at 37 ℃,5% CO 2 Incubate overnight for about 24 hours. TGF-. Beta.1 (10 ng/ml) was diluted with MEM (0.5% FBS, 1% nonessential amino acids, 1mM sodium pyruvate) medium, and the fusion protein was diluted to 200nM, 4-fold dilution, 10 gradients, left at room temperature for 1 hour, and then the original medium of the cell plate was replaced, and incubated at 37 ℃ overnight. Add 100. Mu.l/well of detection reagent Bio-Glo (30 minutes earlier at 25 ℃ water bath thaw equilibration temperature). After incubation for 10 min at room temperature, luminescences were read with SpectraMax i 3.
As shown in FIG. 4, the fusion proteins inhibited the TGF- β -induced activity of the pSMAD3 reporter in a dose-dependent manner, and the IC50 (nM) values of the fusion proteins TV01, TV02, TV03, TV04, TV05 and the positive control 12.7 mAbs were 0.388,0.177,0.255,0.221,0.341 and 0.337, respectively, showing comparable inhibitory activity.
Example 7 cell experiments with the TGF-. Beta./VEGF bifunctional fusion proteins blocking the binding of VEGF to the receptor KDR
KDR cells (purchased from promega, cat. # GA 1082) grown in adherent culture at a density of approximately 80% -90% in log phase were removed from the growth medium. After washing once with DPBS, use
Figure BDA0003218436730000212
Digestion (Sigma, cat. # A6964), neutralization of pancreatin, centrifugation at 200g for 5 minutes, resuspension of cells in DMEM medium containing 10-FBS (purchased from Gibco, cat. # 11995), trypan blue cell counting, adjustment of cell density to 40000/well plating, 50. Mu.l/well, at 37 ℃,5% CO 2 . VEGF was diluted to 30ng/ml with 10% FBS-containing DMEM medium, antibody was diluted two-fold with VEGF-containing medium, 3-fold dilution, 10 gradients. The diluted antibody was added to 25. Mu.l per well of cells (final VEGF concentration 10ng/ml, starting antibody concentration 100 nM), and after incubation at 37 ℃ for 6 hours, 75. Mu.l of detection reagent Bio-Glo (purchased from promega, cat. # G7940) was added to each well. After incubation for 10 min at room temperature, luminescences were read with SpectraMax i3 ×. All data were binoculated, and the signal values averaged using the 4-parameter methodFitting, data analysis was performed with GraphPad Prism 6.
The results of the experiment are shown in FIG. 5: the TGF-beta/VEGF double-function fusion protein and the positive control Bevacizumab can effectively block the interaction between VEGF and a receptor KDR thereof, and the blocking capability of the double-function fusion protein is better. IC of TV03, TV04 and Bevacizumab 50 The (nM) values were 0.096,0.092, and 0.305, respectively.
Example 8 anti-TGF-. Beta./VEGF bifunctional fusion proteins anti-angiogenic effects in model of alkali-burned corneal neovascularization
The model of new vessel of cornea burned by alkali is adopted to verify the function of TGF-beta/VEGF double-function fusion protein in inhibiting new vessel. Firstly, 0.7% sodium pentobarbital is used for intraperitoneal injection, female Balb/c mice (purchased from Slek company) with the diameter of 8 weeks are anesthetized, after the mice are anesthetized, filter paper with the diameter of 2mm is soaked in 1M NaOH, and then the filter paper is placed on the cornea of the mice for burning for 30 seconds; taking away the filter paper, washing the burned eyes with a large amount of physiological saline to prevent NaOH from damaging eyelids, then randomly grouping the animals, and carrying out first administration on the same day (day 1) for 6 days continuously and 5 times daily, wherein 5 mul of TV03 (1 mg/ml) is dripped into the administration group every time, and 5 mul of PBS (0.01M) is dripped into the normal group and the control group; 3 mu l of levofloxacin is firstly dripped before the first administration of the first three days for 30 minutes; on day 7, the mice were euthanized, the cornea was picked off to prepare paraffin sections, and CD31 immunohistochemical staining was performed to evaluate the formation of new blood vessels; neovascular Area (neovasular Area%) = CD31 (+) Area/cornea Area.
The experimental results show in fig. 6 that TV03 significantly inhibited the formation of corneal neovascularization in this model.
Example 9 anti-angiogenic Effect of anti-TGF-. Beta./VEGF bifunctional fusion proteins in a matrigel angiogenesis model
The matrigel containing heparin, VEGF and TGF-beta 1 factors is injected into the subcutaneous part of the mouse, so that the blood vessels under the skin of the mouse are induced to grow into the matrigel, and partial blood is remained in the matrigel. Therefore, measuring the amount of heme in matrigel can be used to characterize the amount of neovasculature in matrigel. We used this model to verify the inhibitory effect of TGF-. Beta./VEGF bifunctional fusion proteins on angiogenesis.
Matrigel (Matrigel Matrix, available from Corning, inc., cat # 356231) was first dispensed, the Matrigel was thawed in a 4 c freezer one night in advance, and a sterile 1.5ml ep tube, 200 μ l tip were placed in a-80 c freezer for use. Matrigel was packed in a clean room at every other day, and then heparin (final concentration 40U), VEGF (final concentration 800 ng/ml), TGF-. Beta.1 (final concentration 800 ng/ml), and TV03 (final concentration 1000. Mu.g/ml), mAb 12.7 (final concentration 860. Mu.g/ml), and fusion protein FC-D2 (a homodimer form of fusion protein constructed by fusing Fc fragment of IgG1 with D2 domain of VEGFR1, whose monomer amino acid sequence is shown in SEQ ID NO:17, final concentration 443.3. Mu.g/ml) were added to matrigel, mixed well and left to work on ice throughout. Wherein the molar amounts of TV03, 12.7, FC-D2 are equal.
8-week male C57BL/6 mice (purchased from Slek) were randomized into 5 groups: blank normal group, n = 10; VEGF + TGF-beta 1 modeling group, n = 15; VEGF + TGF-beta 1+ TV03 administration group, n = 10; VEGF + TGF-beta 1+12.7 administration group, n = 10; VEGF + TGF-beta 1, FC-D2 administration group, n = 10. The mice were anesthetized by intraperitoneal injection with 0.7% sodium pentobarbital, after which the hairs on the inside of the thigh roots on the outer side of the abdomen were shaved off and the matrigel mixture was then injected subcutaneously at this site using an insulin needle (previously frozen at-20 ℃). The mice were removed subcutaneous matrigel after 2 weeks and assayed for heme (hemoglobin) using the Drabkin kit (purchased from sigma, cat # D5941).
As shown in FIG. 7 and Table 6, in this model, the TV03, the 12.7 monoclonal antibody and the FC-D2 can significantly inhibit the angiogenesis induced by VEGF and TGF-beta 1, wherein the efficacy of the TV03 is optimal.
TABLE 6
Figure BDA0003218436730000231
Example 10 anti-tumor Effect of anti-TGF-. Beta./VEGF bifunctional fusion proteins on human pancreatic cancer BxPC-3 nude mouse transplantation tumor model
Collecting the culture in vitroBxPC-3 cells, the concentration of the cell suspension was adjusted to 1X 10 8 Perml, mixed with an equal volume of matrigel. Under sterile conditions, 100. Mu.l of the cell suspension was inoculated subcutaneously into the right flank of BALB/C-nude mice (purchased from Shanghai, inc., laboratory animal technology, wei Tongli, beijing). Measuring the diameter of the transplanted tumor by using a vernier caliper until the average tumor volume grows to 100mm 3 -150mm 3 Animals were randomized left and right. The dose of the test sample TV03 was 46.4mg/kg and the dose of the control mAb 12.7 was set to 40mg/kg. The blank control group was given the same volume of saline. The administration was performed intraperitoneally 2 times a week while measuring the diameter of the transplanted tumor and the body weight of the mouse. The Tumor Volume (TV) is calculated as: TV =1/2 × a × b 2 . Wherein a and b represent length and width, respectively. Calculating Relative Tumor Volume (RTV) according to the measurement result, wherein the calculation formula is as follows: RTV = V t /V 0 . Wherein V 0 When administered in groups (i.e. d) 0 ) Measurement of the resulting tumor volume, V t For the tumor volume at each measurement. The evaluation index of the antitumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows: T/C (%) = (T) RTV /C RTV )×100(T RTV : treatment group RTV; c RTV : negative control group RTV). Tumor inhibition rate TGI (%) =1-T/C (%)
The experimental results are shown in FIG. 8, the TV03 tumor inhibition rate TGI at day 32 is 83.66% in the human pancreatic cancer BxPC-3 nude mouse transplantation tumor model. The results show that TV03 is able to significantly inhibit tumor growth on this model of transplanted tumors.
Example 11 physical stability of anti-TGF-. Beta./VEGF bifunctional fusion proteins
The thermal stability of the TV03 samples in PBS buffer was examined by DSC (Differential scanning calorimetry). Samples were replaced in PBS buffer, controlled at 1mg/ml and tested using MicroCal Vp-Capillary DSC (Malvern). Before detection, the sample and the blank buffer were filtered through a 0.22 μm filter. Add 400. Mu.l sample or blank buffer per well of sample plate (set 6 blank buffer pairs), and finally add ddH to three pairs of well plates 2 O, in preparation forAnd (5) cleaning. And after the sample adding of the sample plate is finished, the plastic soft cover plate is sleeved. The scanning temperature starts from 25 ℃ and ends at 100 ℃ and the scanning speed is 150 ℃/h.
Specific results as shown in table 7, the sample TV03 protein showed good thermal stability.
TABLE 7TV03 thermal stability test data
Figure BDA0003218436730000241
Discussion of the preferred embodiments
Compared with a VEGF receptor and a TGF-beta antibody which are singly used, the TGF-beta/VEGF difunctional antibody fusion protein can block VEGF and TGF-beta signal channels at the same time with high efficiency. Preferably, the bifunctional antibody of the invention can synergistically and remarkably treat ophthalmic diseases such as CNV, PVR, AMD and the like and tumors, and has important clinical application prospects.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
Sequence listing
<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH
<120> TGF-beta/VEGF bifunctional antibody fusion protein
<130> P2021-1643
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 556
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ile Thr Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn Tyr Asn Glu Met Phe
50 55 60
Glu Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asp Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala
435 440 445
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro Phe
450 455 460
Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly
465 470 475 480
Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val
485 490 495
Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg
500 505 510
Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr
515 520 525
Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu
530 535 540
Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
545 550 555
<210> 2
<211> 1725
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgggcgtga aggtgctgtt cgccctgatc tgtattgctg tggctgaggc tcaggtgcag 60
ctggtgcagt ctggagctga ggtgaagaag cctggcgctt ctgtgaaggt gtcttgtaag 120
gcttctggat atatctttat tacctattgg atgaattggg tgagacaggc tcctggccag 180
ggcctggagt ggatcggaca gatttttcca gcttctggct ccacaaatta taatgagatg 240
tttgagggca gagctacact gacagtggat acatctacat ctaccgccta catggaactg 300
tcttctctga gatctgagga tacagctgtg tactattgtg ctagaggcga tggcaattat 360
gctctggatg ctatggatta ttggggccag ggaacactgg tgaccgtgtc ttctgcaagt 420
accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 480
gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 540
tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 600
tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 660
tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtgga gtccaagtac 720
ggcccaccat gtcctccatg tccagcccct gaatttttgg gcgggccttc tgtctttctg 780
tttcctccta aacctaaaga taccctgatg atcagccgca cacccgaagt cacttgtgtg 840
gtcgtggatg tgtctcagga agatcccgaa gtgcagttta actggtatgt cgatggcgtg 900
gaagtgcata atgccaaaac taagccccgc gaagaacagt tcaacagcac ttatcgggtc 960
gtgtctgtgc tcacagtcct ccatcaggat tggctgaatg ggaaagaata taagtgcaag 1020
gtgagcaata agggcctccc cagcagcatc gagaagacta ttagcaaagc caaagggcag 1080
ccacgggaac cccaggtgta cactctgccc ccctctcagg aggagatgac taaaaatcag 1140
gtctctctga cttgtctggt gaaagggttt tatcccagcg acattgccgt ggagtgggag 1200
tctaatggcc agcccgagaa taattataag acaactcccc ccgtcctgga ctctgacggc 1260
agctttttcc tgtattctcg gctgacagtg gacaaaagtc gctggcagga gggcaatgtc 1320
tttagttgca gtgtcatgca tgaggccctg cacaatcact atacacagaa aagcctgtct 1380
ctgagtctgg gcgccggcgg aggcggaagc ggaggaggag gatccgatac tggaaggcca 1440
tttgtggaga tgtacagcga gatcccagag atcattcaca tgacagaagg aagggagctc 1500
gtcatcccat gcagagtgac aagccctaac atcactgtca ctctcaagaa gttcccactc 1560
gacacactca tcccagatgg caagagaatc atttgggaca gcagaaaggg cttcatcatc 1620
tccaacgcca catataagga gatcggactg ctcacttgcg aagctacagt caacggccac 1680
ctctataaga ctaactatct gactcatagg caaacaaact gataa 1725
<210> 3
<211> 218
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly
1 5 10 15
Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Leu Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 4
<211> 708
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgggcgtga aggtgctgtt tgctctgatt tgcattgctg tggctgaggc tgatattgtg 60
ctgacacagt cccctgcttc tctggctgtg tctccaggac agagggctac catcacctgt 120
agagcttctg agtctgtgga ttcttacgga aattctttta tgcattggta tcagcagaag 180
cctggccagc ctcctaagct gctgatctac ctggcttcta atctggagtc tggcgtgcca 240
gctagatttt ctggcagcgg atccggcact gattttacac tgacaattaa tcctgtggag 300
gctgatgaca cagctaatta ctactgtcag cagaataatg aggatccact gacatttgga 360
ggcggcacca aggtggagct gaagagaacc gtcgccgctc ccagcgtctt catcttcccc 420
cccagcgatg agcagctgaa gagcggaacc gccagcgtgg tgtgcctgct gaacaacttc 480
taccccaggg aggccaaggt gcaatggaag gtggacaacg ccctacagag cggcaactcc 540
caggagagcg tgaccgagca ggacagcaag gatagcacct acagcctgag cagcaccctc 600
accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccatcag 660
ggcctgagca gccctgtgac caagagcttc aacaggggcg agtgctga 708
<210> 5
<211> 568
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile
1 5 10 15
Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr
20 25 30
Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu
35 40 45
Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile
50 55 60
Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala
65 70 75 80
Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln
85 90 95
Thr Asn Thr Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly
115 120 125
Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
130 135 140
Ser Gly Tyr Ile Phe Ile Thr Tyr Trp Met Asn Trp Val Arg Gln Ala
145 150 155 160
Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile Phe Pro Ala Ser Gly
165 170 175
Ser Thr Asn Tyr Asn Glu Met Phe Glu Gly Arg Ala Thr Leu Thr Val
180 185 190
Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser
195 200 205
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Asp Gly Asn Tyr Ala
210 215 220
Leu Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
225 230 235 240
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
245 250 255
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
260 265 270
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
275 280 285
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
290 295 300
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
305 310 315 320
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
325 330 335
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
340 345 350
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
355 360 365
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
370 375 380
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
385 390 395 400
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
405 410 415
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
420 425 430
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
435 440 445
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
450 455 460
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
465 470 475 480
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
485 490 495
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
500 505 510
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
515 520 525
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
530 535 540
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
545 550 555 560
Ser Leu Ser Leu Ser Leu Gly Lys
565
<210> 6
<211> 1758
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgggcgtga aggtgctgtt cgccctgatc tgcatcgccg tggccgaggc cgatactgga 60
aggccatttg tggagatgta cagcgagatc ccagagatca ttcacatgac agaaggaagg 120
gagctcgtca tcccatgcag agtgacaagc cctaacatca ctgtcactct caagaagttc 180
ccactcgaca cactcatccc agatggcaag agaatcattt gggacagcag aaagggcttc 240
atcatctcca acgccacata taaggagatc ggactgctca cttgcgaagc tacagtcaac 300
ggccacctct ataagactaa ctatctgact cataggcaaa caaacactat cggtggaggc 360
ggttcaggcg gaggtggcag cggcggtggc gggtcgggag ggggtggctc tcaggtgcag 420
ctggtgcagt ctggagctga ggtgaagaag cctggcgctt ctgtgaaggt gtcttgtaag 480
gcttctggat atatctttat tacctattgg atgaattggg tgagacaggc tcctggccag 540
ggcctggagt ggatcggaca gatttttcca gcttctggct ccacaaatta taatgagatg 600
tttgagggca gagctacact gacagtggat acatctacat ctaccgccta catggaactg 660
tcttctctga gatctgagga tacagctgtg tactattgtg ctagaggcga tggcaattat 720
gctctggatg ctatggatta ttggggccag ggaacactgg tgaccgtgtc ttctgcaagt 780
accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 840
gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 900
tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 960
tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 1020
tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtgga gtccaagtac 1080
ggcccaccat gtcctccatg tccagcccct gaatttttgg gcgggccttc tgtctttctg 1140
tttcctccta aacctaaaga taccctgatg atcagccgca cacccgaagt cacttgtgtg 1200
gtcgtggatg tgtctcagga agatcccgaa gtgcagttta actggtatgt cgatggcgtg 1260
gaagtgcata atgccaaaac taagccccgc gaagaacagt tcaacagcac ttatcgggtc 1320
gtgtctgtgc tcacagtcct ccatcaggat tggctgaatg ggaaagaata taagtgcaag 1380
gtgagcaata agggcctccc cagcagcatc gagaagacta ttagcaaagc caaagggcag 1440
ccacgggaac cccaggtgta cactctgccc ccctctcagg aggagatgac taaaaatcag 1500
gtctctctga cttgtctggt gaaagggttt tatcccagcg acattgccgt ggagtgggag 1560
tctaatggcc agcccgagaa taattataag acaactcccc ccgtcctgga ctctgacggc 1620
agctttttcc tgtattctcg gctgacagtg gacaaaagtc gctggcagga gggcaatgtc 1680
tttagttgca gtgtcatgca tgaggccctg cacaatcact atacacagaa aagcctgtct 1740
ctgagtctgg gcaaatga 1758
<210> 7
<211> 556
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Glu
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn Tyr Asn Glu Met Tyr
50 55 60
Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ile Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala
435 440 445
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro Phe
450 455 460
Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly
465 470 475 480
Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val
485 490 495
Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg
500 505 510
Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr
515 520 525
Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu
530 535 540
Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
545 550 555
<210> 8
<211> 1731
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atggagactg gactcagatg gctgctgctg gtcgctgtgc tgaagggcgt ccaatgccaa 60
gtgcagctgg tgcagagcgg cgccgaggtg aagaagcctg gcgctagcgt gaaggtgagc 120
tgcaaggcta gcggctacac cttcacaagc gagtggatga actgggtgag acaagcccct 180
ggccaaggcc tggagtggat gggacagatc ttccctgccc tgggcagcac caactacaac 240
gagatgtacg agggtagagt caccatgacg accgacacaa gcacaagcac cgcctacatg 300
gagctgagaa gcctgagaag cgacgacacc gccgtgtact actgcgctag aggcatcggc 360
aactacgccc tggacgccat ggactactgg ggccaaggca ccctggtgac agtcagcagc 420
gctagcacca agggccctag cgtgttccct ctggcccctt gcagcagaag cacaagcgag 480
agcaccgccg ccctgggctg tttggtgaag gactacttcc ctgagcctgt gaccgtaagc 540
tggaacagcg gcgccctgac aagcggcgtg cacaccttcc ctgccgtgct gcagagcagc 600
ggcctgtaca gcctgagcag cgtggtgacc gttcctagca gcagcctggg caccaagacc 660
tacacctgca acgtggacca caagcctagc aacaccaagg tggacaagag agtggagagc 720
aagtacggcc cgccatgccc tccttgtcct gccccggagt tcctgggcgg ccctagcgtt 780
ttcctcttcc ctcctaagcc taaggatacg ctaatgatta gcagaacccc tgaggtgacc 840
tgcgtggtgg tggacgtgag ccaagaggac cctgaggtgc agttcaactg gtacgtggac 900
ggcgtggagg tgcacaacgc caagaccaag cctagagagg agcagttcaa cagcacctac 960
agagtggtga gcgtgctgac cgtgctgcac caagactggc tgaacggcaa ggagtacaag 1020
tgcaaggtga gcaacaaggg cctgccgtcc tccatcgaga agaccatcag caaggccaag 1080
ggacagccta gagagcctca agtgtacacc ctgcctccta gccaagagga gatgaccaag 1140
aaccaagtga gcctgacctg tcttgtgaag ggcttttacc ctagcgacat cgccgtggag 1200
tgggagagca acggacagcc tgagaacaac tacaagacca cccctcctgt gctggacagc 1260
gacggcagct tcttcctgta cagcagactg accgtggaca agagcagatg gcaagagggc 1320
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac tcagaaatct 1380
ctgagcctgt cgttaggagc cggcggaggc ggctccggcg gaggcggcag cgacacgggc 1440
agacctttcg tggagatgta cagcgagatc cctgagatca tccacatgac cgagggcaga 1500
gagctggtga tcccttgcag agtaactagc cctaacatca ccgtgaccct gaagaagttc 1560
cctctggaca ccctgatccc tgacggcaag agaatcatct gggacagcag aaagggcttc 1620
atcatcagca acgccaccta caaggagatc ggcctgctga cctgcgaggc caccgtgaac 1680
ggccacctgt acaagaccaa ctacctgacc cacagacaga ccaactgata a 1731
<210> 9
<211> 218
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
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 Arg Ala Ser Glu Ser Val Asp Phe Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Ile
85 90 95
Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 10
<211> 726
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atggatacta gggctcctac acagctgctg ggactgctgc tgctgtggct gcccggcgct 60
agatgtgaca ttcagatgac acagagccct agcagcctga gcgctagcgt gggcgacaga 120
gtgaccatca cctgcagagc tagcgagagc gtggacttct acggcaacag cttcatgcac 180
tggtatcagc agaagcctgg caaggcccct aagctgctga tctacctggc tagcaacctg 240
gagagcggcg tgcctagcag attcagcggc agcggcagcg gcaccgactt caccctgacc 300
atcagcagcc tgcagcctga ggacttcgcc acctactact gtcagcagaa catcgaggac 360
cctctgacct tcggcggcgg caccaaggtg gagatcaaga gaaccgtggc cgcccctagc 420
gtgttcatct tccctcctag cgacgagcag ctgaagagcg gcaccgctag cgtggtgtgc 480
ctgctgaaca acttctaccc tagagaggcc aaggtgcagt ggaaggtgga caacgccctg 540
cagagcggca acagccaaga gagcgtgacc gagcaagaca gcaaggacag cacctacagc 600
ctgagcagca ccctgaccct gagcaaggcc gactacgaga agcacaaggt gtacgcctgc 660
gaggtgaccc accaaggcct gagcagccct gtgaccaaga gcttcaacag aggcgagtgc 720
tgataa 726
<210> 11
<211> 568
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile
1 5 10 15
Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr
20 25 30
Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu
35 40 45
Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile
50 55 60
Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala
65 70 75 80
Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln
85 90 95
Thr Asn Thr Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly
115 120 125
Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
130 135 140
Ser Gly Tyr Thr Phe Thr Ser Glu Trp Met Asn Trp Val Arg Gln Ala
145 150 155 160
Pro Gly Gln Gly Leu Glu Trp Met Gly Gln Ile Phe Pro Ala Leu Gly
165 170 175
Ser Thr Asn Tyr Asn Glu Met Tyr Glu Gly Arg Val Thr Met Thr Thr
180 185 190
Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser
195 200 205
Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ile Gly Asn Tyr Ala
210 215 220
Leu Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
225 230 235 240
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
245 250 255
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
260 265 270
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
275 280 285
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
290 295 300
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
305 310 315 320
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
325 330 335
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
340 345 350
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
355 360 365
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
370 375 380
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
385 390 395 400
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
405 410 415
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
420 425 430
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
435 440 445
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
450 455 460
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
465 470 475 480
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
485 490 495
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
500 505 510
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
515 520 525
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
530 535 540
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
545 550 555 560
Ser Leu Ser Leu Ser Leu Gly Lys
565
<210> 12
<211> 1758
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgggcgtga aggtgctgtt cgccctgatc tgcatcgccg tggccgaggc cgatactgga 60
aggccatttg tggagatgta cagcgagatc ccagagatca ttcacatgac agaaggaagg 120
gagctcgtca tcccatgcag agtgacaagc cctaacatca ctgtcactct caagaagttc 180
ccactcgaca cactcatccc agatggcaag agaatcattt gggacagcag aaagggcttc 240
atcatctcca acgccacata taaggagatc ggactgctca cttgcgaagc tacagtcaac 300
ggccacctct ataagactaa ctatctgact cataggcaaa caaacactat cggtggaggc 360
ggttcaggcg gaggtggcag cggcggtggc gggtcgggag ggggtggctc tcaagtgcag 420
ctggtgcaga gcggcgctga ggtcaaaaag cccggcgcct ccgtgaaggt gagctgtaag 480
gccagcggct acacattcac tagcgagtgg atgaactggg tgagacaagc ccccggccaa 540
ggactggaat ggatgggcca gatcttccca gctctgggct ccactaacta caacgagatg 600
tacgagggaa gggtcactat gactacagac actagcacta gcactgccta catggaactg 660
aggtctctga gaagcgacga tacagccgtg tactactgcg ccagaggcat cggcaactat 720
gctctggatg ccatggacta ctggggccaa ggcactctcg tgactgtgag ctccgcaagt 780
accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 840
gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 900
tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 960
tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 1020
tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtgga gtccaagtac 1080
ggcccaccat gtcctccatg tccagcccct gaatttttgg gcgggccttc tgtctttctg 1140
tttcctccta aacctaaaga taccctgatg atcagccgca cacccgaagt cacttgtgtg 1200
gtcgtggatg tgtctcagga agatcccgaa gtgcagttta actggtatgt cgatggcgtg 1260
gaagtgcata atgccaaaac taagccccgc gaagaacagt tcaacagcac ttatcgggtc 1320
gtgtctgtgc tcacagtcct ccatcaggat tggctgaatg ggaaagaata taagtgcaag 1380
gtgagcaata agggcctccc cagcagcatc gagaagacta ttagcaaagc caaagggcag 1440
ccacgggaac cccaggtgta cactctgccc ccctctcagg aggagatgac taaaaatcag 1500
gtctctctga cttgtctggt gaaagggttt tatcccagcg acattgccgt ggagtgggag 1560
tctaatggcc agcccgagaa taattataag acaactcccc ccgtcctgga ctctgacggc 1620
agctttttcc tgtattctcg gctgacagtg gacaaaagtc gctggcagga gggcaatgtc 1680
tttagttgca gtgtcatgca tgaggccctg cacaatcact atacacagaa aagcctgtct 1740
ctgagtctgg gcaaatga 1758
<210> 13
<211> 448
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Glu
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn Tyr Asn Glu Met Tyr
50 55 60
Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ile Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 14
<211> 336
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
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 Arg Ala Ser Glu Ser Val Asp Phe Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Ile
85 90 95
Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr
225 230 235 240
Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His
245 250 255
Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro
260 265 270
Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro
275 280 285
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser
290 295 300
Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val
305 310 315 320
Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
325 330 335
<210> 15
<211> 453
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
355 360 365
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 Lys
450
<210> 16
<211> 214
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
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
210
<210> 17
<211> 337
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly
225 230 235 240
Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met
245 250 255
Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn
260 265 270
Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp
275 280 285
Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn
290 295 300
Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn
305 310 315 320
Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr
325 330 335
Ile

Claims (12)

1. A bifunctional fusion protein, wherein the bifunctional fusion protein is a dimer formed from two monomers, each monomer comprising:
a first binding domain Z1; and
a second binding domain Z2;
wherein the first binding domain specifically binds to the target molecule TGF-. Beta.s;
the second binding domain specifically binds to the target molecule VEGF.
2. The bifunctional fusion protein of claim 1 wherein each monomer in the bifunctional fusion protein has a structure from N-terminus to C-terminus as shown in formula I:
Figure FDA0003218436720000011
wherein the content of the first and second substances,
t1, T2, T3 are each independently absent or an extracellular region of a type I VEGF receptor (VEGFR 1), and at least one is not absent;
l1, L2, L3 are each independently a bond or linker element;
VL represents the light chain variable region of an anti-TGF-beta antibody;
CL represents the light chain constant region of an anti-TGF-. Beta.antibody;
VH represents the heavy chain variable region of an anti-TGF-beta antibody;
CH represents the heavy chain constant region of an anti-TGF-beta antibody;
"-" represents a disulfide bond or a covalent bond;
"-" represents a peptide bond;
wherein the bifunctional fusion protein has the activity of simultaneously binding VEGF and TGF-beta.
3. The bifunctional fusion protein of claim 1 or 2, wherein the fusion protein is selected from the group consisting of:
(1) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:1, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:3 is shown in the specification;
(2) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:5, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:3 is shown in the specification;
(3) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:7, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:9 is shown in the figure;
(4) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:11, and the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:9 is shown in the figure;
(5) The heavy chain amino acid sequence of the fusion protein is shown as SEQ ID NO:13, the light chain amino acid sequence of the fusion protein is shown as SEQ ID NO:14 is shown in the figure; or
(6) And (2) the polypeptide which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequences in the (1) to (5) and has the activity of simultaneously binding VEGF and TGF-beta.
4. A polynucleotide molecule encoding the fusion protein of any one of claims 1 to 3.
5. An expression vector comprising the polynucleotide molecule of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. A method for preparing the fusion protein according to claim 1, comprising the steps of:
a) Culturing the host cell according to claim 6 under expression conditions, thereby expressing the bifunctional fusion protein;
b) Isolating and purifying the fusion protein of step a).
8. A pharmaceutical composition comprising an effective amount of the fusion protein according to any one of claims 1 to 3 and one or more pharmaceutically acceptable carriers, diluents or excipients.
9. Use of the fusion protein according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 8, for the preparation of a medicament for the treatment of an ophthalmic disease.
10. Use according to claim 9, wherein the ophthalmic disease is selected from: exudative age-related macular degeneration (AMD), choroidal Neovascularization (CNV), proliferative Vitreoretinopathy (PVR), or corneal neovascular disease.
11. Use of the fusion protein according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 8, for the preparation of a medicament for the treatment of a tumor.
12. An immunoconjugate, wherein the immunoconjugate comprises:
(a) The fusion protein of any one of claims 1 to 3; and
(b) A coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
CN202110950470.4A 2021-08-18 2021-08-18 TGF-beta/VEGF difunctional antibody fusion protein Pending CN115707716A (en)

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RU2386638C2 (en) * 2004-03-31 2010-04-20 Дженентек, Инк. Humanised anti-tgf-beta-antibody
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AU2018347521A1 (en) * 2017-10-12 2020-05-07 Immunowake Inc. VEGFR-antibody light chain fusion protein
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