TW201609803A - VEGF/PDGF [beta] fusion protein and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to a VEGF/PDGFR [beta] fusion protein and pharmaceutical use thereof. Furthermore, the present invention relates to a bifunctional fusion protein comprising VEGFR extracellular ligand-binding domain and PDGFR [beta] antibody fragments. It can be used for the treatment of wet age-related macular degeneration (AMD), and any other disease which can be improved by inhibiting VEGF and PDGF signaling pathways, such as cancer and diabetic retinopathy.

Description

VEGF and PDGFR β bispecific fusion protein and use thereof

The present invention relates to a VEGF and PDGFR beta bispecific fusion protein comprising a pharmaceutical composition thereof and use thereof.

Age-related macular degeneration (AMD) is a disabling, blinding disease. It mainly affects the central region of the macular area or the retina responsible for high visual acuity and is the cause of irreversible vision loss in the elderly. According to statistics, more than 30 million people worldwide suffer from the disease. As China's population ages, the disease has seriously jeopardized the lives and health of our people.

AMD comes in two forms, dry AMD and wet AMD. Although dry AMD is more common, it generally results in a lighter, slower loss of vision. New blood vessels are formed under the retina of patients with wet AMD. When photoreceptor cells and retinal pigment epithelial (RPE) cells slowly degenerate, blood vessels tend to develop from a normal position in the choroid to an abnormal position below the retina. This abnormal new blood vessel growth is called choroidal neovascularization (CNV). These abnormal blood vessels leak and bleed, resulting in bleeding, swelling, scar tissue, and severe vision loss. Only about 10% of AMD patients are wet Type, but it accounts for about 90% of the total number of blindness caused by AMD. Wet age-related macular degeneration (AMD) has become a leading threat to blindness in the elderly over 65 years of age.

Currently approved treatment kit for the treatment of wet AMD These include laser coagulation, photodynamic therapy, and anti-vascular drug therapy to reduce the level of excess VEGF or block VEGF production.

Depending on the location, sometimes laser treatment can be used to break the ring. Abnormal blood vessels formed in wet AMD. Only 15% of wet AMD is suitable for laser treatment because the blood vessels cannot be too close to the mid-inner region of the macula. A laser is a beam of light that is absorbed by blood pigments, drugs, and PRE cells and converted into heat to burn abnormal blood vessels. Since the stimuli are still not removed, the formation of new blood vessels is often repeated, resulting in loss of vision. Photodynamic therapy mainly activates photosensitizing drugs by intravenously injecting photosensitizing drugs, and then irradiating choroidal neovascular lesions with non-thermal lasers of specific wavelengths. Treatment of wet AMD with photodynamic therapy can only stabilize or reduce the risk of decreased visual acuity in wet AMD. Because it is not the cause of treatment, photodynamic therapy can not prevent the possibility of recurrence, generally requires multiple treatments. Moreover, it should be protected from light for 48 hours after treatment to avoid photoreaction and skin burns. Therefore, it brings a lot of pain to the patient.

Platelet-derived growth factor family including platelet growth Factor (PDGF) and vascular endothelial cell factor (VEGF). Each growth factor can be produced by a variety of cells, and its receptor is a tyrosine kinase (RTK) type receptor. Platelet-derived growth factor family members include: PDGFA, PDGFB, PDGFC, PDGFD, Placental growth factor, PGF) and vascular endothelial growth factor (VEGF, VEGF41, VEGFB, VEGFC, FIGF (also known as VEGFD)).

VEGF is a powerful and unique vascular endothelial cell Mitogens promote all aspects of the angiogenesis process. In several in vivo experiments investigating angiogenesis, VEGF can induce angiogenesis. In experiments with vascular and lymphatic endothelial cells cultured in vitro, VEGF promotes endothelial cell proliferation, migration, and tubular structure formation. VEGF can increase the permeability of capillaries and posterior venules. Rapid injection of continuous endothelial destruction after skin or muscle injection (Xie K, Wei D, Huag S. Transcriptional anti-angiogenesis therapy of human pancreatic cancer [J]. Cytokine Growth Factor Rev 2006, 17: 147-156) . The role of each VEGF receptor (VEGFR) is different. VEGFR-2 acts to regulate endothelial growth, differentiation, and permeability; while VEGFR-1 is involved in regulating endothelial cell migration and aggregation, and inhibits signal transduction by VEGFR-2. VEGFR-3 may also play a role in vascular development, but more uniquely, it is expressed in lymphatic tissue and may play an important role in lymphoid system formation (Joukov V, Pajusola K, Kaipainen A, et al. A novel vascular endothelial growth factor VEGF-C is a ligand for the Flt4 and KDR receptor tyrosine kinases [J]. EMBO J, 2003, 15(2): 290-298).

VEGF exerts a biological effect by VEGFR. VEGF Binding to VEGFR, dimerizes and autophosphorylates VEGFR, and transmits signals (including MAPK, PI3 kinase, Ras, and phospholipase C pathways) through multiple intracellular pathways, and finally plays a role. So far, VEGF has been found to have 3 Receptors VEGFR-1 (Flt-1), VEGFR-2 (Flk-1) and VEGFR-3 (Flt-4). The first two are mainly expressed in endothelial cells and also in tumor cells, while VEGFR-3 is mainly expressed in lymphatic tissue. The first two receptors have high affinity with VEGF-A and belong to the tyrosine kinase family. Binding to VEGF-A results in its own dimerization, intracellular tyrosine autophosphorylation, and subsequent initiation of downstream signaling proteins to exert its physiological functions. The former two are mainly expressed in endothelial cells and also expressed in tumor cells, and VEGFR-3 is mainly expressed in lymphatic tissue (Oh HA novel molecular mechanism involved neuropilin-1 for vascular endothelial growth factor-induced retinal angiogenesis. Nippon Gakkai Zasshi. 2003-Nov: 107 (11): 651-656).

VEGF is known to be a key regulator of neovascularization associated with intraocular conditions. The level of VEGF in eye drops is closely related to the level of active vascular proliferation in diabetic patients and other patients with ischemic retinopathy. Other studies have demonstrated the presence of VEGF in the choroidal neovascular membrane of AMD patients. The currently approved anti-angiogenic drugs for the treatment of wet AMD aim to neutralize the action of VEGF and block angiogenesis from the surface. For example: Pfaptanib (Pegaptanib, trade name Macugen), its active ingredient, ganbanitan, is a 28-nucleotide "aptamer" with a three-dimensional structure that allows it to interact with extracellular blood vessels. Endothelial growth factor receptor (VEGF) binds and inhibits the binding of VEGF to the corresponding receptor. It needs to be administered by intravitreal injection every six weeks; Ranibizumab (Ranbizumab, trade name Lucentis), the active ingredient of Lucentis, ranibizumab, is an antibody fragment that binds to VEGF and requires intravitreal injection every month. ; VEGF-Trap-eye (trade name Eylea), its active ingredient, Apoxe, is a polypeptide fragment that binds to VEGF and needs to be injected every month for the first three months, followed by injection every two months. .

But the current standard drug treatment for a considerable number of patients Invalid, for example, the current standard Ranibizumab treatment can only improve the visual acuity of 1/3 of patients, and 10% of patients do not respond to the treatment; in addition, about 70% of patients have no obvious visual acuity after anti-VEGF treatment. There is also adaptive resistance in current medical treatment, that is, the patient is resistant to the treatment after a period of treatment. At the same time, because the current treatment is by intraocular injection, each injection not only causes expensive treatment costs, but also causes intraocular infection, bleeding and retinal detachment. The most important complication is intraocular infection. Once infected, the consequences are unimaginable and may be blind. Therefore, the frequency of injection is also important for the development of drugs. Therefore, this field requires more effective treatment, overcomes the resistance to anti-VEGF treatment, and prolongs the duration of anti-VEGF drugs (not requiring one or two months of fundus injection) in order to be more effective and safer.

The platelet-derived factor PDGF is one of the early growth factors that occur during wound healing. It plays an important role in the whole process of wound healing, mainly in promoting wound healing. The common PDGF is a homo- or heterodimer formed by two disulfide bonds of two polypeptide chains, which makes PDGF have various forms of dimer structure, namely PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD. PDGF must bind to the corresponding receptor on the cell membrane to exert its biological effects. The PDGF receptor (PDGFR) is composed of two subunits, α and β, and its molecular weight. It is 170 to 180KD. The binding strength of the two to PDGF is very different. The α unit has a higher affinity with the PDGFa chain and the b chain, while the β subunit has only a high affinity with the b chain. Therefore, the alpha subunit can bind to PDGF-AA, PDGF-AB and PDGF-BB, and the beta subunit binds only to PDGF-BB and PDGF-AB.

PDGFR is a transmembrane glycoprotein with tyrosine egg White kinase activity. It consists of a domain specifically recognized by the extracellular N-terminus and PDGF, a transmembrane intermediate hydrophobic domain, and an intracellular C-terminal domain having tyrosine kinase activity. When PDGF binds to PDGFR on the cell surface, it promotes dimerization of the receptor molecule; initiates autophosphorylation of the tyrosine residue in the intracellular domain, or phosphorylates the tyrosine residue that initiates the specific target protein; In the afferent cells, the cascaded amplification effect regulates the life activities of the cells (including the division and proliferation of target cells). Thus, PDGFR antagonists that block PDGF binding or PDGFR dimerization can be used to treat or prevent diseases associated with the PDGFR pathway.

At present, the means of neutralizing VEGF is considered to have limited effect. It is because the vascular mural cells attached to the blood vessels protect the blood vessels in the absence of VEGF. Therefore, on the basis of neutralizing VEGF, if it can block the protection of vascular cells by pericytes, it can overcome the resistance of anti-VEGF treatment and more effectively treat age-related macular degeneration.

In order to achieve the above object, the present invention provides a bispecific fusion protein comprising a VEGF-binding peptide and a PDGFR β-binding peptide. The VEGF-binding peptide can be located upstream of the PDGFR beta-binding peptide; and vice versa.

In a preferred embodiment of the invention, the double Heteroconjugate proteins, which also include the Fc portion of immunoglobulins. The Fc segment functions to polymerize the fusion protein as a multimerization component. The Fc segment can be located upstream or downstream of the VEGF-binding peptide; can be located upstream or downstream of the PDGFR β-binding peptide; or between the VEGF-binding peptide and the PDGFR β-binding peptide.

In still another preferred embodiment of the invention, the Fc portion of the immunoglobulin is the Fc portion of an immunoglobulin IgG.

In a further preferred embodiment of the invention, the bispecific fusion protein, wherein the amino acid sequence of the Fc segment is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

In still another preferred embodiment of the present invention, the bispecific fusion protein, wherein the VEGF-binding peptide comprises a VEGFR extracellular domain.

In still another preferred embodiment of the invention, the VEGFR is Flt-1.

In still another preferred embodiment of the invention, the VEGFR is Flk-1.

In still another preferred embodiment of the invention, the VEGFR is Flt-4.

In still another preferred embodiment of the invention, the VEGFR extracellular domain is an immunoglobulin domain 2 of Flt1, and an immunoglobulin domain 3 of Flk1 or Flt4.

In still another preferred embodiment of the invention, the amino acid sequence of the VEGFR extracellular domain is set forth in SEQ ID NO: 1.

In still another preferred embodiment of the present invention, the The amino acid sequence of the PDGFR beta binding peptide is selected from one or more of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. Wherein SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 are the heavy chain CDR sequences in the PDGFR β-binding peptide, and SEQ ID NO: 6 is the light chain CDR sequence in the PDGFR β-binding peptide. When selected from a plurality of sequences therein, a combination of SEQ ID NO: 6 and one of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 is preferred.

In still another preferred embodiment of the invention, the pair The specific fusion protein further comprises a spacer, the amino acid sequence of the spacer being selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

In still another preferred embodiment of the invention, the pair The specific fusion protein further includes a spacer represented by an amino acid sequence such as (GGGGS)n, wherein n is 1-10, preferably 2-5.

In still another preferred embodiment of the invention, the pair The amino acid sequence of the specific fusion protein is selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 24.

The present invention also relates to a PDGFR β antibody derivative, Fc segments including PDGFR beta binding peptides and immunoglobulins.

In still another preferred embodiment of the invention, PDGFR β The PDGFR β-binding peptide is defined as above in the antibody derivative; the PDGFR β antibody derivative further includes a spacer as defined above.

In still another preferred embodiment of the present invention, the The amino acid sequence of the PDGFR β antibody derivative is SEQ ID NO: 18.

The present invention also relates to a bispecific fusion egg as described above The preparation method of the white or PDGFR β antibody derivative comprises: constructing an expression vector, transforming the expression vector into a host cell, expressing the expression vector in the host cell to obtain an expression precursor, and expressing the expression precursor to the outside of the cell to obtain the bispecificity Fusion protein. In some embodiments, a polynucleotide encoding a fusion protein as set forth in SEQ ID No. 19-24, or a polynucleotide encoding a PDGFR β antibody derivative as set forth in SEQ ID No. 18, is introduced into a plasmid construction expression vector, The plasmid may be any plasmid suitable for expression in a host cell in the prior art, including but not limited to pcDNA3.1 (+). In some embodiments, the expression vector is transformed into a host cell by methods well known in the art, such as transformation, transfection, and the like. The host cells are cultured under appropriate culture conditions to obtain expression precursors. The expression precursor is secreted outside the cell to obtain a fusion protein as shown in SEQ ID No. 19-24, or a PDGFR β antibody derivative as shown in SEQ ID No. 18.

The present invention also relates to a bispecific fusion egg as described above An expression precursor of a white or PDGFR beta antibody derivative, wherein the expression precursor consists of a signal peptide and the bispecific fusion protein or a PDGFR beta antibody derivative; the amino acid sequence of the signal peptide is selected from the group consisting of SEQ ID NO: 16 Or SEQ ID NO: 17; preferably the signal peptide is located at the N-terminus of the bispecific fusion protein or PDGFR beta antibody derivative.

The invention further provides a table encoding as described above The polynucleotide of the precursor product is preferably DNA.

The invention further provides a plurality containing as described above A nucleotide expression vector.

The invention further provides a table comprising the above The host cell of the vector.

In a preferred embodiment of the invention, the host The cell is a bacterium, preferably Escherichia coli.

In still another preferred embodiment of the present invention, the sink The main cell is a yeast cell, preferably Pichia pastoris or Saccharomyces cerevisiae.

The invention further provides a pharmaceutical composition comprising There is a bispecific fusion protein or PDGFR beta antibody derivative as described above and a pharmaceutically acceptable carrier.

In a preferred embodiment of the invention, wherein The pharmaceutical composition is an injectable solution wherein the bispecific fusion protein, PDGFR beta antibody derivative, or a pharmaceutically acceptable carrier is in dissolved form.

The present invention also relates to the above bispecific fusion protein Use in the manufacture of a medicament for the treatment of a disease or condition associated with inhibition of VEGF and PDGFR beta dual targets in a mammal. The mammal is a human.

The invention also relates to the bispecific fusion protein, PDGFR β Use of antibody derivatives in the preparation of a medicament for inhibiting VEGF and PDGF signaling pathways.

The present invention also relates to the above PDGFR β antibody derivative Use in the manufacture of a medicament for the treatment of a disease or condition associated with inhibition of a PDGFR beta target in a mammal. The mammal is a human.

The invention also relates to the preparation of the PDGFR β antibody derivative Use in a drug that inhibits the PDGF signaling pathway.

In a preferred embodiment of the invention, for pharmaceutical use The disease or condition described on the way is a disease of the eye, or any other disease that can be ameliorated by inhibition of VEGF and/or PDGF signaling pathways, such as tumors and diabetic retinopathy.

In still another preferred embodiment of the invention, the medicament The disease or condition of the eye described in the use is age-related macular degeneration, preferably wet age-related macular degeneration.

The invention also relates to a method of treating wet AMD, It comprises administering to a subject in need thereof a therapeutically effective amount of a bispecific fusion protein or PDGFR beta antibody derivative as described above, or a pharmaceutical composition as described above.

Figure 1 is a graph showing the inhibitory effect of VTEP-0 on PDGFRbb-induced PDGFR β and AKT phosphorylation.

Figure 2 is a graph showing the inhibitory effect of VTEP-13 on PDGFRbb-induced PDGFR β and AKT phosphorylation.

Figure 3 is a graph showing the inhibitory effect of VTEP-15 on PDGFRbb-induced PDGFR β and AKT phosphorylation.

Figure 4 is a graph showing the inhibitory effect of VTEP-17 on PDGFRbb-induced phosphorylation of PDGFR β and AKT.

Figure 5 is a graph showing the inhibitory effect of VTEP-22 on PDGFRbb-induced PDGFR β and AKT phosphorylation.

Figure 6 is a graph showing the inhibition rate of PDGFR-induced PDGFR phosphorylation by VTEP-17.

Figure 7 is a graph showing the inhibition rate of VTEP-17-induced PDGFRbb-induced AKT phosphorylation by VTEP-17.

Figure 8 is a graph showing the inhibitory effect of VTEP-13 on VEGF-induced proliferation of human umbilical vein endothelial cells.

Figure 9 is a graph showing the inhibitory effect of VTEP-17 on VEGF-induced proliferation of human umbilical vein endothelial cells.

Figure 10 is a graph showing the inhibitory effect of VTEP-22 on VEGF-induced proliferation of human umbilical vein endothelial cells.

Figure 11 is a graph of choroidal neovascularization in the mouse model of the eye, showing the inhibitory effect of VTEP-17 on choroidal neovascularization in a mouse model of laser-induced choroidal neovascularization.

In order to more easily understand the present invention, the following specifically defines Certain technical and scientific terms. Unless otherwise expressly defined in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

definition

The three-letter code and the one-letter code for the amino acid used in the present invention are as described in J.biol.chem, 243, p3558 (1968).

The invention provides a VEGF binding comprising a first component a bispecific fusion protein of a peptide and a second component PDGFR beta binding peptide; more specifically, a modified extracellular domain by providing VEGFR (Flt-1, Flk-1, Flt-4), PDGFR A novel fusion protein formed by chimeric peptide binding to the Fc region of IgG.

The term "binding peptide" as used herein refers to the ability to target Bound soluble receptors and fragments thereof and analogs thereof; or antibodies and fragments thereof and analogs thereof.

The VEGF-binding peptide of the present invention preferably contains VEGFR A polypeptide of an extracellular ligand binding domain (also referred to herein as a VEGFR extracellular domain).

The extracellular ligand binding domain is defined as part of the receptor, It is oriented toward the outside of the cell in its natural configuration in the cell membrane. It can contact its cognate ligand. The extracellular domain does not include a hydrophobic amino acid associated with the receptor transmembrane domain or any amino acid associated with the receptor intracellular domain. Von Heijone discloses detailed rules that are frequently cited by those skilled in the art for determining whether an amino acid of a particular receptor belongs to the extracellular, transmembrane or intracellular domain (see Von Heijone, 1995, BioEssay 17:25- 30). In addition, websites such as http://ulrec3.unil.cn/software/TMPRED-form.html can provide protein chemists with information on the prerequisites for protein domain preparation.

The term "PDGFR beta binding peptide" as used herein preferably comprises a polypeptide which specifically recognizes and binds to all or a portion of an antigen binding site of an antibody of the target antigen PDGFR beta; for example, all of the heavy and/or light chain variable regions Or a portion, or at least the HCDR3 region of the heavy chain variable region. The PDGFR beta-binding peptide comprises, without limitation, an antibody, or an antibody fragment, and an immunoglobulin-like domain containing all or part of the antigen binding site of the antibody. More specifically, the relevant definitions in US 61/610905, such as antibody sequences comprising the CDR3 sequence HGGDRSY (SEQ ID NO: 11), may also be employed. The CDR2 sequence of the sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 12) or GILPINKTPNYAQRFQG (SEQ ID NO: 13) is included.

The "PDGFR β antibody derivative" of the present invention includes a PDGFR β-binding peptide and an Fc segment of an immunoglobulin.

The "bispecific fusion protein" as used in the present invention refers to a polypeptide which specifically recognizes and binds to a VEGF epitope and also specifically recognizes and binds to a PDGFR β epitope. The "bispecific fusion protein" of the present invention may further comprise an Fc fragment of human IgG. Any isotype of IgG can be used, including IgGl, IgG2, IgG3, and IgG4. In order to increase the function of the fusion protein, the Fc segment can also be mutated or increased in the hinge region. Methods for making bispecific antibodies are known in the art.

The components of the fusion protein can be directly linked to each other or linked by a spacer (also referred to as a linker peptide). Generally, the term "linker peptide" (or spacer, junction) refers to one or more molecules, such as nucleic acid, amino acid or polypeptide moieties, which can be inserted between one or more of the component domains. Linker peptide sequences can be used to provide desirable target sites between components to facilitate manipulation. Linker peptides can also be provided to enhance expression of the chimeric plurality of components in the host cell, reducing steric hindrance such that the component can take its best tertiary structure to interact better with its target molecule. The "spacer" is used in the present invention for the fusion junction between the VEGF-binding peptide, the PDGFR antibody fragment and the Fc region to ensure proper folding and stability of different functional proteins. In the present invention, the spacer is preferably one or more peptide sequences comprised between one or more components. These polypeptide sequences are between 1 and 50 amino acid lengths, preferably between 1 and 25. "Spacer" is better (GGGGS)n, where n is 1-10, preferably 2-5.

"Precursor of bispecific fusion protein" according to the present invention Signal peptides can also be included to facilitate the secretion of proteins from the cells to the outside of the cells, increasing their yield.

The "antibody" as used in the present invention means that the desired organism is expressed Any form of antibody that is active. Thus, it is used in the broadest sense and specifically includes, but is not limited to, full length antibodies, antibody binding fragments or derivatives. Sources of antibodies include, but are not limited to, strain-only antibodies, multi-pure strain antibodies, genetically engineered antibodies (eg, bispecific antibodies).

The "full length antibody" as used in the present invention means that it contains more than four An immunoglobulin molecule (eg, IgM) of a peptide chain (ie, two heavy chains and two light chains cross-linked to each other by disulfide bonds to form a multimer). Each heavy chain comprises a heavy chain variable region (VH for short) and a heavy chain constant region, and the heavy chain constant region comprises three domains: CH1, CH2 and CH3. Each light chain comprises a stretch of light chain variable region (VL) and a light chain constant region, and the light chain constant region comprises one domain (CL1). The VH region and the VL region may further include a hypervariable region (also referred to herein as a complementarity determining region (CDR)). A more conserved domain interspersed between the complementarity determining regions is called the framework region (FR).

The "antibody-binding fragment or derivative" of the present invention includes any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein which specifically binds to an antigen to form a complex. At least a portion of an antigen binding region or variable region (e.g., one or more CDRs) of a parent antibody is typically included that retains at least some of the binding specificity of the parent antibody. "Antibody binding fragments or derivatives" may be derived from antibodies, for example by suitable standard techniques including proteolysis or recombinant genetic engineering techniques, including manipulation and expression of DNA expressing antibody variable regions and partial constant regions. The full length of the body is modified. "Antibody binding fragment or derivative" includes but is not limited to: (i) Fab fragment; (ii) F(ab') ₂ fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) (vi) a dAb fragment; and (vii) a minimal recognition unit (eg, an isolated complementarity determining region (CDR)) that mimics the amino acid residues of the hypervariable region of the antibody. Other engineering molecules such as bivalent antibodies, trivalent antibodies, tetravalent antibodies, and minibodies are also within the scope of "antibody binding fragments or derivatives."

"Fab Fragment" consists of a complete VH of light and heavy chains And CH1 functional area. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The "Fab'fragment" comprises a VH and CH1 functional region of a light chain and a heavy chain, and also contains a region between the CH1 and CH2 domains such that a chain can be formed between the two heavy chains of the two Fab' fragments. Disulfide bonds to form F(ab') ₂ molecules.

The "F(ab') ₂ fragment" comprises two light chains and two heavy chains containing a partial constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab') ₂ fragment consists of two Fab' fragments held together by a disulfide bond between two heavy chains.

"Fv fragment" contains the variable region VH of the light or/and heavy chain Ribbon. The PDGFR β-binding peptide of the present invention is preferably a Fv fragment, a preferred embodiment is a heavy chain variable region of PDGFR β, and another preferred embodiment is a fusion protein in which a heavy chain variable region is linked to a light chain variable region.

The term "Fc segment" refers to the human immunoglobulin chain constant region, In particular, the carboxy terminus or a part of the immunoglobulin heavy chain constant region has no antigen-binding activity and is a site where an antibody molecule interacts with an effector molecule and a cell. For example, an immunoglobulin Fc region can include a combination of two or more domains of heavy chains CH1, CH2, CH3, CH4 and an immunoglobulin hinge region. According to the amino acid sequence of the heavy chain constant region, immunoglobulins can be classified into different classes, mainly including five types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes) such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2.

"Fc region" preferably includes at least one immunoglobulin hinge Region, as well as the CH2 and CH3 regions of IgG. More preferably, it comprises a CH2 domain of IgG1, a CH3 domain and an immunoglobulin hinge region, and the position of the starting amino acid in the hinge region can be varied. The "Fc segment" has no antigen-binding activity and is a site where an antibody molecule interacts with an effector molecule and a cell.

The "hinge region" is used to link the Fab segment and the Fc segment of the antibody. A bispecific fusion protein can be ligated to the Fc segment in the present invention.

In an embodiment of the invention, the first aspect of the invention The component VEGF-binding peptide is abbreviated as VTE; the second component PDGFR-binding peptide is abbreviated as PDX; and the Fc-segment of the third component IGg is abbreviated as Fc. There are three combinations of the three components: (1) the VTE amino acid sequence is upstream of the Fc amino acid sequence, and the Fc amino acid sequence is upstream of the PDX amino acid sequence, such as VTE---Fc--- PDX; (2) VTE amino acid sequence upstream of the PDX amino acid sequence, PDX amino acid sequence upstream of the Fc amino acid sequence, sequence such as VTE---PDX---Fc; (3) PDX amine Acid sequence in VTE amino acid sequence Upstream, the VTE amino acid sequence is upstream of the Fc amino acid sequence, in the order PDX---VTE---Fc.

Accordingly, in an embodiment of the invention, the encoding VEGF The nucleotide sequence of the binding peptide is located upstream of the nucleotide sequence encoding the PDGFR binding peptide. In another embodiment of the invention, the nucleotide sequence encoding the VEGF-binding peptide is located downstream of the nucleotide sequence encoding the PDGFR-binding peptide.

Application method

The invention provides methods of treatment comprising administering to a subject an effective amount of a bispecific fusion protein of the invention. In a preferred aspect, the fusion protein is substantially purified, i.e., substantially free of substances that limit its effectiveness or produce undesirable side effects. The subject is preferably a mammal, preferably a human.

In a particular embodiment, it is desirable to have the medicament of the invention The composition is administered topically to the area in need of treatment; this can be accomplished, for example, but not by way of limitation, by local perfusion, topical application during surgery, which is by injection, by means of a catheter, for example. Or by means of an implant, wherein the implant is a porous, non-porous or gel-like substance, including a film, such as a sialastic film, fiber or a commercial skin substitute.

Compositions useful in practicing the methods of the invention may be comprised A solution, suspension or both of the reagents of the invention. The term "solution/suspension" refers to a liquid composition in which a first portion of the active agent is present in solution and a second portion of the active agent is in the form of particles which are suspended in a liquid matrix. Liquid compositions also include gels. The liquid composition can be aqueous Or in the form of an ointment. Further, the composition may be placed in the eye in the form of a solid, such as between the eye and the eyelid or in the conjunctival sac, where the bispecific protein disclosed herein is released. Bispecific proteins can generally be released from the above solids to the cornea or directly to the cornea itself, which is typically in direct contact with the cornea. Solids suitable for implantation into the eye typically consist primarily of bioerodible or non-bioerodible polymers. The aqueous solution and/or suspension may be in the form of an eye drop. The desired dose of active agent can be measured by administering a known number of drops to the eye. For example, for a drop of 50 [mu]l volume, administration of 1-3 drops will deliver 50-150 [mu]l of composition.

Aqueous suspension or solution/suspension for carrying out the process of the invention The liquid may comprise one or more polymers as suspending agents. Useful polymers include water soluble polymers such as cellulosic polymers, and water insoluble polymers such as crosslinked carboxyl containing polymers. The aqueous suspensions or solutions/suspensions of the present invention are preferably viscous or mucoadhesive, or more preferably both are viscous or both are mucoadhesive.

Diagnosis and screening methods

The bispecific proteins of the invention are either used diagnostically and/or used in screening methods. For example, the bispecific protein can be used to monitor the level of VEGF and PDGFR during clinical research to evaluate treatment efficiency. In additional embodiments, the methods and compositions of the invention can be used to screen individuals having levels that are, for example, too high or too low for VEGF and PDGFR. It can also be used to screen assays in vivo and in vitro to quantify the amount of unbound VEGF and PDGFR present, for example, in screening methods to identify detection reagents that reduce the expression of VEGF and PDGFR. More generally, the bispecific proteins of the invention are available Any assay or method for quantifying and/or isolating VEGF and PDGFR.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising the bispecific protein of the invention. Such a composition comprises a therapeutically effective amount of one or more of the bispecific proteins provided herein and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" refers to those listed in the Chinese Pharmacopoeia or other pharmacopeia for use in animals and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient that is administered with a drug. Such pharmaceutical carriers can be sterile liquids such as water or oil. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release agents and the like.

Further, a pharmaceutical composition for carrying out the method of the present invention It has a pH compatible with the eye and an osmolality.

Example

The invention is further described in the following examples, which are not intended to limit the scope of the invention.

No specific conditions are indicated in the examples or test examples of the present invention. The experimental method is usually carried out according to conventional conditions or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific source are routine reagents purchased from the market.

General method

Standard methods for molecular biology have been described (Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrood and Russell (2001) Molecular Cloning, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also found in Ausbel et al. (2001) Current Protoclols in Molecular Biology, Vol. 1-4, John Wiley and Sons, Inc. New York, which describes colonization and DNA mutagenesis in bacterial cells (Vol. 1). , in mammalian cells and yeast (volume 2), complex carbohydrate and protein expression (volume 3), and bioinformatics (volume 4).

The invention provides for constructing a plurality of fusion protein molecules A method of nucleotides, the nucleic acid molecule being inserted into a vector capable of expressing the fusion protein after introduction into a suitable host cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. Preferred is Lonza CHO cells. As is well known to those skilled in the art, any method for inserting a DNA fragment into a vector can be used to construct an expression vector that is under the control of a transcription/translation control signal encoding the fusion protein. Such methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination) (see Sambrook et al., Molecular Colonization, Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY).

Table of polynucleotide molecules encoding fusion proteins of the invention The dad can be regulated by another nucleotide sequence such that the fusion protein is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a fusion protein of the invention can be controlled by any promoter/enhancer element known in the art.

Therefore, according to the present invention, the use of the melt disclosed herein is included. An expression vector capable of replicating in a bacterial or fungal host, which is capable of transfecting the host, and thereby controlling expression of the polynucleotide to produce the fusion protein, and then recovering the fusion protein in a biologically active form . In the invention, the biologically active form comprises a form that is capable of binding to both VEGF and PDGFR.

An expression vector comprising the polynucleotide of the present invention may It was identified by three general methods: (a) DNA-DNA hybridization, (b) presence and absence of "marker" gene function, and (c) expression of the inserted sequence. In the first method, the presence of a foreign gene inserted into an expression vector can be detected by DNA-DNA hybridization using a probe containing a sequence homologous to the inserted fusion protein sequence. In the second method, it is possible to identify and select a recombinant vector based on certain "marker" gene functions (eg, resistance against biota, transformation phenotype, etc.) due to insertion of a foreign gene on the vector. /host system. For example, if the fusion protein DNA sequence is inserted into the marker gene sequence of the vector, the recombinant containing the insert can be identified by the lack of function of the marker gene. In a third method, a recombinant expression vector can be identified by detecting a foreign gene product expressed by the recombinant. For example, the detection can be based on the physical or functional properties of the fusion protein.

The cells of the present invention may be transient, preferably The fusion protein is expressed constitutively and permanently.

The fusion protein can be purified by any method to enable It is sufficient to subsequently produce a stable, biologically active fusion protein. For example, but not by way of limitation, the fusion protein may be recovered from the cell as a soluble protein or recovered as an inclusion body. For further purification of the fusion The protein can be subjected to conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration.

Example 1. Bispecific fusion protein

The present invention provides a bispecific fusion protein comprising a VEGF-binding peptide and a PDGFR β-binding peptide; more specifically, a modified extracellular structure by VEGFR (Flt-1, Flk-1, Flt-4) A novel fusion protein formed by chimerization of the domain, the PDGFR β-binding peptide, and the Fc region of IgG.

In an embodiment of the invention, the VEGF-binding peptide amino acid sequence of the invention is referred to as VTE; the second component PDGFR antibody, designated PDX; the Fc region of the third component IGg is referred to as Fc. There are three combinations of the three components: (1) the VTE amino acid sequence is upstream of the Fc amino acid sequence, and the Fc amino acid sequence is upstream of the PDX amino acid sequence, such as VTE---Fc--- PDX, abbreviated as VFP; (2) VTE amino acid sequence upstream of the PDX amino acid sequence, PDX amino acid sequence upstream of the Fc amino acid sequence, sequence such as VTE---PDX---Fc, referred to as VPF (3) The PDX amino acid sequence is upstream of the VTE amino acid sequence, and the VTE amino acid sequence is upstream of the Fc amino acid sequence, in the order of PDX---VTE---Fc, abbreviated as PVF.

The components of the fusion protein can be directly linked to each other or linked by a linker peptide. For example, a hinge region of 5-20 amino acids, preferably 5-15 amino acids, is added upstream of the Fc.

The VEGF-binding peptide in the present invention refers to a polypeptide fragment containing a VEGFR extracellular domain sequence. For example, including the extracellular domain D2 of Flt-1 and the sequence of the extracellular domain D3 of Flk-1 (for convenience, the name is VTE), the sequence is as follows: SEQ ID NO: 1.

The VEGF-binding peptide may also be a VTE plus Fc fragment, abbreviated as VEGF-Trap-Fc: SEQ ID NO: 2.

The PDGFR β-binding peptide in the present invention may be a VH fragment having PDGFR β-binding activity, and the sequence is as follows (for convenience, named PDX, selected from PDX1, PDX2 or PDX3): PDX1 (also known as XB2202 VH, referred to as VH in the present invention) : SEQ ID NO: 3.

PDX2 (XB1115 VH): SEQ ID NO:4.

PDX3 (XB2708 VH): SEQ ID NO:5.

PDX4 (XB2202 VL): SEQ ID NO: 6.

The Fc region of the invention comprises the CH2 and CH3 functional regions of an IgG (such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1), the sequence of which is as follows (designated as Fc for convenience): Fc-1: SEQ ID NO:7.

Fc-2: SEQ ID NO:8.

Fc-3: SEQ ID NO:9.

There may be a hinge region preceding the Fc region, selected from the original sequence or a mutant thereof, and the sequence is as follows: Fc-H1: DKTHT SEQ ID NO: 10. Fc-H2: EPKSSDKTHT SEQ ID NO: 11.

There may be a linker peptide between the components, the sequence is as follows: SPAC1: (GGGGS) n; n = 0-10 SEQ ID NO: 12.

SPAC2: ASTKGPSGGGGSGGGGS SEQ ID NO: 13.

SPAC3: ASTKGPSVFPLAPGGGGS SEQ ID NO: 14.

SPAC4: GGGSEPKSSDKTHTSPPSPAGGGS SEQ ID NO: 15.

The expression precursor of the bispecific fusion protein of the present invention may further have a secretion signal peptide, for example, SIG1: MEFGLSWLFLVAILKGVQC SEQ ID NO: 16.

SIG2: MDMRVPAQLLGLLLLWFPGSRC SEQ ID NO: 17.

The bispecific fusion protein of the present invention is expressed by cells to obtain the following preferred sequence: VTEP-0: SEQ ID NO: 18.

Sequence description: Arrangement of Fc (hIgG1)-XB2202 VH, Fc (human IgG1, Fc-3, SEQ ID NO: 9) is linked to the XB2202 heavy chain variable region (PDX1) by a linker peptide (SPAC1, n=2). SEQ ID NO: 3), the order of combination is: Fc-PDX; VTEP-13: SEQ ID NO: 19.

Sequence Description: Alignment of VEGF-Trap-XB2202-FC, VEGF Trap (VTE) links the XB2202 heavy chain variable region (PDX1, SEQ ID NO: 3) and Fc (Fc-) via a linker peptide (SPAC1, n=2) 1, SEQ ID NO: 7), having a hinge region between PDX1 and Fc (Fc-H2, SEQ ID NO: 11) in the order of: VTE-PDX-Fc; VTEP-15: SEQ ID NO:20.

Sequence Description: Alignment of VEGF-Trap-Fc-(G4S)4-XB2202, VEGF-Trap Fc fusion protein (VF1 sequence, by hinge region Fc-H1, SEQ ID NO: 10, and Fc-2, SEQ ID NO :6 ligation fusion), linked to the XB2202 heavy chain variable region (PDX1, SEQ ID NO: 3) by a linker peptide (SPAC1, n=4) in the order of: VTE-Fc-PDX; VTEP-17: SEQ ID NO:21.

Sequence Description: Alignment of VEGF-Trap-FC-(G4S)5-XB2202VL-Linker-VH, VEGF-Trap Fc fusion protein linked to the light chain variable region of XB2202 (PSX4, via a linker peptide (SPAC1, n=4) SEQ ID NO: 6) and heavy chain variable region (PDX1, SEQ ID NO: 3) in the order of: VTE-Fc-PDX; VTEP-22: SEQ ID NO:22.

Description of sequence: Alignment of XB2708VH-linker-1- VEGF-Trap-Fc, XB2708 heavy chain variable region (PDX3, SEQ ID NO: 5) linked to VEGF-Trap Fc fusion protein by a linker peptide (SPAC2), combination The order is: PDX-VTE-Fc VTEP-25: SEQ ID NO:23.

Sequence Description: The sequence is such as XB2202VH-(G4S)5-VEGF-Trap-Fc, and the XB2202 heavy chain variable region is ligated to the VEGF-Trap Fc fusion protein by a linker peptide (SPAC1, n=5) in the order of PDX. -VTE-Fc; VTEP-26: SEQ ID NO:24.

Sequence description: Arrange as XB2202VH-(G4S)3-VL- (G4S)5-VEGF-Trap-Fc, which is a XB2202 heavy chain variable region and a light chain variable region linked to a VEGF Trap Fc fusion protein by a linker peptide (SPAC1, n=3) in the order of PDX- VTE-Fc.

Example 2 Vector Construction

All PCR and colonization-related operations were performed according to molecular selection criteria, with reference to Molecular Colonization (Sambrook et al., Cold Spring Harbor Laboratory).

Experimental Materials:

Eukaryotic expression vector pcDNA3.1(+) (Life technologies, Cat. No. V790-20); nucleotide sequence encoding VTE protein (SEQ ID NO: 1): synthesized by gene synthesis company (Jin Weizhi, Suzhou); encoding Fc Protein fragment (SEQ ID NO: 7, SEQ ID NO: 8 or Nucleotide sequence of SEQ ID NO: 9): human antibody heavy chain γ 1 constant region Fc fragment, synthesized by Gene Synthesis Corporation (Jin Weizhi, Suzhou); PDX protein fragment (SEQ ID NO: 3, SEQ ID NO: 4. Nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6): synthesized by Gene Synthesis Corporation (Jin Weizhi, Suzhou).

experimental method: 1, fragment stitching

According to the order of VTE, Fc, PDX in each sequence of VTEP-0, VTEP-13, VTEP-15, VTEP-17, VTEP-22, VTEP-25, VTEP-26, and adding the linker modification sequence, translated into corresponding The nucleotide sequence was subjected to overlap PCR, and each fragment was spliced into a nucleotide sequence expression fragment of the corresponding protein.

2. Introduction of the enzyme cleavage site and signal peptide sequence:

A restriction endonuclease KpnI site, a Kozak sequence, and a signal peptide sequence were introduced at the 5' end of each expression fragment by PCR; a stop codon TGA and a NotI restriction endonuclease site were introduced at the 3' end of each fragment, respectively. . The resulting DNA sequence is from the 5' to the 3' end: KpnI site-Kozak sequence-signal peptide sequence-expression fragment (structural sequence includes Fc-PDX, VTE-Fc-PDX, PDX-VTE-Fc or VTE- PDX-Fc)-TGA stop codon-NotI site. In an embodiment of the invention, the signal peptide of VTE-0 is preferably the sequence of SIG2, and other embodiments are preferably the sequence of SIG1.

3. Construct an expression vector

Each fragment was inserted into plasmid pcDNA3.1(+) using KpnI and NotI restriction enzyme sites, respectively, to construct an expression vector.

Example 3. Protein expression experimental method:

The expression vector constructed in Example 2 was transiently transfected using FreeStyle 293 cells (GIBCO, Cat# R79007).

FreeStyle 293 cell suspension culture was added to the medium (Freestyle 293 expression medium, GIBCO, Cat#12338018), and a final concentration of 1% ultra low immunoglobulin fetal bovine serum (Ultra Low IgG Fetal Bovine Serum, GIBCO, Cat#16250078) was added. .

The expression vector and transfection reagent PEI (Polysciences, Cat#239662) constructed in Example 2 were prepared, and the expression vector amount was 100 μg/100 ml of cells, and the quality ratio of the expression vector to PEI was 1:2. The cell density on the day of transfection was 1 × 10 ⁶ /ml. 1L of FreeStyle 293 was transfected into cells, and 50 ml of Opti-MEM medium (GIBCO, Cat#11058021) was mixed with the expression vector, allowed to stand for 5 min, and filtered; 50 ml of Opti-MEM medium was mixed with PEI and allowed to stand for 5 min. filter. The expression vector and PEI were mixed and allowed to stand for 15 min.

The expression vector and the PEI mixture were slowly added to the cells, and cultured at 37 ° C, 8% CO ₂ , and a 130 rpm shaker incubator. After 5 days, the supernatant was collected by centrifugation for protein purification.

Example 4 Protein Purification experimental method: 1. Affinity chromatography

The cell culture medium was centrifuged at a high speed, and the supernatant was collected, and the first step chromatography was carried out by affinity chromatography. Chromatographic conditions: The chromatographic medium is Protein A or a derivative filler that interacts with Fc, such as Ma's Mabselect; the equilibration buffer is 1×PBS (NaCl 137 mmol/L, KCl 2.7 mmol/L, Na ₂ HPO ₄ 10 mmol/L). , KH ₂ PO ₄ 2mmol / L, pH 7.4), balance 5 column volume; cell supernatant loading, flow rate control on the sample column retention time ≧ 1min; after the end of the sample, using 1 × PBS ( pH 7.4) Wash until UV absorption falls back to baseline; elution buffer is 0.1M glycine (pH 3.0) for chromatographic elution, elution samples are collected according to UV absorption peak, and finally 1M Tris (pH9) .0) Neutralization.

2, size exclusion.

The first eluted sample was subjected to ultrafiltration and concentration, and subjected to size exclusion chromatography. The exclusion chromatography conditions were as follows: buffer was 1×PBS (NaCl 137 mmol/L, KCl 2.7 mmol/L, Na ₂ HPO ₄ 10 mmol/L, KH ₂ PO _{4 2} mmol/L, pH 7.4), chromatography column XK26/60 Superdex 200 (GE), flow rate 4 ml/min, loading volume less than 5 ml. The target protein peak is combined according to UV absorption.

3: Purity detection.

The purity was detected by SEC-HPLC, the column for detection was TSK-Gel 2000-SWXL (TOSOH), the flow rate was 0.7 ml/min, the loading amount was 50 μl, and the mobile phase was: 1×PBS (NaCl 137 mmol/L, KCl 2.7 mmol). /L, Na ₂ HPO ₄ 10 mmol/L, KH ₂ PO _{4 2} mmol/L, pH 7.4). The SEC-HPLC purity is greater than 95%.

Test case Test Example 1 PDGFR Phosphorylation Cell Experiment First, the purpose of the test:

The fusion protein of the present invention was evaluated by Western blotting method to inhibit PDGFR β and AKT (S473) phosphorylation in Caki cells, and the test was performed. The fusion protein of Ming has the function of PDGFR antibody.

Second, the test materials:

Samples of the invention: VTEP-0, VTEP-13, VTEP-15, VTEP-17 and VTEP-22.

Caki cells: purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, article number TCHu135; Phospho-PDGF receptor β (Tyr751) (88H8) Mouse mAb (PDGFR β antibody): purchased from Cell Signalling Technology, article number #3166; PDGF-BB CF (10 UG) (PDGFR ligand PDGFRbb): purchased from R&D, Cat. No. 220-BB-010; Protease inhibitor: Complete Mini EDTA-free from Roche, Cat. No. 04693159001; Phosphorylase inhibitor: PhosSTOP was purchased from Roche, Cat. No. 04906837001; McCOY's 5A Medium: purchased from Life Technologies, Cat. No. 16600-082; Cellular RIPA Lysate: purchased from Biyuntian Biotechnology Institute, Catalog No. P0013B.

Third, the test method:

1. Take 4 6-well plates, add 0.8×10 ⁶ Caki cells to each well, incubate at 37 ° C for 6 hours, and change to McCOY's 5A serum-free medium for 37 ° C overnight culture;

2. The cells were exchanged for the next day, and 2 ml of serum-free medium was added to each well.

3. Prepare different concentration sample solutions: VTEP-0, VTEP-13, VTEP-15, VTEP-17, VTEP-22, diluted with McCOY's 5A serum-free medium. The concentration of each sample is shown in Figure 1 - Figure 5. Line 1. For example, the loading mode of VTEP-13 is 200, 66.7, 22.2, 7.4, 0, 200 nM, 1 ml per well.

4. Incubate for 50 minutes at 37 ° C, add PDGFR ligand PDGFRbb (ligand), the final concentration is 40 ng / ml, incubate for 10 minutes at 37 ° C; for each sample plus ligand, see the second line on Figure 1 - Figure 5 "+" indicates that the sample shown is added to the ligand as described above, and "-" indicates that the sample is not added to the ligand;

5. Wash twice with PBS (pH 7.4), lyse the cells with cell RIPA lysate (containing protease and phosphorylase inhibitor), and quantify BCA protein;

6. Electrophoresis, 8% SDS-PAGE, Western blot.

Fourth, the test results:

The results are shown in Fig. 1 - Fig. 5: p-PDGFR β in Fig. 1 - Fig. 5 indicates that the electropherogram of the line indicates the phosphorylated PDGFR β content in the cell lysate after the test is added; p-AKT ( S473) indicates that the electrophoresis of the line is the content of phosphorylated AKT (S473) in the cell lysate after the sample is added for testing; PDGFR β indicates that the cell lysate phosphorylates PDGFR β and non-phosphorylated PDGFR after the sample is added for testing. The total content of β; GAPDH indicates that the electrophoresis of the line is the content of the housekeeping gene GAPDH protein in the cell lysate after the sample is added for testing.

The results showed that the sample of the present invention has the function of PDGFR antibody, and both can inhibit the phosphorylation of PDGFR β and AKT (S473), have a significant dose effect on the inhibition of PDGFR β phosphorylation, and inhibit the phosphorylation of downstream AKT (S473). The system also has a certain dose effect. And the antibody itself does not have a PDGFR beta phosphorylation agonist effect. The inhibition rate of GFEP-17 on PDGFR-induced PDGFR phosphorylation was 3.6 nm, as shown in Figure 6; the inhibition rate of VTEP-17 on PDGFR ligand-induced PDGFRbb-induced AKT (S473) phosphorylation was 3.2nm, as shown in Figure 7.

Test Example 2 HUVEC Cell Proliferation Experiment First, the purpose of the test:

The inhibition of proliferation of HUVEC cells by the samples of the invention was examined.

Second, the test materials:

Samples of the present invention: VTEP-13, VTEP-15 and VTEP-22; HUVEC cells (human umbilical vein endothelial cells): available from ATCC, Product Number ^{CRL-1730 TM; Low Serum Growth} Supplement (LSGS, low serum growth supplement) : purchased from GIBCO, item number S-003-10; Medium 200 medium: purchased from GIBCO, article number M-200-500; VEGF165: purchased from R&D, article number 293-VE-010; CCK8: purchased from Tongren Chemical, article number CK04.

Third, the test method:

1. In a 96-well plate, 100 μl of Medium200+ Low Serum Growth Supplement medium (with LSGS Medium 200 medium) containing 2000 HUVEC cells was added to each well, and the plate was incubated at 37 ° C in a 5% CO ₂ incubator.

2. After 24 hours, change Medium200+ Low Serum Growth Supplement to medium 200 medium with 0.5% FBS. Hole 90 μl.

3. The sample was diluted three times with the serum-free medium 200 medium from the original concentration, and a total of 9 points were diluted. The diluted samples were incubated with 200 ng/ml VEGF165, 37 ° C, 5% CO ₂ incubator for 1 hour.

4. Add a mixture of samples of different dilutions and VEGF configured in step 3 to the cells, 10 μl per well, and incubate the plates in a 37 ° C, 5% CO ₂ incubator.

After 5 and 72 hours, 10 μl of CCK8 was added to each well for color development, and CD450 was detected 4 hours later.

Fourth, the test results:

Results As shown in Fig. 8 to Fig. 10, the three samples had different inhibitory effects on HUVEC cell proliferation, as shown in Table 2.

Test Example 3 Reaction Affinity Test of Sample with PDGFR β Protein First, the purpose of the test:

The reaction affinity of the samples of the invention and the PDGFR β protein was determined using a Biacore, GE instrument.

Second, experimental instruments, materials and reagents

Samples of the invention: VTEP-0, VTEP-13, VTEP-15, VTEP-17, VTEP-22, VTEP-25 and VTEP-26.

Experimental instrument: Biacore X100, GE; experimental material: biosensing wafer CM5 (Cat.# BR-1000-12, GE); experimental reagent: 1), amine coupling kit (Cat.# BR-1000-50 , GE); 2), human anti-capture kit (Cat. # BR-1008-39, GE); 3), PDGFR β (Cat. # 10514-H08H, Sino Biological); 4), glycine hydrochloride ( pH 1.5) regeneration solution (Cat. # BR-1003-54, GE); 5), HBS-EP + 10 times buffer solution (Cat. # BR-1006-69, GE) diluted to 1 time with DIWater (pH 7.4 ).

Third, the test method:

Experimental samples VTEP-0, VTEP-13, VTEP-15: Covalently couple human anti-capture antibodies to CM5 biochips according to the instructions in the human anti-capture kit instructions, thereby affinity capture of a certain amount of VTE protein, and then The surface of the wafer flows through a series of concentration gradients of PDGFR β protein, and the binding signal is obtained by Biacore instrument to obtain the binding and dissociation curves. After each cycle of dissociation was completed in the experiment, the biochip was washed and regenerated using the regeneration solution disposed in the human anti-capture kit.

Experimental samples VTEP-17, VTEP-22, VTEP-25, VTEP-26

PDGFR β protein was covalently coupled to a CM5 biochip according to the method described in the description of the amine coupling kit, and then a series of concentrations of VTE protein were flowed on the surface of the wafer to detect the reaction immediately using a Biacore instrument. The signal acquires a binding and dissociation curve. The data obtained were analyzed using a 1:1 (Langmuir) binding model using GE's BIAevaluation software. The affinity Kd values determined by this method are shown in Table 3 below.

Fourth, the experimental results

The sample of the present invention has a high reaction affinity with the PDGFR β protein, and the results are shown in Table 3.

Test Example 4 Reaction Affinity Test of Sample with VEGF Protein

First, the purpose of the test:

The reaction affinity of the samples of the invention and the VEGF protein was determined using a Biacore, GE instrument.

Second, experimental instruments, materials and reagents

Samples of the invention: VTEP-17 and VTEP-22.

Experimental instrument: Biacore X100, GE; experimental material: biosensing wafer CM5 (Cat.# BR-1000-12, GE);

Experimental reagents: 1) Amine coupling reagent kit (Cat. # BR-1000-50, GE); 2) Human anti-capture kit (Cat. # BR-1008-39, GE); 3) VEGF165/VEGFA ( Cat. # 11066-HNAB, Sino Biological); 4) HBS-EP + 10-fold buffer solution (Cat. #BR-1006-69, GE) diluted to 1 fold (pH 7.4) with DIWater.

Third, the test method:

Human anti-capture antibody was covalently coupled to a CM5 biochip according to the method described in the human anti-capture kit, thereby affinity-capturing a certain amount of VTE protein, and then flowing through a series of gradient concentrations of VEGF165 protein on the surface of the wafer. The Biacore instrument instantly detects the reaction signal to obtain binding and dissociation curves. The data obtained were analyzed using a 1:1 (Langmuir) binding model using GE's BIAevaluation software. The affinity Kd values determined by this method are shown in Table 4 below.

Fourth, the experimental results

The sample of the invention has high reaction affinity with VEGF protein, and the results are shown in Table 4:

Test Example 5 Half-life detection in vivo First, the purpose of the experiment:

To test the pharmacokinetic parameters of VTEP-17 of the present invention in rats.

Second, experimental materials and reagents:

Animals: 180±10 g SD rats, male and female (provided by Sipple-Beikai Experimental Animal Co., Ltd., animal production license number SCXK (Shanghai) 2008-0016), 6 in each group.

PDGFR-His: an extracellular fragment consisting of PDGFR β (human CD140b/PDGFRb gene functional region from www.uniprot.org, SEQ ID NO: 25) plus a Flag tag and a his tag (marked by a horizontal line in the sequence) It can be prepared by well-known methods in the field, such as establishing a pure strain, transposing a plasmid and purifying the protein with a nickel column, and verifying the desired protein by electrophoresis. In this test, it can be used to bind to a fragment having the function of a PDGFR β antibody, and the sequence is as follows: SEQ ID NO: 25.

Goat anti-human IgG peroxidase conjugated antibody: Jackson Cat. No.: 109-035-088; microplate reader: Thermo Scientific.

Third, the experimental method: 1. In vivo administration

Six SD rats, male and female, were intraperitoneally injected. Under sterile conditions, VTEP-17 was dissolved in physiological saline to a final concentration of 20 μg/mL. Each rat was administered IP at a dose of 100 μg/kg. The IP group received blood from the fundus vein of the rats at 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 11 hr, 24 hr, 48 hr, 72 hr, 200 μL each time (equivalent to taking 100 μL of serum); the collected blood samples were at room temperature. Place for half an hour until agglutination, then centrifuge at 10,000 xg for 5 minutes at 4 °C. The supernatant was collected and immediately subjected to an experiment or sample aliquoting - 80 ° C storage. Avoid repeated freezing and thawing.

2. ELISA test using the serum sample obtained in step 1.

1) directly coated with 100 ng/ml of PDGFR at 4 ° C overnight; 2) blocked the plate with 300 μl of PBS containing 5% skim milk, and sealed at 37 ° C for 2 h, while blocking the uncoated blank well as a control; 3) PBST was washed 3 times; 4) 100 μl of VTEP-17-containing rat serum sample was added to each well (a rat serum sample was taken before the experiment and diluted according to different ratios to obtain the best antibody concentration in the serum in the middle of the standard curve. Dilution ratio, the serum sample was diluted according to the optimal dilution ratio), incubate at 37 °C for 2 h; 5) Wash with PBST 3 times; 6) Add 100 μl of goat anti-human IgG peroxidase-conjugated antibody per well (1:2500) Incubate at 37 ° C for 1 h; 7) Wash with PBST 3 times. Add 100 μl of TMB matrix to each well, incubate at 37 ° C for 5-10 min, then add 100 μl of 1.25 MH ₂ SO ₄ per well to stop the reaction; 8) ELISA plate reader to read the OD value at 450 nm wavelength.

The actual concentration of the sample is calculated based on the standard curve equation and the OD value of the sample.

Standard curve: The VTE-17 concentration (5, 10, 20, 50 and 100 ng/ml) is plotted on the abscissa, and the OD value corresponding to different concentrations of VTE-17 is plotted on the ordinate. The typical standard curve equation is obtained. The linear range of the standard curve is 5-100 ng/ml.

Fourth, the experimental results:

The VTEP-17 obtained in Example 2 was tested by the above method, and the pharmacokinetic calculation was carried out using the non-compartment model of Phoenix ^TM WinNonlin 6.1 software to obtain the in vivo half-life (T _1/2 ). The results are as follows:

The inventors introduced a VH fragment against PDGFR β based on neutralization of VEGF. The fusion protein has the dual specificity of neutralizing VEGF and blocking the signaling pathway of PDGFR β, so that it can effectively treat people who are not responding to VEGF treatment and overcome the resistance of neutralizing VEGF drug therapy. In addition, it is possible to prolong the period of intraocular injection required by the patient, thereby better treating the age-related macular degeneration on an existing basis.

Test Example 6 Inhibitory effect of VTEP protein on choroidal neovascularization in a mouse model of laser-induced choroidal neovascularization First, the purpose of the test:

This test was used to investigate the inhibitory effect of VTEP protein on choroidal neovascularization in a mouse model of laser-induced choroidal neovascularization.

Test principle: This experiment destroys the local Bruch's membrane in the mouse retina by a certain energy laser, so that the choroidal blood supply system and the retinal blood supply system of the barrier are communicated, choroidal capillary endothelial cells, pericytes, fibroblasts. And inflammatory cells enter the subretinal and pigment epithelial layers, accompanied by inflammatory factors, pro-angiogenic factors and changes in extracellular matrix components, breaking the balance between local pro-angiogenic and anti-angiogenic factors, and promoting local neovascularization Among them, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-mediated endothelial cell growth and extracellular matrix, pericyte envelopment play an important role in choroidal neovascularization.

Second, experimental materials and reagents:

Experimental animals: adult (6-8 weeks old) C57BL/6J female rats purchased from Shanghai Lake Test Animals LLC.

Test equipment: Image analyzer: KS 400 type from Zeiss, Germany

Argon laser launcher: Lumenis Selecta Duet, Lumenis, USA

Slit lamp: Suzhou Liuliu

Ophthalmic surgery equipment: Huaiyin medical equipment

Ophthalmic Surgery Microscope: LEICA, USA

Slides, coverslips: Shitai

Microinjector and Syringe: Parker Hannifin PICOSPRITZER III, Parker, USA

Reagent preparation: Mido Eye Drops: Shentian Pharmaceutical Co., Ltd.

Proparacaine Hydrochloride Eye Drops: Alcon Corporation

Dikro Eye Cream: Xingqi Pharmaceutical Co., Ltd.

4% paraformaldehyde: Shanghai Shenggong

Chloral hydrate: Shanghai Shenggong

Sodium dextran fluorescein-labeled dextran: Sigma-Aldrich, USA

Fluorescent Mounting Medium: DAKO

VTEP-17 (SEQ ID NO: 21) and VEGF-Trap (SEQ ID NO: 2) proteins: diluted with the required dilution concentration buffer (PBS buffer) according to the designed grouping concentration, and the operation in the ultra-clean bench is completed. Packed in 20ul / tube 4 degrees cryopreservation, ready for use.

GONIC Binder: US DOW

The test samples are grouped as follows:

Group 1 (g1): PBS buffer

Group 2(g2): VEGF-Trap 10ug

Group 6(g6): VTEP-17 5.1ug

Group 7 (g7): VTEP-17 15.4ug

Group8(g8): VTEP-17 46.2ug

Third, the experimental method:

6-8 weeks old C57/BL6 mice were fed with free water and fed in 12 hours of light and dark alternately. They were randomly divided into 5 groups, 8 rats in each group, half male and half female, and anesthetized with intraperitoneal injection of 3.5% chloral hydrate at 0.01 ml/g body weight. , Dolly eye drops applied to the ocular surface dilated, after the anesthesia is sufficient, the fundus laser photocoagulation (wavelength 532nm, energy 120mw, time 0.1s, spot diameter 100nm) under the slit lamp, the bubble generator can be seen after the laser It is considered to be effective, and the effective laser spot distance is 1-2PD from the nipple, which is located in different quadrants, 4-6 per eye, that is, the modeling is successful. Each group was intraperitoneally injected with chloral hydrate on the first day and the seventh day after model establishment, and the corresponding proteins were administered by microinjection in a vitreous cavity (see experimental group design). On the 14th day after the laser, anesthetized with chloral hydrate was intraperitoneally injected, the thoracic cavity was dissected, and the heart was exposed. The heart was perfused with 0.4 ml of 25 mg/ml luciferin sodium per mouse. After the mice were sacrificed by excessive anesthesia, the eyeballs were taken. % Formalin was fixed at room temperature for 5 hours. Remove the fixed eyeball, cut it along the limbus, remove the crystal, cornea, and vitreous. The remaining eye cup is cut radially with the nipple as the center, flatten it on the slide, carefully remove the retina, and add the seal. The cover is covered with a cover slip. Tissue sheet at -20 degrees Preservation, observation under a fluorescent microscope every other day, photographing the laser plaque with a 20-fold magnification objective, and counting the area of choroidal neovascularization with Image pro Plus.

Data processing: SAS 9.0 statistical software was used for analysis. One-way ANOVA was used to compare the mean values between the two groups with t-test. P < 0.05 was considered statistically significant.

Fourth, the experimental results:

One-way analysis of variance showed statistically significant differences between the groups (P < 0.02), of which g1, g2 and the other groups were significantly different, there was no significant difference between g6, g7, g8. The results are shown in Figure 11: choroidal neovascularization in the mouse eye of the model, and the histogram shows the mean ± standard deviation.

Discussion: In this experiment, VEGF-Trap has a neutralizing effect on VEGF, and VTEP-17 has a dual role of neutralizing PDGF and neutralizing VEGF. The results suggest that both proteins can effectively inhibit choroidal neovascularization (G1 is statistically different from the other groups), and there is statistically significant difference between G2 (only VEGF-Trap) and G6-8 (different concentration VTEP-17). The difference indicates that the anti-angiogenic effect of VTEP-17 is stronger than that of VEGF-Trap, but the measurement-effect relationship is not obvious in this concentration span. There is no significant difference in anti-angiogenic effects between different concentrations of VTEP-17.

<110> Shanghai Hengrui Pharmaceutical Co., Ltd., Jiangsu Hengrui Pharmaceutical Co., Ltd.

<120> VEGF and PDGFR β bispecific fusion protein and use thereof

<130> 340006CG

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<170> PatentIn version 3.3

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<223> VEGF-Trap-Fc, VEGF-binding peptide can also be VTE plus Fc fragment

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<223> PDX1, VH fragment with PDGFR β binding activity XB2202 VH

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<223> PDX2, VH fragment with PDGFR β binding activity XB1115 VH

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<223> PDX3, VH fragment with PDGFR β binding activity XB2708 VH

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<223> PDX4, VL fragment XB2202 VL with PDGFR β binding activity

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<213> Human Fc

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<223> Fc variant of human IgG1, Fc-2

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<223> Fc region front hinge region, Fc-H1

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<223> Fc region front hinge region, Fc-H2.

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<223> Fusion Protein Spacer SPAC1

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<223> Fusion Protein Spacer SPAC2

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<223> Fusion Protein Spacer SPAC3

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<223> Fusion Protein Spacer SPAC4

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<223> Fusion protein expression precursor secretion signal peptide SIG1

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<223> Fusion protein expression precursor secretion signal peptide SIG2

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<223> VTEP-0, arranged as FC (hIgG1)-XB2202 VH is Fc(Fc-3) linked to XB2202 heavy chain variable region (PDX1) by a spacer (SPAC1, n=2) in the order of Fc-PDX

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<223> VTEP-13, arranged as VEGF-Trap-XB2202-FC, is a VEGF Trap (VTE) linked to the XB2202 heavy chain variable region (PDX1) and Fc (Fc-1) by a spacer (SPAC1, n=2) ), there is a hinge region (Fc-H2) between PDX1 and Fc, and the order of combination is: VTE-PDX-Fc

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<223> VTEP-15: Alignment, such as VEGF-Tarp-FC-(G4S)4-XB2202, is a VEGF-Trap Fc fusion protein (VTE sequence, fused to Fc-2 by the hinge region Fc-H1) The XB2202 heavy chain variable region (PDX1) was ligated by a spacer (SPAC1, n=4) in the order of: VTE-Fc-PDX

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<223> VTEP-17: Arranges VEGF-Tarp-FC-(G4S)5-XB2202VL-Linker-VH, which is a VEGF-Trap Fc fusion protein linked to the light chain of XB2202 by a spacer (SPAC1, n=4). Variable region (PDX4) and heavy chain variable region (PDX1), the order of combination is: VTE-Fc-PDX

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<223> VTEP22: Arranged as XB2708VH-linker-1-VEGF-Tarp-FC, the XB2708 heavy chain variable region (PDX3) is linked to the VEGF-Trap Fc fusion protein by a spacer (SPAC2) in the order of: PDX-VTE-Fc

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<223> VTEP25: Arranged as XB2202VH-(G4S)5-VEGF-Tarp-FC, the XB2202 heavy chain variable region is linked to the VEGF-Trap Fc fusion protein by a spacer (SPAC1, n=5), in combination order For: PDX-VTE-Fc

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<223> VTEP26: Alignment such as XB2202VH-(G4S)3-VL-(G4S)5-VEGF-Tarp-FC, which is a heavy chain variable region and a light chain variable region of XB2202 by a spacer (SPAC1, n= 3) Linked to VEGF Trap Fc fusion protein in the order of: PDX-VTE-Fc

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Claims (55)

A bispecific fusion protein comprising a VEGF binding peptide and a PDGFR beta binding peptide. The bispecific fusion protein of claim 1, further comprising an Fc segment of an immunoglobulin. The bispecific fusion protein of claim 2, wherein the Fc portion of the immunoglobulin is the Fc portion of an immunoglobulin IgG. The bispecific fusion protein of claim 3, wherein the amino acid sequence of the Fc segment is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The bispecific fusion protein of claim 1, wherein the VEGF-binding peptide comprises a VEGFR extracellular domain. The bispecific fusion protein of claim 5, wherein the VEGFR is Flt-1. The bispecific fusion protein of claim 5, wherein the VEGFR is Flk-1. The bispecific fusion protein of claim 5, wherein the VEGFR is Flt-4. The bispecific fusion protein of claim 5, wherein the VEGFR extracellular domain comprises an immunoglobulin domain 2 of Flt1, and an immunoglobulin domain 3 of Flk1 or Flt4. The bispecific fusion protein of claim 5, wherein the amino acid sequence of the VEGFR extracellular domain is set forth in SEQ ID NO: 1. The bispecific fusion protein according to claim 1, wherein the amino acid sequence of the PDGFR β-binding peptide is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: One or more of 6. The bispecific fusion protein of claim 1, further comprising a spacer, the amino acid sequence of the spacer selected from the group consisting of: SEQ ID NO: 12, multiple repeats of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The bispecific fusion protein according to claim 1, which further comprises a spacer represented by an amino acid sequence such as (GGGGS) _n , wherein n is 1-10. The bispecific fusion protein of claim 13, wherein n is 2-5. The bispecific fusion protein according to any one of claims 1 to 14, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 24. The method for preparing a bispecific fusion protein according to any one of claims 1 to 15, comprising: constructing an expression vector, transforming the expression vector into a host cell, and expressing in the host cell to obtain an expression precursor. The bispecific fusion protein is secreted extracellularly to obtain the bispecific fusion protein. A method for preparing any one of claims 1 to 15 An expression precursor of a bispecific fusion protein, wherein the expression precursor consists of a signal peptide and the bispecific fusion protein of any one of claims 1 to 14; the amino acid of the signal peptide The sequence is selected from the group consisting of SEQ ID NO: 16 or SEQ ID NO: 17. The expression precursor of claim 17, wherein the signal peptide is located at the N-terminus of the bispecific fusion protein. A polynucleotide encoding an expression precursor as described in claim 17 or 18. An expression vector comprising the polynucleotide of claim 19 of the patent application. A host cell comprising the expression vector of claim 20 of the patent application. The host cell of claim 21, wherein the host cell is a eukaryotic cell. The host cell of claim 22, wherein the host cell is a yeast cell. The host cell of claim 23, wherein the host cell is Pichia pastoris or Saccharomyces cerevisiae. A pharmaceutical composition comprising: the bispecific fusion protein according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 25, wherein the pharmaceutical composition is an injectable solution, wherein the bispecific fusion protein and/or the pharmaceutically acceptable carrier is in a dissolved form. Use of a bispecific fusion protein according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of a disease or condition associated with inhibition of VEGF and PDGFR β dual targets in a mammal. Use as described in claim 27, for the preparation of a medicament for inhibiting VEGF and PDGF signaling pathways. The use of claim 27, wherein the mammal is a human. The use of claim 27, wherein the disease or condition is a tumor or an eye disease. The use of claim 27, wherein the disease or condition is diabetic retinopathy or age-related macular degeneration. The use of claim 31, wherein the disease is wet age-related macular degeneration. A method of treating wet age-related macular degeneration comprising: administering to a patient a therapeutically effective amount of the bispecific fusion protein of any one of claims 1 to 15 or as claimed in claim 25 The pharmaceutical composition described in the item. A PDGFR beta antibody derivative comprising a PDGFR beta binding peptide and an Fc portion of an immunoglobulin. The PDGFR β antibody derivative according to claim 34, wherein the amino acid sequence of the PDGFR β-binding peptide is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO :6. The PDGFR β antibody derivative as described in claim 34, Also included therein is a spacer having an amino acid sequence selected from the group consisting of: SEQ ID NO: 12, multiple repeats of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. . The PDGFR β antibody derivative according to claim 34, which further comprises a spacer represented by an amino acid sequence such as (GGGGS) _n , wherein n is 1-10. The PDGFR β antibody derivative according to claim 37, wherein n is 2-5. The PDGFR β antibody derivative according to claim 34, wherein the amino acid sequence is SEQ ID NO: 18. A method for producing a PDGFR β antibody derivative according to any one of claims 34 to 39, which comprises: constructing an expression vector, transforming the expression vector into a host cell, and expressing in a host cell to obtain an expression precursor The PDGFR β antibody derivative is secreted outside the cell to obtain the PDGFR β antibody derivative. An expression precursor for the preparation of a PDGFR β antibody derivative according to any one of claims 34 to 39, wherein the expression precursor is composed of a signal peptide and any of claims 34 to 39 A composition of the PDGFR beta antibody derivative; the amino acid sequence of the signal peptide is selected from the group consisting of SEQ ID NO: 16 or SEQ ID NO: 17. The expression precursor of claim 41, wherein the signal peptide is located at the N-terminus of the PDGFR β antibody derivative. A polynucleotide encoding a precursor as described in claim 41 or 42 of the patent application. An expression vector comprising the polynucleotide of claim 43 of the patent application. A host cell comprising the expression vector of claim 44 of the patent application. The host cell of claim 45, wherein the host cell is a eukaryotic cell, preferably a yeast cell, more preferably Pichia pastoris or Saccharomyces cerevisiae. A pharmaceutical composition comprising: the PDGFR β antibody derivative according to any one of claims 34 to 39, and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 47, wherein the pharmaceutical composition is an injectable solution, wherein the PDGFR β antibody derivative and/or a pharmaceutically acceptable carrier is in a dissolved form. Use of a PDGFR beta antibody derivative according to any one of claims 34 to 39 for the manufacture of a medicament for the treatment of a disease or condition associated with inhibition of a PDGFR beta target in a mammal. The use according to claim 49 of the patent application is for the preparation of a medicament for inhibiting the PDGF signaling pathway. The use of claim 49, wherein the mammal is a human. The use as described in claim 49, wherein the disease or disease The disease is a disease of the tumor or the eye. The use of claim 52, wherein the disease or condition is diabetic retinopathy or age-related macular degeneration. The use according to claim 53, wherein the disease is wet age-related macular degeneration. A method of treating wet age-related macular degeneration comprising: administering to a patient a therapeutically effective amount of a PDGFR β antibody derivative according to any one of claims 34 to 39, or as in claim 47 The pharmaceutical composition described in the item.