CN115246886A - anti-EGFR/VEGF (epidermal growth factor receptor/vascular endothelial growth factor) bifunctional fusion protein and application thereof - Google Patents

anti-EGFR/VEGF (epidermal growth factor receptor/vascular endothelial growth factor) bifunctional fusion protein and application thereof Download PDF

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CN115246886A
CN115246886A CN202110458717.0A CN202110458717A CN115246886A CN 115246886 A CN115246886 A CN 115246886A CN 202110458717 A CN202110458717 A CN 202110458717A CN 115246886 A CN115246886 A CN 115246886A
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fusion protein
vegf
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邓岚
黄浩旻
朱祯平
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Dansheng Pharmaceutical Technology Shanghai Co ltd
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Priority to TW111115786A priority patent/TW202300530A/en
Priority to CN202280030685.7A priority patent/CN117242097A/en
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Abstract

The invention relates to the technical field of fusion proteins, in particular to an anti-EGFR/VEGF bifunctional fusion protein and application thereof. The anti-EGFR/VEGF bifunctional fusion protein comprises: (a) An anti-EGFR antibody, or antigen-binding fragment thereof, and (b) a D2 domain of the extracellular region of VEGFR 1. The fusion protein has great clinical application prospect in treating diseases related to high expression or abnormal expression of EGFR and VEGF.

Description

anti-EGFR/VEGF (epidermal growth factor receptor/vascular endothelial growth factor) bifunctional fusion protein and application thereof
Technical Field
The invention relates to the technical field of fusion proteins, in particular to an anti-EGFR/VEGF bifunctional fusion protein and application thereof.
Background
EGFR (Epidermal Growth Factor Receptor) is a Receptor for Epidermal Growth Factor (EGF) and belongs to the ErbB Receptor family. EGFR is a transmembrane glycoprotein with a molecular weight of 170KDa, belongs to receptor tyrosine kinase, and is activated by converting a monomer into a dimer under the action of related ligands such as EGF and transforming growth factor- α (TGF α), so as to further activate downstream signaling pathways and regulate cell proliferation. Numerous studies have shown that there is high or abnormal expression of EGFR in most tissues of tumors such as glial cell carcinoma, kidney cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, and the like. Abnormalities in EGFR function are associated with the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. The dysfunction of the plant mainly appears in two aspects: one is the excessive abnormal expression in tumor tissues, and the persistent activation of EGFR mutants in tumor cells (no ligand stimulation or formation of self-circulating stimulation pathways are required). In tissues from colon cancer patients, the expression rate of EGFR is about 25-77%. Relevant clinical data show that the degree of EGFR expression is closely related to the malignancy of tumors and the prognosis of tumor patients.
There are two phases of tumor growth, from the slow avascular growth phase to the rapid angiogenic proliferation phase. If no blood vessels are formed inside the tumor, the primary tumor grows slowly and metastasis cannot be achieved. Inhibition of tumor angiogenesis is therefore considered to be one of the currently promising approaches to tumor therapy. Among the Vascular Endothelial Growth Factor (VEGFs) family, VEGF-A165 (hereinafter referred to as VEGF) is the most abundant active subtype. VEGF, by binding to the type II receptor VEGFR2, activates a signaling pathway to undergo a cascade of reactions that promote neovascularization and maintain its integrity. However, the type I receptor VEGFR1 binds VEGF much more than VEGFR2, and the site of action is mainly the extracellular domain D2 domain of VEGFR 1. VEGFR1-D2 blocks the combination of VEGFR2 and VEGF by competing with the combination of VEGF, thereby blocking a signal path, inhibiting the proliferation and angiogenesis of endothelial cells, and inhibiting the rapid proliferation and metastasis of tumors.
At present, patients still have no response or poor response to the treatment of anti-EGFR antibody, anti-VEGF antibody or VEGF-Trap, and a need exists for developing a bifunctional fusion protein capable of targeting EGFR and VEGF simultaneously to meet clinical needs.
Disclosure of Invention
The invention aims to provide a novel anti-EGFR/VEGF difunctional fusion protein which can be simultaneously combined with EGFR and VEGF so as to block EGFR and VEGF signal paths. It is also an object of the present invention to provide nucleic acid molecules encoding said fusion proteins; providing an expression vector comprising said nucleic acid molecule; providing a host cell comprising the expression vector; providing a method for preparing the fusion protein; providing a pharmaceutical composition comprising said fusion protein; provides the application of the fusion protein or the pharmaceutical composition in preparing a medicament for treating cancer; methods of providing the fusion protein or the pharmaceutical composition for treating cancer are provided.
In order to achieve the purpose, the invention provides the following technical scheme:
the first aspect of the invention provides an anti-EGFR/VEGF bifunctional fusion protein, comprising: (a) An anti-EGFR antibody, or antigen-binding fragment thereof, and (b) a D2 domain of the extracellular region of VEGFR 1.
In a preferred embodiment, the anti-EGFR antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:16, the amino acid sequence of HCDR2 is shown as SEQ ID NO:17, the amino acid sequence of the HCDR3 is shown as SEQ ID NO:18 is shown in the figure; the amino acid sequence of LCDR1 is shown as SEQ ID NO:19, the amino acid sequence of LCDR2 is shown as SEQ ID NO:20, the amino acid sequence of LCDR3 is shown as SEQ ID NO: shown at 21.
In a preferred embodiment, the anti-EGFR antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the amino acid sequence of VH is as set forth in SEQ ID NO:22, and the amino acid sequence of VL is shown in SEQ ID NO: as shown at 24.
In a preferred embodiment, the heavy chain variable region VH comprises at least one deglycosylation mutation; preferably, the mutation is located within the framework region FR of the heavy chain variable region VH.
In a preferred embodiment, the mutation is at position 85 of the VH of the heavy chain variable region, the mutations being numbered according to the Kabat numbering system; preferably, the mutation is N85E.
In a preferred embodiment, the amino acid sequence of the VH of the heavy chain variable region is as set forth in SEQ ID NO:23, the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: as shown at 24.
In a preferred embodiment, the anti-EGFR antibody comprises a heavy chain constant region and a light chain constant region; more preferably, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3, or human IgG4 and the light chain constant region is selected from human Kappa (Kappa) or human Lambda (Lambda).
In a preferred embodiment, the anti-EGFR antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, scFv, fv, fab, or F (ab') 2.
In a preferred embodiment, the anti-EGFR antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
In a preferred embodiment, the anti-EGFR antibody is a monoclonal antibody.
In a preferred embodiment, the anti-EGFR antibody comprises an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3, and the amino acid sequence is as shown in SEQ ID NO:7, or a light chain as shown in figure 7.
In a preferred embodiment, the D2 domain of the extracellular region of VEGFR1 comprises an amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO: 8.
In a preferred embodiment, the anti-EGFR antibody or antigen-binding fragment thereof is linked to the D2 domain of the extracellular region of VEGFR1 directly by a peptide bond or by a peptide linker.
In a preferred embodiment, the peptide linker comprises the amino acid sequence (GGGGS) n, said n being selected from 1, 2, 3, 4 or 5.
In a preferred embodiment, the anti-EGFR antibody is a full-length antibody comprising 2 heavy chains and 2 light chains; the N-terminus or C-terminus of the extracellular domain D2 domain of VEGFR1 is linked to the N-terminus or C-terminus of the heavy chain or light chain of the anti-EGFR antibody by a peptide linker.
In a preferred embodiment, the N-terminus of the D2 domain of the extracellular region of VEGFR1 is linked to the C-terminus of the heavy chain of the anti-EGFR antibody by a peptide linker.
In a preferred embodiment, the fusion protein is selected from the group consisting of:
a) The fusion protein comprises an amino acid sequence shown as SEQ ID NO:5, and the amino acid sequence is as shown in SEQ ID NO: 7; or the like, or, alternatively,
b) The fusion protein comprises an amino acid sequence shown as SEQ ID NO:6, and the amino acid sequence is as shown in SEQ ID NO: 7; or the like, or, alternatively,
c) The fusion protein comprises an amino acid sequence shown as SEQ ID NO:9, and the amino acid sequence is as shown in SEQ ID NO: 7; or the like, or a combination thereof,
d) The fusion protein comprises an amino acid sequence shown as SEQ ID NO:10, and the amino acid sequence is as shown in SEQ ID NO:7, or a light chain as shown.
In a preferred embodiment, the fusion protein is selected from the group consisting of: 602-D2, 602dN-D2, 602-D2-M2 or 602dN-D2-M2.
In a second aspect, the invention provides a nucleic acid molecule encoding the above-mentioned anti-EGFR/VEGF bifunctional fusion protein.
In a preferred embodiment, the nucleic acid molecule comprises:
a) Encoding the heavy chain of the fusion protein as shown in SEQ ID NO:11, and a nucleic acid sequence as set forth in SEQ ID NO: 13; or the like, or, alternatively,
b) Encoding the heavy chain of the fusion protein as shown in SEQ ID NO:12, and a nucleic acid sequence as set forth in SEQ ID NO: 13; or the like, or, alternatively,
c) Encoding the heavy chain of the fusion protein as shown in SEQ ID NO:14, and a nucleic acid sequence as set forth in SEQ ID NO: 13; or the like, or a combination thereof,
d) A heavy chain encoding a fusion protein as set forth in SEQ ID NO:15, and a nucleic acid sequence as set forth in SEQ ID NO:13, or a nucleic acid sequence as set forth in seq id no.
In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule as described above.
In a fourth aspect, the invention provides a host cell comprising a nucleic acid molecule as described above.
The fifth aspect of the invention provides a preparation method of an anti-EGFR/VEGF bifunctional fusion protein, which comprises the following steps:
a) Culturing the host cell under expression conditions so as to express the anti-EGFR/VEGF double-function fusion protein;
b) Isolating and purifying the anti-EGFR/VEGF bifunctional fusion protein of step a).
In a sixth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned anti-EGFR/VEGF bifunctional fusion protein and one or more pharmaceutically acceptable carriers, diluents or excipients.
In a seventh aspect, the invention provides a use of the above-mentioned anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition in the preparation of a medicament for treating cancer, preferably, cancer that highly or abnormally expresses EGFR and/or highly or abnormally expresses VEGF.
In a preferred embodiment, the cancer is selected from: colorectal cancer, skin cancer, or squamous cell carcinoma; more preferably, the cancer is selected from colon cancer or cutaneous squamous cell carcinoma.
In a preferred embodiment, the treatment comprises administering to the subject an effective amount of an anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition, which is administered by injection; more preferably, the injection is selected from intravenous, intramuscular, or subcutaneous injection.
In a preferred embodiment, the treatment comprises administering to the subject an effective amount of an anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition, said effective amount referring to a dose of 0.5-30 mg/kg; more preferably, the interval between the administrations of the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is selected from the group consisting of: a) Once per week; b) Twice a week; c) Three times per week; even more preferably, the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is administered at a time selected from the group consisting of: a) At least 1 week; b) At least 2 weeks; c) For at least 3 weeks.
In an eighth aspect, the present invention provides a method for treating cancer, comprising administering to a subject an effective amount of the above-mentioned anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition, preferably, the cancer highly or aberrantly expresses EGFR and/or highly or aberrantly expresses VEGF.
In a preferred embodiment, the cancer is selected from the group consisting of: colorectal cancer, skin cancer, or squamous cell carcinoma; more preferably, the cancer is selected from colon cancer or cutaneous squamous cell carcinoma.
In a preferred embodiment, the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is administered by injection; more preferably, the injection is selected from intravenous, intramuscular, or subcutaneous injection.
In a preferred embodiment, the effective amount refers to a dose of 0.5-30 mg/kg; more preferably, the interval between the administrations of the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is selected from the group consisting of: a) Once per week; b) Twice a week; c) Three times per week; even more preferably, the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is administered at a time selected from the group consisting of: a) At least 1 week; b) At least 2 weeks; c) For at least 3 weeks.
The invention has the following positive effects: the fusion protein can be simultaneously combined with EGFR and VEGF at a high affinity at a molecular level, and has good thermal stability; at a cellular level, the polypeptide can effectively inhibit A431 cell proliferation, and the capability of blocking the combination of VEGF and a receptor KDR is better than that of anti-VEGF monoclonal antibody Bevacizumab; at the animal level, the compound plays a remarkable tumor inhibition effect in both LIM1215 transplanted tumor and SW48 transplanted tumor models, and the tumor inhibition effect in both LIM1215 transplanted tumor and SW48 transplanted tumor is better than that of control anti-EGFR monoclonal antibody 602 and anti-VEGF monoclonal antibody Bevacizumab, and surprisingly, the compound also has a tumor inhibition effect in SW48 transplanted tumor which is better than that of the combination of Bevacizumab and 602, and a synergistic effect is shown. The fusion protein has great clinical application prospect in treating diseases related to high expression or abnormal expression of EGFR and VEGF.
Drawings
FIG. 1: structural schematic diagram of the anti-EGFR/VEGF bi-functional fusion protein.
FIG. 2A: HPLC detection result chart of the anti-EGFR/VEGF bifunctional fusion protein 602-D2.
FIG. 2B: HPLC detection result chart of the anti-EGFR/VEGF bifunctional fusion protein 602-D2-M2.
FIG. 2C: HPLC detection result chart of the anti-EGFR/VEGF bifunctional fusion protein 602 dN-D2.
FIG. 2D: HPLC detection result chart of the anti-EGFR/VEGF bifunctional fusion protein 602dN-D2-M2.
FIG. 3: and (3) an ELISA detection result chart of the affinity of the anti-EGFR/VEGF bifunctional fusion protein and the EGFR.
FIG. 4: and (3) an ELISA detection result chart of the affinity of the anti-EGFR/VEGF bifunctional fusion protein and VEGF.
FIG. 5: and (3) an ELISA detection result graph of the anti-EGFR/VEGF dual-function fusion protein simultaneously combining EGFR and VEGF.
FIG. 6: FACS detection result graph of the combination of the anti-EGFR/VEGF dual-function fusion protein and the target cell surface antigen EGFR.
FIG. 7 is a schematic view of: the result of the detection of the inhibiting activity of the anti-EGFR/VEGF bifunctional fusion protein on the proliferation of A431 cells is shown.
FIG. 8: and (3) a detection result graph of the anti-EGFR/VEGF bifunctional fusion protein blocking the combination of VEGF and a receptor KDR.
FIG. 9: the result of the anti-tumor effect of the anti-EGFR/VEGF dual-function fusion protein on the LIM1215 transplantation tumor model is shown.
FIG. 10: the result of the anti-tumor effect of the anti-EGFR/VEGF bifunctional fusion protein on an SW48 transplantation tumor model is shown.
Detailed Description
The following experimental examples are further illustrative of the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional or routine methods in the art, such as methods of making nucleic acid molecules, methods for constructing vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids or introducing plasmids into host cells, methods of culturing host cells, and the like, such methods being well known to those having ordinary skill in the art and described in numerous publications, including Sambrook, j., fritsch, e.f. and maniis, t. (1989) Molecular Cloning: a Laboratory Manual,2nd edition, cold spring Harbor Laboratory Press.
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 linkage by peptide bonds, either directly or via one or more connecting peptides (peptide linkers). The term "linker peptide" refers to a short peptide that can link two polypeptide sequences, typically a peptide of 2-30 amino acids in length.
In the present invention, the term "antibody" refers to a full-length antibody, and the term "antigen-binding fragment" refers to a fragment derived from an antibody and capable of binding an epitope of an antigen, including but not limited to scFv, fv, fab, or F (ab') 2.
In the present invention, the term "full-length antibody" refers to an isotetraglycan protein of about 150000 daltons with the same structural features, comprising a variable region (V) and a constant region (C), which consists of two identical heavy chains (H) and two identical light chains (L). Each heavy chain has at one end a heavy chain variable region (VH) followed by a heavy chain constant region consisting of three domains, CH1, CH2, and CH 3. Each light chain has a light chain variable region (VL) at one end and a light chain constant region at the other end, the light chain constant region comprising a domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain variable region is paired with the heavy chain variable region. The constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit various 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 κ (Kappa) or λ (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 "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, vol.I, 647-669 (1991)). The CDRs of the heavy chain variable region (VH) and light chain variable region (VL) are called HCDR and LCDR, respectively.
In the present invention, the term "humanized antibody" refers to an antibody in which the Complementarity Determining Regions (CDRs) of the antibody are derived from a non-human species (e.g., rodent), and the remainder of the antibody molecule (including the framework region FRs and the constant region C) is derived from a human. Wherein the framework region FR residues may be altered to maintain binding affinity. In the present invention, the term "chimeric antibody" refers to an antibody in which the variable region (V) is derived from a non-human species (e.g., rodent) and the constant region (C) is derived from a human.
In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population comprising identical individual antibodies, except for a few possible natural mutations. Monoclonal antibodies are highly specific to a single determinant on the antigen, and to a single epitope site. Monoclonal antibodies can be synthesized by hybridoma culture and are not contaminated with other immunoglobulins.
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 tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the measurement of antibodies and anti-antibodies in a BIACORE instrument using Surface Plasmon Resonance (SPR for short)The binding affinity of the antigen or the relative affinity of the antibody for antigen binding using ELISA.
In the present invention, the terms "affinity", "binding" or "binding affinity" are used interchangeably and refer to the strength of a binding interaction between two molecules.
In the present invention, the term "D2 domain of extracellular region of VEGFR 1" includes the native sequence and its variants (comprising at least 1 amino acid mutation) as long as the mutation does not alter the biological activity of the D2 domain.
In the present invention, the term "expression vector" refers to an expression vector conventional in the art, which may be a virus or a plasmid, comprising appropriate regulatory sequences, such as a promoter, a terminator, an enhancer, and the like. The expression vector preferably comprises: pcDNA3.4, pDR1, pDHFR or pTT5.
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 is stably self-replicating and the carried nucleic acid molecule 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) or 293F cells.
In the present invention, the term "effective amount" refers to the amount or dose of the active ingredient that produces the desired effect in the treated individual, including improvement of the condition of the individual, after administration of the fusion protein or pharmaceutical composition of the present invention to a patient.
In the present invention, the term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, rats and mice, and the like.
In the present invention, the terms "determining" or "detecting" are used interchangeably.
In the present invention, the term "TIW" refers to three times a week and the term "mpk" refers to mg/kg (mg/kg).
In the present invention, the term "SEM" refers to a scanning electron microscope.
In the present invention, the term "mAU (m Absorbance Unit)" refers to milliabsorbance units.
The sequence information referred to in the present invention is summarized in table 1.
TABLE 1 sequence listing
Figure BDA0003041456690000081
Figure BDA0003041456690000091
Figure BDA0003041456690000101
Figure BDA0003041456690000111
Figure BDA0003041456690000121
Figure BDA0003041456690000131
The positive control anti-EGFR mab 602 used in the following examples was a human-mouse chimeric monoclonal antibody that was expressed using a CHO cell expression system according to the amino acid sequence of Erbitux (Cetuximab, IMC-C225), and prepared after cell culture and production. The positive control anti-VEGF monoclonal antibody Bevacizumab (601) is a humanized monoclonal antibody prepared according to the sequence and method disclosed in US patent 6884879B 1.
The EGFR-ECD-Fc protein used in the following examples was obtained by linking the extracellular domain (Met 1-Ser 645) of human EGFR protein (NP-005219) to an FC tag and expressing the protein by transient transfection into 293F cells. EGFR-ECD-Fc-biotin is obtained by biotinylating EGFR-ECD-Fc protein with biotinylation reagent (thermofisher, cat. # 20217). VEGF referred to in the following examples is VEGF-A165.
HRP referred to in the following examples is horseradish peroxidase.
The fusion proteins or antibodies referred to in the following examples are numbered or defined according to the Kabat system.
The sources of the test materials and the test reagents used in the following examples are specifically described below. Unless otherwise specified, all are commercially available.
Experimental materials:
a431 cell: human skin squamous carcinoma cell line, purchased from ATCC.
KDR cells: purchased from promega, cat. # GA1082.
LIM1215: human colorectal cancer cell line, purchased from Ji Ni Ohio Biotech, inc., guangzhou.
SW48: human colon cancer cell line, purchased from ATCC.
SD rat: purchased from Zhejiang Uetonglihua laboratory animal technology, inc.
BALB/C nude mice: purchased from laboratory animal science and technology ltd, viton, beijing.
Experimental reagent:
pcDNA3.4 vector: purchased from thermofisher under item number a14697.
Recombinant VEGF-a165 protein: purchased from acrobiosystems, cat. # VE5-H4210.
HRP-labeled goat anti-human Fab antibodies: purchased from abcam, cat. # ab87422.
Goat anti-human IgG-FITC: purchased from sigma, cat. # F9512.
Goat anti-human FC antibody: purchased from Abcam, cat. # ab97225.
1 HBS working solution: purchased from GE HealthCare, cat. # BR100669.
Protein A chip: purchased from GE HealthCare, cat. #29127558.
EDC: from Fortebio, cat. #18-1033.
s-NHS: from Fortebio, cat. #18-1067.
AR2G probe: from Fortebio, cat. #18-5092.
Sodium acetate: from Fortebio, cat. #18-1070.
Ethanolamine: purchased from Fortebio, cat. #18-1071.
1, kinetic Buffer: purchased from Fortebio, cat. #18-1072.
Figure BDA0003041456690000141
solution: purchased from Sigma, cat. # a6964.
DMEM medium: purchased from Gibco, cat. #11995.
Bio-Glo: purchased from promega, cat. # G7940.
TMB: available from BD corporation under the designation 555214.
HRP-labeled Streptavidin (Streptavidin): purchased from BD Pharmingen, cat # 554066.
HRP-labeled murine anti-human Fab antibodies: purchased from underwriters, cat. # a01855.
EGFR-his: purchased from SINO, #10001-H08H.
protein A: purchased from therofisher, cat. #21184.
The nucleic acid sequences used in the following examples were synthesized by Biotechnology engineering (Shanghai) Inc. or Jinzhi Biotechnology Inc.
Example 1 anti-EGFR/VEGF bifunctional fusion protein construction
The invention adopts a mode of connecting the anti-EGFR monoclonal antibody and the D2 structural domain of VEGFR1 in series to construct the anti-EGFR/VEGF difunctional fusion protein, and the structural schematic diagram is shown in figure 1.
Fusion protein 602-D2
The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 1) and the C-terminus of the heavy chain of anti-EGFR monoclonal antibody 602 (SEQ ID NO: 2) were joined by peptide linker L (SEQ ID NO: 4) to give the heavy chain of fusion protein 602-D2 (SEQ ID NO: 5), and the light chain sequence of fusion protein 602-D2 was SEQ ID NO:7.
fusion protein 602dN-D2
The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 1) and the C-terminus of the heavy chain of anti-EGFR monoclonal antibody 602dN (SEQ ID NO: 3) were linked by a peptide linker L (SEQ ID NO: 4) to give the heavy chain of fusion protein 602dN-D2 (SEQ ID NO: 6), and the light chain sequence of fusion protein 602dN-D2 is SEQ ID NO:7.
fusion protein 602-D2-M2
The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 8) and the C-terminus of the heavy chain of anti-EGFR monoclonal antibody 602 (SEQ ID NO: 2) were linked by peptide linker L (SEQ ID NO: 4) to give the heavy chain of fusion protein 602-D2-M2 (SEQ ID NO: 9), and the light chain sequence of fusion protein 602-D2-M2 is SEQ ID NO:7.
fusion protein 602dN-D2-M2
The N-terminus of the D2 domain of VEGFR1 (SEQ ID NO: 8) and the C-terminus of the heavy chain of anti-EGFR monoclonal antibody 602dN (SEQ ID NO: 3) were linked by a peptide linker L (SEQ ID NO: 4) to give the heavy chain of fusion protein 602dN-D2-M2 (SEQ ID NO: 10), the light chain sequence of fusion protein 602dN-D2-M2 is SEQ ID NO:7.
wherein the D2 domain of VEGFR1 (SEQ ID NO: 8) in fusion proteins 602-D2-M2 and 602dN-D2-M2 is truncated at the C-terminus by 2 amino acids relative to the D2 domain of VEGFR1 (SEQ ID NO: 1) of fusion proteins 602-D2 and 602dN-D2, which are easily shed during fermentation and are removed without affecting potency.
Wherein, the 85-asparagine of the heavy chain (SEQ ID NO: 3) of the anti-EGFR monoclonal antibody 602dN is mutated into glutamic acid (N85E) relative to the heavy chain (SEQ ID NO: 2) of the anti-EGFR monoclonal antibody 602, so that the charge heterogeneity caused by glycosylation is reduced, the development of a subsequent production preparation process is facilitated, and the deglycosylation has NO obvious influence on the activity of the bifunctional fusion protein.
Example 2 expression and purification of anti-EGFR/VEGF bifunctional fusion proteins
The fusion protein 602-D2 heavy chain nucleic acid sequence is SEQ ID NO:11, the light chain nucleic acid sequence is SEQ ID NO:13. the fusion protein 602dN-D2 heavy chain nucleic acid sequence is SEQ ID NO:12, the light chain nucleic acid sequence is SEQ ID NO:13. the fusion protein 602-D2-M2 heavy chain nucleic acid sequence is SEQ ID NO:14, the light chain nucleic acid sequence is SEQ ID NO:13. the fusion protein 602dN-D2-M2 heavy chain nucleic acid sequence is SEQ ID NO:15, the light chain nucleic acid sequence is SEQ ID NO:13. the DNA fragments of the heavy chain and the light chain of the 4 anti-EGFR/VEGF double-function fusion proteins are respectively subcloned into a pcDNA3.4 vector, 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 is loaded on a HiTrap MabSelect SuRe column, and protein is eluted by 100mM citric acid (pH 3.5) eluent 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 of fig. 2A-2D show that the 4 fusion proteins have uniform molecular state and the monomer purity reaches more than 97%.
Example 3 determination of affinity of anti-EGFR/VEGF bifunctional fusion protein to antigen by enzyme-Linked immunosorbent assay (ELISA)
3.1 ELISA (enzyme-Linked immuno sorbent assay) for detecting affinity of anti-EGFR (epidermal growth factor receptor)/VEGF (vascular endothelial growth factor receptor) dual-function fusion protein and EGFR (epidermal growth factor receptor)
The recombinant EGFR-ECD-Fc protein was diluted to 1. Mu.g/ml with the coating solution, and 50. Mu.l/well was added to the microplate at 4 ℃ overnight. PBST washing plate 3 times, adding 200 u l/hole of blocking liquid, 37 degrees after 1 hours PBST washing plate 1 times for use. Diluting the anti-EGFR/VEGF dual-function fusion protein to 60nM with a diluent, diluting by 4 times to form 8 concentration gradients, sequentially adding the sealed ELISA plates, 100 mu l/hole, and standing at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and HRP-labeled mouse anti-human Fab antibody 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 ℃); stop solution is added into each hole to stop the reaction of the substrate, the OD value is read at 450nm of an enzyme labeling instrument, and GraphPad Prism7 software is used for data analysis, plotting and calculating EC50.
The experimental results are shown in fig. 3, the EC50 of the binding of the 4 anti-EGFR/VEGF bifunctional fusion proteins 602-D2, 602-D2-M2, 602dN-D2-M2 and the positive control anti-EGFR mab 602 to the EGFR-ECD is 0.633nM, 0.749nM, 0.738nM, 0.633nM and 0.656nM, respectively, which indicates that the fusion proteins 602-D2, 602-D2-M2, 602dN-D2-M2 can specifically bind to the EGFR, and the affinity to the EGFR is equivalent to that of the anti-EGFR mab 602.
3.2 ELISA (enzyme-Linked immuno sorbent assay) for detecting affinity of anti-EGFR (epidermal growth factor receptor)/VEGF (vascular endothelial growth factor receptor) dual-function fusion protein and VEGF
Recombinant VEGF-A165 protein was coated onto microtiter plates 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 anti-EGFR/VEGF double-function fusion protein by using a diluent to 100nM, and diluting by 4 times to form 8 concentration gradients, sequentially adding the blocked ELISA plates, 100 mu l/hole, and standing at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and HRP-labeled goat anti-human Fab antibody 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 ℃); stop solution is added into each hole to stop the reaction of the substrate, the OD value is read at 450nm of an enzyme labeling instrument, and GraphPad Prism7 software is used for data analysis, plotting and calculating EC50.
The experimental results are shown in fig. 4, the anti-EGFR/VEGF bifunctional fusion proteins 602dN-D2, 602-D2-M2, 602dN-D2-M2 and the positive control anti-VEGF mab Bevacizumab (601) have binding EC50 of 0.497nM, 0.603nM, 0.422nM and 0.747nM, respectively, which indicates that the fusion proteins 602dN-D2, 602-D2-M2 and 602dN-D2-M2 can specifically bind VEGF, and the affinity of the fusion proteins to VEGF is equivalent to that of the anti-VEGF Bevacizumab (601).
3.3 ELISA (enzyme-Linked immuno sorbent assay) for detecting capability of anti-EGFR/VEGF (epidermal growth factor receptor/vascular endothelial growth factor receptor) bifunctional fusion protein for simultaneously binding EGFR and VEGF
Steric hindrance may affect the ability of the anti-EGFR/VEGF bifunctional fusion protein to bind to both antigens simultaneously. To test the ability of the anti-EGFR/VEGF bifunctional fusion protein to bind both EGFR and VEGF, the recombinant VEGF-A165 protein was incubated at 20 ng/Kong Baoban, 4 ℃ overnight. PBST washing plate 3 times, adding 200 u l/hole of blocking liquid, 37 degrees after 1 hours PBST washing plate 1 times for use. Diluting the anti-EGFR/VEGF dual-function fusion protein by using a diluent to 200nM, diluting by 2 times to form 12 concentration gradients, sequentially adding the closed ELISA plates, 100 mu l/hole, and placing for 1 hour at 37 ℃. The plates were washed 3 times with PBST, EGFR-ECD-Fc-biotin was added at 150 ng/well, and left at 37 ℃ for 1 hour. After 3 PBST washes, streptavidin labeled with HRP (Streptavidin) was added and the plate was left at 37 ℃ for 30 minutes. After PBST washing the plate for 3 times, beating the residual liquid drops on absorbent paper as dry as possible, adding 100 mu l of TMB into each hole, and placing for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. Mu.l per well2M H 2 SO 4 The substrate reaction was stopped with stop solution, the OD was read at 450nm with a microplate reader, and the data were analyzed with GraphPad Prism7 software, plotted and the EC50 was calculated.
The experimental result is shown in FIG. 5, the anti-EGFR/VEGF bifunctional fusion proteins 602dN-D2 and 602dN-D2-M2 can simultaneously and specifically bind to EGFR and VEGF, and the EC50 is 4.599nM and 4.518nM respectively.
Example 4 measurement of binding affinity of anti-EGFR/VEGF bifunctional fusion proteins to the target cell surface antigen EGFR by FACS method
In this experiment, A431 cells expressing EGFR on the cell surface were used as target cells, and the target cells were arranged at 2X 10 5 Perwell was inoculated in a 96-well plate, washed three times with PBS containing 0.5% BSA, centrifuged at 300g each for 5 minutes, and the supernatant was discarded. The anti-EGFR/VEGF bifunctional fusion protein and the control antibody were diluted to 50nM,3 fold diluted to form 10 gradients as primary antibodies were added to 96-well plates, and the cells were suspended and incubated at 4 ℃ for 1 hour. The cells were washed twice with PBS containing 0.5% BSA to remove unbound antibody, and incubated with 100. Mu.l of 1g/ml goat anti-human IgG-FITC for 30 min at 4 ℃. The cells were centrifuged at 300g for 5min, washed twice with PBS containing 0.5% BSA to remove unbound secondary antibodies, and finally resuspended in 200. Mu.l PBS and the binding affinity of the fusion protein to the A431 cell surface EGFR was determined by Beckman Co. Mu. Lter Cytoflex flow cytometer. The data obtained were analyzed by GraphPad Prism7 software fitting.
The experimental results are shown in FIG. 6, the anti-EGFR/VEGF bifunctional fusion proteins 602dN-D2, 602dN-D2-M2 and the positive control anti-EGFR monoclonal antibody 602 can be specifically bound with the EGFR expressed on the cell surface, the EC50 is 1.784nM, 1.840nM and 1.831nM respectively, and the affinity is equivalent. Among them, the negative control (negative control) is an isotype control which does not bind to the EGFR antigen.
Example 5 determination of affinity dissociation constant KD of anti-EGFR/VEGF bifunctional fusion proteins to antigen EGFR
Measuring kinetic parameters of the combination and dissociation of the anti-EGFR/VEGF difunctional fusion Protein and the antigen EGFR by using a Biacore 8K molecular interaction analyzer and a capture method, diluting the fusion Protein to 3 mu g/ml by using 1 x HBS working solution, and capturing the fusion Protein by using a Protein A chip; antigen EGFR-his was diluted with 1 × HBS working solution, and 6 concentration gradients with the highest concentration of 25nM were set to bind to the fusion protein and dissociate in HBS working solution.
The experimental results are shown in Table 2, the anti-EGFR/VEGF bifunctional fusion proteins 602dN-D2-M2, 602-D2-M2 and 602-D2 are equivalent to the positive control anti-EGFR monoclonal antibody 602 in affinity dissociation constant for EGFR-his.
TABLE 2 affinity dissociation constants
Figure BDA0003041456690000181
Note: KD is the affinity constant; kon is the binding rate constant; kdis the dissociation rate constant.
Example 6 determination of affinity dissociation constant KD of anti-EGFR/VEGF bifunctional fusion proteins to antigen VEGF
Kinetic parameters of the binding dissociation of the anti-EGFR/VEGF bifunctional fusion protein and antigen VEGF-A165 were determined using the 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. The fusion proteins were diluted with 1 × kinetic Buffer working solution and dissociated in 1 × kinetic Buffer working solution with 5 concentration gradients with a maximum concentration of 12.5 nM.
The experimental result is shown in Table 3, the affinity of the anti-EGFR/VEGF bifunctional fusion protein 602dN-D2-M2 for VEGF165 is slightly better than that of a positive control anti-VEGF monoclonal antibody Bevacizumab.
TABLE 3 affinity dissociation constants
Figure BDA0003041456690000182
Note: KD is the affinity constant; kon is the binding rate constant; kdis the dissociation rate constant.
Example 7 inhibitory Activity of anti-EGFR/VEGF bifunctional fusion proteins on A431 cell proliferation
Adherent cultured log-phase grown A431 cells were taken and the growth medium RPMI 1640 (10% FBS, 1% streptomycin) was discarded. After one wash with DPBS, it was digested with pancreatin and neutralized with growth medium. Trypan blue cells were counted and centrifuged at 300g for 5min. Resuspending in RPMI 1640 (1% FBS, 1% streptomycin) medium, counting, plating, adjusting the density to 5000/well, 100. Mu.l/well, placing at 37 ℃ and 5% CO 2 Incubate overnight for about 24 hours. The next day RPMI 1640 pure medium was prepared to dilute the fusion protein to 100nM as starting concentration, 3-fold gradient dilution 10 gradients. Then, 90. Mu.l of each well was added, and after further incubation in an incubator at 37 ℃ for 72 hours, 80. Mu.l/well of Cell-Titer Glo (25 ℃ water bath thawing equilibrium temperature 30 minutes earlier) was added. After incubation at room temperature for 10 minutes, the luminescences (fluorescence intensity) were read with spectramax i 3.
The experimental results are shown in fig. 7, the anti-EGFR/VEGF bifunctional fusion proteins 602-D2-M2, 602dN-D2-M2 and the positive control 602 monoclonal antibody can effectively inhibit proliferation of a431 cells, and the IC50 is 2.942nM, 3.976nM and 3.556nM, respectively, which indicates that the inhibition activity of the anti-EGFR/VEGF bifunctional fusion protein on proliferation of a431 cells is equivalent to that of the positive control anti-EGFR monoclonal antibody 602.
Example 8 cell experiment of anti-EGFR/VEGF bifunctional fusion protein blocking binding of VEGF and receptor KDR
KDR cells growing in the logarithmic phase of adherent culture with the density of about 80% -90% are taken, and the growth medium is discarded. After washing once with DPBS, use
Figure BDA0003041456690000191
Digestion, neutralization of pancreatic enzymes, 200g centrifugation for 5 minutes, heavy suspension of cells in 10% FBS-containing DMEM medium, trypan blue cell count, cell density adjusted to 40000/well plating, 50. Mu.l/well. VEGF-A165 to 30ng/ml was diluted in DMEM medium containing 10% FBS, fusion protein was diluted in VEGF-containing medium at a fold ratio, 3 fold dilution, 10 gradients. The diluted fusion protein was added to the cell wells at 25. Mu.l/well (final VEGF concentration 10ng/ml, initial fusion protein concentration 50 nM), and after incubation at 37 ℃ for 6 hours, each well was added75 μ l of detection reagent Bio-Glo was added. After incubation at room temperature for 10 minutes, luminescences (fluorescence intensity) were read with SpectraMax i3 ×. All data were in duplicate wells, and the signal values were averaged and fitted to the 4-parameter method and analyzed using GraphPad Prism7 software.
The experimental results are shown in FIG. 8, the anti-EGFR/VEGF dual-function fusion protein and the positive control anti-VEGF monoclonal antibody Bevacizumab can effectively block the interaction between VEGF and a receptor KDR thereof, and the blocking abilities of the fusion proteins 602dN-D2 and 602dN-D2-M2 are better than the IC of the positive control anti-VEGF monoclonal antibodies Bevacizumab,602 dN-D2-M2 and Bevacizumab 50 0.147nM, 0.120nM and 0.448nM, respectively.
Example 9 pharmacokinetic study of anti-EGFR/VEGF bifunctional fusion proteins
4 SD rats for experiments are female, the weight of the SD rats is about 200g, the SD rats are adjusted in light/dark for 12/12 hours, the temperature is 20-26 ℃, the humidity is 40-70%, and the SD rats can freely eat and drink water. Each rat was injected with the fusion protein via tail vein at a dose of 10mg/kg and an injection volume of 5ml/kg. Blood was collected from the fundus vein of rats at about 200. Mu.l each time for 1h, 4h, 24h, 72h, 96h, 168h, 336h, and 504h after administration. The collected blood samples were allowed to stand at room temperature for half an hour until agglutination, and then centrifuged at 8000rpm at 4 ℃ for 10 minutes to obtain serum. The serum was immediately stored at-80 ℃. The concentration of the fusion protein in the serum was measured by ELISA.
1) Coating the ELISA plate with EGFR-ECD-Fc at 4 deg.C overnight, with a coating amount of 100 ng/well; after coating was completed, the plates were washed 3 times with PBST and then blocked with PBS +2% BSA at 37 ℃ for 2 hours; then adding 500 times diluted rat serum to incubate for 1 hour; after PBST washing for 3 times, adding goat anti-human FC antibody marked by HRP, and standing for 30 minutes at 37 ℃; 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 The substrate reaction is stopped by the stop solution, and the OD value is read at 450nm of an enzyme-labeling instrument.
2) Protein A is coated, and EGFR is detected. Coating ELISA plate with protein A overnight at 4 deg.C in an amount of 100 ng/well; after coating, the plates were washed 3 times with PBST and then with PBS +2%BSA blocking at 37 ℃ for 2 hours; then adding 500 times diluted rat serum to incubate for 1 hour; washing the PBST for 3 times, adding a mouse anti-human Fab antibody marked by HRP, and standing 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 2M H per well 2 SO 4 The substrate reaction is stopped by the stop solution, and the OD value is read at 450nm of an enzyme-labeling instrument.
The half-life of the anti-EGFR/VEGF dual-function fusion protein medicament in the rat body is calculated by using Phoenix software, pharmacokinetic parameters are shown in a table 4, and PK analysis results show that the half-life of the fusion protein 602dN-D2-M2 in the rat body is about 138 hours.
TABLE 4 602dN-D2-M2 pharmacokinetic parameters in rat
Figure BDA0003041456690000201
Example 10 anti-tumor Effect of anti-EGFR/VEGF bifunctional fusion proteins on LIM1215 transplantation tumor model
Collecting LIM1215 cells cultured in vitro (the surface of LIM1215 cells expresses EGFR, and an anti-EGFR antibody can effectively inhibit the proliferation of LIM1215 cells), and adjusting the concentration of the cell suspension to 2X 10 7 Perml, mixed with an equal volume of matrigel. Under sterile conditions, 100. Mu.l of cell suspension was inoculated subcutaneously into the right flank of nude mice. Measuring the diameter of the transplanted tumor by using a vernier caliper until the average tumor volume grows to 200mm 3 Animals were randomized into groups after left and right.
The test sample 602dN-D2-M2 was administered at doses of 5.65mg/kg and 28.25mg/kg, the CONTROL drug 602 was administered at doses of 5mg/kg and 25mg/kg, the CONTROL drug Bevacizumab was administered at doses of 5mg/kg and 25mg/kg, and the blank CONTROL group (CONTROL) was administered with the same volume of physiological saline. The administration is performed 3 times per week by intraperitoneal injection for 3 weeks. Throughout the experiment, the diameter of the transplanted tumor was measured 2 times a week, and the body weight of the mice was measured. The formula for Tumor Volume (TV) is: TV =1/2 × a × b 2 . Wherein a and b represent length and width, respectively. Relative Tumor Volume (RTV) is calculated from the measurement results.
The experimental results are shown in fig. 9, the tumor inhibition effect of 602dN-D2-M2 at two doses is significantly better than that of the control anti-EGFR mab 602 and the anti-VEGF mab Bevacizumab on the LIM1215 transplanted tumor model, which indicates that 602dN-D2-M2 inhibits tumor growth by blocking EGFR and VEGF on the LIM1215 transplanted tumor model.
Example 11 anti-tumor Effect of anti-EGFR/VEGF bifunctional fusion proteins on SW48 transplantation tumor model
Collecting SW48 cells cultured in vitro (the surface of SW48 cells expresses EGFR, and anti-EGFR antibody can effectively inhibit the proliferation of SW48 cells), and adjusting the concentration of cell suspension to 6 × 10 7 Perml, mixed with an equal volume of matrigel. Under sterile conditions, 100. Mu.l of cell suspension was inoculated subcutaneously into the right flank of nude mice. Measuring the diameter of the transplanted tumor by using a vernier caliper until the average tumor volume grows to 200mm 3 Animals were then administered randomly in groups.
The test sample 602dN-D2-M2 was administered at a dose of 11.3mg/kg, the CONTROL drug 602 and Bevacizumab were each administered at a dose of 10mg/kg, and the combination group was administered 602 and Bevacizumab (10 mg/kg and 10 mg/kg) simultaneously, and the blank CONTROL group (CONTROL) was administered with the same volume of physiological saline. The administration is performed 3 times per week by intraperitoneal injection for 3 weeks. Throughout the experiment, the diameter of the transplanted tumor was measured 2 times per week, and the body weight of the mice was weighed. The formula for Tumor Volume (TV) is: TV =1/2 × a × b 2 . Wherein a and b represent length and width, respectively.
The experimental results are shown in fig. 10, and the control anti-VEGF mab Bevacizumab and anti-EGFR mab 602 both significantly inhibited tumor growth in the SW 48-transplanted tumor model. Under the condition of equimolar dose, the tumor inhibition effect of 602dN-D2-M2 is better than that of Bevacizumab or 602, and the combination of the Bevacizumab and the Bevacizumab 602 shows a synergistic effect.
Example 12 physical stability of anti-EGFR/VEGF bifunctional fusion proteins
DSC (Differential scanning calorimetry) was used to examine the thermal stability of 602dN-D2-M2 samples in PBS buffer. Samples were replaced in PBS buffer, controlled at 1mg/ml and tested using MicroCal Vp-Capillary DSC (Malvern). Before detection, the sample and 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, and preparing for cleaning. And after the sample adding of the sample plate is finished, the plastic soft cover plate is sleeved on the sample plate. The scanning temperature starts from 25 ℃ and ends at 100 ℃ and the scanning speed is 150 ℃/h. As shown in Table 5, the sample 602dN-D2-M2 protein showed good thermal stability.
TABLE 5 602dN-D2-M2 thermal stability assay data
Figure BDA0003041456690000221
Sequence listing
<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH
<120> anti-EGFR/VEGF difunctional fusion protein and application thereof
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 100
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
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
100
<210> 2
<211> 449
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 3
<211> 449
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 5
<211> 559
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro
450 455 460
Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu
465 470 475 480
Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr
485 490 495
Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys
500 505 510
Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr
515 520 525
Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His
530 535 540
Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile
545 550 555
<210> 6
<211> 559
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro
450 455 460
Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu
465 470 475 480
Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr
485 490 495
Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys
500 505 510
Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr
515 520 525
Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His
530 535 540
Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile
545 550 555
<210> 7
<211> 214
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 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> 8
<211> 98
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
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
<210> 9
<211> 557
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro
450 455 460
Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu
465 470 475 480
Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr
485 490 495
Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys
500 505 510
Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr
515 520 525
Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His
530 535 540
Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
545 550 555
<210> 10
<211> 557
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Gly Arg Pro
450 455 460
Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu
465 470 475 480
Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr
485 490 495
Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys
500 505 510
Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr
515 520 525
Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His
530 535 540
Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
545 550 555
<210> 11
<211> 1683
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
caagtgcagc tgaagcaaag cggccccgga ctggtgcaac caagccagtc tctgagcatc 60
acatgcacag tcagcggctt ctctctgact aattacggag tgcactgggt gaggcaatcc 120
cccggcaagg gcctcgaatg gctgggagtg atttggtccg gcggcaacac agactacaac 180
actccattca cttccagact gtccatcaat aaggacaact ccaagtccca agtgtttttt 240
aagatgaact ccctccagag caacgacaca gccatctact actgtgccag agccctcaca 300
tactacgact atgagttcgc ttactggggc caaggcactc tggtcacagt gtccgctgct 360
agcactaaag gccctagcgt gttcccactg gccccttcca gcaagagcac atccggaggc 420
actgccgctc tgggatgtct ggtgaaggac tatttcccag agccagtgac agtcagctgg 480
aacagcggcg ctctgactag cggcgtgcat acttttccag ccgtcctcca gtcctccgga 540
ctgtattctc tgtccagcgt ggtcactgtg ccatcctcct ctctgggcac tcaaacatac 600
atctgtaacg tcaatcacaa gccaagcaat acaaaggtgg acaagagggt cgagcctaag 660
agctgcgaca agactcacac atgcccacca tgcccagccc cagagctcct cggaggacca 720
agcgtctttc tgtttccacc aaagccaaag gacacactca tgatctctag gacaccagag 780
gtgacttgtg tcgtggtgga cgtctcccac gaggacccag aagtcaagtt caactggtac 840
gtcgacggag tcgaggtcca caacgctaag actaagccta gggaagaaca gtacaatagc 900
acttacagag tcgtctccgt gctcacagtc ctccaccaag attggctgaa tggcaaggag 960
tacaagtgca aggtctccaa caaggctctg ccagccccaa tcgagaagac tatcagcaag 1020
gccaagggcc agcctagaga gcctcaagtg tacacactgc caccatctag ggaagagatg 1080
actaagaacc aagtgtctct gacttgtctg gtcaagggct tttatccatc cgacatcgcc 1140
gtggaatggg agagcaacgg ccaaccagag aacaactaca agactactcc tccagtgctc 1200
gactccgatg gcagcttttt tctgtactcc aagctgacag tggacaagtc cagatggcag 1260
caaggcaatg tcttcagctg tagcgtgatg catgaggccc tccacaacca ttacactcag 1320
aaatccctct ccctcagccc cggcgctggc ggaggcggaa gcggaggagg aggatccgat 1380
actggaaggc catttgtgga gatgtacagc gagatcccag agatcattca catgacagaa 1440
ggaagggagc tcgtcatccc atgcagagtg acaagcccta acatcactgt cactctcaag 1500
aagttcccac tcgacacact catcccagat ggcaagagaa tcatttggga cagcagaaag 1560
ggcttcatca tctccaacgc cacatataag gagatcggac tgctcacttg cgaagctaca 1620
gtcaacggcc acctctataa gactaactat ctgactcata ggcaaacaaa cactatctga 1680
taa 1683
<210> 12
<211> 1683
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
caagtgcagc tgaagcaaag cggccccgga ctggtgcaac caagccagtc tctgagcatc 60
acatgcacag tcagcggctt ctctctgact aattacggag tgcactgggt gaggcaatcc 120
cccggcaagg gcctcgaatg gctgggagtg atttggtccg gcggcaacac agactacaac 180
actccattca cttccagact gtccatcaat aaggacaact ccaagtccca agtgtttttt 240
aagatgaact ccctccagag cgaggacaca gccatctact actgtgccag agccctcaca 300
tactacgact atgagttcgc ttactggggc caaggcactc tggtcacagt gtccgctgct 360
agcactaaag gccctagcgt gttcccactg gccccttcca gcaagagcac atccggaggc 420
actgccgctc tgggatgtct ggtgaaggac tatttcccag agccagtgac agtcagctgg 480
aacagcggcg ctctgactag cggcgtgcat acttttccag ccgtcctcca gtcctccgga 540
ctgtattctc tgtccagcgt ggtcactgtg ccatcctcct ctctgggcac tcaaacatac 600
atctgtaacg tcaatcacaa gccaagcaat acaaaggtgg acaagagggt cgagcctaag 660
agctgcgaca agactcacac atgcccacca tgcccagccc cagagctcct cggaggacca 720
agcgtctttc tgtttccacc aaagccaaag gacacactca tgatctctag gacaccagag 780
gtgacttgtg tcgtggtgga cgtctcccac gaggacccag aagtcaagtt caactggtac 840
gtcgacggag tcgaggtcca caacgctaag actaagccta gggaagaaca gtacaatagc 900
acttacagag tcgtctccgt gctcacagtc ctccaccaag attggctgaa tggcaaggag 960
tacaagtgca aggtctccaa caaggctctg ccagccccaa tcgagaagac tatcagcaag 1020
gccaagggcc agcctagaga gcctcaagtg tacacactgc caccatctag ggaagagatg 1080
actaagaacc aagtgtctct gacttgtctg gtcaagggct tttatccatc cgacatcgcc 1140
gtggaatggg agagcaacgg ccaaccagag aacaactaca agactactcc tccagtgctc 1200
gactccgatg gcagcttttt tctgtactcc aagctgacag tggacaagtc cagatggcag 1260
caaggcaatg tcttcagctg tagcgtgatg catgaggccc tccacaacca ttacactcag 1320
aaatccctct ccctcagccc cggcgctggc ggaggcggaa gcggaggagg aggatccgat 1380
actggaaggc catttgtgga gatgtacagc gagatcccag agatcattca catgacagaa 1440
ggaagggagc tcgtcatccc atgcagagtg acaagcccta acatcactgt cactctcaag 1500
aagttcccac tcgacacact catcccagat ggcaagagaa tcatttggga cagcagaaag 1560
ggcttcatca tctccaacgc cacatataag gagatcggac tgctcacttg cgaagctaca 1620
gtcaacggcc acctctataa gactaactat ctgactcata ggcaaacaaa cactatctga 1680
taa 1683
<210> 13
<211> 648
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gacattttac tgacccagtc ccccgtgatt ctctccgtgt cccccggtga gcgggtgagc 60
ttctcttgtc gtgccagcca gtccatcggc accaacatcc actggtacca gcagaggacc 120
aacggatccc cccggctgct gatcaagtac gccagcgagt ccatcagcgg catcccttct 180
cgtttctccg gatccggatc cggcaccgac ttcactttaa gcatcaacag cgtggagagc 240
gaggacatcg ccgattacta ctgccagcag aacaacaact ggcccaccac cttcggcgct 300
ggcaccaagc tggagctgaa aaggaccgtg gccgccccct ccgtgttcat cttccccccc 360
tccgatgaac agctgaagag cggaaccgcc tccgtggtgt gtttactgaa caacttctac 420
ccccgggagg ccaaggtgca gtggaaggtg gacaacgctt tacagagcgg caactcccaa 480
gaaagcgtga ccgagcaaga tagcaaggac agcacctact ctttatccag cactttaact 540
ttaagcaagg ccgactacga gaaacacaag gtgtacgctt gtgaggtgac acaccaaggt 600
ttatcctccc ccgttacaaa gtccttcaat cggggcgagt gctgataa 648
<210> 14
<211> 1677
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
caagtgcagc tgaagcaaag cggccccgga ctggtgcaac caagccagtc tctgagcatc 60
acatgcacag tcagcggctt ctctctgact aattacggag tgcactgggt gaggcaatcc 120
cccggcaagg gcctcgaatg gctgggagtg atttggtccg gcggcaacac agactacaac 180
actccattca cttccagact gtccatcaat aaggacaact ccaagtccca agtgtttttt 240
aagatgaact ccctccagag caacgacaca gccatctact actgtgccag agccctcaca 300
tactacgact atgagttcgc ttactggggc caaggcactc tggtcacagt gtccgctgct 360
agcactaaag gccctagcgt gttcccactg gccccttcca gcaagagcac atccggaggc 420
actgccgctc tgggatgtct ggtgaaggac tatttcccag agccagtgac agtcagctgg 480
aacagcggcg ctctgactag cggcgtgcat acttttccag ccgtcctcca gtcctccgga 540
ctgtattctc tgtccagcgt ggtcactgtg ccatcctcct ctctgggcac tcaaacatac 600
atctgtaacg tcaatcacaa gccaagcaat acaaaggtgg acaagagggt cgagcctaag 660
agctgcgaca agactcacac atgcccacca tgcccagccc cagagctcct cggaggacca 720
agcgtctttc tgtttccacc aaagccaaag gacacactca tgatctctag gacaccagag 780
gtgacttgtg tcgtggtgga cgtctcccac gaggacccag aagtcaagtt caactggtac 840
gtcgacggag tcgaggtcca caacgctaag actaagccta gggaagaaca gtacaatagc 900
acttacagag tcgtctccgt gctcacagtc ctccaccaag attggctgaa tggcaaggag 960
tacaagtgca aggtctccaa caaggctctg ccagccccaa tcgagaagac tatcagcaag 1020
gccaagggcc agcctagaga gcctcaagtg tacacactgc caccatctag ggaagagatg 1080
actaagaacc aagtgtctct gacttgtctg gtcaagggct tttatccatc cgacatcgcc 1140
gtggaatggg agagcaacgg ccaaccagag aacaactaca agactactcc tccagtgctc 1200
gactccgatg gcagcttttt tctgtactcc aagctgacag tggacaagtc cagatggcag 1260
caaggcaatg tcttcagctg tagcgtgatg catgaggccc tccacaacca ttacactcag 1320
aaatccctct ccctcagccc cggcgctggc ggaggcggaa gcggaggagg aggatccgat 1380
actggaaggc catttgtgga gatgtacagc gagatcccag agatcattca catgacagaa 1440
ggaagggagc tcgtcatccc atgcagagtg acaagcccta acatcactgt cactctcaag 1500
aagttcccac tcgacacact catcccagat ggcaagagaa tcatttggga cagcagaaag 1560
ggcttcatca tctccaacgc cacatataag gagatcggac tgctcacttg cgaagctaca 1620
gtcaacggcc acctctataa gactaactat ctgactcata ggcaaacaaa ctgataa 1677
<210> 15
<211> 1677
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
caagtgcagc tgaagcaaag cggccccgga ctggtgcaac caagccagtc tctgagcatc 60
acatgcacag tcagcggctt ctctctgact aattacggag tgcactgggt gaggcaatcc 120
cccggcaagg gcctcgaatg gctgggagtg atttggtccg gcggcaacac agactacaac 180
actccattca cttccagact gtccatcaat aaggacaact ccaagtccca agtgtttttt 240
aagatgaact ccctccagag cgaggacaca gccatctact actgtgccag agccctcaca 300
tactacgact atgagttcgc ttactggggc caaggcactc tggtcacagt gtccgctgct 360
agcactaaag gccctagcgt gttcccactg gccccttcca gcaagagcac atccggaggc 420
actgccgctc tgggatgtct ggtgaaggac tatttcccag agccagtgac agtcagctgg 480
aacagcggcg ctctgactag cggcgtgcat acttttccag ccgtcctcca gtcctccgga 540
ctgtattctc tgtccagcgt ggtcactgtg ccatcctcct ctctgggcac tcaaacatac 600
atctgtaacg tcaatcacaa gccaagcaat acaaaggtgg acaagagggt cgagcctaag 660
agctgcgaca agactcacac atgcccacca tgcccagccc cagagctcct cggaggacca 720
agcgtctttc tgtttccacc aaagccaaag gacacactca tgatctctag gacaccagag 780
gtgacttgtg tcgtggtgga cgtctcccac gaggacccag aagtcaagtt caactggtac 840
gtcgacggag tcgaggtcca caacgctaag actaagccta gggaagaaca gtacaatagc 900
acttacagag tcgtctccgt gctcacagtc ctccaccaag attggctgaa tggcaaggag 960
tacaagtgca aggtctccaa caaggctctg ccagccccaa tcgagaagac tatcagcaag 1020
gccaagggcc agcctagaga gcctcaagtg tacacactgc caccatctag ggaagagatg 1080
actaagaacc aagtgtctct gacttgtctg gtcaagggct tttatccatc cgacatcgcc 1140
gtggaatggg agagcaacgg ccaaccagag aacaactaca agactactcc tccagtgctc 1200
gactccgatg gcagcttttt tctgtactcc aagctgacag tggacaagtc cagatggcag 1260
caaggcaatg tcttcagctg tagcgtgatg catgaggccc tccacaacca ttacactcag 1320
aaatccctct ccctcagccc cggcgctggc ggaggcggaa gcggaggagg aggatccgat 1380
actggaaggc catttgtgga gatgtacagc gagatcccag agatcattca catgacagaa 1440
ggaagggagc tcgtcatccc atgcagagtg acaagcccta acatcactgt cactctcaag 1500
aagttcccac tcgacacact catcccagat ggcaagagaa tcatttggga cagcagaaag 1560
ggcttcatca tctccaacgc cacatataag gagatcggac tgctcacttg cgaagctaca 1620
gtcaacggcc acctctataa gactaactat ctgactcata ggcaaacaaa ctgataa 1677
<210> 16
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Asn Tyr Gly Val His
1 5
<210> 17
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser
1 5 10 15
<210> 18
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr
1 5 10
<210> 19
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His
1 5 10
<210> 20
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Tyr Ala Ser Glu Ser Ile Ser
1 5
<210> 21
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gln Gln Asn Asn Asn Trp Pro Thr Thr
1 5
<210> 22
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 23
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 24
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105

Claims (27)

1. An anti-EGFR/VEGF bifunctional fusion protein, comprising: (a) An anti-EGFR antibody, or antigen-binding fragment thereof, and (b) a D2 domain of the extracellular region of VEGFR 1.
2. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1, wherein the anti-EGFR antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:16, the amino acid sequence of HCDR2 is shown as SEQ ID NO:17, the amino acid sequence of the HCDR3 is shown as SEQ ID NO:18 is shown in the figure; the amino acid sequence of LCDR1 is shown as SEQ ID NO:19, the amino acid sequence of LCDR2 is shown as SEQ ID NO:20, the amino acid sequence of LCDR3 is shown as SEQ ID NO: shown at 21.
3. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1 or claim 2, wherein the anti-EGFR antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the amino acid sequence of VH is as set forth in SEQ ID NO:22, and the amino acid sequence of VL is shown as SEQ ID NO: as shown at 24.
4. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1 or 2, wherein the heavy chain variable region VH comprises at least one deglycosylation mutation; preferably, the mutation is located within the framework region FR.
5. The anti-EGFR/VEGF bifunctional fusion protein of claim 4, wherein the mutation is at position 85 of the VH of the heavy chain variable region, the mutations being numbered according to the numbering system of Kabat; preferably, the mutation is N85E.
6. The anti-EGFR/VEGF bifunctional fusion protein of claim 5, wherein the VH of the heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO:23, the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: as shown at 24.
7. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1, wherein the anti-EGFR antibody comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3, or human IgG4, and the light chain constant region is selected from human Kappa (Kappa) or human Lambda (Lambda).
8. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1, wherein the anti-EGFR antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
9. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1, wherein the anti-EGFR antibody or antigen-binding fragment thereof is selected from a full-length antibody, scFv, fv, fab or F (ab') 2.
10. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1, wherein the anti-EGFR antibody comprises an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3, and the amino acid sequence is as shown in SEQ ID NO:7, or a light chain as shown in figure 7.
11. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1, wherein the D2 domain of the extracellular region of VEGFR1 comprises the amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO: 8.
12. The anti-EGFR/VEGF bifunctional fusion protein according to claim 1, wherein the anti-EGFR antibody or antigen-binding fragment thereof is linked to the D2 domain of the extracellular region of VEGFR1 directly by a peptide bond or by a peptide linker.
13. The anti-EGFR/VEGF bifunctional fusion protein according to claim 12, wherein the peptide linker comprises an amino acid sequence (GGGGS) n selected from 1, 2, 3, 4 or 5.
14. The anti-EGFR/VEGF bifunctional fusion protein according to any one of claims 1 to 13, wherein the anti-EGFR antibody is a full-length antibody comprising 2 heavy chains and 2 light chains, and the N-terminus or the C-terminus of the D2 domain of the extracellular region of the VEGFR1 is linked to the N-terminus or the C-terminus of the heavy chain or the light chain of the anti-EGFR antibody by a peptide linker.
15. The anti-EGFR/VEGF bifunctional fusion protein according to claim 14, wherein the N-terminus of the D2 domain of the extracellular region of VEGFR1 is linked to the C-terminus of the heavy chain of the anti-EGFR antibody by a peptide linker.
16. The anti-EGFR/VEGF bifunctional fusion protein according to claim 15, wherein the fusion protein is selected from the group consisting of:
a) The fusion protein comprises an amino acid sequence shown as SEQ ID NO:5, and the amino acid sequence is as shown in SEQ ID NO: 7; or the like, or, alternatively,
b) The fusion protein comprises an amino acid sequence shown as SEQ ID NO:6, and the amino acid sequence is as shown in SEQ ID NO: 7; or the like, or, alternatively,
c) The fusion protein comprises an amino acid sequence shown as SEQ ID NO:9, and the amino acid sequence is as shown in SEQ ID NO: 7; or the like, or a combination thereof,
d) The fusion protein comprises an amino acid sequence shown as SEQ ID NO:10, and the amino acid sequence is as shown in SEQ ID NO:7, or a light chain as shown in figure 7.
17. A nucleic acid molecule encoding the anti-EGFR/VEGF bifunctional fusion protein of any one of claims 1 to 16.
18. The nucleic acid molecule of claim 17, wherein said nucleic acid molecule comprises:
a) Encoding the heavy chain of the fusion protein as shown in SEQ ID NO:11, and a nucleic acid sequence as set forth in SEQ ID NO: 13; or the like, or, alternatively,
b) Encoding the heavy chain of the fusion protein as shown in SEQ ID NO:12, and a nucleic acid sequence as set forth in SEQ ID NO: 13; or the like, or, alternatively,
c) Encoding the heavy chain of the fusion protein as shown in SEQ ID NO:14, and a nucleic acid sequence as set forth in SEQ ID NO: 13; or the like, or, alternatively,
d) Encoding the heavy chain of the fusion protein as shown in SEQ ID NO:15, and a nucleic acid sequence as set forth in SEQ ID NO:13, or a nucleic acid sequence as set forth in seq id no.
19. An expression vector comprising the nucleic acid molecule of claim 17 or 18.
20. A host cell comprising the expression vector of claim 19.
21. A method for preparing an anti-EGFR/VEGF bifunctional fusion protein according to any one of claims 1 to 16, comprising the steps of:
a) Culturing the host cell of claim 20 under expression conditions such that the anti-EGFR/VEGF bifunctional fusion protein is expressed;
b) Isolating and purifying the anti-EGFR/VEGF bifunctional fusion protein of step a).
22. A pharmaceutical composition comprising an anti-EGFR/VEGF bifunctional fusion protein according to any one of claims 1 to 16 and one or more pharmaceutically acceptable carriers, diluents or excipients.
23. Use of the anti-EGFR/VEGF bifunctional fusion protein according to any one of claims 1 to 16, or the pharmaceutical composition according to claim 22 for the preparation of a medicament for the treatment of cancer.
24. The use of claim 23, wherein the cancer expresses EGFR with high or abnormal expression and/or VEGF with high or abnormal expression.
25. The use of claim 24, wherein the cancer is selected from the group consisting of: colorectal cancer, skin cancer, or squamous cell carcinoma; preferably, the cancer is selected from colon cancer or cutaneous squamous cell carcinoma.
26. The use of claim 23, wherein the treatment comprises administering to the subject an effective amount of an anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition, wherein the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is administered by injection; preferably, the injection is selected from intravenous, intramuscular, or subcutaneous injection.
27. The use of claim 23, wherein the treatment comprises administering to the subject an effective amount of an anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition, wherein the effective amount is a dose of 0.5-30 mg/kg; preferably, the interval of administration of the pharmaceutical composition is selected from: a) Once per week; b) Twice a week; c) Three times per week; more preferably, the pharmaceutical composition is administered at a time selected from the group consisting of: a) At least 1 week; b) At least 2 weeks; c) For at least 3 weeks.
CN202110458717.0A 2021-04-27 2021-04-27 anti-EGFR/VEGF (epidermal growth factor receptor/vascular endothelial growth factor) bifunctional fusion protein and application thereof Pending CN115246886A (en)

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PCT/CN2022/089244 WO2022228424A1 (en) 2021-04-27 2022-04-26 Anti-egfr/vegf bifunctional fusion protein and use thereof
TW111115786A TW202300530A (en) 2021-04-27 2022-04-26 Anti-EGFR/VEGF bifunctional fusion protein and use thereof
CN202280030685.7A CN117242097A (en) 2021-04-27 2022-04-26 anti-EGFR/VEGF (epidermal growth factor receptor)/VEGF (vascular endothelial growth factor receptor) dual-function fusion protein and application thereof

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WO2017186950A1 (en) * 2016-04-28 2017-11-02 Biomunex Pharmaceuticals Bispecific antibodies targeting egfr and her2
EP3668893A4 (en) * 2017-08-16 2021-08-04 Dragonfly Therapeutics, Inc. Proteins binding nkg2d, cd16, and egfr, hla-e ccr4, or pd-l1
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