CN111196855B - anti-EGFR/PD-1 bispecific antibodies - Google Patents

Abstract

The invention belongs to the field of tumor treatment, and discloses an anti-EGFR/PD-1 bispecific antibody, a preparation method and an application thereof in tumor resistance. More specifically, the scFv and IgG are connected by a peptide linker L2 to obtain a bispecific antibody with similar whole antibody structure and function, which has the functions of targeting the tumor surface antigen EGFR and activating tumor immunity, and can effectively exert the ADCC function of the Fc segment of the antibody. Compared with monoclonal antibodies, the molecule has obvious synergistic effect, and the anti-tumor effect is superior to that of antibody combination.

Description

anti-EGFR/PD-1 bispecific antibodies

Technical Field

The invention belongs to the technical field of tumor treatment and biology, and relates to a preparation method and application of a bispecific antibody molecule for resisting EGFR and PD-1.

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 functional abnormality is mainly manifested 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%. The relevant clinical data show that the expression level of EGFR is closely related to the malignancy degree of the tumor and the prognosis of tumor patients.

Erbitux (Cetuximab, IMC-C225) is a human murine chimeric monoclonal antibody directed against EGFR, specifically binds to EGFR, competitively blocks binding of EFGR to its ligand, thereby inhibiting EGFR signaling. Erbitux was approved for marketing in 2 months 2004 for EGFR positivity in combination with irinotecan, metastatic colorectal cancer for which irinotecan chemotherapy was ineffective, and for treatment of regional early head and neck Squamous Cell Carcinoma (SCCHN). The EGFR monoclonal antibody independently developed by Sanshengkou Jian is a human-mouse chimeric monoclonal antibody which is obtained by expressing according to the amino acid sequence of Erbitux by a CHO cell expression system and using an independently developed cell culture production process. In vivo and in vitro biological activity and anti-tumor activity researches show that the EGFR monoclonal antibody independently developed by the Sansheng Jian pharmaceutical industry has very similar biological activity with the positive control drug Erbitux, and the biological activity of the EGFR monoclonal antibody is slightly superior to that of the positive control drug Erbitux in certain aspects.

Human programmed cell death receptor-1 (PD-1) is a type I membrane protein consisting of 288 amino acids, with the extracellular segment being the Ig variable (V-type) domain responsible for binding ligands and the intracellular segment being the cytoplasmic tail responsible for binding signal transduction molecules. The PD-1 cytoplasmic tail contains two tyrosine-based signal transduction motifs, the ITIM (immunoreceptor tyrosine inhibition motif) and ITSM (immunoreceptor tyrosine transduction motif), respectively. PD-1 is expressed on the surface of activated T lymphocytes, and the binding of the PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death-Ligand 2) can inhibit the activity of the T lymphocytes and the related in vivo cellular immune response. Numerous studies have shown that the interaction of PD-1 and PD-L1 not only maintains the balance of the immune system in vivo, but also is the primary mechanism that leads to evasion of immune surveillance by tumor cells that are positive for PD-L1 expression. By blocking a PD-1/PD-L1 signal path, the immune system can be activated, and the killing of T cells to tumor cells is promoted.

(pembroliz μ Mab) was the first humanized monoclonal antibody to PD-1 marketed and approved by the FDA for the treatment of melanoma on month 9 2014, and the approved indications by 2018 include: melanoma, non-small cell lung cancer, hodgkin's lymphoma, head and neck squamous cell carcinoma, bladder cancer, gastric cancer, and solid tumors bearing MSI-H or dMMR.

(nivol. Mu. Mab) is a PD-1 monoclonal antibody from Mich-Shi Guibao, calif., which was approved by FDA and marketed in 12 months 2014, and the indications include: melanoma, non-small cell lung cancer, renal cell carcinoma, classical hodgkin's lymphoma, head and neck squamous carcinoma, bladder cancer, colorectal cancer, and hepatocellular carcinoma. Chinese patent application CN201710054783.5 discloses a brand new anti-PD-1 humanized monoclonal antibody independently developed by the Sansheng Guojian pharmaceutical industry. In vivo and in vitro biological activity and anti-tumor activity studies show that the biological activity of the PD-1 monoclonal antibody developed by the Sansheng healthcare industry is between that of a positive control drug Opdivo and Keytruda, and is slightly superior to the positive control drug Opdivo in some aspects.

Bispecific antibodies (BsAb) refer to antibody molecules that bind two (or more) different epitopes simultaneously. Bispecific antibodies have a unique mechanism of action compared to traditional monoclonal antibodies: 1) Bispecific antibodies can simultaneously bind to 2 or more different antigenic molecules or different epitopes of the same molecule, resulting in a synergistic effect. The antibody combination often does not have this effect. 2) Mediate cell-cell interactions. Bispecific antibodies can bind to two antigens on effector and target cells, respectively, and bridge between effector and target cells, facilitating cell-cell interactions, e.g., mediating killing of tumor cells by immune cells. Bispecific antibodies therefore have unique advantages not possessed by traditional monoclonal antibodies.

The patent provides a bispecific antibody capable of targeting tumor cell surface molecules EGFR and T lymphocyte surface molecules PD-1. The bispecific antibody exhibits a synergistic effect compared to the monoclonal antibody, and the anti-tumor effect of the bispecific antibody is superior to that of the antibody combination compared to the antibody combination.

Disclosure of Invention

The invention provides a novel bispecific antibody capable of specifically binding with EGFR and PD-1, and also provides a preparation method and application of the bispecific antibody.

Accordingly, it is an object of the present invention to provide a bispecific antibody capable of specifically binding to EGFR and PD-1; providing a nucleotide molecule encoding said bispecific antibody; providing an expression vector comprising said nucleotide molecule; a host cell providing the expression vector; provides a method for preparing the bispecific antibody; providing a pharmaceutical composition comprising the bispecific antibody; provides the use of said bispecific antibody in the manufacture of a medicament.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a bispecific antibody capable of specific binding to EGFR and PD-1, comprising an immunoglobulin antibody IgG and two identical single chain variable fragment scfvs, wherein each scFv comprises a variable region VH and a variable region VL, VH is linked to VL by a peptide linker L1, and the C-terminus of each scFv is linked to the N-terminus of the IgG heavy chain via a peptide linker L2.

The "bispecific antibody" of the present invention refers to a bispecific antibody having two different antigen binding sites capable of simultaneously binding to EGFR and PD-1, comprising two single chain variable fragment scfvs and an immunoglobulin antibody IgG conjugated thereto, the C-terminus of each scFv being linked to the N-terminus of each heavy chain of the IgG via a peptide linker L2, forming a heavy chain fusion protein of the bispecific antibody, wherein each scFv comprises a variable region VH and a variable region VL, the VH being linked to the VL via a peptide linker L1.

The present invention "single chain variable fragment scFv" refers to a fusion protein comprising an immunoglobulin heavy chain VH and light chain VL variable regions, the VH being fused to the VL by a peptide linker, wherein the fusion protein retains the same antigen specificity as an intact immunoglobulin.

The "immunoglobulin antibody IgG" of the present invention is an approximately 150kDa molecule consisting of four peptide chains, comprising two identical gamma heavy chains of approximately 50kDa and two identical light chains of approximately 25kDa, thereby having a tetrameric quaternary structure. The two heavy chains are interconnected by disulfide bonds and are each linked to one light chain. The resulting tetramer has two identical halves that form a fork or Y-like shape, with each end of the fork containing an identical antigen binding site. IgG antibodies can be divided into subclasses (e.g., igG1, 2, 3, 4) based on minor differences in amino acid sequences in the constant region of the heavy chain.

Preferably, the VH comprises the complementarity determining regions HCDR1-3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3;

the VL comprises complementarity determining regions LCDR1-3, wherein the amino acid sequence of LCDR4 is SEQ ID NO:4,lcdr5 has the amino acid sequence of SEQ ID NO:5,LCDR6 has the amino acid sequence of SEQ ID NO:6;

the heavy chain of the IgG comprises the complementarity determining region HCDR4-6, wherein the amino acid sequence of HCDR4 is SEQ ID NO:7, wherein the amino acid sequence of HCDR5 is SEQ ID NO:8, wherein the amino acid sequence of HCDR6 is SEQ ID NO:9;

the light chain of the IgG comprises a complementarity determining region LCDR4-6, wherein the amino acid sequence of LCDR4 is SEQ ID NO:10, wherein the amino acid sequence of LCDR5 is SEQ ID NO:11, wherein the amino acid sequence of LCDR6 is SEQ ID NO:12.

in the art, the binding region of an antibody typically comprises a light chain variable region and a heavy chain variable region, each variable region comprising three domains of 3 CDRs. The CDR domains of the heavy and light chains of an antibody are referred to as HCDR and LCDR, respectively. Thus, a conventional antibody antigen-binding site comprises six CDRs, including sets of CDRs from heavy and light chain V regions, respectively.

Preferably, the amino acid sequence of VH is SEQ ID NO:13,vl has the amino acid sequence of SEQ ID NO:14;

the amino acid sequence of the heavy chain variable region of the IgG is SEQ ID NO:15, the amino acid sequence of the light chain variable region is SEQ ID NO:16.

preferably, the amino acid sequence of the peptide linker L1 is SEQ ID NO:17.

preferably, the amino acid sequence of the peptide linker L2 is SEQ ID NO:18.

preferably, the amino acid sequence of the single-chain variable fragment scFv is SEQ ID NO:19.

preferably, the heavy chain amino acid sequence of the bispecific antibody is SEQ ID NO:20, the light chain amino acid sequence of which is SEQ ID NO:21.

in constructing the bispecific antibodies of the present invention, the problems associated with the chemical and physical stability of the bispecific antibodies are also solved, such as expression of physically stable molecules, increased heat and salt dependent stability, reduced aggregation, increased solubility at high concentrations, and maintenance of affinity for the two antigens EGFR and PD-1, respectively.

In another aspect, the present invention provides a nucleotide molecule encoding a bispecific antibody according to any one of the above.

Preferably, the nucleotide sequence of the heavy chain of the bispecific antibody capable of specifically binding to EGFR and PD-1 encoded by the nucleotide molecule is SEQ ID NO:22, the nucleotide sequence encoding the light chain thereof is SEQ ID NO:23.

the preparation method of the nucleotide molecule is a conventional preparation method in the field, and preferably comprises the following preparation methods: the nucleotide molecules encoding the monoclonal antibodies are obtained by gene cloning techniques such as the PCR method, or the like, or by artificial full-sequence synthesis.

Those skilled in the art know that the nucleotide sequence encoding the amino acid sequence of the above-described bispecific antibody may be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homolog. The polynucleotide homologue of the present invention may be produced by substituting, deleting or adding one or more bases of the gene encoding the bispecific antibody within a range in which the activity of the antibody is maintained.

In another aspect, the present invention provides an expression vector, wherein the expression vector comprises the nucleotide molecule.

Wherein the expression vector is conventional in the art, refers to an expression vector comprising appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate sequences. The expression vector may be a virus or a plasmid, such as a suitable phage or phagemid, for more technical details see for example Sambrook et al, molecular Cloning: a Laboratory Manual, second edition, cold Spring Harbor Laboratory Press,1989. Many known techniques and Protocols for nucleic acid manipulation are described in Current Protocols in Molecular Biology, second edition, ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.

The present invention further provides a host cell containing the above-described expression vector.

The host cell of the present invention is any host cell that is conventional in the art, as long as it can stably replicate itself and can efficiently express the nucleotide. Wherein the host cell includes prokaryotic expression cells and eukaryotic expression cells, the expression vector preferably includes: COS, CHO (Chinese hamster Ovary), NS0, sf9, sf21, DH5 α, BL21 (DE 3) or TG1, more preferably e.coli TG1, BL21 (DE 3) cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies). The recombinant expression transformant of the present invention can be obtained by transforming the expression vector into a host cell. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.

Preferably, the host cell is a eukaryotic cell. Preferably selected from CHO cells and 293E cells.

In another aspect, the present invention provides a method for preparing the bispecific antibody capable of specifically binding to EGFR and PD-1, comprising the steps of:

a) Culturing the above host cell under expression conditions to express the bispecific antibody capable of specifically binding to EGFR and PD-1;

b) Isolating and purifying the bispecific antibody of step a).

The culture method of the host cell and the separation and purification method of the antibody are conventional methods in the field, and for the specific operation method, reference is made to the corresponding cell culture technical manual and antibody separation and purification technical manual. The preparation method of the anti-EGFR/PD-1 bispecific antibody disclosed by the invention comprises the following steps: culturing the above host cell under expression conditions to express the bispecific antibody capable of specifically binding to EGFR and PD-1; isolating and purifying the anti-EGFR/PD-1 bispecific antibody. Using the above method, the recombinant protein can be purified as a substantially homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis.

The anti-EGFR/PD-1 bispecific antibody disclosed in the present invention can be isolated and purified by affinity chromatography, and the anti-EGFR/PD-1 bispecific antibody bound to the affinity column can be eluted by conventional methods such as high salt buffer, pH change, etc., depending on the characteristics of the affinity column used. The inventor of the invention carries out detection experiments on the obtained anti-EGFR/PD-1 bispecific antibody, and the experimental results show that the anti-EGFR/PD-1 bispecific antibody can be well combined with target cells and antigens and has higher affinity.

In another aspect, the present invention provides a composition comprising a bispecific antibody capable of specifically binding to EGFR and PD-1 as described above and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The bispecific antibody provided by the invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a curative effect more stably, and the preparations can ensure the conformation integrity of the amino acid core sequence of the bispecific antibody disclosed by the invention and simultaneously protect the multifunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation). In general, it is generally stable for at least one year at 2 ℃ to 8 ℃ for liquid formulations and at least six months at 30 ℃ for lyophilized formulations. The bispecific antibody preparation can be suspension, hydro-acupuncture, freeze-drying and other preparations commonly used in the pharmaceutical field.

For hydro-acupuncture or lyophilization formulations of the bispecific antibodies disclosed herein, pharmaceutically acceptable carriers preferably include, but are not limited to: one or a combination of a surfactant, a solution stabilizer, an isotonicity adjusting agent, and a buffer. Wherein the surfactant preferably includes, but is not limited to: nonionic surfactants such as polyoxyethylene sorbitol fatty acid esters (tween 20 or 80); poloxamer (such as poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; tetradecyl, oleyl, or octadecyl sarcosine; pluronics; MONAQUATTM, etc., in an amount to minimize the tendency of the anti-EGFR/PD-1 bispecific antibody to granulate. Solution stabilizers preferably include, but are not limited to, one or a combination of the following list: saccharides, for example, reducing sugars and non-reducing sugars; amino acids, such as monosodium glutamate or histidine; alcohols, for example: trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol, and the like, the solution stabilizer should be added in an amount such that the resulting formulation remains stable for a period of time deemed stable by one skilled in the art. The isotonicity adjusting agent preferably includes, but is not limited to, one of sodium chloride, mannitol, or a combination thereof. Buffers preferably include, but are not limited to: tris, histidine buffer, phosphate buffer, or a combination thereof.

In another aspect, the present invention provides the use of the bispecific antibody capable of specifically binding to EGFR and PD-1, or the pharmaceutical composition as described above, for the manufacture of a medicament, characterized in that said medicament is for the treatment of cancer or tumor.

The medicament for treating cancer or tumor refers to a medicament for inhibiting and/or treating tumor, and can include delay of development of symptoms related to tumor and/or reduction of severity of the symptoms, further include reduction of existing tumor symptoms and prevention of other symptoms, and also include reduction or prevention of tumor metastasis and the like.

The tumors targeted by the drugs of the present invention preferably include, but are not limited to: lung cancer, bone cancer, stomach cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, rectal cancer, colon cancer, cancer of the anal region, breast cancer, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, cancer of the urethra, cancer of the penis, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphatic cancer, transitional cell cancer, bladder cancer, kidney or ureter cancer, renal cell cancer, renal pelvis cancer, hodgkin's disease, non-hodgkin's lymphoma, soft tissue sarcoma, childhood solid tumor, lymphocytic lymphoma, central Nervous System (CNS) tumor, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, melanoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic or acute leukemia, and combinations of said cancers.

When the bispecific antibody and the composition thereof of the present invention are administered to animals including human, the dose to be administered varies depending on the age and body weight of the patient, the nature and severity of the disease, and the route of administration, and the results of animal experiments and various cases can be referred to, and the total dose of administration cannot exceed a certain range. The dosage of intravenous injection is 1-1800 mg/day.

The bispecific antibody and the composition thereof disclosed by the invention can also be combined with other antitumor drugs for administration so as to achieve the purpose of more effectively treating tumors, and the antitumor drugs include but are not limited to: 1. cytotoxic drugs: 1) Drugs that act on the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosoureas, methyl sulfonates; platinum compounds such as Cisplatin (cissplatin), carboplatin (Carboplatin), and Oxaliplatin (Oxaliplatin); antibiotics such as Adriamycin (Adriamycin/Doxorubicin), actinomycin D (Dactinom ycin D), daunorubicin (Daunorubicin), epirubicin (Epirubicin), mithramycin (Mithramycin), etc.; 2) Drugs that affect nucleic acid metabolism: dihydrofolate reductase inhibitors such as Methotrexate (MTX) and Pemetrexed (Pemetrexed); thymidine synthase inhibitors such as fluorouracils (5-fluorouracil, capecitabine), etc.; purine nucleoside synthase inhibitors such as 6-mercaptopurine and the like; ribonucleotide reductase inhibitors such as hydroxyurea (Hydroxyarbamide) and the like; DNA polymerase inhibitors such as cytarabine (cytisine arabinoside) and Gemcitabine (Gemcitabine), etc.; 3) Tubulin-acting drugs: docetaxel (Docetaxel), vinblastine (vinchristine), vinorelbine (Vinorelbine), podophylline, homoharringtonine, etc.; 2. hormone drugs: antiestrogens such as Tamoxifen (Tamoxifen), droloxifene (Droloxifene), exemestane (Exemestane), etc.; aromatase inhibitors such as Aminoglutethimide (Aminoglutethimide), formestane (Formestane), letrozole (Letrozl e), anastrozole (Anastrozole), etc.; anti-androgen: flutamide RH-LH agonists/antagonists: norrad, etalone, and the like; 3. biological response modifier drugs: the medicine has anti-tumor effect mainly by regulating body immunity function, such as Interferon (Interferon); interleukin-2 (Interleuki n-2); thymosin peptides (Thymosins), and the like; 4. monoclonal antibody drugs: trastuzumab (Trast uzumab), rituximab (Rituximab), cetuximab (Cetuximab), bevacizumab (Bevacizumab), and the like; 5. other classes of anti-tumor drugs: including some drugs whose current mechanism is not clear and which are to be further studied, etc. The bispecific antibody and the composition thereof disclosed by the invention can be combined with one of the antitumor drugs or the combination thereof.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

1. compared with the monoclonal antibody, the anti-EGFR/PD-1 bispecific antibody provided by the invention shows a synergistic effect, can simultaneously bind to EGFR and PD-1, has close affinity to a target cell and an antigen compared with the monoclonal antibody, has good biological activity, can inhibit the growth of a colon cancer cell SW-48 and a skin squamous carcinoma cell A431, activates a T lymphocyte and promotes the killing of the T cell on the tumor cell.

2. In contrast to antibody combinations, which require the injection of two separate products or a single injection of a co-formulation of two different antibodies, it is difficult to find (at relatively high concentrations) formulation conditions that have acceptable viscosities and promote chemical and physical stability of the two antibodies, and the way in which the two antibodies are combined involves the cumulative cost of both drugs, which increases the patient's cost of payment. The anti-EGFR/PD-1 bispecific antibody provided by the invention can simultaneously target a tumor cell surface molecule EGFR and a T lymphocyte surface molecule PD-1, and has better killing effect on tumor cells than the combination of antibodies.

3. The Fab deglycosylation of the anti-EGFR/PD-1 bispecific antibody has no influence on the activity, and the industrialization difficulty of the subsequent process is reduced.

4. The anti-EGFR/PD-1 bispecific antibody can be applied to tumor or cancer treatment alone or in combination with other antitumor drugs, namely can be applied to preparation of the antitumor or cancer drugs.

Drawings

FIG. 1: the invention discloses a molecular structure schematic diagram of an anti-EGFR/PD-1 bispecific antibody.

FIG. 2A: the invention relates to an anti-EGFR/PD-1 bispecific antibody HPLC detection map.

FIG. 2B: the result of SDS-PAGE detection of the anti-EGFR/PD-1 bispecific antibody of the invention. From left to right, three lanes are the non-reduced sample antibody, the molecular weight marker, and the reduced sample antibody.

FIG. 3A: the ELISA detects the binding of the anti-EGFR/PD-1 bispecific antibody of the invention to EGFR-ECD-Fc.

FIG. 3B: ELISA detects the binding of the anti-EGFR/PD-1 bispecific antibody of the present invention to PD-1-ECD-Fc.

FIG. 3C: ELISA detects the combination of the anti-EGFR/PD-1 bispecific antibody of the invention with EGFR-ECD-Fc and PD-1-ECD-Fc at the same time.

FIG. 4A: the anti-EGFR/PD-1 bispecific antibody of the invention has the binding activity with a cell surface antigen EGFR.

FIG. 4B: the anti-EGFR/PD-1 bispecific antibody of the invention has the binding activity with a cell surface antigen PD-1.

FIG. 5A: the invention discloses a detection result of the dynamic parameter characteristics of the combination of an anti-EGFR/PD-1 bispecific antibody and EGFR-ECD-his.

FIG. 5B: the invention discloses a detection result of the dynamic parameter characteristics of the combination of an anti-EGFR/PD-1 bispecific antibody and PD-1-ECD-his.

FIG. 6: the results of the experiment on the inhibition of the proliferation of A431 cells in vitro by the anti-EGFR/PD-1 bispecific antibody are shown.

FIG. 7: the invention discloses a cell experiment result of the anti-EGFR/PD-1 bispecific antibody for blocking the combination of PD-1 and PD-L1.

FIG. 8A: the invention discloses an ADCC effect experiment result of an anti-EGFR/PD-1 bispecific antibody on A431 tumor cells.

FIG. 8B: the invention discloses an ADCC effect experiment result of an anti-EGFR/PD-1 bispecific antibody on CD4+ T lymphocytes.

FIG. 9: the invention relates to pharmacodynamic research of an anti-EGFR/PD-1 bispecific antibody on a nude mouse SW48 colon cancer model.

FIG. 10: the invention relates to pharmacodynamic research of an anti-EGFR/PD-1 bispecific antibody on a humanized PD-1 mouse MC38 colon cancer model.

FIG. 11: the anti-EGFR/PD-1 bispecific antibody of the invention mediates the killing research of SW-48 tumor cells by PBMC. By using ^* Indicating that there is a significant difference from the group without the antibody ^# Shows that the significant difference exists compared with the combined drug group, wherein ^* P<0.05, ^** P<0.01, ^*** P<0.001， ^# P<0.05, ^## P<0.01, ^### P<0.001。

FIG. 12: results of pharmacokinetic parameters of the anti-EGFR/PD-1 bispecific antibody of the present invention in rats.

FIG. 13A: ELISA detects the combination of the anti-EGFR/PD-1 bispecific antibody b and the anti-EGFR/PD-1 bispecific antibody c of the invention and EGFR-ECD-Fc.

FIG. 13B: ELISA detects the combination of the anti-EGFR/PD-1 bispecific b antibody and the anti-EGFR/PD-1 bispecific antibody c of the invention and PD-1-ECD-Fc.

FIG. 13C: fab molecular weight deconvolution profiles of the anti-EGFR/PD-1 bispecific antibody b and the anti-EGFR/PD-1 bispecific antibody c of the present invention.

FIG. 13D: capillary isoelectric focusing profiles of the anti-EGFR/PD-1 bispecific antibodies of the invention.

FIG. 13E: capillary isoelectric focusing profiles of the anti-EGFR/PD-1 bispecific antibody b of the invention.

FIG. 13F: capillary isoelectric focusing profiles of the anti-EGFR/PD-1 bispecific antibody c of the invention.

Detailed Description

The following examples and experimental examples are intended to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells ^nd edition,Cold spring Harbor Laboratory Press.

The experimental materials and sources used in the following examples and the methods of formulating the experimental reagents are specifically described below.

Experimental materials:

CHO cells: available from Thermo fisher corporation under the designation a29133.

293E cells: from the NRC biotechnology Research Institute.

Colon cancer cell a431: purchased from ATCC.

PD-L1aAPC/CHO-K1 cells: purchased from Promega corporation under the designation J1252.

CD4+ T cells: available from Allcellels under the trade designation LP180329.

NK cells: purchased from Allcellels, cat # PB012-C.

SW48 colon cancer cell line: purchased from Shanghai Life sciences research institute of Chinese academy of sciences.

BALB/C nude mice: purchased from Shanghai Ling Biotech, inc.

MC38 mouse colon cancer cell line: purchased from and Meta-Biotechnology (Shanghai) Inc.

Mouse strain C57BJ/6J-PDCD1em1 (Hpdcd 1)/Smoc: purchased from shanghai, south china, modular biotechnology, ltd, product number: NM-KI-00015.

PBMC cells: purchased from Allcellels, cat # PB005-C.

SD rat: SCXK (Zhe) 2018-0001, a production license, available from laboratory animal technology, inc. of Tonglihua, wei, zhejiang.

Experimental reagent:

PBS: purchased from Biotechnology engineering (Shanghai) Inc., cat # B548117.

BSA: purchased from Biotechnology (Shanghai) Inc., cat # A60332.

ELISA coating solution: 0.1M NaHCO ₃ ，pH9.5。

ELISA blocking solution: PBS +2% bsa.

ELISA diluent: PBS +0.5% BSA.

ELISA stop solution: 50 μ l 2M H ₂ SO ₄ 。

PBST：PBS+0.05％Tween 20。

CCK-8 color developing solution: purchased from Dojindo, cat # CK04.

Bio-Glo: purchased from Promega, cat # G7940.

FBS: purchased from Gibco, cat # 10099.

TMB: available from BD corporation under the designation 555214.

Streptavidin: available from BD corporation under the designation 554061.

HRP-labeled murine anti-human Fab antibodies: purchased from sigma, cat # A0293.

anti-CD 28 antibody: purchased from Abcam under accession number ab213043.

anti-CD 3 antibodies: r & D, # mab100.

IL-2: purchased from R & D, cat # 202-IL.

Goat anti-human IgG-FITC: purchased from sigma, cat # F4143.

anti-MHC I positive control antibody: purchased from Invivogen, cat # Hla-c1.

Protein a chip: label No. 29139131-AA; lot:10261132.

HBS-EP working solution: available from Life science, BR-1006-69.

An experimental instrument:

SpectraMax i3x microplate reader: purchased from Molecular Devices, inc.

Spectra maxm5 microplate reader: purchased from Molecular Devices, inc.

Beckman Co. Mu. Lter CytoFLEX flow cytometer: purchased from Beckman corporation.

HiTrap MabSelectSuRe column: purchased from GE company.

The EGFR monoclonal antibodies in the embodiment of the invention are human and mouse chimeric monoclonal antibodies which are obtained by the Sansheng Guojian pharmaceutical industry according to the Erbitux amino acid sequence, expressed by a CHO cell expression system and independently developed by a cell culture production process. The PD-1 monoclonal antibodies in the embodiment of the invention are all brand-new anti-PD-1 humanized monoclonal antibodies which are disclosed in Chinese patent application CN201710054783.5 and independently developed by the health pharmaceutical industry of three kingdoms.

Example 1 molecular construction of anti-EGFR/PD-1 bispecific antibody

The invention adopts a series connection mode of scFv and IgG to construct an anti-EGFR/PD-1 bispecific antibody, and the specific form is scFv-L2-IgG, as shown in figure 1. Wherein, the scFv adopts a molecular form of VH-L1-VL. The heavy chain variable region VH (SEQ ID NO: 13) and the light chain variable region VL (SEQ ID NO: 14) of the anti-PD-1 monoclonal antibody were linked via L1 (SEQ ID NO: 17) to give a single-chain antibody fragment scFv (SEQ ID NO: 19) against PD-1. The single-chain antibody fragment was linked to the heavy chain of the anti-EGFR monoclonal antibody using L2 (SEQ ID NO: 18) to give the heavy chain of the anti-EGFR/PD-1 bispecific antibody molecule (SEQ ID NO: 20), while the light chain of the EGFR monoclonal antibody (SEQ ID NO: 21) remained unchanged. To increase the expression efficiency of the antibody molecules in CHO cells, jin Weizhi company was entrusted with codon optimization of the nucleic acid sequence of the anti-EGFR/PD-1 bispecific antibody molecule. Optimization mainly considers factors such as codon preference, GC content, mRNA secondary structure, repetitive sequence and the like, and then entrusts Jin Wei intelligent company to synthesize. The anti-EGFR/PD-1 bispecific antibody heavy chain nucleic acid sequence is SEQ ID NO:22, the light chain nucleic acid sequence is SEQ ID NO:23.

example 2 expression and purification of anti-EGFR/PD-1 bispecific antibody

The DNA fragments of the heavy chain and the light chain of the anti-EGFR/PD-1 bispecific antibody are respectively subcloned into a pTT5 vector, and recombinant plasmids are extracted to co-transfect CHO cells and/or 293E cells. After 7 days of cell culture, the culture fluid was subjected to high-speed centrifugation, vacuum filtration through a microfiltration membrane, and then applied to a HiTrap MabSelectSuRe column, and the protein was eluted in one step with an eluent of 100mM citric acid and pH3.5, and the target sample was recovered and dialyzed into PBS. The purified protein was detected by HPLC, as shown in fig. 2A, the antibody molecules were homogeneous, and the monomer purity was more than 97%. Reduced and non-reduced protein electrophoretic loading buffers were added, respectively, and detection was performed after boiling, and the results are shown in fig. 2B, where the full-length protein molecule was at 250kD (theoretical molecular weight 198 kD), the heavy chain was at 76kD, and the light chain was at 23 kD.

Example 3 determination of affinity of anti-EGFR/PD-1 bispecific antibody for antigen by enzyme-Linked immunosorbent assay (ELISA)

3.1 ELISA for detecting affinity of anti-EGFR/PD-1 bispecific antibody and EGFR

The recombinant EGFR-ECD-Fc protein was diluted to 3. Mu.g/ml with the coating solution, and 50. Mu.l/well was added to the microplate at 4 ℃ overnight. The plates were washed 3 times with PBST, 200. Mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The anti-EGFR/PD-1 bispecific antibody was diluted to 100. Mu.g/ml with a diluent, diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02 ng/ml), and the resultant mixture was sequentially added to the blocked ELISA plate at 100. Mu.l/well and allowed to stand at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and an HRP-labeled mouse anti-human Fab antibody was added thereto and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); adding stop solution into each hole to stop the reaction of the substrate, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC ₅₀ . The experimental results are shown in FIG. 3A shows that the anti-EGFR/PD-1 bispecific antibody and the positive control EGFR monoclonal antibody are combined with the EC of the EGFR-ECD ₅₀ 0.2091nM and 0.2090nM, respectively, with comparable affinities.

3.2 ELISA for detecting affinity of anti-EGFR/PD-1 bispecific antibody and PD-1

To test the binding capacity of the anti-EGFR/PD-1 bispecific antibody to PD-1, the recombinant PD-1-ECD-Fc protein was diluted to 0.4. Mu.g/ml with coating solution, 50. Mu.l/well was added to the plate, and overnight at 4 ℃. The plates were washed 3 times with PBST, 200. Mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The anti-EGFR/PD-1 bispecific antibody was diluted to 100. Mu.g/ml with a diluent, diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02 ng/ml), and the resultant mixture was sequentially added to the blocked ELISA plate at 100. Mu.l/well and allowed to stand 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 ℃); 50 μ l of 2M H was added to each well ₂ SO ₄ Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC ₅₀ . The results are shown in FIG. 3B, EC of anti-EGFR/PD-1 bispecific antibody and positive control PD-1 monoclonal antibody combined with PD-1-ECD ₅₀ The affinity of the anti-EGFR/PD-1 bispecific antibody is equivalent to that of the PD-1 monoclonal antibody at 0.228nM and 0.149nM, respectively.

3.3 ELISA for detecting the ability of anti-EGFR/PD-1 bispecific antibody to simultaneously bind to PD-1 and EGFR

Steric hindrance may affect the ability of an anti-EGFR/PD-1 bispecific antibody to bind both antigens simultaneously. To test the ability of the anti-EGFR/PD-1 bispecific antibody to bind both PD-1 and EGFR, the recombinant PD-1-ECD-Fc protein was diluted to 0.4. Mu.g/ml with coating solution, 50. Mu.l/well was added to the plate, 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/PD-1 bispecific antibody with diluent to 100 μ g/ml, diluting at 4 times ratio to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02 ng/ml), sequentially adding blocking solution, and blockingThe plate (5) was left at 37 ℃ for 1 hour in a volume of 100. Mu.l/well. 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 PBST washing the plate 3 times, HRP-labeled Streptavidin was added and 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 2M H per well ₂ SO ₄ Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC ₅₀ . Experimental results As shown in FIG. 3C, the anti-EGFR/PD-1 bispecific antibody can bind to EGFR and PD-1 simultaneously, and EC ₅₀ 0.2863nM.

Example 4FACS method for determining the binding affinity of an anti-EGFR/PD-1 bispecific antibody to a target cell surface antigen

4.1 FACS method for determining binding affinity of anti-EGFR/PD-1 bispecific antibody to EGFR on A431 cell surface

In the experiment, the colon cancer cell A431 with high EGFR expression on the cell surface is taken as a target cell, and the A431 cell is treated according to the proportion of 2 multiplied by 10 ⁵ Perwell was inoculated into 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. Mu.l of 12-gradient anti-EGFR/PD-1 bispecific antibody serially diluted from 3. Mu.g/ml according to a 3-fold gradient was used as a primary antibody, and 12-gradient EGFR monoclonal antibody serially diluted from 1. Mu.g/ml according to a 3-fold gradient was used as a positive control to be added to a 96-well plate, and cells were suspended and incubated at 4 ℃ for 1h. The cells were washed twice with PBS to remove unbound anti-EGFR/PD-1 bispecific antibody and incubated with 100. Mu.l of 1g/ml goat anti-human IgG-FITC for 30 minutes at 4 ℃. The cells were centrifuged at 300g for 5 min, washed twice with PBS to remove unbound secondary antibodies, and finally resuspended in 200. Mu.l PBS and the binding affinity of the anti-EGFR/PD-1 bispecific antibody to the cells was determined by Beckman Co. Mu. Lter Cytoflex flow cytometer. The data obtained were analyzed by GraphPad Prism6 software fitting. The results are shown in FIG. 4A, and the anti-EGFR/PD-1 bispecific antibody and the positive control EGFR monoclonal antibody can specifically bind to the EGFR, EC expressed on the cell surface ₅₀ 4.377nM and 4.976nM, respectively, with comparable affinities.

4.2 FACS method for determining binding affinity of anti-EGFR/PD-1 bispecific antibody to PD-1 on surface of PD-1 stable cell strain

Similarly, using CHO-stable cells expressing PD-1 on the cell surface as target cells, the binding affinity of the anti-EGFR/PD-1 bispecific antibody to PD-1 on the cell surface was determined by flow cytometry, and the data obtained was analyzed by fitting GraphPad Prism6 software as described previously. The results are shown in FIG. 4B, and the anti-EGFR/PD-1 bispecific antibody and the positive control PD-1 monoclonal antibody can specifically bind to cell surface-expressed PD-1,EC ₅₀ The affinity of the two was equivalent to 1.528nM and 1.618nM, respectively.

Example 5Biacore ^TM 8K determination of affinity of anti-EGFR/PD-1 bispecific antibodies to antigens

Kinetic parameters of the binding of the anti-EGFR/PD-1 bispecific antibody and the antigen EGFR-ECD-his were determined using the proteinA capture method. An anti-EGFR/PD-1 bispecific antibody with a concentration of 1. Mu.g/ml was bound to a Protein A chip, and the antigen EGFR-ECD-his was bound to the antibody with 6 concentration gradients diluted 2-fold from 50nM to the lower with 1X HBS-EP working solution, and dissociated in HBS-EP working solution.

Kinetic parameters of the binding of the anti-EGFR/PD-1 bispecific antibody and the antigen PD-1-ECD-his were determined using the proteinA capture method. An anti-EGFR/PD-1 bispecific antibody was bound to the Protein A chip at a concentration of 1. Mu.g/ml, and the antigen PD-1-ECD-his was bound to the antibody in HBS-EP working solution after being diluted 2-fold from 250nM with 1X HBS-EP working solution and in 5 concentration gradients.

The kinetic parameters of the anti-EGFR/PD-1 bispecific antibody combined with EGFR-ECD-his and PD-1-ECD-his are shown in Table 1, and the detection results of the kinetic characteristic parameters are respectively shown in FIGS. 5A and 5B.

TABLE 1 kinetic parameters of anti-EGFR/PD-1 bispecific antibodies

KD is the affinity constant; ka is the antigen-antibody binding rate; kd is the antigen-antibody dissociation rate; KD = KD/ka.

Document ANTICANCER RESEARCH (2007) reports Cetuximab binding kinetic parameters determined by Biacore as ka:2.2E +05M ^-1 s ^-1 ，kd：1.1E-03s ^-1 KD:5.2E-09M. The anti-EGFR part of the anti-EGFR/PD-1 bispecific antibody disclosed by the invention comprises an amino acid sequence consistent with the variable region of the Cetuximab antibody, and the experimental result shows that the binding kinetic parameters of the anti-EGFR/PD-1 bispecific antibody and the antigen EGFR are consistent with the literature reports. The binding kinetic parameters measured by Biacore for Nivolumab (No. 5C 4) reported in patent document EP2161336A1 are ka:4.32E +05M ^-1 s ^-1 ，kd：3.15E-04s ^-1 KD:0.73E-09M. The experimental results show that the binding kinetic parameters of the anti-EGFR/PD-1 bispecific antibody and the antigen PD-1 are approximately equivalent to those of the marketed drug Nivolumab. In conclusion, the anti-EGFR/PD-1 bispecific antibody has good binding kinetic properties with the antigens EGFR and PD-1.

Example 6 in vitro proliferation inhibition of squamous carcinoma A431 cells by anti-EGFR/PD-1 bispecific antibody

EGFR is highly expressed on the surface of A431 cells, and the anti-EGFR antibody can effectively inhibit the in vitro proliferation of the A431 cells. A431 cells in logarithmic growth phase were grown at 2.5X 10 depending on the cell growth rate ³ Inoculating the cells into a 96-well culture plate, and adding EGFR monoclonal antibodies or anti-EGFR/PD-1 bispecific antibodies with different concentrations after adherent growth for 24 h. The highest acting concentration of the EGFR monoclonal antibody is 60nM, the EGFR monoclonal antibody is diluted three times, 10 different concentrations are provided, each concentration is provided with double wells, and a blank control and a cell-free zeroing well are provided. Cells at 37 deg.C, 5% ₂ After further culturing for 72h under the condition, adding 20 mul/hole of CCK-8 color development solution, and incubating for 1.5h at room temperature. Reading on a SpectraMaxM5 microplate reader with the detection wavelength of 450nm and the reference wavelength of 650nm, analyzing data by GraphPad Prism6, plotting and calculating IC ₅₀ . The results are shown in FIG. 6, IC of anti-EGFR/PD-1 bispecific antibody and positive control EGFR monoclonal antibody on A431 cells ₅₀ The inhibitory activity was equivalent to 0.809nM and 0.931nM, respectively.

Example 7 cell experiment of anti-EGFR/PD-1 bispecific antibody blocking the binding of PD-1 to PD-L1

Taking logarithmic phase-grown PD-L1aAPC/CHO-K1 cells, trypsinizing into single cells, transferring to white background 96-well plate, 100 μ L/well, 40000 cells/well, placing at 37 deg.C, and 5% ₂ And incubated overnight. The anti-EGFR/PD-1 bispecific antibody, the positive control PD-1 monoclonal antibody and the isotype negative control antibody IgG1 are diluted into 2 Xworking solution according to a triple gradient, the highest concentration is 600nM, the lowest concentration is 0.09nM, and 9 concentration gradients are obtained. Simultaneously, the density is 1.4-2 multiplied by 10 ⁶ The pancreatic digestion of PD-1 effector cells with the cell viability of more than 95 percent into 1.25 multiplied by 10 ⁶ Cells/ml single cell suspension. PD-L1aAPC/CHO-K1 cells paved the day before are taken, supernatant is discarded, 40 mu L of antibody working solution diluted in a gradient way is added, and equal volume of PD-1 effector cells is added. Standing at 37 deg.C, 5% CO ₂ And incubated for 6 hours. After 6 hours incubation of the cells at 37 ℃, 80 μ l of detection reagent Bio-Glo was added to each well. After incubation for 10 minutes at room temperature, the luminescences were read with SpectraMax i3 ×. All data were pooled in duplicate wells, the signal values averaged and fitted using the 4-parameter method, data analyzed using GraphPad Prism6, plotted and IC calculated ₅₀ . The experimental results are shown in FIG. 7, the anti-EGFR/PD-1 bispecific antibody and the positive control PD-1 monoclonal antibody block the IC of the PD-1 and PD-L1 combination ₅₀ The inhibitory activity of the anti-EGFR/PD-1 bispecific antibody was comparable to that of PD-1 monoclonal antibody at 1.425nM and 0.891nM, respectively.

Example 8 ADCC Effect of anti-EGFR/PD-1 bispecific antibodies on A431 tumor cells and T lymphocytes

8.1 ADCC Effect of anti-EGFR/PD-1 bispecific antibodies on A431 tumor cells

Diluting A431 cells to 2X 10 with 1640 medium containing 5% FBS ⁵ After one/ml, 50. Mu.l were taken up in a 96-well flat-bottom plate overnight. The highest concentration of 40. Mu.g/ml anti-EGFR/PD-1 bispecific antibody and the highest concentration of 30. Mu.g/ml EGFR monoclonal antibody were diluted in 4-fold gradient for 9 concentration gradients, 50. Mu.l of different concentrations of positive control EGFR monoclonal antibody or anti-EGFR/PD-1 bispecific antibody were placed in 96-well plates, with duplicate wells for each concentration. Cells were assayed at 37 ℃ and 5% CO ₂ After further culturing for 15 minutes under the above conditions, effector cells (NK cells in this experiment) and target cells (A431 cells in this experiment) = target cells5:1 ratio NK cells were added, and the% CO was 5% at 37 ℃% ₂ The culture was continued for 3 hours under the conditions. After centrifugation at 300g, 100. Mu.l of the supernatant was added with 50. Mu.l of LDH and reacted for 15 minutes at room temperature in the dark. OD values were read at 490nm with a microplate reader, and data were analyzed by GraphPad Prism6, plotted and the killing rate was calculated. The experimental results are shown in FIG. 8A, the anti-EGFR/PD-1 bispecific antibody has obvious antibody-dependent cell killing effect on A431 tumor cells, and compared with positive control EGFR monoclonal antibody, the anti-EGFR/PD-1 bispecific antibody and EC of EGFR monoclonal antibody ₅₀ The activity of the two is 1.367nM and 1.731nM respectively.

8.2 ADCC Effect of anti-EGFR/PD-1 bispecific antibodies on CD4+ T cells

Activation of CD4+ T cells: coating 5. Mu.g/mL of anti-CD 3 antibody into 24-well plates, seeding 0.5M/well of CD4+ T cells, and adding 2. Mu.g/mL of anti-CD 28 antibody and 100U/mL of IL-2, 5% CO at 37% ₂ The culture was carried out under the conditions for 72 hours. Activated CD4+ T cells were collected and the PD-1 expression level was examined by FACS. Activated CD4+ T cells were diluted to 2X 10% with 1640 medium containing 5% FBS ⁵ After one/ml, take 50. Mu.l to a 96-well flat bottom plate. The anti-EGFR/PD-1 bispecific antibody with the highest concentration of 80. Mu.g/ml and the anti-MHC I positive control antibody with the highest concentration of 10. Mu.g/ml were diluted by 4-fold gradient for 10 concentration gradients, and 50. Mu.l of samples with different concentrations were put in a 96-well plate, and duplicate wells were set for each concentration. Cells were assayed at 37 ℃ and 5% CO ₂ After further culturing for 15 minutes under the conditions, NK cells were added in such a proportion that effector cells (NK cells in this experiment) and target cells (CD 4+ T cells in this experiment) =5:1, and the content of CO was 5% at 37 ℃ ₂ The culture was continued for 3 hours under the conditions. After centrifugation at 300g, 100. Mu.l of the supernatant was added with 50. Mu.l of LDH and reacted at room temperature for 15 minutes in the dark. OD values were read at 490nm with a microplate reader, and data were analyzed using GraphPad Prism 6. The results of the experiment are shown in fig. 8B, the anti-EGFR/PD-1 bispecific antibody has no significant antibody-dependent cell killing effect on activated CD4+ T cells.

Example 9 anti-tumor Effect of anti-EGFR/PD-1 bispecific antibodies on SW48 transplantation tumor model

This experiment was used to evaluate anti-EGFR activity in animal models of anti-EGFR/PD-1 bispecific antibodies. SW48 junctionEGFR is expressed on the surface of intestinal cancer cells, and the anti-EGFR antibody can effectively inhibit the proliferation of SW48 cells. The dose of the positive control EGFR mab was set at 25mg/kg, the dose of the test sample anti-EGFR/PD-1 bispecific antibody was designed at an equimolar dose to the EGFR mab, i.e., 34mg/kg, and one low dose group was set at 6.8mg/kg. The control group was given the same volume of saline. Collecting in vitro cultured colon cancer SW48 cells, adjusting the cell suspension concentration to 3 × 10 ⁷ And/ml. Under sterile conditions, 100. Mu.l of cell suspension was inoculated subcutaneously into the right flank of BALB/C nude mice. Measuring the diameter of the transplanted tumor by using a vernier caliper until the average tumor volume grows to 100-200mm ³ Animals were then randomized into groups. EGFR monoclonal antibody and anti-EGFR/PD-1 bispecific antibody were administered according to the above dosage, and control group was administered with equal amount of physiological saline by intraperitoneal injection 3 times per week 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. Calculating Relative Tumor Volume (RTV) according to the measurement result, wherein the calculation formula is as follows: RTV = V _t /V ₀ . Wherein V ₀ When administered in groups (i.e. d) ₀ ) Measurement of the resulting tumor volume, V _t For the tumor volume at each measurement. The evaluation index of the antitumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows: T/C (%) = (T) _RTV /C _RTV )×100(T _RTV : treatment group RTV; c _RTV : negative control group RTV). The evaluation standard of curative effect is as follows: T/C (%)>40% is invalid; T/C (%). Is less than or equal to 40, and p is less than or equal to 0.05 after statistical treatment. The experiment was repeated twice. As shown in FIG. 9, the anti-EGFR/PD-1 bispecific antibody showed 53.7% of tumor inhibition at a dose of 34mg/kg, 63.6% of tumor inhibition of EGFR mab (25 mg/kg) at approximately the same molar dose, and 48.2% of tumor inhibition of anti-EGFR/PD-1 bispecific antibody at a low dose of 6.8mg/kg. The results show that anti-EGFR/PD-1 bispecific antibodies inhibit tumor growth by blocking EGFR on the SW48 transplanted tumor model. Under the condition of equal molar dose, the anti-EGFR/PD-1 bispecific antibody and the positive control EGFR monoclonal antibody have equivalent tumor inhibition effect and lowAt the dose (6.8 mg/kg), the anti-EGFR/PD-1 bispecific antibody also had anti-tumor activity.

Example 10 anti-EGFR/PD-1 bispecific antibody anti-tumor Effect on humanized PD-1 mouse MC38 transplantation tumor model

This experiment was used to evaluate anti-PD-1 activity of anti-EGFR/PD-1 bispecific antibodies in animal models. Adopts humanized PD-1 mouse strain C57BJ/6J-PDCD1 ^em1(Hpdcd1) and/Smoc. The dose of the positive control PD-1 monoclonal antibody is set to 10mg/kg, the dose of the tested anti-EGFR/PD-1 bispecific antibody is designed to be equimolar with the dose of the PD-1 monoclonal antibody, namely 16mg/kg, and the control group is given the same volume of physiological saline. Collecting MC38 cells cultured in vitro, adjusting the cell suspension concentration to 1 × 10 ⁷ And/ml. Under sterile conditions, 100. Mu.l of the cell suspension was inoculated subcutaneously into the right flank of the humanized PD-1 mouse. Measuring the diameter of the subcutaneous transplanted tumor of the humanized PD-1 mouse by using a vernier caliper until the average tumor volume grows to 100-200mm ³ Animals were then randomized into groups. PD-1 monoclonal antibody, anti-EGFR/PD-1 bispecific antibody according to dosage, control group give equal amount of normal saline, weekly intraperitoneal injection administration 2 times, continuous administration 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. Calculating Relative Tumor Volume (RTV) according to the measurement result, wherein the calculation formula is as follows: RTV = V _t /V ₀ . Wherein V ₀ When administered in groups (i.e. d) ₀ ) Measurement of the resulting tumor volume, V _t For the tumor volume at each measurement. The evaluation index of the antitumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows: T/C (%) = (T) _RTV /C _RTV )×100(T _RTV : treatment group RTV; c _RTV : negative control group RTV). The evaluation standard of the curative effect is as follows: T/C (%)>40% is invalid; T/C (%) is less than or equal to 40, and p is less than or equal to 0.05 by statistical treatment. The experiment was repeated twice. The results are shown in FIG. 10, on day 21, the anti-EGFR/PD-1 bispecific antibody showed 91.5% of tumor inhibition at 16mg/kg dose, and the PD-1 monoclonal antibody (10 mg/kg) showed about the same molar dose85.2 percent. The results show that the anti-EGFR/PD-1 bispecific antibody can inhibit the tumor growth by blocking PD-1 on the humanized PD-1 mouse MC38 transplantation tumor model, and the tumor inhibition effect is equivalent to that of the positive control PD-1 monoclonal antibody.

Example 11 anti-EGFR/PD-1 bispecific antibody mediates killing of SW-48 tumor cells by PBMCs

Cultured SW-48 cells were collected, diluted in RPMI 1640 medium and plated at 1X 10 per well in 96-well plates ⁴ Cells, 37 ℃,5% ₂ Incubated under conditions overnight. PBMCs were diluted in RPMI 1640 medium in suspension (PBMC donor numbers # D180198 and # D180199, respectively) at a ratio of effector cells (PBMC cells in this experiment) = target cells (SW-48 cells in this experiment) =1, 2 × 10 cells per well ⁵ PBMC cells. The experimental groups were prepared by adding the anti-EGFR/PD-1 bispecific antibody to the final concentrations of 100nM, 10nM and 1nM, the antibody combination group was prepared by mixing the EGFR monoclonal antibody and the PD-1 monoclonal antibody according to 1:1 with the final concentrations of 100nM, the drug combination group alone was prepared by mixing the EGFR monoclonal antibody or the PD-1 monoclonal antibody with the final concentration of 100nM, and the blank control group was prepared without adding any antibody and was prepared by adding 5% CO at 37 ℃ in the presence of 5% CO ₂ The culture was continued for 5 days under the conditions. Wash 3 times with PBS and add 200. Mu.l PBS and 50. Mu.l detection reagent Bio-Glo per well. After 5 min incubation at room temperature, luminescences were read with SpectraMax i3 ×, data were analyzed with GraphPad Prism6, and ^* indicating that there is a significant difference from the group without the antibody ^# Indicating that the significant difference exists compared with the combined drug group. Experimental data are shown in FIG. 11, and 100nM anti-EGFR/PD-1 bispecific antibody significantly mediated killing of SW-48 tumor cells by PBMC compared to the negative blank control group ( ^*** P<0.001,p is the bias), and the killing effect appears dose-dependent, with the high dose group (final concentration of 100 nM) being significantly better than the low dose group (final concentrations of 10nM and 1 nM). Compared with a single drug group, the anti-EGFR/PD-1 bispecific antibody is obviously superior to the EGFR monoclonal antibody and the PD-1 monoclonal antibody, and compared with the antibody combination group, the anti-EGFR/PD-1 bispecific antibody with the length of 100nM is also obviously superior to the drug combination of the EGFR monoclonal antibody and the PD-1 monoclonal antibody (the two experiments are respectively the drug combination of the EGFR monoclonal antibody and the PD-1 monoclonal antibody) ^# P<0.05 and ^### P<0.001 Showing that the anti-EGFR/PD-1 bispecific antibody mediates the PBMC on SW-48 tumor cellsHas synergistic effect in killing process.

Example 12 pharmacokinetic Studies of anti-EGFR/PD-1 bispecific antibodies

The general experimental procedure and procedure were as follows, 4 SD rats per group weighing about 200g, each rat being injected via the tail vein with a dose of 2mg of anti-EGFR/PD-1 bispecific antibody; blood was collected from the orbit at a specific time after administration, and serum was collected by centrifugation at 8000rpm/min after the blood had naturally coagulated. The concentration of the antibody drug in serum is detected by three measurement methods, the principle of the method 1 and the method 2 depends on the capacity of the anti-EGFR/PD-1 bispecific antibody to simultaneously combine two antigens and can reflect the content change of the complete antibody molecules in the plasma, and the method 3 is used for detecting the content change of the total anti-EGFR/PD-1 bispecific antibody and the anti-EGFR/PD-1 bispecific antibody fragment in the plasma. Method 1) EGFR was coated and PD-1 was detected. Coating the ELISA plate with EGFR-ECD-Fc overnight at 4 ℃ in a coating amount of 150 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 2000 times diluted rat serum to incubate for 1 hour, and taking rat blank serum as a control; after PBST washing for 3 times, adding PD-1-ECD-Fc-biotin to incubate for 1 hour, wherein the coating amount is 7.5 ng/hole; washing the plate, finally adding the mouse anti-human 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 of 2M H per well ₂ SO ₄ Stopping the substrate reaction by the stop solution, reading the OD value at 450nm of an enzyme-labeling instrument, and deducting the OD value of blank plasma during data processing. Method 2) coating PD-1, detecting EGFR. Coating the ELISA plate with PD-1-ECD-Fc overnight at 4 ℃, wherein the coating amount is 20 ng/hole; 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 2000 times diluted rat serum to incubate for 1 hour, and taking rat blank serum as a control; washing the plate for 3 times by PBST, adding HRP-labeled Streptavidin, 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 Stopping the substrate reaction by the stop solution at 450nm of the microplate readerOD values were read and blank plasma OD values were subtracted at data processing. Method 3) coating protein A and detecting the Fab fragment of the antibody. Coating ELISA plate with protein A overnight at 4 deg.C in an 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 2000 times diluted rat serum to incubate for 1 hour, and taking rat blank serum as a control; 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 of 2M H per well ₂ SO ₄ Stopping the substrate reaction by the stop solution, reading the OD value at 450nm of the microplate reader, and deducting the OD value of blank plasma during data processing. The half-life of the antibody drug in rats was calculated using Phoenix software and data was compiled and plotted using GraphPad Prism 6. Pharmacokinetic parameters are shown in table 2, and experimental results are shown in table 2 and fig. 12, the in vivo half-lives of the rats detected by the methods 1 and 2 are 173 and 171 hours, respectively, and the in vivo half-life of the rats detected by the method 3 is 298 hours. The half-life of method 3 assay is longer, a possible explanation being: 1) The anti-EGFR/PD-1 bispecific antibody can be degraded into antibody fragments such as scFv and IgG in plasma, and the total content change of the anti-EGFR/PD-1 bispecific antibody and the IgG antibody fragments in the plasma is detected by using the method 3; 2) The method 3 is more sensitive, and the experimental result contains systematic errors.

TABLE 2 pharmacokinetic parameters of anti-EGFR/PD-1 bispecific antibodies in rats

group	HL_Lambda_z(hr)	Cmax(ng/μl)	AUClast(hr*ng/μl)	AUCINF_obs(hr*ng/μl)	Vz_obs(μl/kg)	Cl_obs(μl/hr/kg)	MRTlast(hr)
								1	173.824885	548	480	65872.95925	78278.5545	32461.149	130.832355
2	171.4581033	656	56840.5	75046.37925	45154.8225	137.52142	159.2041925
								3	298.304435	1264.5	191849.7225	286251.695	14937.14625	35.2596775	181.62501

Example 13 Effect of Fab deglycosylation of anti-EGFR/PD-1 bispecific antibodies on Activity

The anti-EGFR/PD-1 bispecific antibody molecule has glycosylation at the 347 th asparagine of the heavy chain, which increases the charge heterogeneity of the antibody molecule. In order to improve the homogeneity of the antibody molecule and facilitate the development of the production process, the asparagine at position 347 is mutated into glutamic acid to obtain the anti-EGFR/PD-1 bispecific antibody b (heavy chain amino acid sequence SEQ ID NO: 24) of the antibody molecule, and the asparagine at position 347 is mutated into aspartic acid to obtain the anti-EGFR/PD-1 bispecific antibody c (heavy chain amino acid sequence SEQ ID NO: 25) of the antibody molecule. Mass spectrometric identification indicated no glycosylation at position 347 after mutation, and experimental data as shown in FIG. 13C and Table 4. The protein expression purification method is the same as that of example 2, and the ELISA method for detecting the binding of the anti-EGFR/PD-1 bispecific antibody b and the anti-EGFR/PD-1 bispecific antibody c to the antigen is the same as that of example 3. The results of the antigen affinity experiments are shown in table 3 and fig. 13A and 13B. The affinity of the anti-EGFR/PD-1 bispecific antibody b and the anti-EGFR/PD-1 bispecific antibody c to the antigen did not change significantly compared to the anti-EGFR/PD-1 bispecific antibody, indicating that Fab deglycosylation had no significant effect on the activity of the anti-EGFR/PD-1 bispecific antibody. Capillary isoelectric focusing (i-CIEF) experiments are shown in FIGS. 13D, 13E and 13F, and compared with the anti-EGFR/PD-1 bispecific antibody, the charge heterogeneity of the anti-EGFR/PD-1 bispecific antibody b and the anti-EGFR/PD-1 bispecific antibody c is obviously reduced, the difficulty of subsequent process development is reduced, and the possibility is provided for subsequent large-scale production.

Table 3 results of antigen affinity assay by ELISA

TABLE 4 Fab molecular weight determination of anti-EGFR/PD-1 bispecific antibody b and anti-EGFR/PD-1 bispecific antibody c

Sample name	Theoretical molecular weight of heavy chain Fab	Heavy chain Fab test molecular weight
			anti-EGFR/PD-1 double antibody b	50582.27	50581.70
anti-EGFR/PD-1 dual anti-c	50581.29	50580.80

As can be seen from the above experiments, the anti-EGFR/PD-1 bispecific antibody of the present invention has affinity to target cells and antigens, inhibitory activity to squamous carcinoma A431 cells, and tumor inhibition rate on SW48 transplantation tumor model, all equivalent to that of the positive anti-EGFR monoclonal antibody or anti-PD-1 monoclonal antibody; the anti-EGFR/PD-1 bispecific antibody has no obvious antibody-dependent cell killing effect on activated CD4+ T cells; on a humanized PD1 mouse MC38 transplanted tumor model, the anti-EGFR/PD-1 bispecific antibody can block the growth of a PD1 transplanted tumor, and the tumor inhibition rate is slightly superior to that of a positive control.

Furthermore, 100nM of anti-EGFR/PD-1 bispecific antibody significantly mediated the killing of SW-48 tumor cells by PBMC: ( ^*** P<0.001 Compared with an antibody combination group, the 100nM anti-EGFR/PD-1 bispecific antibody is also obviously superior to the drug combination of the EGFR monoclonal antibody and the PD-1 monoclonal antibody (the two experiments are respectively ^# P<0.05 and ^### P<0.001 And the killing effect appears dose-dependent, with the high dose group (final concentration of 100 nM) being significantly better than the low dose group (final concentrations of 10nM and 1 nM).The anti-EGFR/PD-1 bispecific antibody was shown to have a synergistic effect in mediating the killing process of SW-48 tumor cells by PBMC.

The Fab deglycosylation of the anti-EGFR/PD-1 bispecific antibody has no influence on the activity, reduces the difficulty of subsequent process development, and provides possibility for subsequent large-scale production.

Sequence listing

<110> Shenyang Sansheng pharmaceutical Limited liability company

<120> anti-EGFR/PD-1 bispecific antibody

<130> 201811

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Ser Tyr Asp Met Ser

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<210> 2

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Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys

1 5 10 15

<210> 3

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<212> PRT

<213> Composite

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Pro Tyr Gly Gly Tyr Phe Asp Val

1 5

<210> 4

<211> 11

<212> PRT

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<400> 4

Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Composite

<400> 5

Tyr Ala Ser Gln Ser Ile Ser

1 5

<210> 6

<211> 9

<212> PRT

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<400> 6

Gln Gln Ser Asn Ser Trp Pro His Thr

1 5

<210> 7

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<212> PRT

<213> Composite

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Leu Thr Asn Tyr Gly Val His

1 5

<210> 8

<211> 21

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<400> 8

Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser

1 5 10 15

Arg Leu Ser Ile Asn

20

<210> 9

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Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr

1 5 10

<210> 10

<211> 11

<212> PRT

<213> Composite

<400> 10

Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His

1 5 10

<210> 11

<211> 7

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<213> Composite

<400> 11

Tyr Ala Ser Glu Ser Ile Ser

1 5

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<211> 9

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Gln Gln Asn Asn Asn Trp Pro Thr Thr

1 5

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Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

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<211> 119

<212> PRT

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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> 16

<211> 107

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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

<210> 17

<211> 20

<212> PRT

<213> Composite

<400> 17

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 18

<211> 15

<212> PRT

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<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 19

<211> 244

<212> PRT

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<400> 19

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

130 135 140

Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile

180 185 190

Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe

210 215 220

Cys Gln Gln Ser Asn Ser Trp Pro His Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Val Glu Ile Lys

<210> 20

<211> 708

<212> PRT

<213> Composite

<400> 20

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

130 135 140

Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile

180 185 190

Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe

210 215 220

Cys Gln Gln Ser Asn Ser Trp Pro His Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gly Gly Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln

260 265 270

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

275 280 285

Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu

290 295 300

Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr

305 310 315 320

Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln

325 330 335

Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr

340 345 350

Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp

355 360 365

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro

370 375 380

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

385 390 395 400

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

405 410 415

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

420 425 430

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

435 440 445

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

450 455 460

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

465 470 475 480

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

485 490 495

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

500 505 510

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

515 520 525

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

530 535 540

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

545 550 555 560

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

565 570 575

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

580 585 590

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

595 600 605

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

610 615 620

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

625 630 635 640

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

645 650 655

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

660 665 670

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

675 680 685

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

690 695 700

Ser Pro Gly Lys

705

<210> 21

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<212> PRT

<213> Composite

<400> 21

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> 22

<211> 2124

<212> DNA

<213> Composite

<400> 22

gaagtcaagc tggtggaatc cggaggcgga ctcgtgcagc ccggaggatc tttaaggctg 60

tcttgtgctg ccagcggctt tgccttctcc agctacgaca tgagctgggt gcggcaagcc 120

cccggtaaga ggctcgaatg ggtggctacc atttccggcg gcggacggta cacctactat 180

cccgacacag tcaagggtcg tttcaccatc tcccgggata acgccaagaa ttcccactat 240

ttacagatga atagcctccg ggccgaggat accgccgtgt atttttgcgc ctccccctac 300

ggcggctact ttgacgtgtg gggtcaaggt acactggtca cagtgtctag cggaggcgga 360

ggaagcggag gtggcggcag cggcggaggt ggtagcggag gaggaggatc cgaaatcgtg 420

ctgacccaga gccccgctac actgtcttta tcccccggag agagggctac cctcagctgt 480

cgggccagcc agtccatcag caacttcctc cactggtatc agcagaaacc cggccaagct 540

cctaggttat taatcaagta cgccagccag agcatctccg gcattcccgc tcgtttttcc 600

ggcagcggaa gcggaaccga ctttacttta accatctcca gcctcgaacc cgaagacttc 660

gctgtgtact tttgccagca gtccaacagc tggccccaca cattcggcca aggaaccaag 720

gtcgagatca aaggaggcgg cggctctggt ggcggaggct ccggtggcgg aggatcccaa 780

gttcagctga agcagagcgg acccggttta gtgcaaccca gccaatcttt atccatcact 840

tgtacagtct ccggcttcag cctcacaaac tacggcgtgc actgggtgag gcaatctccc 900

ggtaagggac tggagtggct gggagtgatt tggtccggcg gcaacaccga ttacaacacc 960

cccttcacat ctcgtctgag catcaacaag gacaatagca agagccaagt tttcttcaaa 1020

atgaactctt tacagtccga ggacaccgcc atttactatt gcgctcgtgc tctcacctac 1080

tatgactatg aattcgccta ctggggccaa ggcactttag tcaccgtgtc cgccgctagc 1140

acaaaaggcc cctccgtgtt tcctctcgcc cctagcagca aatccaccag cggaggcaca 1200

gctgctctgg gctgcctcgt gaaggactat tttcccgagc ccgtgaccgt ctcttggaac 1260

tccggagctc tcaccagcgg cgtgcacaca ttccccgctg tgctgcagag cagcggttta 1320

tactctttat ccagcgtggt caccgtgccc agcagctctt taggaaccca gacatacatc 1380

tgcaacgtga atcacaagcc cagcaacacc aaagtcgata agcgggtgga acctaaatct 1440

tgtgataaga cccatacttg tcctccttgt cccgctcccg aactcctcgg cggcccttcc 1500

gtgttcctct tccctcccaa gcctaaggat accctcatga tcagccggac acccgaagtg 1560

acttgtgtgg tggtcgatgt gagccatgag gaccccgagg tgaagttcaa ctggtacgtg 1620

gacggcgtgg aggtgcacaa cgctaagacc aagcctcggg aagagcagta caacagcaca 1680

tatagggtgg tcagcgtcct caccgtgctc catcaagatt ggctgaacgg caaggaatac 1740

aagtgtaagg tgtccaacaa ggctttaccc gcccccattg agaagaccat ctccaaagct 1800

aagggccaac ctcgggaacc ccaagtttat accctccccc ctagccggga ggagatgacc 1860

aaaaatcaag tttctttaac atgtctggtg aagggcttct accccagcga catcgccgtg 1920

gaatgggagt ccaacggcca acccgagaac aactataaga ccaccccccc cgttttagac 1980

tccgatggtt cttttttttt atatagcaag ctcaccgtgg ataagagccg gtggcagcaa 2040

ggtaatgtgt tttcttgttc cgtgatgcac gaggctttac acaatcacta cacccagaag 2100

tctttatccc tctcccccgg taag 2124

<210> 23

<211> 642

<212> DNA

<213> Composite

<400> 23

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 gc 642

<210> 24

<211> 708

<212> PRT

<213> Composite

<400> 24

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

130 135 140

Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile

180 185 190

Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe

210 215 220

Cys Gln Gln Ser Asn Ser Trp Pro His Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gly Gly Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln

260 265 270

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

275 280 285

Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu

290 295 300

Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr

305 310 315 320

Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln

325 330 335

Val Phe Phe Lys Met Asn Ser Leu Gln Ser Glu Asp Thr Ala Ile Tyr

340 345 350

Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp

355 360 365

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro

370 375 380

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

385 390 395 400

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

405 410 415

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

420 425 430

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

435 440 445

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

450 455 460

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

465 470 475 480

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

485 490 495

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

500 505 510

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

515 520 525

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

530 535 540

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

545 550 555 560

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

565 570 575

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

580 585 590

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

595 600 605

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

610 615 620

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

625 630 635 640

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

645 650 655

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

660 665 670

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

675 680 685

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

690 695 700

Ser Pro Gly Lys

705

<210> 25

<211> 708

<212> PRT

<213> Composite

<400> 25

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

130 135 140

Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile

180 185 190

Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe

210 215 220

Cys Gln Gln Ser Asn Ser Trp Pro His Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gly Gly Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln

260 265 270

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

275 280 285

Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu

290 295 300

Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr

305 310 315 320

Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln

325 330 335

Val Phe Phe Lys Met Asn Ser Leu Gln Ser Gln Asp Thr Ala Ile Tyr

340 345 350

Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp

355 360 365

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro

370 375 380

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

385 390 395 400

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

405 410 415

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

420 425 430

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

435 440 445

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

450 455 460

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

465 470 475 480

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

485 490 495

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

500 505 510

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

515 520 525

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

530 535 540

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

545 550 555 560

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

565 570 575

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

580 585 590

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

595 600 605

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

610 615 620

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

625 630 635 640

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

645 650 655

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

660 665 670

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

675 680 685

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

690 695 700

Ser Pro Gly Lys

705

Claims (17)

1. A bispecific antibody capable of specifically binding to EGFR and PD-1, characterized in that it comprises an immunoglobulin antibody IgG and two identical single chain variable fragment scfvs, wherein each scFv comprises a variable region VH and a variable region VL, VH being linked to VL by a peptide linker L1, the C-terminus of each scFv being linked to the N-terminus of the IgG heavy chain via a peptide linker L2;

the VH comprises complementarity determining regions HCDR1-3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2,HCDR3 has the amino acid sequence of SEQ ID NO:3;

the VL comprises complementarity determining regions LCDR1-3, wherein the amino acid sequence of LCDR4 is SEQ ID NO:4,lcdr5 has the amino acid sequence of SEQ ID NO:5,LCDR6 has the amino acid sequence of SEQ ID NO:6;

the heavy chain of the IgG comprises the complementarity determining region HCDR4-6, wherein the amino acid sequence of HCDR4 is SEQ ID NO:7, wherein the amino acid sequence of HCDR5 is SEQ ID NO:8, wherein the amino acid sequence of HCDR6 is SEQ ID NO:9;

the light chain of the IgG comprises complementarity determining regions LCDR4-6, wherein the amino acid sequence of LCDR4 is SEQ ID NO:10, wherein the amino acid sequence of LCDR5 is SEQ ID NO:11, wherein the amino acid sequence of LCDR6 is SEQ ID NO:12.

2. the bispecific antibody of claim 1, wherein the amino acid sequence of VH is SEQ ID NO:13,vl has the amino acid sequence of SEQ ID NO:14;

the amino acid sequence of the heavy chain variable region of the IgG is SEQ ID NO:15, the amino acid sequence of the light chain variable region is SEQ ID NO:16.

3. the bispecific antibody of claim 1, wherein the amino acid sequence of peptide linker L1 is SEQ ID NO:17.

4. the bispecific antibody of claim 1, wherein the amino acid sequence of peptide linker L2 is SEQ ID NO:18.

5. the bispecific antibody of claim 1, wherein the amino acid sequence of said single chain variable fragment scFv is SEQ ID NO:19.

6. the bispecific antibody of claim 1, wherein the heavy chain amino acid sequence of said bispecific antibody is SEQ ID NO:20, having the light chain amino acid sequence of SEQ ID NO:21.

7. a nucleotide molecule encoding a bispecific antibody according to any one of claims 1 to 6.

8. The nucleotide molecule of claim 7, wherein the nucleotide sequence encoding the heavy chain of the bispecific antibody that specifically binds to EGFR and PD-1 is SEQ ID NO:22, the nucleotide sequence encoding the light chain thereof is SEQ ID NO:23.

9. an expression vector comprising the nucleotide molecule of any one of claims 7 or 8.

10. The expression vector of claim 9, wherein the expression vector is selected from the group consisting of pDR1, pcdna3.1 (+), pcdna3.1/ZEO (+), pDHFR and pTT5.

11. The expression vector of claim 9, wherein the expression vector is pTT5.

12. A host cell comprising the expression vector of claim 9.

13. The host cell of claim 12, wherein the host cell is a eukaryotic cell.

14. The host cell of claim 12, wherein the host cell is selected from the group consisting of CHO cells and 293E cells.

15. A method of producing a bispecific antibody capable of specifically binding to EGFR and PD-1 according to any one of claims 1 to 6, said method comprising the steps of:

a) Culturing the host cell of any one of claims 12-14 under expression conditions, thereby expressing the bispecific antibody that specifically binds to EGFR and PD-1;

b) Isolating and purifying the bispecific antibody of step a).

16. A composition comprising a bispecific antibody capable of specifically binding to EGFR and PD-1 according to any one of claims 1 to 6 and one or more pharmaceutically acceptable carriers, diluents or excipients.

17. Use of a bispecific antibody capable of specifically binding to EGFR and PD-1 according to any one of claims 1 to 6, or a composition according to claim 16, for the preparation of a medicament for the treatment of cancer or tumor.

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