CN106519036B

CN106519036B - The bifunctional protein and the preparation method and application thereof of anti-CD47 and EGFR

Info

Publication number: CN106519036B
Application number: CN201610966053.8A
Authority: CN
Inventors: 杨赟
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2016-11-04
Filing date: 2016-11-04
Publication date: 2019-06-11
Anticipated expiration: 2036-11-04
Also published as: CN106519036A

Abstract

The present invention relates to tumor immunology fields, specifically disclose the bifunctional protein and the preparation method and application thereof of anti-CD47 and EGFR a kind of.The bifunctional protein EGFR/CD47-BsAb-Ig (abbreviation Bi-SP) includes heavy chain, the light chain of the anti-side EGFR and the Fc fusion protein of the anti-side CD47 of the anti-side EGFR.The bifunctional protein Bi-SP prepared using preparation method provided by the invention, can have the function of Panitumumab and SIRP alpha-mutant simultaneously, can block the proliferation for the tumour cell that CD47 is mediated and EGFR is induced.It in addition, bifunctional protein Bi-SP can farthest remain conventional monoclonal antibody structure as traditional IgG molecule, because there is the presence of Fc segment, can be purified with common Protein A column affinity chromatography, be conducive to large-scale production purifying.

Description

The bifunctional protein and the preparation method and application thereof of anti-CD47 and EGFR

Technical field

The present invention relates to tumor immunology fields, specifically, being related to the bifunctional protein of anti-CD47 and EGFR a kind of.

Background technique

Malignant tumour is to seriously endanger the disease of human health, is in second in the disease of various lethals.In recent years Coming, the disease incidence of malignant tumour obviously rises, meanwhile, therapeutic effect is poor, and advanced stage easily shifts, and prognosis is bad.At present Clinically, used conventional treatments such as chemicotherapy and operative treatment largely alleviates pain, but these therapies Side effect and limitation are still very big, and curative effect is difficult to further increase.

The proliferation of normal cell is strictly to activate its growth factor receptors to control by respective ligand, for example grow Factor receptor tyrosine kinases.Cancer cell is also to be proliferated under the activation of growth factor receptors, but when losing normal proliferative Strict control.It is this it is out of control may be such as overexpression or the growth factor receptor of growth factor as caused by several factors The overexpression of body, and the Spontaneous Activation of the biochemical route by growth factor regulation.Participating in carcinogenic receptor includes epidermis Growth factor receptors (EGFR), from the growth factor receptors (PDGFR) of blood platelet, insulin-like growth factor receptor (IGFR), trk C (NGFR) and fibroblast growth factor receptor (FGF) etc..

EGF-R ELISA (i.e. EGFR) is also referred to as c-erbB l/Herl, and family member is growth factor receptor Body tyrosine kinase, they interact in cell surface and special growth factor or native ligand, such as with EGF or TGFα phase Interaction, thus activated receptor tyrosine kinase.EGFR is adjusting growth, reparation and the existence of tumour cell, new vessels life At playing an important role in, invasion and transfer, while there is expression in significant component of human tumor.Many pernicious In tumour, the expression of EGFR is often related to poor prognosis and lower survival rate.It follows that if there is drug can block The activity of EGFR, that will inhibit its phosphorylation and signal transduction, to play the antitumor action of multiple pathways, while also can Increase the antitumor curative effect of chemotherapy and radiation.In some researchs, EGFR inhibitor and Treated with Chemotherapeutic Drugs object and radiotherapeutic drug join When cooperation is for some tumor cell lines, shows cumulative and act synergistically.

The occurrence and development of tumour are the processes that the multifactor a variety of factors of multimachine system participate in.CD47, that is, integrin associated protein (Integrin-Associated Protein, IAP), is expressed in cell surface, in combination with the Signal Regulation of Macrophage Surface Immunity receptor tyrosine in the cytoplasm of protein-alpha (Signal-Reg μ Latory Protein α, SIRP α) and then phosphorylation SIRP α Inhibitory motifs (Immunoreceptor Tyrosine-based Inhibition Motifs, ITIMs), recruit phosphatases The inhibition signal of " do not eat my (Don ' t eat me) " is issued after SHP-1 to macrophage.CD47 molecule is in tumour cell table The wide expression in face inhibits the Phagocytosis of Macrophages For Tumor.Accordingly, researcher, which develops, can activate macrophage The CD47 antagonist (CD47 antagonists) of cell Phagocytosis.CD47 antagonist will pass through closing CD47 and SIRP α knot It closes and the phagocytosis of activating macrophage, enhances anti tumor immune response, be finally reached the purpose for inhibiting tumour growth.

It can be seen that can not only inhibit tumour caused by EGFR signal activation if CD47 and EGFR can be blocked simultaneously Cell Proliferation, the tumor-inhibitory signal that will also CD47 be blocked to mediate activate the immune response to tumour, therefore may be than independent It blocks EGFR or CD47 to have the synergistic effect for preferably inhibiting tumor growth and proliferation, will be of great significance to the treatment of tumour.

CD47 antagonist mainly include target CD47 antibody and two kinds of SIRP alpha-mutant.Studies have shown that targeting CD47 Monoclonal antibody Ab400 test in vitro and in vivo in all show to hepatocellular carcinoma growth significantly inhibit effect.Equally, exist When for treatment of solid tumor such as breast cancer, colon cancers, targeting CD47 antibody also shows effective tumor killing effect.Research recently Show that engineered SIRP alpha-mutant (SIRP α-Fc) has to the higher affinity of CD47 and show to a variety of swollen The depression effect of tumor is also a kind of CD47 antagonist of great potential.EGFR inhibitor mainly includes monoclonal antibody, tyrosine Kinase inhibitor, ligand-toxin and immunotoxin are coupled the vaccine etc. that object and GEM 132 and EGFR/ ligand are dominated.Target The growth for expressing the tumor cell line of EGFR can be inhibited to show in the treatment of lung cancer and colorectal cancer is treated to the antibody of EGFR Apparent tumor killing effect is shown.The appropriate wooden monoclonal antibody (Panitumumab, ABX-EGF, AMG954, trade name: VECTIBIX) of pa is complete The IgG2 class monoclonal antibody of source of people, affinity is about 5 × 10^-11mol/L.The antibody is produced and is sold by Amgen company It sells, the appropriate wooden monoclonal antibody of the FDA of in September, 2006 approval pa is used to treat the metastatic colorectal carcinoma of expression EGFR.Its main mechanism It is the combination for blocking EGF and tumor cell surface EGFR, induction EGFR internalization, and then the growth of inhibition tumour cell.

But monoclonal antibody use in conjunction has various limitations.Firstly, clinically applying the safety and effect of two kinds of antibody Fruit does not have elaborate report so far, it is understood that there may be security risk；In addition, two kinds of antibody of use in conjunction are there is also insufficiency of accommodation and make The too high defect of valence, limits its clinical application.

Therefore, the bifunctional protein that can block two target spots simultaneously is constructed using technique for gene engineering, can hindered Disconnected EGFR, and CD47 can be blocked, and then more effectively inhibit the growth and proliferation of tumour, this is always those skilled in the art Urgent problem to be solved.

Summary of the invention

In order to solve the problems in the existing technology, the object of the present invention is to provide double function of anti-CD47 and EGFR a kind of Can albumen and preparation method thereof, the bifunctional protein can both inhibit EGFR, also can antagonism CD47, can be used for preparing antitumor Drug.

In order to achieve the object of the present invention, technical scheme is as follows:

In a first aspect, the present invention provides a kind of bifunctional protein EGFR/CD47-BsAb-Ig of anti-CD47 and EGFR (letters Claim Bi-SP), the bifunctional protein includes the Fc fusion of the heavy chain of the anti-side EGFR, the light chain of the anti-side EGFR and the anti-side CD47 Albumen.

The Fc fusion protein of the heavy chain of the anti-side EGFR, the light chain of the anti-side EGFR and the anti-side CD47 can be by CHO It is intracellular formed disulfide bond form to obtain it is a kind of can inhibit EGFR, and can antagonism CD47 bifunctional protein.

In order to avoid occur in the anabolic process of said elements the anti-side EGFR antibody or the anti-side CD47 fusion protein from I connects or combination, and the present invention is to the area constant region CH3 in the heavy chain of the above-mentioned anti-side EGFR and the Fc fusion protein of the anti-side CD47 Rite-directed mutagenesis has been carried out, it is specific as follows: to introduce four mutation in the anti-side EGFR heavy chain constant region CH3, including in SEQ ID NO.2 348th is changed into tryptophan by threonine, and the 350th is changed into alanine by leucine, and the 474th tyrosine is changed into figured silk fabrics Propylhomoserin, the 330th tyrosine are changed into cysteine.Two mutation, including SEQ ID are introduced in the anti-side the CD47 area Fc of Bi-SP The 166th is changed into tryptophan by threonine and the 152nd serine is changed into cysteine in NO.12.It is anti-after mutated Fc sections of the side CD47 and the anti-side EGFR heavy chain constant region can be formed by these amino acid is similar to " key structure (Knob-in- Hole binding pattern) " considerably increases the probability correctly matched, reduces a possibility that forming self pair.

Based on above-mentioned adjustment, the bifunctional protein specifically includes SEQ ID NO.14, SEQ ID NO.16 and SEQ ID Amino acid sequence shown in NO.18.

Second aspect, the present invention provides the preparation methods of aforementioned bifunctional protein, include the following steps:

S1, building are respectively containing nucleotides sequence shown in SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO.17 The carrier of column, and it is cultivated with liposome method cotransfection into host cell, it screens and stablizes expression bifunctional protein Cell clone；

S2, cell culture is isolated and purified, obtains bifunctional protein Bi-SP.

It should be noted that carrier of the present invention can be carrier commonly used in the art, such as pCDNA3.1, PDHFF etc..It include the fusion dna sequence for being connected with suitable transcription and translation and adjusting sequence in expression vector.

Optionally, as in a specific embodiment of the invention, eukaryotic expression vector pcDNA3.1 is used.

Further, the host cell for expressing the bifunctional protein can be prokaryotic cell, such as DH5 α, BL21 (DE3), TG1 etc.；It can also be mammal or insect host cell, COS, CHO, NSO, sf9 and sf21 etc. may be applicable to this Invention.

Preferably, the present invention selects CHO-K1 cell (ATCC) as the expression of host cell progress bifunctional protein.

Further, the condition of culture of host cell is this field conventional culture conditions, such as using containing 10% serum DMEM culture medium is cultivated.

Further, it in S2, is screened and is stablized with the Selective agar medium containing 600 μ g/mL G418 and 250 μ g/mL Zeocin Express the cell clone of bifunctional protein.Using the above method, bifunctional protein can be purified as substantially uniform substance, such as It is single band on SDS-PAGE electrophoresis.Further, it can use the method for affinity chromatography to disclosed by the invention double Functional protein is isolated and purified, according to the characteristic of the affinity column utilized, can be used conventional method such as high-salt buffer, Change the methods of pH bifunctional protein of the elution of bound on affinity column.

The present invention carries out affinity detection to Bi-SP, it is found that it fully remains Panitumumab and SIRP α mutation The affinity of body.Next step experiment, including tumor cell proliferation, internal tumor suppression and rush macrophage phagocytosis are carried out using Bi-SP Deng experiment, the experimental results showed that, bifunctional protein Bi-SP disclosed by the invention has Panitumumab and SIRP α mutation simultaneously The function of body, it can block the proliferation for the tumour cell that CD47 is mediated and EGFR is induced.In addition, bifunctional protein Bi- SP can farthest remain conventional monoclonal antibody structure as traditional IgG molecule, because there is depositing for Fc segment It can purified with common Protein A column affinity chromatography, be conducive to large-scale production purifying.Experiment shows identical Under dosage, bifunctional protein Bi-SP has the antineoplaston effect for being substantially better than Panitumumab.

Three aspects, are based on preceding solution, can express SEQ ID NO.14, SEQ ID NO.16 and SEQ ID simultaneously The host cell of amino acid sequence shown in NO.18, and including respectively contain SEQ ID NO.13, SEQ ID NO.15 and The carrier of the carrier of nucleotide sequence shown in SEQ ID NO.17 combines also within protection scope of the present invention.

Fourth aspect, the present invention also provides the compositions for containing aforementioned bifunctional protein Bi-SP.

And provide aforementioned bifunctional protein Bi-SP or composition the answering in the preparation of antitumor drugs containing it With.

Above-mentioned bifunctional protein Bi-SP disclosed by the invention, and pharmaceutically acceptable auxiliary material forms drug system together For agent composition to more stably play curative effect, these preparations can guarantee human antibody amino acid core disclosed by the invention The conformation integrality of sequence, while the polyfunctional group of protected protein matter also being wanted to prevent its degradation (including but not limited to cohesion, deamination Or oxidation).Under normal conditions, for liquid preparation, it can usually save and at least stablize 1 year under the conditions of 2 DEG C -8 DEG C, for Lyophilized preparation is stablized in 30 DEG C of holdings at least six months.Herein preparation can commonly be suspended for pharmaceutical field, water needle, freeze-drying Equal preparations, preferably water needle or lyophilized preparation, for the water needle or lyophilized preparation of above-mentioned human antibody disclosed by the invention, pharmacy Upper acceptable auxiliary material includes one or a combination set of surfactant, solution stabilizer, isotonic regulator and buffer, wherein Surfactant includes nonionic surface active agent such as Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80)； Poloxamer (such as poloxamer 188)；Triton；Lauryl sodium sulfate (SDS)；Sodium Laurylsulfate；Myristyl, Asia Oil base or octadecyl sarcosine；Pluronics；MONAQUATTM etc., additional amount should make the granulating of bifunctional protein become Gesture is minimum, and solution stabilizer can be carbohydrate, including reducing sugar and nonreducing sugar, amino acids include monosodium glutamate or Histidine, alcohols include one or a combination set of trihydroxylic alcohol, advanced sugar alcohol, propylene glycol, polyethylene glycol, the additional amount of solution stabilizer The preparation those skilled in the art eventually formed should be made to think to reach stable time interior holding stable state, isotonic tune Saving agent can be one of sodium chloride, mannitol, and buffer can be one of TRIS, histidine buffering liquid, phosphate buffer.

Above-mentioned preparation is the composition comprising bifunctional protein, after to animal administration including people, antitumor effect Fruit is obvious.Specifically, effectively to the prevention of tumour and/or treatment, it can be used as anti-tumor drug use.

So-called anti-tumor drug in the present invention refers to the drug for inhibiting and/or treating tumour, may include with swollen Tumor grows the delay of related symptoms development and/or the reduction of these severity of symptom, it further comprises already present swollen The mitigation of tumor growth simultaneous phenomenon and the appearance for preventing other symptoms, also reduce or prevent transfer.

Bifunctional protein Bi-SP and the composition containing it are administered to animal including people in the present invention, administration Age and weight of the dosage because of patient, disease traits and seriousness and administration route and it is different, can refer to zoopery knot Fruit and various situations, total dosage is no more than a certain range.

Bifunctional protein Bi-SP disclosed by the invention and it can also combine with other antineoplastics containing its composition Administration, for the treatment of tumour, these antineoplastics include:

1, cytotoxic drug (1) acts on the drug of DNA chemical structure: alkylating agent such as nitrogen mustards, nitrous urine class, methyl Sulfonic acid esters；Platinum-like compounds such as cis-platinum, carboplatin and oxalic acid platinum etc.；Mitomycin (MMC)；(2) drug of nucleic acid synthesis is influenced: Dihydrofolate reductase inhibitor such as methopterin (MTX) and Alimta etc.；Thymus nucleoside synthetase inhibitors such as fluorouracil Class (5FU, FT-207, capecitabine) etc.；Purine nucleosides synthetase inhibitors such as Ismipur (6-MP) and 6-TG etc.； Ribonucleotide reductase inhibitor such as hydroxycarbamide (HU) etc.；DNA poly enzyme inhibitor such as cytarabine (Ara-C) and gemzar (Gemz) Deng；(3) act on the drug of transcribed nucleic acid: selectively acting inhibits DNA dependenc RNA polymerase, to inhibit in DNA profiling RNA synthesis drug such as: actinomycin D, daunorubicin, adriamycin, Epi-ADM, aclacinomycin, mithramycin；(4) Mainly act on the drug of tubulin synthesis: taxol, taxotere, vinblastine, vinorelbine, Podophyllum emodi var chinense alkali class, high tricuspid China fir ester alkali；(5) other Cytotoxic drugs: L-Asparaginasum mainly inhibits the synthesis of protein；

2, steroids antiestrogenic: tamoxifen, Droloxifene, Exemestane etc.；Arimedex: ammonia Rumi It is special, Lactel is grand, Letrozole, auspicious Ningde etc.；Antiandrogen: its ammonia RH-LH agonist/antagonist of fluorine: Zoladex, enatone etc.；

3, biological response modifiers: main that tumour interferon is inhibited by body's immunity；Interleukin 2；Chest Gland peptides；

4, monoclonal antibody: western appropriate former times Cetuximab (C225)；Trastuzumab monoclonal antibody (Trastuzumab) bevacizumab (Avastin)；

5, other include that some current mechanism are unknown and need the drug further studied；Cell-differentiation inducers such as Wei Jia Class；Cell death inducer.Bifunctional protein Bi-SP disclosed by the invention and combinations thereof can be with above-mentioned anti-tumor drug One or a combination set of drug combination.

The beneficial effects of the present invention are:

The present invention constructs the bifunctional protein that can block two target spots simultaneously using technique for gene engineering, can hinder Disconnected EGFR, and CD47 can be blocked, and then more effectively inhibit the growth and proliferation of tumour.

Detailed description of the invention

Fig. 1 is bifunctional protein structural schematic diagram；

Fig. 2 is the SDS-PAGE verification test of bifunctional protein；First swimming lane is protein molecular weight reference substance, the second swimming Road is bifunctional protein Bi-SP, and third swimming lane is the appropriate wooden monoclonal antibody (Panitumumab) of pa, and the 4th swimming lane is SIRP alpha-mutant egg It is white；

Fig. 3 is bifunctional protein activity experiment result in conjunction with EGFR and CD47；

Fig. 4 is flow cytometry verification result of the bifunctional protein in conjunction with EGFR and CD47 double positive cells；

Fig. 5 is the immunofluorescence experiment of bifunctional protein activating macrophage；

Fig. 6 is that bifunctional protein inhibits tumor-bearing mice A431 cell tumour to generate experiment；

Fig. 7 is that bifunctional protein inhibits tumor-bearing mice H292 cell tumour to generate experiment.

Specific embodiment

The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The present invention is further detailed in following embodiment, experimental example, should not be construed as limiting the invention.It is real Applying example does not include detailed descriptions of conventional methods, such as the method that those are used for carrier construction and plasmid, will encode the base of albumen Method because of carrier as being inserted into and plasmid or method that plasmid is introduced into host cell.Such method is for this field In be well-known with the personnel of ordinary skill, and be all described in many publications, including Molec μ Lar Cloning (Sambrook, J., Fritsch, E.F.and Maniais, T., 1989): A Laboratory Manual, 2nd Edition, Cold spring Harbor Laboratory Press.

It is defined as follows involved in the present invention:

Bi-SP: the bifunctional protein of targeting CD47 and EGFR；Panitumumab: Victibix；SIRP alpha-mutant-Fc: Signal adjusting protein-α crystallizable fragment fusion protein；CD47: integrin associated protein；EGFR: EGF-R ELISA； TKI: tyrosine kinase inhibitor；IgG: immunoglobulin G；ELISA: enzyme-linked immunosorbent assay；SPR: surface plasma is total Vibration technology；CI: confidence interval；LC, antibody light chain；HC, heavy chain of antibody；CRC: colon cancer；ECD: extracellular space；SEC: molecule Sieve chromatography；CCK-8: viable count kit 8；SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The clone of embodiment 1 human antibody light and heavy chain constant region and the area Fc gene

With lymphocyte separation medium separating health human lymphocyte, mentioned with Trizol reagent (Invitrogen Products) Take total serum IgE, according to document (Cell, 1980,22:197-207) and document (Nucleic Acids Research, 1982,10: 4071-4079) sequence reported separately designs primer amplification IgG1 type heavy chain of antibody and light chain constant region gene, and PCR reaction is equal Using thermal starting, reaction condition: 94 DEG C 5 minutes；94 DEG C 45 seconds, 60 DEG C 45 seconds, 72 DEG C 70 seconds, 30 circulation；72 DEG C 10 minutes. PCR product is through agarose gel electrophoresis purification and recovery and is cloned into pGEM-T carrier (Promega Products), sequence verification Confirmation obtains correct clone afterwards.SEQ ID NO.1 and SEQ ID NO.2 respectively illustrates the nucleosides of heavy chain constant region (CH) Acid and amino acid sequence.SEQ ID NO.3 and SEQ ID NO.4 respectively illustrates the nucleotide and amino of constant region of light chain (CL) Acid sequence.SEQ ID NO.11 and SEQ ID NO.12 respectively illustrates the nucleotide and amino acid sequence of constant region fc section (Fc) Column.Correct clone in this example is denoted as pGEM-T/CH, pGEM-T/CL, pGEM-T/Fc.

The building of 2 bifunctional protein Bi-SP of embodiment

Specific construction method: full genome synthesizes the weight of Panitumumab, light-chain variable region gene, Panitumumab's Light and heavy chain variable region sequences refer to patent document (WO9850433), the sequence of SIRP alpha-mutant referring to Science 341, 88(2013).The heavy chain variable region of Panitumumab is connect with the heavy chain constant region of human IgG1's antibody, constitutes the anti-of Bi-SP EGFR stresses chain gene；PCR clone SIRP α and human IgG1's antibody Fc sections of heavy chain constant region are connect, and constitute the anti-of Bi-SP The side CD47 antigen-4 fusion protein gene.The method and human IgG1's antibody that the light chain variable region of Panitumumab passes through overlap PCR Constant region of light chain connection, constitutes the anti-side the EGFR light chain gene of Bi-SP.By above-mentioned heavy, light chain gene and antigen-4 fusion protein gene It is respectively charged into eukaryotic expression vector pcDNA3.1 (Invitrogen Products).Above-mentioned plasmid one reinstates liposome transfection CHO-K1 cell (ATCC), and stablize expression with the Selective agar medium screening containing 600 μ g/mL G418 and 250 μ g/mL Zeocin The cell clone of bifunctional protein.It is difunctional from the purifying of the supernatant of cell culture by affinity chromatography using Protein A column Protein B i-SP.

Process is expressed in the building that bifunctional protein is described in detail by taking Bi-SP as an example below:

The heavy chain and light chain variable region and the anti-side CD47 SIRP alpha-mutant of the anti-side EGFR Bi-SP are respectively Bi-SPVH, Bi- SPVL and Bi-SPSIRP α recycles purpose band after Overlap PCR synthesis and is cloned into pGEM-T carrier, and screening is positive Cloning and sequencing respectively obtains pGEM-T/Bi-SPVH (nucleotide sequence and amino acid sequence such as SEQ ID NO.5 and SEQ ID NO.6), pGEM-T/Bi-SPVL (nucleotide sequence and amino acid sequence such as SEQ ID NO.7 and SEQ ID NO.8) and pGEM- T/Bi-SPSIRP α (nucleotide sequence and amino acid sequence such as SEQ ID NO.9 and SEQ ID NO.10).PCR uses Roche The high-fidelity amplification system of company, reaction condition are equal are as follows: 95 DEG C 5 minutes；94 DEG C 50 seconds, 55 DEG C 45 seconds, 72 DEG C 55 seconds, 30 are followed Ring；72 DEG C 10 minutes.

PGEM-T/Bi-SPVH gene is connect with the weight chain constant area gene in pGEM-T/CH using Overlap PCR, And be cloned into pGEM-T carrier, it selects after correctly cloning with HindIII and EcoRI digestion, is purified through agarose gel electrophoresis Target fragment is recycled, is attached with the plasmid pCDNA3.1 (+) of same digestion with T4DNA ligase, carrier for expression of eukaryon is constructed PCDNA3.1/ZEO (+) (Bi-SPVHCH) (nucleotide sequence and amino acid sequence such as SEQ ID NO.15 and SEQ ID NO.16)。

PGEM-T/Bi-SIRP α gene is connect with the Fc section gene in pGEM-T/Fc using Overlap PCR, and gram It is grand into pGEM-T carrier, select after correct clone with HindIII and EcoRI digestion, through agarose gel electrophoresis purification and recovery Target fragment is attached with the plasmid pCDNA3.1 (+) of same digestion with T4DNA ligase, constructs carrier for expression of eukaryon PCDNA3.1/ZEO (+) (Bi-SPSIRP α Fc) (nucleotide sequence and amino acid sequence such as SEQ NO:17 and SEQ NO:18).

Using Overlap PCR by pGEM-T/Bi-SPVL Bi-SPVL gene and pGEM-T/CL in chain constant Area's gene is connected, and is cloned into pGEM-T carrier, selects with HindIII and EcoRI digestion after correctly cloning, solidifying through agarose Gel electrophoresis purification and recovery target fragment is attached with the plasmid pCDNA3.1 (+) of same digestion with T4 DNA ligase, and building is true Nuclear expression carrier pCDNA3.1/ZEO (+) (Bi-SPVLCL) (nucleotide sequence and amino acid sequence such as SEQ ID NO:13 and Shown in SEQ ID NO:14).

3 × 10 are inoculated in 3.5cm tissue culture dishes⁵A CHO-K1 cell, cell culture to 90%-95% merge when into Row transfection: 9 μ g of plasmid (plasmid pCDNA3.1 (+) pCDNA3.1/ZEO (+) (Bi-SPVHCH) 3 μ g, pCDNA3.1/ZEO (+) is taken (Bi-SPVLCL) 3 μ g, pCDNA3.1/ZEO (+) (Bi-SP SIRP α Fc) 3 μ g) and 20 μ L Lipofectamine2000Reagent [Invitrogen Products] is dissolved in 500 μ L plasma-free DMEM mediums, room respectively Temperature stands 5 minutes, and above 2 kinds of liquid is mixed, and is incubated at room temperature 20 minutes so that DNA- liposome complex is formed, uses therebetween Serum-containing media in the DMEM culture medium replacement culture dish of 3mL serum-free, then by the DNA- liposome complex of formation It is added in plate, CO₂The DMEM complete medium that 2mL contains 10% serum is added in incubator culture after 4 hours, be placed in CO₂Incubator relaying Continuous culture.Cell, which is changed, after transfection carries out for 24 hours screens resistance containing the Selective agar medium of 600 μ g/mLG418 and 250 μ g/mL Zeocin Clone.The high-expression clone that screening is obtained is expanded with serum free medium to be cultivated, and with Protein A affinity column, (GE company is produced Product) isolate and purify bifunctional protein Bi-SP.Bi-SP is dialysed with PBS, finally with UV absorption standard measure.Bi-SP's For structure as shown in Figure 1, the anti-EGFR that left side is Bi-SP stresses chain and light chain, right side is that the SIRP α of the anti-side CD47 of Bi-SP is prominent Variant Fc fusion protein.

The verification test of 1 bifunctional protein purity of experimental example

The polyacrylamide gel containing 10% acrylamide concentration of SDS is prepared, by antibody Panitumumab, merges egg White SIRP α and bifunctional protein Bi-SP difference loading carries out gel electrophoresis assay, to analyze the purity of each component.As a result such as Fig. 2 Shown, the purity of Bi-SP is 90% or more.

The combination Activity determination of 2 bifunctional protein Bi-SP and EGFR and CD47 of experimental example

EGFR (R&D Products) is diluted to 2 μ with 0.05mmol/L sodium carbonate-bicarbonate buffer (pH 9.6) G/mL, 50 holes μ L/, 4 DEG C of coatings are overnight.After 10% defatted milk room temperature closes 2h, the bifunctional protein of various concentration, SIRP is added Alpha-mutant and Panitumumab (Amgen Products), 50 holes μ L/, each concentration take 3 parallel holes, are incubated at room temperature 2h.It abandons Supernatant, PBS are washed 3 times, the CD47 of the HRP label diluted by potency are added, 50 holes μ L/ are incubated at room temperature 45min.PBS fills After dividing washing, after TMB colour developing, sets and measure 450nm absorbance in microplate reader (enzyme mark colour comparatour, BIO-Rad company, the U.S.) (A450) value.As shown in figure 3, ordinate is the OD value (or absorbance value) of 450nm in figure, abscissa is experimental result The logarithm of antibody concentration.SIRP α and Panitumumab are only capable of combining CD47 or EGFR, therefore readings is close to 0, and Bi-SP group It shows and is increased with addition antibody concentration, the concentration dependent effect that corresponding OD value also increases.Bi-SP is confirmed by this test Not only the binding characteristic in combination with CD47 but also in combination with EGFR.The results showed that because bifunctional protein Bi-SP is in combination with EGFR In combination with CD47, and concentration dependant effect is shown in S curve.Panitumumab and SIRP alpha-mutant is because that can only combine EGFR Or CD47, therefore it is active without combining.The experimental results showed that, the bifunctional protein Bi-SP of building remains parent simultaneously above The combination activity of Panitumumab and SIRP alpha-mutant.

3 flow cytometry of experimental example

The lung cancer H292 of the bis- positives of EGFR and CD47 and epidermal carcinoma A431 cell 2%FCS-PBS are resuspended at 1 × 10⁶ A/mL is separately added into the Bi-SP and reference substance IgG of different dilutions, is placed in 4 DEG C of incubation 1h, washes cell 2 with 2%FCS-PBS Time.Add FITC label goat anti-human igg (H+L) secondary antibody in 4 DEG C of incubation 1h, after washing cell 2 times with 2%FCS-PBS It is analyzed on flow cytometer, calculates the relative affinity of the percentage of staining positive cells and each antibody of comparison.Experiment knot For fruit as shown in figure 4, abscissa is the concentration of antibody in figure, ordinate is average fluorescent strength.Flow cytometry the result shows that, The cell combination of the Bi-SP positives bis- to CD47 and EGFR is combined with concentration dependent, and S curve is presented.Control group is that nothing is added Close IgG group.The experimental results showed that Bi-SP can be in conjunction with A431 the and H292 cell of the bis- positives of EGFR and CD47, i.e. Bi-SP In combination with the EGFR and CD47 of cell surface.

4 bifunctional protein Bi-SP of experimental example promotes Macrophages For Tumor phagocytosis experiment in vitro

Using serum free medium lavation mouse peritoneal, irrigating solution prepares mouse peritoneal macrophage, and the specific method is as follows: right Three NOD-SCID mouse shift to an earlier date 5 days intraperitoneal injection 1-2mL thioglycolate mediums；After 5 days, cervical dislocation is put to death small Mouse.5mL, which is extracted, with syringe contains dual anti-RPMI-1640 serum free medium injection abdominal cavity；Abdomen 1-2 points are gently rubbed with cotton balls Cell suspension is drawn after clock；After 1000rpm is centrifuged 5min, it washed once with serum-free RPMI-1640 culture medium, be centrifuged again, Cell is resuspended to cultivate in the RPMI-1640 culture solution containing 10% fetal calf serum；It swallows and imitates followed by fluorescence microscope Fruit, the specific method is as follows: with 5 × 10⁴The mouse macrophage in abdominal cavity source is layered in 24 orifice plates and cultivated by the density in a/hole Night；By 5 × 10⁶Fluoresceincarboxylic acid acetoacetic ester (CFSE) in a A431 or H292 cell marking.Macrophage will be cultivated Culture medium is changed to the culture medium for not adding serum, and Nature enemy 2 hours.Every hole is added 2 × 10⁵The tumour of a CFSE label is thin Born of the same parents；Next the following other antibody of group, every group of three multiple holes, final concentration of 10 μ g/mL:(1 are added) IgG group；(2) Bi-SP group, 37 DEG C incubation 2-3 hours.Cell is washed three times with culture medium, observes and takes pictures under confocal fluorescent microscopic, calculates every 100 The ratio of CFSE positive cell in macrophage (three visuals field).Experimental result as shown in figure 5, A figure be Bi-SP group processing after, Compared with the processing of control group IgG antibody, macrophage significantly increases A431 cell phagocytosis efficiency.B figure is statistical data, display There were significant differences compared with IgG group for the cell number that Bi-SP processing group is swallowed.C figure is after Bi-SP group is handled, compared with right According to a group IgG antibody processing, macrophage significantly increases H292 cell phagocytosis efficiency.D figure is statistical data, it is shown that Bi- There were significant differences compared with IgG group for the cell number that SP processing group is swallowed.The experimental results showed that Bi-SP can be effectively facilitated The phagocytosis of Macrophages For Tumor.

Inhibit growth of tumour cell experiment in 5 bifunctional protein Bi-SP body of experimental example

With epidermal carcinoma A431 cell or lung cancer H292 cell subcutaneous inoculation female NOD-SCID mouse, after inoculated tumour cell Reach 80mm to tumor tissues³-120mm³, following 3 groups of intravenous injection, every group of 6 mouse: 1. 2. Bi-SP is 3. for human IgG control group Panitumumab.It injects 2 times, co-injection 4 weeks weekly.In administration process, every observation tumour growth situation on the 2nd, tumour is measured Size is to evaluate the tumor-inhibiting action of Bi-SP.Experimental result is as shown in Figure 6 and Figure 7, and abscissa is the lotus knurl of A431 tumour in Fig. 6 Mouse receives the time of drug-treated, and ordinate is gross tumor volume, and arrow is the time of drug injection twice, shows by one section After the drug Bi-SP treatment of time, compared with unrelated IgG treatment group and Panitumumab group, Bi-SP group shows significant swollen Tumor recession trend, difference is statistically significant, in Fig. 7 abscissa be H292 tumour tumor-bearing mice receive drug-treated when Between, ordinate is gross tumor volume, and arrow is the time of drug injection twice, is shown through drug Bi-SP treatment after a period of time Afterwards, compared with unrelated IgG treatment group and Panitumumab group, Bi-SP group shows that significant tumor regression trend, difference have system Meter learns meaning.The experimental results showed that Bi-SP can effectively inhibit the tumour growth with tumor-bearing mice, and its tumor killing effect is aobvious Work is better than Pan group and IgG control group group.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Xinxiang College of Medical Science

<120>bifunctional protein and the preparation method and application thereof of anti-CD47 and EGFR

<130> KHP161116843.4Q

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 1596

<212> DNA

<213>human IgG1's heavy chain constant region

<400> 1

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttggtgag 300

aggccagcac agggagggag ggtgtctgct ggaagccagg ctcagcgctc ctgcctggac 360

gcatcccggc tatgcagccc cagtccaggg cagcaaggca ggccccgtct gcctcttcac 420

ccggaggcct ctgcccgccc cactcatgct cagggagagg gtcttctggc tttttcccca 480

ggctctgggc aggcacaggc taggtgcccc taacccaggc cctgcacaca aaggggcagg 540

tgctgggctc agacctgcca agagccatat ccgggaggac cctgcccctg acctaagccc 600

accccaaagg ccaaactctc cactccctca gctcggacac cttctctcct cccagattcc 660

agtaactccc aatcttctct ctgcagagcc caaatcttgt gacaaaactc acacatgccc 720

accgtgccca ggtaagccag cccaggcctc gccctccagc tcaaggcggg acaggtgccc 780

tagagtagcc tgcatccagg gacaggcccc agccgggtgc tgacacgtcc acctccatct 840

cttcctcagc acctgaactc ctggggggac cgtcagtctt cctcttcccc ccaaaaccca 900

aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg gacgtgagcc 960

acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca 1020

agacaaagcc gcgggaagag cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg 1080

tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc aacaaagccc 1140

tcccagcccc catcgagaaa accatctcca aagccaaagg tgggacccgt ggggtgcgag 1200

ggccacatgg acagaggccg gctcggccca ccctctgccc tgagagtgac cgctgtacca 1260

acctctgtcc ctacagggca gccccgagaa ccacaggtgt acaccctgcc cccatgccgg 1320

gatgagctga ccaagaacca ggtcagcctg tggtgcctgg tcaaaggctt ctatcccagc 1380

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1440

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1500

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1560

tacacgcaga agagcctctc cctgtctccc ggtaaa 1596

<210> 2

<211> 524

<212> PRT

<213>human IgG1's heavy chain constant region

<400> 2

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Gly Glu Arg Pro Ala Gln Gly Gly Arg Val Ser Ala Gly Ser

100 105 110

Gln Ala Gln Arg Ser Cys Leu Asp Ala Ser Arg Leu Cys Ser Pro Ser

115 120 125

Pro Gly Gln Gln Gly Arg Pro Arg Leu Pro Leu His Pro Glu Ala Ser

130 135 140

Ala Arg Pro Thr His Ala Gln Gly Glu Gly Leu Leu Ala Phe Ser Pro

145 150 155 160

Gly Ser Gly Gln Ala Gln Ala Arg Cys Pro Pro Arg Pro Cys Thr Gln

165 170 175

Arg Gly Arg Cys Trp Ala Gln Thr Cys Gln Glu Pro Tyr Pro Gly Gly

180 185 190

Pro Cys Pro Pro Lys Pro Thr Pro Lys Ala Lys Leu Ser Thr Pro Ser

195 200 205

Ala Arg Thr Pro Ser Leu Leu Pro Asp Ser Ser Asn Ser Gln Ser Ser

210 215 220

Leu Cys Arg Ala Gln Ile Leu Gln Asn Ser His Met Pro Thr Val Pro

225 230 235 240

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

245 250 255

Ser Ser Leu His Pro Gly Thr Gly Pro Ser Arg Val Leu Thr Arg Pro

260 265 270

Pro Pro Ser Leu Pro Gln His Leu Asn Ser Trp Gly Asp Arg Gln Ser

275 280 285

Ser Ser Ser Pro Gln Asn Pro Arg Thr Pro Ser Ser Pro Gly Pro Leu

290 295 300

Arg Ser His Ala Trp Trp Trp Thr Ala Thr Lys Thr Leu Arg Ser Ser

305 310 315 320

Ser Thr Gly Thr Trp Thr Ala Trp Arg Cys Ile Met Pro Arg Gln Ser

325 330 335

Arg Gly Lys Ser Ser Thr Thr Ala Arg Thr Val Trp Ser Ala Ser Ser

340 345 350

Pro Ser Cys Thr Arg Thr Gly Met Ala Arg Ser Thr Ser Ala Arg Ser

355 360 365

Pro Thr Lys Pro Ser Gln Pro Pro Ser Arg Lys Pro Ser Pro Lys Pro

370 375 380

Lys Val Gly Pro Val Gly Cys Glu Gly His Met Asp Arg Gly Arg Leu

385 390 395 400

Gly Pro Pro Ser Ala Leu Arg Val Thr Ala Val Pro Thr Ser Val Pro

405 410 415

Thr Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg

420 425 430

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

435 440 445

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

450 455 460

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

465 470 475 480

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

485 490 495

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

500 505 510

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

515 520

<210> 3

<211> 318

<212> DNA

<213>people Kappa constant region of light chain

<400> 3

actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60

actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120

aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180

aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240

cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300

ttcaacaggg gagagtgt 318

<210> 4

<211> 106

<212> PRT

<213>people Kappa constant region of light chain

<400> 4

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 5

<211> 357

<212> DNA

<213> pGEM-T/Bi-SPVH

<400> 5

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagc agtggtgatt actactggac ctggattcgg 120

cagtccccag ggaagggact ggagtggatt ggacacatct attacagtgg gaacaccaat 180

tataacccct ccctcaagag cagactcacc atatcaattg acacgtccaa gactcagttc 240

tccctgaagc tgagttctgt gaccgctgcg gacacggcca tttattactg tgtgcgagat 300

cgagtgactg gtgcttttga tatctggggc caagggacaa tggtcaccgt ctcttca 357

<210> 6

<211> 119

<212> PRT

<213> pGEM-T/Bi-SPVH

<400> 6

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 7

<211> 324

<212> DNA

<213> pGEM-T/Bi-SPVL

<400> 7

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacatcagc aactatttaa attggtatca gcagaaacca 120

gggaaagccc ctaaactcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240

gaagatattg caacatattt ctgtcaacac tttgatcatc tcccgctcgc tttcggcgga 300

gggaccaagg tggagatcaa acga 324

<210> 8

<211> 108

<212> PRT

<213> pGEM-T/Bi-SPVL

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu

85 90 95

Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 9

<211> 354

<212> DNA

<213>the anti-side the CD47 SIRP alpha-mutant of Bi-SP

<400> 9

gaagaagaac tgcagatcat ccagccggac aaatctgttt ctgttgcggc gggtgaatct 60

gcgatcctgc actgcaccat cacctctctg ttcccggttg gtccgatcca gtggttccgt 120

ggtgcgggtc cggcgcgtgt tctgatctac aaccagcgtc agggtccgtt cccgcgtgtt 180

accaccgttt ctgaaaccac caaacgtgaa aacatggact tctctatctc tatctctaac 240

atcaccccgg cggacgcggg cacctactac tgcatcaaat tccgtaaagg ttctccggac 300

accgaattta aatctggtgc gggcaccgaa ctgtctgttc gtgcgaaacc gtct 354

<210> 10

<211> 118

<212> PRT

<213>the anti-side the CD47 SIRP alpha-mutant of Bi-SP

<400> 10

Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu

35 40 45

Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser

115

<210> 11

<211> 696

<212> DNA

<213>anti-the side CD47 Fc sections of Bi-SP

<400> 11

gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360

atctccaaag ccaaagggca gccccgagaa ccacaggtgt gcaccctgcc cccatcccgg 420

gatgagctga ccaagaacca ggtcagcctg tcctgcgccg tcaaaggctt ctatcccagc 480

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540

cccgtgctgg actccgacgg ctccttcttc ctcgtgagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacgcaga agagcctctc cctgtccccg ggtaaa 696

<210> 12

<211> 232

<212> PRT

<213>anti-the side CD47 Fc sections of Bi-SP

<400> 12

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

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

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

35 40 45

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

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

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

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

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

115 120 125

Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

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

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

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

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 13

<211> 642

<212> DNA

<213>the anti-side the EGFR light chain of Bi-SP

<400> 13

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacatcagc aactatttaa attggtatca gcagaaacca 120

gggaaagccc ctaaactcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240

gaagatattg caacatattt ctgtcaacac tttgatcatc tcccgctcgc tttcggcgga 300

gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<210> 14

<211> 214

<212> PRT

<213>the anti-side the EGFR light chain of Bi-SP

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu

85 90 95

Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 15

<211> 1953

<212> DNA

<213>anti-EGFR stresses chain in Bi-SP

<400> 15

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagc agtggtgatt actactggac ctggattcgg 120

cagtccccag ggaagggact ggagtggatt ggacacatct attacagtgg gaacaccaat 180

tataacccct ccctcaagag cagactcacc atatcaattg acacgtccaa gactcagttc 240

tccctgaagc tgagttctgt gaccgctgcg gacacggcca tttattactg tgtgcgagat 300

cgagtgactg gtgcttttga tatctggggc caagggacaa tggtcaccgt ctcttcagct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tggtgagagg 660

ccagcacagg gagggagggt gtctgctgga agccaggctc agcgctcctg cctggacgca 720

tcccggctat gcagccccag tccagggcag caaggcaggc cccgtctgcc tcttcacccg 780

gaggcctctg cccgccccac tcatgctcag ggagagggtc ttctggcttt ttccccaggc 840

tctgggcagg cacaggctag gtgcccctaa cccaggccct gcacacaaag gggcaggtgc 900

tgggctcaga cctgccaaga gccatatccg ggaggaccct gcccctgacc taagcccacc 960

ccaaaggcca aactctccac tccctcagct cggacacctt ctctcctccc agattccagt 1020

aactcccaat cttctctctg cagagcccaa atcttgtgac aaaactcaca catgcccacc 1080

gtgcccaggt aagccagccc aggcctcgcc ctccagctca aggcgggaca ggtgccctag 1140

agtagcctgc atccagggac aggccccagc cgggtgctga cacgtccacc tccatctctt 1200

cctcagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca aaacccaagg 1260

acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac gtgagccacg 1320

aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat aatgccaaga 1380

caaagccgcg ggaagagcag tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc 1440

tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc 1500

cagcccccat cgagaaaacc atctccaaag ccaaaggtgg gacccgtggg gtgcgagggc 1560

cacatggaca gaggccggct cggcccaccc tctgccctga gagtgaccgc tgtaccaacc 1620

tctgtcccta cagggcagcc ccgagaacca caggtgtaca ccctgccccc atgccgggat 1680

gagctgacca agaaccaggt cagcctgtgg tgcctggtca aaggcttcta tcccagcgac 1740

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1800

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1860

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1920

acgcagaaga gcctctccct gtctcccggt aaa 1953

<210> 16

<211> 643

<212> PRT

<213>anti-EGFR stresses chain in Bi-SP

<400> 16

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

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

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

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

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Gly Glu Arg Pro Ala Gln Gly

210 215 220

Gly Arg Val Ser Ala Gly Ser Gln Ala Gln Arg Ser Cys Leu Asp Ala

225 230 235 240

Ser Arg Leu Cys Ser Pro Ser Pro Gly Gln Gln Gly Arg Pro Arg Leu

245 250 255

Pro Leu His Pro Glu Ala Ser Ala Arg Pro Thr His Ala Gln Gly Glu

260 265 270

Gly Leu Leu Ala Phe Ser Pro Gly Ser Gly Gln Ala Gln Ala Arg Cys

275 280 285

Pro Pro Arg Pro Cys Thr Gln Arg Gly Arg Cys Trp Ala Gln Thr Cys

290 295 300

Gln Glu Pro Tyr Pro Gly Gly Pro Cys Pro Pro Lys Pro Thr Pro Lys

305 310 315 320

Ala Lys Leu Ser Thr Pro Ser Ala Arg Thr Pro Ser Leu Leu Pro Asp

325 330 335

Ser Ser Asn Ser Gln Ser Ser Leu Cys Arg Ala Gln Ile Leu Gln Asn

340 345 350

Ser His Met Pro Thr Val Pro Arg Ala Ser Pro Gly Leu Ala Leu Gln

355 360 365

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

370 375 380

Ser Arg Val Leu Thr Arg Pro Pro Pro Ser Leu Pro Gln His Leu Asn

385 390 395 400

Ser Trp Gly Asp Arg Gln Ser Ser Ser Ser Pro Gln Asn Pro Arg Thr

405 410 415

Pro Ser Ser Pro Gly Pro Leu Arg Ser His Ala Trp Trp Trp Thr Ala

420 425 430

Thr Lys Thr Leu Arg Ser Ser Ser Thr Gly Thr Trp Thr Ala Trp Arg

435 440 445

Cys Ile Met Pro Arg Gln Ser Arg Gly Lys Ser Ser Thr Thr Ala Arg

450 455 460

Thr Val Trp Ser Ala Ser Ser Pro Ser Cys Thr Arg Thr Gly Met Ala

465 470 475 480

Arg Ser Thr Ser Ala Arg Ser Pro Thr Lys Pro Ser Gln Pro Pro Ser

485 490 495

Arg Lys Pro Ser Pro Lys Pro Lys Val Gly Pro Val Gly Cys Glu Gly

500 505 510

His Met Asp Arg Gly Arg Leu Gly Pro Pro Ser Ala Leu Arg Val Thr

515 520 525

Ala Val Pro Thr Ser Val Pro Thr Gly Gln Pro Arg Glu Pro Gln Val

530 535 540

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Gly Lys

<210> 17

<211> 1050

<212> DNA

<213>the anti-side the CD47 Fc fusion protein of Bi-SP

<400> 17

gaagaagaac tgcagatcat ccagccggac aaatctgttt ctgttgcggc gggtgaatct 60

gcgatcctgc actgcaccat cacctctctg ttcccggttg gtccgatcca gtggttccgt 120

ggtgcgggtc cggcgcgtgt tctgatctac aaccagcgtc agggtccgtt cccgcgtgtt 180

accaccgttt ctgaaaccac caaacgtgaa aacatggact tctctatctc tatctctaac 240

atcaccccgg cggacgcggg cacctactac tgcatcaaat tccgtaaagg ttctccggac 300

accgaattta aatctggtgc gggcaccgaa ctgtctgttc gtgcgaaacc gtctgagccc 360

aaatcttctg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 420

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 540

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 600

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720

aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 780

ctgaccaaga accaggtcag cctgtcctgc gccgtcaaag gcttctatcc cagcgacatc 840

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900

ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 960

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020

cagaagagcc tctccctgtc cccgggtaaa 1050

<210> 18

<211> 350

<212> PRT

<213>the anti-side the CD47 Fc fusion protein of Bi-SP

<400> 18

Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu

35 40 45

Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

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

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

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

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

Claims

1. a kind of bifunctional protein Bi-SP of anti-CD47 and EGFR, which is characterized in that the bifunctional protein includes the anti-side EGFR Heavy chain, the light chain of the anti-side EGFR and the Fc fusion protein of the anti-side CD47.

2. bifunctional protein Bi-SP according to claim 1, which is characterized in that the bifunctional protein includes SEQ ID Amino acid sequence shown in NO.14, SEQ ID NO.16 and SEQ ID NO.18.

3. the preparation method of bifunctional protein as claimed in claim 1 or 2, which comprises the steps of:

S1, building are respectively containing nucleotide sequence shown in SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO.17 Carrier, and its cotransfection is cultivated into host cell, screen the cell clone for stablizing expression bifunctional protein；

S2, cell culture is isolated and purified, obtains bifunctional protein Bi-SP.

4. according to the method described in claim 3, it is characterized in that, the carrier is eukaryotic expression vector pcDNA3.1.

5. according to the method described in claim 4, it is characterized in that, the host cell is CHO-K1 cell.

6. according to the described in any item methods of claim 3~5, which is characterized in that with containing 600 μ g/mL G418 and 250 μ g/mL The cell clone of expression bifunctional protein is stablized in the Selective agar medium screening of Zeocin.

7. a kind of host cell, which is characterized in that while expressing SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO.18 Shown in amino acid sequence.

8. a kind of carrier combination, which is characterized in that including containing SEQ ID NO.13, SEQ ID NO.15 and SEQ ID respectively The carrier of nucleotide sequence shown in NO.17.

9. containing the composition of bifunctional protein Bi-SP of any of claims 1 or 2.

10. bifunctional protein Bi-SP application in preparation of anti-tumor drugs of any of claims 1 or 2.