CN105175545B - A kind of anti-PD-1 bifunctional antibody of anti-vegf-and its application - Google Patents
A kind of anti-PD-1 bifunctional antibody of anti-vegf-and its application Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract
The invention discloses a kind of anti-PD-1 bifunctional antibody of anti-vegf-and its applications, belong to molecular immunology technical field.The anti-PD-1 bifunctional antibody of anti-vegf-provided by the present invention includes light chain and heavy chain, wherein the amino acid sequence of light chain is as shown in SEQ ID NO.2, and the amino acid sequence of heavy chain is as shown in SEQ ID NO.4 or SEQ ID NO.6.Meanwhile the present invention also provides the applications of the gene and bifunctional antibody of coding bifunctional antibody.Bifunctional antibody provided by the present invention can be in conjunction with PD-1 and VEGF, with very high affinity, T cell secretion IL2 and inducing T cell secretion of gamma-IFN can be effectively stimulated simultaneously, and can significantly inhibit mice tumors grew, and there are huge potentiality in the application prepared in anti-tumor drug.
Description
Technical field
The present invention relates to a kind of anti-PD-1 bifunctional antibody of anti-vegf-and its applications, belong to molecular immunology technical field.
Background technique
According to WHO scholarly forecast, the year two thousand twenty is arrived, the tumor invasion number of population in the world is up to 20,000,000 people, death toll
12,000,000 people are up to, therefore, tumour will become the big killer of this century mankind first, and the prestige of most serious is constituted to human survival
The side of body.With Chinese population aging, the population for suffering from tumor disease can be more and more, therefore developing anti-tumor medicaments also become country
The emphasis of healthy cause.
Vascular endothelial growth factor is called VEGF.Vegf protein is the Liang Jian biotechnology company in 1989 by the U.S.
Scientist distinguish successful purification and identification, and clone and determine its gene order, it was demonstrated that VPF and VEGF are that same gene is compiled
The same albumen of code.The types (isoforms) such as there are six VEGF: VEGF-A ,-B ,-C ,-D, and-E；Its molecular weight from 35 to
44kDa etc., it is each with waiting type specificities specific with three " vascular endothelial growth factor receptors " (VEGFR-1, -2, and -3)
Combination combines.VEGF is highly conserved homodimer glycoprotein.Two molecular weight are respectively the single-stranded with disulfide bond of 24kDa
Form dimer.The monomer that VEGF is decomposed is inactive, removes 2 glycosyl of N to biological effect without influence, but may secrete in cell
In work.Due to the different cut mode of mRNA, produce respectively VEGF121, VEGF145, VEGF165, VEGF185,
At least five kinds of protein forms such as VEGF206, wherein VEGF121, VEGF145, VEGF165 are secreting type soluble proteins, can be directly
Vasoactive endothelial cell promotes vascular endothelial cell proliferation, increases vasopermeability.Nineteen ninety, Harvard University
Doctor Folkman proposes that famous Folkman is theoretical, i.e. tumor tissue growth, it is necessary to provide foot by new vessels generation
Enough oxygen and nutriment maintains.It is considered as the basis of VEGF clinical application.Monoclonal of the anti-vegf in conjunction with VEGFR
Antibody can inhibit vascular endothelial growth factor, for treating all kinds of metastatic cancers.
Programmed death receptor 1 (programmed death 1, PD-1) is CD28 superfamily member.PD-1 is expressed in work
The T cell of change, B cell and myeloid cell, there are two ligands, i.e. Programmed death ligand-1 (programmed death
Ligand 1, PD-L1) and PD-L2.PD-L1/L2 is expressed in antigen presenting cell, and PD-L1 also has expression in Various Tissues.
The coinhibitory signals of the combination mediating T-cell activation of PD-1 and PD-L1, regulatory T-cell activation and proliferation, function similarly to
The down regulation of CTLA-4.Scientist of Chinese origin displays flat laboratory first discovery PD-L1 and expresses in tumor tissues height, and adjusts
Save the function of tumor-infiltrated cd8 t cell.Therefore, there is important meaning to antitumor using PD-1/PD-L1 as the immunological regulation of target spot
Justice.In recent years, carry out rapidly there are many Anti-PD-1/PD-L1 antibody in the clinical research of immunotherapy of tumors.At present
Pembrolizumab and Nivolumab is used for advanced melanoma by FDA approval, and nearest Nivolumab is also by the U.S.
FDA approval is used for the treatment of advanced stage squamous non-small cell lung cancer.In addition, MPDL3280A (anti-PD-L1 monoclonal antibody), Avelumab
(anti-PD-L1 monoclonal antibody) etc. has also entered in multiple late phase clinical researchs, covers non-small cell carcinoma, melanoma, bladder cancer etc.
Multiple tumor kinds.Due to the broad-spectrum anti-tumor prospect and surprising drug effect of PD-1 antibody, industry is generally believed for PD-1 access
Antibody will bring the breakthrough progress for the treatment of kinds of tumors treatment: for treating lung cancer in non-cellule type, clear-cell carcinoma, ovary
Cancer, melanoma, leukaemia and anemia etc..In American Cancer Society's (AACR) annual meetings in 2012 and 2013 and
After the clinical efficacy data about PD-1 antibody drug made known in U.S. clinical tumour association (ASCO) annual meeting, PD-1 antibody at
Antibody drug is being ground for global pharmaceutical industry is most very powerful and exceedingly arrogant.
Immunotherapy of tumors is applied immunology principle and method, improves the immunogenicity and pairing effect cell of tumour cell
The sensibility of killing, excitation and enhancing antitumor immunity of organism response, and application immunocyte and effector molecule are transfused host
Interior, collaboration body immune system killing tumour inhibits tumour growth.Immunotherapy of tumors is concerned recently, is oncotherapy
The focus in field.In recent years, the good news of immunotherapy of tumors was continuous, non-at present in some tumor types such as melanoma
Powerful anti-tumor activity is illustrated in the treatment of Small Cell Lung Cancer etc., and existing immunotherapy of tumors drug obtains the U.S.
FDA (Food and Drug Administration, FDA) ratifies clinical application.Immunotherapy of tumors is due to its brilliant treatment
Effect and novelty were chosen as year most important scientific breakthrough by " science " magazine in 2013.Immunotherapy of tumors is expected to become
After operation, chemotherapy, radiotherapy, an innovation of therapeutic field of tumor after targeted therapy.
Bifunctional antibody is called bispecific antibody, is a kind of non-natural antibody, it can target two different targets simultaneously
Point or albumen.Bispecific antibody can specifically bind two different antigens simultaneously, due to its specificity and bi-functional
Effect in immunotherapy of tumors is more and more important.Double spies of U.S. FDA on December 03 examination & approval Amgen research and development in 2014
Heterogenetic antibody Blincyto (Blinatumomab) listing, the treatment for acute lymphoblastic leukemia.Blinatumomab
For CD19, CD3 bispecific antibody, Blincyto (Blinatumomab) is first double characteristic antibody of U.S. FDA examination & approval.
It has been demonstrated to have more than more than 40 currently, developing existing bifunctional antibody form, but since production efficiency is low and medicine generation is dynamic
The problems such as poor mechanical property, the research and development of bispecific antibody are difficult all the time.Currently, both at home and abroad still without a kind of anti-
The anti-PD-1 bifunctional antibody of VEGF-.
Summary of the invention
To solve the above problems, the present invention provides a kind of anti-PD-1 bifunctional antibody of anti-vegf-, the technical side taken
Case is as follows:
The purpose of the present invention is to provide a kind of anti-PD-1 bifunctional antibodies of anti-vegf-.The bifunctional antibody includes light chain
And heavy chain, wherein the amino acid sequence of light chain as shown in SEQ ID NO.2, the amino acid sequence of heavy chain such as SEQ ID NO.4 or
Shown in SEQ ID NO.6.
Preferably, the bifunctional antibody is the anti-PD-1 bifunctional antibody BsAbB7 of anti-vegf-, the amino acid sequence of light chain
As shown in SEQ ID NO.2, the amino acid sequence of heavy chain is as shown in SEQ ID NO.4.
Preferably, the bifunctional antibody is the anti-PD-1 bifunctional antibody BsAbB8 of anti-vegf-, the amino acid sequence of light chain
As shown in SEQ ID NO.2, the amino acid sequence of heavy chain is as shown in SEQ ID NO.6.
The present invention also provides a kind of gene for encoding the anti-PD-1 bifunctional antibody of the anti-vegf-, the gene encoded lights
The nucleotide sequence of chain is as shown in SEQ ID NO.1, the nucleotide sequence of encoding heavy chain such as SEQ ID NO.3 or SEQ ID
Shown in NO.5.
Preferably, it is anti-to be separately encoded anti-vegf-by the gene nucleotide sequence SEQ ID NO.1 and SEQ ID NO.3
The light chain and heavy chain of PD-1 bifunctional antibody BsAbB7.
Preferably, it is anti-to be separately encoded anti-vegf-by the gene nucleotide sequence SEQ ID NO.1 and SEQ ID NO.5
The light chain and heavy chain of PD-1 bifunctional antibody BsAbB8.
Application of any antibody in the drug of preparation prevention, diagnosis, treatment or adjuvant therapy of tumors also belongs to this
The protection scope of invention.
Any antibody is preparing the application in anti-tumor drug also within protection scope of the present invention.
Preferably, the application is specifically in the drug, blocking VEGF signal path, the drug for inhibiting tumour for combining VEGF
Middle application.
Preferably, the application is specifically in the drug for combining PD-1, the drug for blocking PD-1 in conjunction with PDL-1, activation T
The drug of lymphocyte improves IL-2 in T lymphocyte, IFN-γ expression, inhibits to apply in the drug of tumour.
Specifically, the tumour is selected from lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, nephroncus, oophoroma, preceding
Column gland cancer, bladder cancer, breast cancer, the cancer of the esophagus, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical carcinoma, leukemia, osteocarcinoma, lymph cancer, pancreas
Cancer etc..
What the present invention obtained has the beneficial effect that:
Method used in the present invention is the new leading edge method of one kind of current treatment tumour, and immunotherapy of tumors is expected to become
After operation, chemotherapy, radiotherapy, an innovation of therapeutic field of tumor after targeted therapy.
The newfound bifunctional antibody BsAbB7 and BsAbB8 of the present invention can be in conjunction with PD-1, and difunctional BsAbB7 is affine
Power is 0.24nM, and difunctional BsAbB8 affinity is 0.24nM.Simultaneously also can be in conjunction with VEGF, bifunctional antibody BsAbB7 parent
It is 0.15nM with power, bifunctional antibody BsAbB8 affinity is 0.12nM.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis result of PD-1 and VEGF antigen；
(wherein, A is VEGF antigen；B is PD-1 antigen).
Fig. 2 is the SDS-PAGE electrophoresis result figure of BsAb7 and BsAb8；
(wherein, A is the SDS-PAGE electrophoresis result of BsAb7；B is the SDS-PAGE electrophoresis result of BsAb8).
Fig. 3 is that bifunctional antibody BsAb7 and BsAb8 inducer T lymphocyte secretes IL2 ability measurement result.
Fig. 4 is bifunctional antibody BsAb7 and BsAb8 inducer T lymphocyte secretion of gamma-IFN ability measurement result.
Specific embodiment
The present invention will be further described combined with specific embodiments below, but the present invention should not be limited by the examples.
Material therefor, reagent, instrument and method in following embodiment are the routine in this field without specified otherwise
Material, reagent, instrument and method can be obtained by commercial channel.
The preparation of embodiment 1 VEGF and PD1 albumen
1, the expression vector establishment of PD-1 antigen
From the cDNA of the PD-1 of Nanjing Jin Sirui company synthesis people, Gene ID is that 5133, cDNA ID is NM_
005018.2.Fc purification tag is added after the extracellular region PD-1 gene of synthesis, and introduces Xba I, Bam H I two at both ends
Restriction enzyme site is connected to pTT5 expression plasmid, correct through sequence verification.The plasmid transfection Trans10 being sequenced (is purchased from
Beijing Quanshijin Biotechnology Co., Ltd), picking monoclonal is inoculated into 1 liter of LB liquid medium, until OD600When being 1, centrifugation
Thallus is collected, extracts plasmid with the big extraction reagent kit of plasmid (being purchased from Qiagen company).
2, the expression vector establishment of VEGF antigen
To the Fc protein fragments mFc of amino acid and mouse IgG corresponding to gene VEGF (NCBI Gene ID:7422)
(Ig gamma-2A chain C region) carries out Combined design and obtains VEGF-mFc.To improve target gene in 293F cell
Expression efficiency in expression system, sequence is optimized, and introduces Xba I, the restricted digestion position Bam H I two at both ends
Point is connected to pTT5 expression plasmid, correct through sequence verification.The plasmid transfection Trans10 being sequenced is (purchased from the full Shi Jinsheng in Beijing
Object Technology Co., Ltd.), picking monoclonal is inoculated into 1 liter of LB liquid medium, until OD600When being 1, thalline were collected by centrifugation, uses
The big extraction reagent kit of plasmid (being purchased from Qiagen company) extracts plasmid.
3, the expression and purifying of PD-1 and VEGF antigen
Sequencing is identified that correct expression vector transfects 293F cell (being purchased from Invitrogen company), 37 degree, 5%CO2,
After 130rpm/min is cultivated 7 days, supernatant is collected by centrifugation.Supernatant is centrifuged 10min in 4000rpm, then with 0.45 μm of membrane filtration；
400mM NaCl is added in filtrate；Adjust PH to 8.0.Sample is after 0.2 μm of filter membrane filters again, and loading is to having used PBS (137mM
NaCl,2.7mM KCl,10mM Na2HPO4,2mM KH2PO4, pH7.4) and the 5ml HiTrap Protein A column that has balanced；To
On sample it is complete after rinsed with PBS, flow velocity 5ml/min, ultraviolet monitoring be level.Buffer B (1M Glycine, pH 3.5) is washed
De-, flow velocity 1ml/min collects eluting peak with Tris and is neutralized to pH 7.5, and carries out SDS-PAGE detection, SDS-PAGE electrophoresis knot
Fruit is as shown in Figure 1.Liquid is changed into PBS with pipe concentration eluting peak is concentrated by ultrafiltration, and thus obtains antigen.
The preparation of 2 bifunctional antibody BsAb7 and BsAb8 albumen of embodiment
1. bifunctional antibody expression vector establishment
The cDNA (its sequence is as shown in SEQ ID NO.1) of the light chain of artificial synthesized antibody BsAb7, artificial synthesized antibody
The cDNA (its sequence is as shown in SEQ ID NO.3) of BsAb7 heavy chain, (its sequence is such as by the cDNA of artificial synthesized antibody BsAb8 heavy chain
Shown in SEQ ID NO.5), the cDNA of synthesis is cloned into respectively in pTT5 plasmid, and determine that plasmid construction is correct by being sequenced.
The plasmid transfection Trans10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd) being sequenced, picking monoclonal is inoculated into 1 liter
LB liquid medium, until OD600When being 1, thalline were collected by centrifugation, extracts matter with the big extraction reagent kit of plasmid (being purchased from Qiagen company)
Grain.
2. bifunctional antibody BsAb7 and BsAb8 protein expression and purifying
Sequencing is identified that correct BsAb7 and BsAb8 heavy chain expression vector and light chain expression vector (1:1) cotransfection arrive
In 293F cell, 37 degree, 5%CO2, after 130rpm/min is cultivated 7 days, supernatant is collected by centrifugation.Supernatant 4000rpm is centrifuged
10min, and with 0.45 μm of membrane filtration, collect filtrate；400mM NaCl is added in filtrate；Adjust PH to 8.0.Sample is through 0.2 μm
After filter membrane filters again, loading is to having used PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4,2mM KH2PO4,
PH7.4 the 5ml HiTrap MabSelect column) balanced (purchased from GE company)；It is rinsed after complete on sample with PBS, flow velocity
5ml/min, ultraviolet monitoring are level.Buffer B (1M Glycine, pH 3.5) elution, flow velocity 1ml/min collect eluting peak
It is neutralized to pH 7.5 with Tris, and carries out SDS-PAGE detection, the non-reduced electrophoresis detection result of SDS-PAGE is shown in Fig. 2.Use ultrafiltration
Eluting peak is concentrated in concentration tube, changes liquid into PBS with desalting column, thus obtains antibody BsAb7 and BsAb8 albumen.
3 bifunctional antibody BsAb7 and BsAb8 of embodiment is to PD-1 and VEGF affinity determination
Pass through the table of 3000 instrument of Biacore (being purchased from GE company) analysis anti-PD-1/VEGF antibody BsAbB7 and BsAbB8
Levy affinity and binding kinetics.Recombination fusion protein and biotin are total to using biotin labeling reagent box (Pierce company)
Valence coupling then passes through the SA chip (purchased from GE company) of Avidin label, response value RU is made to reach 450 or so.By that will resist
Body is flowed in PBS buffer solution with the flow velocity of 0.0133,0.0266,0.0532,0.1064,0.2128 μM of concentration and 50 μ l/min
It moves and measures and combine.It tracks Ag-Ab binding kinetics 3 minutes and tracks Dissociation 10 minutes.It uses
BIAevaluation software is by association and dissociation curve matching to 1: 1 Lang Gemiaoer (Langmuir) binding model.In order to make parent
Effect of the resultant force when assessing binding constant minimizes, and is intended using only the initial data section for corresponding to the association and dissociation phase
It closes, Kd, Kon and Koff value of measurement the results are shown in Table 1.
1 bifunctional antibody BsAb7 and BsAb8 of table is to PD-1, VEGF affinity costant (Kd) measurement result
| Kn(105M-1s-1) | Koff(10-5s-1) | Kd(nm) | |
| BsAb7-PD-1 | 1.02 | 2.42 | 0.24 |
| BsAb8-PD-1 | 0.96 | 2.33 | 0.24 |
| BsAb7-VEGF | 1.44 | 2.10 | 0.15 |
| BsAb8-VEGF | 1.89 | 2.31 | 0.12 |
4 competitive ELISA method of embodiment measures bifunctional antibody BsAb7 and BsAb8 respectively and PDL-1 competitive binding is anti-
Former PD-1
With PD-1-mFc coated elisa plate, 1%BSA closing, respectively by the antibody BsAb7 and BsAb8 of various concentration with
PDL-1-hFc mixing is added 37 DEG C of ELIAS secondary antibody after 37 DEG C of incubations and is incubated for 30 minutes.The extinction of 450nm is detected in microplate reader
Value (is shown in Table 2).Bifunctional antibody BsAb7 and BsAb8 is in conjunction with antigen PD-1 the results show that bifunctional antibody BsAb7 and BsAb8
Can effectively with PDL-1 competitive binding PD-1 albumen, and its joint efficiency be in dose-dependence.By to the double of combination
The competitive ELISA interpretation of result of function antibody BsAb7 and BsAb8, the combination of curve simulation bifunctional antibody BsAb7 and BsAb8
Efficiency EC50 is respectively as follows: 2.5nM and 2.9nM.
2 competitive ELISA of table measures bifunctional antibody BsAb7 and BsAb8 and blocks PD-1/PDL-1 binding ability result
| Antibody concentration | OD450(BsAb7) | OD450(BsAb8) |
| (stoste) 2 μ g/ml | 0.096 | 0.107 |
| 1:3 | 0.101 | 0.110 |
| 1:9 | 0.323 | 0.323 |
| 1:27 | 0.733 | 0.802 |
| 1:81 | 0.852 | 0.831 |
| 1:243 | 0.857 | 0.854 |
| 1:729 | 0.898 | 0.866 |
| Control/0 | 0.901 | 0.899 |
The external evoked T cell of 5 bifunctional antibody BsAb7 and BsAb8 of embodiment secretes IL2
Using Ficoll centrifugal process (being purchased from GE company) and CD4+T cell enrichment column (being purchased from R&D Systems company), system
Standby fresh PBMC, purifies human T-cell.By plating cells into 96 hole flat undersides, after overnight incubation, addition 25nmol,
The tetanus toxin (TT) of tri- kinds of various concentration bifunctional antibody BsAb7 and BsAb8 and 80ng/ml of 5nmol, 1nmol is added
The Isotype control antibodies of tri- kinds of concentration of 25nmol, 5nmol, 1nmol collect supernatant after 3 days as negative control, culture, use
Luminex instrument (being purchased from LifeTechnology company) and cell factor IL2 detection kit are (public purchased from BD Biosciences
Department) detection supernatant IL2 secretion level.As a result as shown in Figure 3, the results showed that: bifunctional antibody BsAb7 and BsAb8 can have
The function of effect stimulation T cell, secretes IL2, and related with antibody concentration, and isotype control Ab cannot promote T cell proliferation and
IL2 secretion.
The 6 external evoked T cell secretion of gamma-IFN of bifunctional antibody BsAb7 and BsAb8 of embodiment
With Ficoll centrifugal process (being purchased from GE company) and CD4+T cell enrichment column (being purchased from R&D Systems company), preparation
Fresh PBMC purifies human T-cell.Using Miltenyi CD14 monocyte Purification Kit monocyte, and
Monocyte and GM-CSF and IL-4 (being purchased from PeproTech company) generate DC cell after cultivating 7 days together.By plating cells
Into 96 hole flat undersides, after overnight incubation, every part of culture in the total volume of 200 μ l comprising 10e5 purifying T cell with
10e4 dendritic cells.Tri- kinds of various concentration bifunctional antibody BsAb7 and BsAb8 antibody of 25nmol, 5nmol, 1nmol are added,
The Isotype control antibodies of tri- kinds of concentration of 25nmol, 5nmol, 1nmol are added as negative control.Cell is cultivated 5 in 37 DEG C
It.After 5 days, 100 μ l culture mediums are taken out from every part of culture and are measured for cell factor IFN-γ.Utilize OptEIA ELISA
Kit (be purchased from BD Biosciences company) measures the level of IFN-γ.As a result display (as shown in Figure 4) is difunctional anti-
Body BsAb7 and BsAb8 can effective stimulus T cell function secrete cytokines IFN-γ, it is and related with concentration, and homotype pair
It cannot promote T cell proliferation and IFN-γ secretion according to antibody.
Embodiment 7 bifunctional antibody BsAb7 and BsAb8 inhibit mice tumors grew
People Caki-1 cell is cultivated, collects cell in the cell log phase, being made into concentration is (1.0 × 107)/ml cell is outstanding
Liquid takes 6-8 weeks nude mice, injects 1.0 × 10 in nude mice right fore oxter6A (about 0.1ml) cell suspension, tumour is long within 10 days or so
To diameter about 5mm, tumorigenesis success is divided into 4 groups at random: negative control group (intraperitoneal injection of saline group), BsAb7 group (tail
Be injected intravenously 3mg/kg group), BsAb8 group (tail vein injection 3mg/kg), positive controls Avastin (tail vein injection
2mg/kg).It is administered once within every 3 days, successive administration 21 days.After 21 days, puts to death nude mice and claim tumor weight, tumour inhibiting rate=[1- experiment
Group average knurl weight (B, C, D group are experimental group)/A group average knurl weight)] × 100%.Experimental result is as shown in table 3, BsAb7 and
BsAb8 can inhibit mice tumors grew.
Table 3 bifunctional antibody BsAb7 and BsAb8 inhibit mice tumors grew result
| Group | Average knurl weight (g) | Average tumour inhibiting rate (%) |
| Negative control | 2.544±0.316 | / |
| BsAb7 | 0.932±0.224 | 63.4 |
| BsAb8 | 0.867±0.255 | 65.9 |
| Avastin | 0.912±0.213 | 64.2 |
Although the present invention has been disclosed in the preferred embodiment as above, it is not intended to limit the invention, any to be familiar with this
The people of technology is not departing from spirit and scope of the invention, can do various change and modification, therefore, guarantor of the invention
Shield range should subject to the definition of the claims.
Claims (10)
1. a kind of anti-PD-1 bifunctional antibody of anti-vegf-includes light chain and heavy chain, which is characterized in that the amino acid sequence of light chain
As shown in SEQ IDNO.2, the amino acid sequence of heavy chain is as shown in SEQIDNO.4 or SEQIDNO.6.
2. the anti-PD-1 bifunctional antibody of a kind of anti-vegf-according to claim 1, which is characterized in that the amino acid sequence of light chain
Column are as shown in SEQIDNO.2, and the amino acid sequence of heavy chain is as shown in SEQ ID NO.4.
3. the anti-PD-1 bifunctional antibody of a kind of anti-vegf-according to claim 1, which is characterized in that the amino acid sequence of light chain
Column are as shown in SEQIDNO.2, and the amino acid sequence of heavy chain is as shown in SEQID NO.6.
4. a kind of gene of the anti-PD-1 bifunctional antibody of anti-vegf-described in a kind of coding claim 1, which is characterized in that coding
The nucleotide sequence of light chain is as shown in SEQIDNO.1, the nucleotide sequence of encoding heavy chain such as SEQIDNO.3 or SEQIDNO.5 institute
Show.
5. gene according to claim 4, which is characterized in that the nucleotide sequence SEQIDNO.1 and SEQIDNO.3 difference
Encode the light chain and heavy chain of the anti-PD-1 bifunctional antibody of anti-vegf-.
6. gene according to claim 4, which is characterized in that the nucleotide sequence SEQIDNO.1 and SEQIDNO.5 difference
Encode the light chain and heavy chain of the anti-PD-1 bifunctional antibody of anti-vegf-.
7. the answering in the drug of preparation prevention, diagnosis, treatment or adjuvant therapy of tumors of any antibody described in claim 1-3
With.
8. any antibody described in claim 1-3 is preparing the application in anti-tumor drug.
9. applying according to claim 8, which is characterized in that in the drug for combining VEGF, blocking VEGF signal path, inhibit
It is applied in the drug of tumour.
10. applying according to claim 8, which is characterized in that in the drug for combining PD-1, block PD-1 in conjunction with PDL-1
Drug, activated T lymphocytes drug, improve T lymphocyte in IL-2, IFN-γ expression drug in apply.
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| CN106977602B (en) * | 2016-08-23 | 2018-09-25 | 中山康方生物医药有限公司 | A kind of anti-PD1 monoclonal antibodies, its medical composition and its use |
| CN106967172B (en) | 2016-08-23 | 2019-01-08 | 康方药业有限公司 | The anti-PD-1 bifunctional antibody of anti-CTLA 4-, its medical composition and its use |
| WO2018035710A1 (en) | 2016-08-23 | 2018-03-01 | Akeso Biopharma, Inc. | Anti-ctla4 antibodies |
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