CN106554416B - A kind of application of anti-PD-L1 Humanized monoclonal antibodies joint interferon gene stimulates the protein (STING) agonist in antitumor - Google Patents
A kind of application of anti-PD-L1 Humanized monoclonal antibodies joint interferon gene stimulates the protein (STING) agonist in antitumor Download PDFInfo
- Publication number
- CN106554416B CN106554416B CN201510909216.4A CN201510909216A CN106554416B CN 106554416 B CN106554416 B CN 106554416B CN 201510909216 A CN201510909216 A CN 201510909216A CN 106554416 B CN106554416 B CN 106554416B
- Authority
- CN
- China
- Prior art keywords
- cancer
- cell
- sting
- agonist
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Abstract
The invention belongs to pharmaceutical technology field, the application in treatment tumour is used in combination in specially anti-PD-L1 Humanized monoclonal antibodies and interferon gene stimulates the protein (STING) agonist.The present invention research shows that, PD-L1 Humanized monoclonal antibodies joint interferon gene stimulates the protein (STING) agonist can inhibit tumour growth, activate T cell, and better than exclusive use PD-L1 Humanized monoclonal antibodies or interferon gene stimulates the protein (STING) agonist, therefore, PD-L1 Humanized monoclonal antibodies joint interferon gene stimulates the protein (STING) agonist can be used for preparing anti-tumor drug.
Description
Technical field
The invention belongs to pharmaceutical technology field, specially anti-PD-L1 Humanized monoclonal antibodies and interferon gene stimulation
The application in treatment tumour is used in combination in albumen (STING) agonist.
Background technique
PD-1, also known as programmed death receptor 1 are CD28 superfamily member, help professor by Kyoto Univ Japan's sheet is multitudinous
It was found in 1992.The receptor PD-L1 of PD-1 is called apoptosis-ligand 1 or surface antigen differentiation cluster 274
(cluster of differentiation 274, CD274) or B7 autoploid (B7 homolog 1, B7-H1), is the mankind
A kind of intracorporal protein, is encoded by CD274 gene.PD-L1 is the transmembrane protein that size is 40kDa, is pressed down with immune system
It is formed with pass.Under normal conditions, immune system can generate reaction to the exotic antigen for being gathered in lymph node or spleen, inspire tool antigen
The hyperplasia of the cell poisoning property CD8+T cell of specificity.Programmed cell death receptor -1(PD-1) match with apoptosis -
Body 1(PD-L1) it combines, the signal of inhibition can be conducted, the hyperplasia of lymph node CD8+ T cell is lowered, and by blocking PD-1
In conjunction with PD-L1, T lymphocyte can effectively be prevented to inhibit signal, activate killer T cell or effector T cell.Use list
Clonal antibody blocks PD-L1 in conjunction with PD-1, can be with activating effect T cell, and then kills tumour using the effector T cell of activation
Cell, to become the critical treatment means of immunotherapy for cancer.
PD-L1 albumen is Immune target inhibitor, and the immunization therapy for directly acting on these target spots can be to immune system
Brake signal is discharged, attacks it to tumour cell.The inhibitor of anti-PD-L1 albumen includes monoclonal antibody a variety of swollen
It is effective in cure in tumor type, such as lung cancer.Research has shown that PD-L1 monoclonal antibody is either in non-small cell lung cancer still small
The overall survival of patient can be effectively improved in cell lung cancer, and extend patient's service life.
It is announced in world's lung cancer meeting in 2013, PD-L1 antibody just in clinical studies is exactly company, Roche Group
MPDL3280A(Atezolizumab), Overall response rate of the medicine in all Patients with Non-small-cell Lung reaches 23%.And most
The health giving quality safety of new MPDL3280A comparison two/tri- line of docetaxel treatment non-small cell lung cancer and predictive biology mark
The II phase clinical research of will object result indicates, PD-L1 monoclonal antibody significantly improves existence with increasing of expressing of PD-L1, PD-L1 table
It can be used as the prediction index of MPDL3280A treatment up to result.The medicine, which treats advanced Non-small cell lung, has significant curative effect,
Disease control rate reaches 42%, and objective remission rate 16%, furthermore has controllable safety, 3-4 grades of drug-associateds do not occur
Death incident caused by pneumonia etc..PD-L1 monoclonal antibody drug MPDL3280A has clinically been shown be widely applied before
Scape and surprising oncotherapy effect, can be used for the treatment of multiple tumor types, therefore, exploitation and PD-L1 monoclonal antibody medicine
Object MPDL3280A equally has the antibody drug of high-affinity, and combines other drugs for oncotherapy, for cancer
Immunization therapy, makes it have better therapeutic effect, and lower toxic side effect has very important significance.
T lymphocyte derives from the multipotential stem cell of marrow.In human embryo's phase and nascent phase, a part in marrow is more
Energy stem cell or pre-T cell move in thymus gland, and the differentiation and maturation under the induction of thymin becomes with immunocompetent T
Cell.Mature T cell is settled down through the thymus dependent area of blood distribution to peripheral immune organ, and can be through lymphatic vessel, peripheral blood
It is recycled with tissue fluid etc., plays the functions such as cellular immunity and immunological regulation.T lymphocyte is according to function and surface marker
It is segmented into many types: 1, cytotoxic T cell (cytotoxic T cell) or effector T cell: eliminating infected thin
Born of the same parents.The function of these cells is just as one " killer " or cytotoxin, because they can be to the special antigen reactive mesh of generation
Mark cell is killed.The major surfaces mark of cytotoxic T cell is CD8, also referred to as killer T cell.2, T helper cell
(helper T cell) plays the part of the role of pilot process in immune response: it can be other types of to activate with hyperplasia diffusion
Generate the immunocyte of direct immunization reaction.The major surfaces mark of T helper cell is CD4.3, adjusting/inhibition T cell
(regulatory/suppressor T cell): it is responsible for adjusting organism immune response.It usually plays and maintains self tolerance and keep away
It is immunoreacted the important function of excessive damage body.4, memory T cell (memory T cell): in secondary immune response
It plays an important role.
Interferon is one group of reactive protein (mainly glycoprotein) with multiple functions, be it is a kind of by monocyte and
The cell factor that lymphocyte generates.Interferon is a kind of broad-spectrum disease resistance toxic agent, not direct killing or inhibition virus, and main
It is so that cell is generated antiviral protein by cell surface receptor effect, to inhibit the duplication of hepatitis B, type is divided into
Three classes, α-(leucocyte) type, β-(fibroblast) type, γ-(lymphocyte) type；Interferon can also enhance natural kill simultaneously
The vigor of cell (NK cell), macrophage and T lymphocyte, to play immunoregulation effect, and enhances anti-virus ability.
They have a variety of biologies such as the antiviral of wide spectrum, influence cell growth, and differentiation, adjusting immune function on allogenic cell
Activity.Interferon has antiviral, inhibition tumor cell proliferation, inducing apoptosis of tumour cell effect, to kill tumour cell.
Interferon has immunoregulation effect to humoral immunity, cellular immunity, also there is certain immune increasing to macrophage and NK cell
Pretend use.
Interferon is found by Younger and Salvin, since antigenicity is different from the interferon of discovery, names II type then
Interferon IFN or IFN-γ.Interferon is one of most important Th1 cytokines, can be by a plurality of types of immunological effects
Cell, CD4+Th1 cell, CD8+CTL cell, NK cell, B cell, NKT cell and APC are generated.Interferon has direct
Inhibit tumor cell proliferation, increases surface MHC antigen and expression of tumor necrosis factor, Antineoplastic angiogenesis etc. are a variety of antitumor
Effect.Tumour cell can be expressed by the Fas/FasL of modulate tumor cell and be enhanced to Recent study discovery, IFN-γ
To the sensibility of Fas institute mediated apoptosis approach, the ability for making tumour cell escape immune system attack is reduced, to inhibit tumour
The malignant proliferation of cell.
IL-2, that is, interleukin 2 (interleukin-2) also known as t cell growth factor (T cell growth
Factor, TCRF).The cell factor with extensive bioactivity mainly generated by the CD4+Th1 cell activated.It can promote
The proliferation of Th0 and CTL, therefore be the important factor of regulation immune response, also assist in antibody response, hematopoiesis and oncological surveillance.
Interferon gene stimulates the protein (STING) is a kind of transmembrane protein, usually in 152-173 regions
(dimerization domain, DD) handover forms dimer and in self suppression state.When by some ligands (such as
CDN molecular configuration changes and is activated after stimulation), recruits the TANK combination kinases 1(TANK-binding in cytoplasm
Kinase 1, TBK1), TBK1 is mediated to the phosphorylation of IRF3, causes interference with element (interferon, IFN)-β and other a variety of
The formation of cytokine (cytokines).The generation of IFN β is mark (the Yasuo Tanaka & Zhijian J. of STING activation
Chen. Sci Signal. 2012 Mar 6; 5(214)).Ring dinucleotides cGAMP, be up to the present find it is unique
One kind can directly activate source of mouse but also activate the agonist of source of people STING albumen.
Agonist, which refers to, to be combined with the protein molecular of receptor on cells or signal transduction pathway, and generates natural materials
Characteristic physiological efficiency chemicals or drug.Ring dinucleotides cGAMP can induce I as the native agonist of STING
Property interferon generate (X Cai, YH Chiu, ZJ Chen, Molecular cell, Volume 54, Issue 2,24
April 2014, Pages 289-296).STING activator is ring dinucleotides, comprising: c-di-AMP, c-di-GMP,
C-di-IMP, c-AMP-GMP, c-AMP-IMP, c-GMP-IMP etc..STING agonist being capable of inducing interferon
The generation of (interferon, IFN)-β can play the effect for inhibiting tumour.It is special due to STING agonist effect mechanism
Property, STING agonist can combine other anti-tumor drugs and treat tumour together.
Summary of the invention
The present invention provides a kind of anti-PD-L1 Humanized monoclonal antibodies, the technical solution taken is as follows:
The purpose of the present invention is to provide a kind of anti-PD-L1 Humanized monoclonal antibodies, include light chain and heavy chain.Wherein,
The amino acid sequence of light chain is as shown in SEQ ID NO.1, and the amino acid sequence of heavy chain is as shown in SEQ ID NO.2.
Preferably, the anti-PD-L1 Humanized monoclonal antibodies are anti-PD-L1 Humanized monoclonal antibodies 11D8, include
Light chain and heavy chain, wherein the amino acid sequence of light chain is as shown in SEQ ID NO.1, the amino acid sequence of heavy chain such as SEQ ID
Shown in NO.2.
Application of the antibody in preparation prevention, diagnosis, treatment or adjuvant therapy of tumors.
Application of the antibody in the drug of preparation prevention, diagnosis, treatment or adjuvant therapy of tumors.
Application of the antibody in the medicine material of preparation prevention, diagnosis, treatment or adjuvant therapy of tumors.Specifically,
It is applied in preparation PD-L1 access blocking agent.
Application of the antibody combined STING agonist in treatment or adjuvant therapy of tumors.
Application of the antibody combined STING agonist in the drug of preparation treatment or adjuvant therapy of tumors.
STING agonist prepares the application in the medicine material of prevention, diagnosis, treatment or adjuvant therapy of tumors.
More specifically, monoclonal antibody of the present invention or monoclonal antibody conjugates are in preparing following drug
Purposes: the drug of high-affinity combination PD-L1, block drug of the PD-1 in conjunction with PDL1, activated T lymphocytes drug, mention
The drug that IL-2, IFN-γ are expressed in high T lymphocyte.
More specifically, monoclonal antibody of the present invention or monoclonal antibody conjugates joint STING agonist exist
It prepares the purposes in following drug: inhibiting the drug of tumour growth, the drug of activated T lymphocytes, improves in T lymphocyte
The drug that IL-2, IFN-γ are expressed.
More specifically, STING agonist of the present invention is as the purposes prepared in following medicine material: inhibiting tumour
The drug of growth, activated T lymphocytes drug, improve the drug of IL-2 in T lymphocyte, IFN-γ expression.
More specifically, the tumour be selected from lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, nephroncus, oophoroma,
Prostate cancer, bladder cancer, breast cancer, the cancer of the esophagus, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical carcinoma, leukemia, osteocarcinoma, lymph cancer, pancreas
Dirty cancer etc..
PD-L1 is mentioned above, be specific protein title, explanation is such as not added, with most open source literatures and NCBI data
Library, European gene database are consistent.Its GENE are as follows: CD274；GENE ID are as follows: 29126；Other name packets disclosed in PD-L1
It includes: B7-H, B7H1, PDCD1L1, PDCD1LG1, PDL1.
STING is mentioned above, be specific protein title, explanation is such as not added, with most open source literatures and NCBI data
Library, European gene database are consistent.Its GENE are as follows: TMEM173；GENE ID are as follows: 340061；Other lives disclosed in STING
Name includes: Transmembrane Protein 173, ERIS, MITA, MPYS, NET23, SAVI, STING,
hMITA, hSTING。
The STING agonist being mentioned above, including but not limited to c-di-AMP, c-di-GMP, c-di-IMP, c-
AMP-GMP, c-AMP-IMP, c-GMP-IMP and sulphur-substitutive derivative and its mixture.
STING the agonist CDN, including but not limited to c-AMP-GMP, c-di-AMP, c-di-GMP being mentioned above
And sulphur-substitutive derivative and its mixture.
What the present invention obtained has the beneficial effect that:
The PD-L1 humanized antibody that the present invention develops is to utilize humanized antibody phage library and display technique of bacteriophage
It carries out antibody screening and affinity maturation obtains, there is high affinity, can more effectively play the work of antagonism PD-L1 function
With.
New discovery of the present invention a kind of humanized antibody 11D8 that can block PD-L1 function, humanized antibody 11D8 and PD-
L1 antigentic specificity combines, and it is 69 pM that SPR method, which measures humanized antibody 11D8 affinity, and affinity is better than Roche Group
The PD-L1 antibody A tezolizumab of company's exploitation.The resistance of humanized antibody 11D8 of the invention as PD-1/PD-L1 access
Disconnected agent can inhibit tumour growth with activated T lymphocytes, to become the key component in immunotherapy of tumors novel drugs.
A kind of humanized antibody 11D8,11D8 that can block PD-L1 function of new discovery of the present invention combines STING agonist
It uses, tumor effect is inhibited to be better than independent medication, better than the PD-L1 antibody A tezolizumab of company, Roche Group exploitation.
Humanized antibody 11D8 of the invention combines STING agonist, and activated T lymphocytes can be enhanced, and enhancing inhibits tumour growth,
To become component crucial in immunotherapy of tumors novel drugs.
Detailed description of the invention
Fig. 1 is human PD-L 1 antigen SDS-PAGE electrophoresis detection result.
Fig. 2 is the SDS-PAGE electrophoresis detection result of humanized antibody 11D8.
Fig. 3 is the result that SPR method measures humanized antibody 11D8 affinity.
Fig. 4 is that humanized antibody 11D8 and STING agonist induction T cell IL-2 secretes result.
Fig. 5 is humanized antibody 11D8 and STING agonist induction T cell IFN-γ secretion.
Fig. 6 is humanized antibody 11D8 and STING agonist inhibits tumor growth delay.
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 1. PD-L1 antigen of embodiment
The cDNA of the PD-L1 of people is synthesized by Nanjing Jin Sirui company, Gene ID is ID:29126.In the extracellular of synthesis
His label is added after area's PD-L1 gene, is connected to pcDNA3.1 expression plasmid, it is correct through sequence verification.The plasmid being sequenced turns
DH5 α is contaminated, picking monoclonal is inoculated into LB liquid medium, until thalline were collected by centrifugation, is mentioned greatly with plasmid when OD600 is 1-1.5
Kit (being purchased from Qiagen company) extracts plasmid.Sequencing is identified that correct expression vector transfection Expi293 cell (is purchased from
Thermo company), 37 degree, 5 % CO2, after 130 rpm/min are cultivated 5 days, collected by suction supernatant.Sample through 0.2 μm of filter membrane again
After secondary filtering, loading to the 5ml HisTrap for having used combination buffer (TrisCl, pH7.4,400 mM NaCl) to balance
FF column (is purchased from GE life science)；It is rinsed after complete on sample with the same buffer of the imidazoles containing 20mM, 5 ml/min of flow velocity.Most
It is eluted afterwards with elution buffer (Tris.HCl, pH 7.4,400 mM NaCl, 250 mM Imidazole), 1 ml/ of flow velocity
Min collects eluting peak, changes liquid into PBS with pipe concentration eluting peak is concentrated by ultrafiltration, thus obtains the His unlabelled antigen of PD-L1.
PD-L1 antigen SDS-PAGE electrophoresis detection the result is shown in Figure 1.
The expression and purification of 2 PD-L1 antibody 11D8 of embodiment
By light chain cdna (its translated amino acid sequence such as SEQ ID NO.1 of Nanjing Jin Sirui company synthetic antibody 11D8
It is shown), its translated amino acid sequence of the cDNA(of antibody 11D8 heavy chain is as shown in SEQ ID NO.2).The cDNA of synthesis is distinguished
It is cloned into pcDNA3.1 plasmid, and correct by sequence verification plasmid construction.Correct 11D8 heavy chain expression is identified into sequencing
Carrier and light chain expression vector (1:1) cotransfection are into expi293 cell, and 37 degree, 8% CO2,130 rpm/min, culture 5
After it, supernatant is collected by centrifugation.By supernatant 6000rpm, 4 degree of centrifugation 30min, and with 0.22 μm of membrane filtration, filtrate is collected；Filter
400mM NaCl is added in liquid；Adjust PH to 7.5.Sample is after 0.22 μm of filter membrane filters again, and loading is to having used PBS (137 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4,2 mM KH2PO4, pH7.4) the 5 ml HiTrap that have balanced
Protein A column；It is rinsed after complete on sample with PBS, 5 ml/min of flow velocity, ultraviolet monitoring is level.Buffer B(1 M
Glycine, pH 3.5) it elutes, 1 ml/min of flow velocity, eluting peak, which is collected, with Tris is neutralized to pH 7.5.With being concentrated by ultrafiltration, pipe is dense
Contracting eluting peak changes liquid into PBS with desalting column, thus obtains antibody 11D8 albumen.The SDS-PAGE electrophoresis detection of antibody 11D8
As a result see Fig. 2.
The affinity Kd of 3 SPR method of embodiment measurement PD-L1 humanized antibody 11D8
The acquisition of PD-L1 antigen is purchased from GE life referring to embodiment 1,3000 instrument of Biacore and correlation SA chip
Science.The binding kinetics of anti-PD-L1 antibody 11D8 are analyzed in this experiment using Biacore 3000.Utilize biotin labeling reagent
Recombined human PD-L1 extracellular region protein and biotin covalent coupling are then passed through the SA of Avidin label by box (Pierce company)
Chip makes RU response value to 450.By antibody with various concentration (including: 0.01,0.02,0.04,0.08,0.16 μM) and 50 μ
The flow velocity of l/min flows in PBS buffer solution, detects and measures binding constant.Tracking Ag-Ab binding kinetics 3 minutes
And track Dissociation 10 minutes.Using BI Aevaluation software by association and dissociation curve matching to 1: 1 Lang Gemiao
That (Langmuir) binding model.Experimental result (Kd, Kon and Koff value of measuring in triplicate) is shown in Fig. 3.
The external evoked T cell of 4 humanized antibody 11D8 of embodiment and STING activator CDN secretes IL-2
Ficoll reagent is purchased from GE life science, and STING activator CDN is purchased from Sigma or Invivogen company, broken
Wind toxin element be purchased from Sigma company, human cell factor IL-2 detection kit be purchased from R&D company, T cell, DC cell enrichment column,
Monocyte purification kit is purchased from Miltenyi Biotech company, and GM-CSF and IL-4 are purchased from PeproTech public affairs
Department.Illustrate to extract with the reference of Ficoll centrifugal process and prepare fresh human PBMC, separates T cell and DC cell, use Miltenyi
CD14 monocyte Purification Kit monocyte, and after monocyte cultivates 7 days together with GM-CSF and IL-4
Generate DC cell.By plating cells into 96 hole flat undersides, after overnight incubation, 1 nmol, 5 nmol, tri- kinds of 25 nmol is added
Various concentration 11D8 antibody or the STING activator (CDN) that 1 mM is added together, the broken of 80 ng/ml is added in all holes
The Isotype control antibodies of 5 nmol concentration are added as negative control in wind toxin element (TT), and culture collected supernatant after 3 days, used
Luminex instrument (being purchased from LifeTechnology company) and human cell factor IL-2 detection kit detect culture medium supernatant IL-
2 secretion level, data are handled with SPSS10.0 software, are examined using one-way analysis of variance (one-way ANOVA)
Compare the conspicuousness of each group difference, significance a=0.05, negative control group (* < 0.01 * < 0.05, *).As a result (see Fig. 4)
Show humanized antibody 11D8 can effective stimulus T cell function, secrete IL-2, and, humanized antibody related with antibody concentration
11D8 joint STING activator CDN can enhance IL-2 secretion, and then enhance T cell activation effect.
5 humanized antibody 11D8 of embodiment and the external evoked T cell secretion of gamma-IFN of STING activator CDN
Ficoll reagent is purchased from GE life science, and STING activator CDN is purchased from Sigma or Invivogen company, GM-
CSF and IL-4 is purchased from PeproTech company, and OptEIA ELISA kit is purchased from BD Biosciences company.T is thin
Born of the same parents and the separation of DC cell and induction are referring to embodiment 4.By plating cells into 96 hole flat undersides, after overnight incubation, every part of culture
T cell and 10000 DC cells of the object comprising 100000 purifying in the total volume of 200 μ l.1 nmol, 5 is added
Nmol, 25 nmol, tri- kinds of various concentration 11D8 antibody, or STING activator (CDN) the addition Isotype control of 1 mM is added together
Antibody is as negative control.Cell is cultivated 5 days in 37 DEG C.After 5 days, 100 μ l culture mediums are taken out from every part of culture and are used for
The measurement of cell factor IFN-γ.Utilize the level of OptEIA ELISA kit measurement IFN-γ, data SPSS10.0 software
It is handled, the conspicuousness for comparing each group difference, significance is examined using one-way analysis of variance (one-way ANOVA)
A=0.05, negative control group (* < 0.01 * < 0.05, *).As a result display humanized antibody 11D8 (as shown in Figure 5) can be effective
The function secrete cytokines IFN-γ of T cell is stimulated, and related with concentration, when being used in conjunction with STING activator CDN, Neng Gouzeng
Strong stimulation T cell secrete cytokines IFN-γ.
6 humanized antibody 11D8 of embodiment and STING activator CDN inhibits tumour growth
SCID beige, SPF grade, 6-8 week old tie up experimental animal company, tonneau China purchased from Beijing, and mouse raising is SPF
Grade.A549 human lung adenocarcinoma cell line, A375 melanoma cell strain are purchased from ATCC, cell culture and passage referring to common cell
Culture and propagating method.Tumor model method for building up: immune deficiency SCID-beige mouse 80 is randomly divided into 10 groups, every group 8
Only, lung gland cell A549 or A375 melanoma cells about 5 × 10e6 are subcutaneously injected, tumour is long to 1-3 after injection 1 week
Mm, tumorigenesis success；Take the human peripheral blood mononuclear cell PBMC(separation method of separator well referring to embodiment 4 and 5), with 1640 cells
Culture solution is resuspended into cell suspension, is injected in SCID-beige mouse body.Antybody therapy: respectively after injecting PBMC cell, the 1st
It is 5 mg/kg, 10 mg/kg respectively that it the 7th day, the 14th day injects 11D8 or STING activator CDN, 11D8 injection dosage respectively
With 20 mg/kg.STING activator CDN injection dosage is 20 mg/kg.Positive antibody (Atezolizumab) injection dosage is
10 mg/kg, negative control group injecting normal saline.Observation tumour growth situation daily, at data SPSS10.0 software
Reason is examined using one-way analysis of variance (one-way ANOVA) and compares the conspicuousness of each group difference, and significance a=
0.05, negative control group (* < 0.01 * < 0.05, *).The experimental results showed that (Fig. 6): 11D8 is able to suppress tumour growth, and with
Dosage is positively correlated, and STING activator CDN can enhance the therapeutic effect of 11D8.
Although the present invention is disclosed as above with embodiment, it is not intended to limit the invention, therefore, protection of the invention
Range should subject to the definition of the claims.
Sequence table
Amino acid sequence
A kind of > anti-PD-L1 monoclonal antibody and its application
> SEQ ID NO.1
> artificial sequence 1-214
AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGlyAspArgValThr IleThrC
ysGlnAlaSerGlnAspValTyrArgTyrIleAlaTrpTyrGlnGlnLysPro GlyLysAlaProLysLeuLeuI
leHisTyrSerThrThrLeuGluThrGlyValProSer ArgPheSerGlySerGlySerGlyThrAspPheThrP
heThrIleSerSerLeuGlnPro GluAspIleGlnThrTyrTyrCysValGlnTyrGluAsnValLeuProSerP
heGlyGly GlyThrLysLeuGluIleLysArgThrValAlaAlaProSerValPheIlePheProPro SerAsp
GluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsnPheTyr ProArgGluAlaLysValGln
TrpLysValAspAsnAlaLeuGlnSerGlyAsnSerGln GluSerValThrGluGlnAspSerLysAspSerThr
TyrSerLeuSerSerThrLeuThr LeuSerLysAlaAspTyrGluLysHisLysValTyrAlaCysGluValThr
HisGlnGly LeuSerSerProValThrLysSerPheAsnArgGlyGluCys***
> SEQ ID NO.2
> artificial sequence 1-445
GlnValGlnLeuVal GlnSerGlyAlaGlu ValLysLysProGlyAlaSerValLysVal SerCy
sLysAlaSerAlaTyrAsnIleArgAspSerTyrIleHisTrpValArgGlnAla ProGlyGlnGlyLeuGluTr
pIleGlyArgIleGluProAlaAsnAspAsnSerSerTyr AlaGlnLysPheGlnGlyArgValThrMetThrAr
gAspThrSerThrSerThrValTyr MetGluLeuSerSerLeuArgSerGluAspThrAlaValTyrTyrCysAl
aLysGlyLeu LysValLysTyrPheAspValTrpGlyAlaGlyThrThrValThrValSerSerAlaSer ThrL
ysGlyProSerValPheProLeuAlaProCysSerArgSerThrSerGluSerThr AlaAlaLeuGlyCysLeuV
alLysAspTyrPheProGluProValThrValSerTrpAsn SerGlyAlaLeuThrSerGlyValHisThrPheP
roAlaValLeuGlnSerSerGlyLeu TyrSerLeuSerSerValValThrValProSerSerSerLeuGlyThrL
ysThrTyrThr CysAsnValAspHisLysProSerAsnThrLysValAspLysArgValGluSerLysTyr Gly
ProProCysProSerCysProAlaProGluPheLeuGlyGlyProSerValPheLeu PheProProLysProLys
AspThrLeuMetIleSerArgThrProGluValThrCysVal ValValAspValSerGlnGluAspProGluVal
GlnPheAsnTrpTyrValAspGlyVal GluValHisAsnAlaLysThrLysProArgGluGluGlnPheAsnSer
ThrTyrArgVal ValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLys Va
lSerAsnLysGlyLeuProSerSerIleGluLysThrIleSerLysAlaLysGlyGln ProArgGluProGlnVa
lTyrThrLeuProProSerGlnGluGluMetThrLysAsnGln ValSerLeuThrCysLeuValLysGlyPheTy
rProSerAspIleAlaValGluTrpGlu SerAsnGlyGlnProGluAsnAsnTyrLysThrThrProProValLe
uAspSerAspGly SerPhePheLeuTyrSerArgLeuThrValAspLysSerArgTrpGlnGluGlyAsnVal P
heSerCysSerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSer LeuSerLeuGlyLys
Claims (7)
1. a kind of Humanized monoclonal antibodies of anti-PD-L1 include light chain and heavy chain, which is characterized in that the amino acid sequence of light chain
Column are as shown in SEQ ID NO.1, and the amino acid sequence of heavy chain is as shown in SEQ ID NO.2.
2. application of the antibody described in claim 1 in the drug of preparation prevention, diagnosis, treatment or adjuvant therapy of tumors, special
Sign is that the tumour includes lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, nephroncus, oophoroma, prostate cancer, wing
Guang cancer, breast cancer, the cancer of the esophagus, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical carcinoma, osteocarcinoma, cancer of pancreas.
3. application of the antibody described in claim 1 in the medicine material of preparation prevention, diagnosis, treatment or adjuvant therapy of tumors；
It is characterized in that, the tumour includes lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, nephroncus, oophoroma, prostate
Cancer, bladder cancer, breast cancer, the cancer of the esophagus, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical carcinoma, osteocarcinoma, cancer of pancreas.
4. being applied according to Claims 2 or 3, which is characterized in that applied in preparation PD-L1 access blocking agent, Yi Ji
IL-2 and IFN- Υ secretion is activated, the application in T cell is activated.
5. antibody combined interferon gene stimulates the protein agonist answering in the preparation of antitumor drugs according to claim 1
With, wherein the tumour, including lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, nephroncus, oophoroma, prostate cancer,
Bladder cancer, breast cancer, the cancer of the esophagus, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical carcinoma, osteocarcinoma, cancer of pancreas.
6. application according to claim 5, which is characterized in that the interferon gene stimulates the protein agonist includes c-
AMP-GMP, c-di-AMP, c-di-GMP and sulphur-substitutive derivative and its mixture.
7. application according to claim 5, which is characterized in that the interferon gene stimulates the protein agonist routinely medicine
Agent length of schooling at various dosage forms, the dosage form include tablet, capsule, granule, suspension, emulsion, solution, syrup or
One of injection etc. is a variety of, and oral or injection administration approach is taken to carry out antitumor and its directly related cancer prevention
Or treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510909216.4A CN106554416B (en) | 2015-12-09 | 2015-12-09 | A kind of application of anti-PD-L1 Humanized monoclonal antibodies joint interferon gene stimulates the protein (STING) agonist in antitumor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510909216.4A CN106554416B (en) | 2015-12-09 | 2015-12-09 | A kind of application of anti-PD-L1 Humanized monoclonal antibodies joint interferon gene stimulates the protein (STING) agonist in antitumor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106554416A CN106554416A (en) | 2017-04-05 |
| CN106554416B true CN106554416B (en) | 2019-03-15 |
Family
ID=58418210
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510909216.4A Active CN106554416B (en) | 2015-12-09 | 2015-12-09 | A kind of application of anti-PD-L1 Humanized monoclonal antibodies joint interferon gene stimulates the protein (STING) agonist in antitumor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106554416B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016130616A1 (en) | 2015-02-11 | 2016-08-18 | The Johns Hopkins University | Bacteria over-expressing c-di-amp and therapeutic methods |
| HRP20221263T1 (en) | 2016-03-18 | 2023-03-03 | Immune Sensor, Llc | Cyclic di-nucleotide compounds and methods of use |
| CN109970857B (en) * | 2017-12-27 | 2022-09-30 | 信达生物制药(苏州)有限公司 | anti-PD-L1 antibodies and uses thereof |
| CN113813375B (en) * | 2020-06-19 | 2023-06-16 | 杭州星鳌生物科技有限公司 | Composition of novel anti-novel coronavirus complex and application of novel anti-novel coronavirus complex in medicines for preventing and treating coronavirus infection diseases |
| CN114159429A (en) * | 2021-11-05 | 2022-03-11 | 山东第一医科大学附属省立医院(山东省立医院) | Application of PD-1 inhibitor and STING agonist in tumor treatment |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104640A (en) * | 2006-07-10 | 2008-01-16 | 苏州大学 | Preparation for anti human PD-L1 monoclonal antibody and application thereof |
| SG11201502796RA (en) * | 2012-12-13 | 2015-05-28 | Aduro Biotech Inc | Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use |
| EP2996472B1 (en) * | 2013-05-18 | 2019-03-27 | Aduro Biotech, Inc. | Compositions and methods for inhibiting "stimulator of interferon gene" dependent signalling |
-
2015
- 2015-12-09 CN CN201510909216.4A patent/CN106554416B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106554416A (en) | 2017-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jiang et al. | cGAS-STING, an important pathway in cancer immunotherapy | |
| CN106554416B (en) | A kind of application of anti-PD-L1 Humanized monoclonal antibodies joint interferon gene stimulates the protein (STING) agonist in antitumor | |
| Hamid et al. | Intratumoral immunotherapy—update 2019 | |
| US10815303B2 (en) | Fusion protein for restoring the functions of failing immune cells and application thereof | |
| CN105175545B (en) | A kind of anti-PD-1 bifunctional antibody of anti-vegf-and its application | |
| US9872909B2 (en) | Method for enhancing specific immunotherapies in cancer treatment | |
| CN109071632A (en) | Magnetic target therapy agent and application thereof | |
| US20200354458A1 (en) | Bifunctional Fusion Protein Targeting CD47 and PD-L1 | |
| WO2016180034A1 (en) | Anti-ctla-4 and anti-pd-1 dual variable domain immunoglobulin | |
| CN106755023A (en) | Chimeric antigen receptor immunocyte with safety switch and preparation method and application | |
| CN110520438A (en) | Oncolytic viral therapy | |
| Freiser et al. | The immune system and head and neck squamous cell carcinoma: from carcinogenesis to new therapeutic opportunities | |
| US10980859B2 (en) | In vivo individualized systemic immunotherapeutic method and device | |
| US11788093B2 (en) | Chimeric antigen receptor t-cells expressing interleukin-8 receptor | |
| US20220111069A1 (en) | Immunoswitch nanoparticles for reprogrammed t cell responses | |
| CN101072582B (en) | Alpha thymosin peptides as cancer vaccine adjuvants | |
| JP2021525283A (en) | A pharmaceutical composition for preventing or treating cancer, which comprises an expression inhibitor or activity inhibitor of CD300c. | |
| CN108495865A (en) | Chimeric antigen receptor with cytokine receptor activation or blocking-up structure domain | |
| CN114870009A (en) | Anti-tumor combined composition, application thereof and anti-tumor medicine | |
| US20230287075A1 (en) | Dual cytokine fusion proteins comprising multi-subunit cytokines | |
| ZA200703528B (en) | Immunotherapeutic formulations with Interleukin-2-neutralising capacity | |
| WO2021062332A1 (en) | Combination of inhibitors of il4 signaling and immune checkpoints for treating cancer | |
| Bryniarski | The influence of cyclophosphamide on immune function of murine macrophages | |
| JP2005124568A (en) | Method for activating cell, method for producing the cell using the same, and pharmaceutical composition | |
| CN114014938B (en) | Chimeric Antigen Receptor (CAR) and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190426 Address after: 311200 C-806, Kaidi Road, Xiaoshan Economic and Technological Development Zone, Xiaoshan District, Hangzhou City, Zhejiang Province Patentee after: Hangzhou star bioscience Co., Ltd. Address before: 252000 New District Office of Dongchangfu District, Liaocheng City, Shandong Province, Building 2-504, No. 15, No. 13 East Road Patentee before: Liaocheng city run bio Pharmaceutical Technology Co., Ltd. |
|
| TR01 | Transfer of patent right |