CN109232740A - A kind of anti-PD-L1 antibody and its application in antineoplaston - Google Patents
A kind of anti-PD-L1 antibody and its application in antineoplaston Download PDFInfo
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- 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
- C07K16/2827—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 B7 molecules, e.g. CD80, CD86
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C07—ORGANIC CHEMISTRY
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The present invention provides a kind of anti-PD-L1 antibody or its antigen-binding fragment, can specifically bind PD-L1 molecule, in conjunction with can block the combination of PD-L1 and PD-1 later, and can generate T cell activation and antitumor equal biological effects.
Description
Technical field
The invention belongs to medical domains, and in particular to a kind of antibody or its antigen-binding fragment, the antibody or its antigen knot
Close 1 ligand i type of fragments specific recognizer death molecule (programmed cell death 1ligand 1, PD-
L1), the immune response that body can be stimulated as immune activation object, to generate the function and effect of the diseases such as antitumor.
Background technique
2011, cancer was more than heart disease, became the global first big cause of death.WHO is announced in December, 2013, the whole world
Newly-increased cancer patient's number alreadys exceed 14,000,000 every year, this is compared with 12,700,000 people of statistical result in 2008, and number is substantially
Increase.The death toll of the same period, cancer patient also increased, and increase to 8,200,000 people from past 7,600,000 people.Exempt within 2013
Epidemic disease anti-cancer therapies are chosen as first of annual 10 big technological breakthroughs by Science magazine, and since then, tumour immunotherapy research is continuous
Breakthrough is obtained, it is controlling for current cancer therapies research field most prospect that clinical application, which also achieves immense success,
Treatment means are expected to become the new conventional treatments after operation, chemicotherapy method.
In early stage the 1980s, Allison and other researchers have determined and have been responsible for identifying antigen on T cell surface
The gene structure of α β T cell receptor (TCR).The later period eighties, Boone, Rosenberg, Old et al. are studied and are found respectively, no
With there are some tumour specific antigens in tumour patient body, simultaneously specific killing tumour cell can be identified by T cell,
So that the hope of immunotherapy of tumors rekindles, numerous studies are dedicated to the research and development of tumor therapeutic vaccine.However,
The study found that only TCR signal is not sufficient to activation antigen specific T-cells, the activation of T cell also needs Schwartz et al.
The participation of other molecules, that is, the synergistic effect of so-called second signal " costimulatory molecules ".Simultaneously the study found that only specific
Antigen presenting cell (APCs) can express costimulatory molecules, and most cells, including tumour cell can not mention
For costimulatory molecules signal.In early stage the 1990s, Allison etc. has found CD28 molecule, is capable of providing t cell activation
Required second signal.The discovery of Linsley et al. research later expression is CD28 molecule in the B7 molecule of APCs cell surface
Ligand, and Allison etc. is studied by mouse model, and can express the tumour cell of B7 molecule after transformation can be small
Mouse immune system is removed rapidly.Therefore, the missing of tumour cell B7 developed by molecule may be that body can not effective activated t cell
Immune key factor.
Show Cytotoxic T lymphocyte associated antigen-4 (cytotoxic T in the research of the 1990s
Lymphocyte-associated antigen-4, CTLA-4) play in vivo with the antipodal function of CD28, if
CD28 this kind molecule is compared to be an automobile " throttle ", then CTLA-4 this kind molecule play be " brake " function
Energy.After body T cell activation, this kind of molecule meeting " inspection " immune cell activation degree, in the cell of activation in expression
Immune suppression function is adjusted and plays, so that the T cell of body is unlikely to hyper-proliferative and activates and injuring normal cell, because
This this kind of molecule is otherwise known as " immunologic test point " molecule.Cancer cell utilizes the immunosuppression mechanism of this kind of molecule, escapes body
Immune system killing.Studies have shown that the signal of CTLA-4 is blocked using CTLA-4 monoclonal antibody specific, it can be significant
T cell activity is improved, and is found in the research of the mouse model of kinds of tumors, it can be big after monoclonal antibodies block CTLA-4
It is big to improve mouse to the rejection ability of tumour.Other than CTLA-4, immunologic test point molecule further includes programmed death receptor
Molecule -1 (Programmed cell death receptor 1, PD-1) and its ligand PD-L1 (PD-1 ligand 1),
The B7 such as TIM-3, LAG-3, TIGIT superfamily and CD28 superfamily molecule.These " suppressions are blocked by monoclonal antibody specific
System " signal, can discharge the activity of T cell, so that these T cells can play antitumor action again.Tumour immunity
Checkpoint therapy is the contribution of antitumor strategy: on the one hand, immunologic test point therapy is not directly targeted tumour cell, and
It is the immune system for acting on patient, the signal functioned by the T cell that lifts restrictions is to discharge T cell activity；It is another
Aspect, this activation to T cell does not simultaneously have antigentic specificity, but reactivates to entire immune system, therefore energy
Enough it is suitable for the treatment of a variety of different tumours, can be used as the conventional therapies of tumour.Moreover, CTLA-4 antibody blocking therapy
Success, started immunosupress relevant molecule and blocked development and application in oncotherapy, be based on PD-1 and PD-L1 etc.
The blocking antibody of the immunosuppression molecule exploitation of representative equally achieves important breakthrough.
Ligand of the PD-L1 as PD-1 molecule has played important function during T cell plays effect.Study table
It is bright, high expression in tumour cell can in inducing tumor-specific T cell PD-1 molecule expression up-regulation so that
T cell function is inhibited by certain, causes the immunologic escape of tumour.Therefore, it targets PD-L1 and blocks between PD-1/PD-L1
Interaction, T cell inhibition signal that can be antagonism PD-1 numerator mediated, and then restore the anti-tumor activity of T cell,
Have the function that treat tumour, is the important target spot of immunotherapy of tumors.
Summary of the invention
One aspect of the present invention be to provide it is a kind of can in conjunction with PD-L1 molecular specific anti-PD-L1 antibody or its
Antigen-binding fragment, the anti-PD-L1 antibody include as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5
Heavy chain CDRs；And the light chain CDRs as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.Wherein described
Section is selected from Fab, Fab ', Fab '-SH, Fv, scFv, F (ab ')2, double antibody and the polypeptide comprising CDRs, the anti-PD-L1 antibody
Or its antigen-binding fragment can block the combination of PD-L1 and PD-1.
In a particular embodiment, the anti-PD-L1 antibody includes sequence of heavy chain and SEQ shown in SEQ ID NO:9
Sequence of light chain shown in ID NO:10 or the anti-PD-L1 antibody include sequence of heavy chain and SEQ ID shown in SEQ ID NO:1
Sequence of light chain shown in NO:2.
In specific embodiments, the anti-PD-L1 antibody includes heavy chain variable region shown in SEQ ID NO:11
With light chain variable region shown in SEQ ID NO:12, or, the anti-PD-L1 antibody include SEQ ID NO:13 shown in heavy chain can
Become light chain variable region shown in area and SEQ ID NO:14.
In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment are source of mouse or the anti-PD-L1 of humanization
Monoclonal antibody, the preferably described humanization PD-L1 monoclonal antibody include the region people Fc, the area Fc of more preferably IgGl.
In specific embodiments, the present invention relates to polypeptide, sequence is as shown in SEQ ID NO:11, wherein described more
Peptide is a part for specifically binding the antibody of PD-L1 molecule, and wherein the antibody also includes shown in SEQ ID NO:12
Polypeptide.
In specific embodiments, the present invention relates to polypeptide, sequence is as shown in SEQ ID NO:13, wherein described more
Peptide is a part for specifically binding the antibody of PD-L1 molecule, and wherein the antibody also includes shown in SEQ ID NO:14
Polypeptide.
In specific embodiments, the present invention relates to polypeptide, sequence is as shown in SEQ ID NO:12, wherein described more
Peptide is a part for specifically binding the antibody of PD-L1 molecule, and wherein the antibody also includes shown in SEQ ID NO:11
Polypeptide.
In specific embodiments, the present invention relates to polypeptide, sequence is as shown in SEQ ID NO:14, wherein described more
Peptide is a part for specifically binding the antibody of PD-L1 molecule, and wherein the antibody also includes shown in SEQ ID NO:13
Polypeptide.
The second aspect of the present invention is to provide the more of the separation for encoding the anti-PD-L1 antibody or its antigen-binding fragment
Nucleotide, wherein the anti-PD-L1 antibody includes light shown in sequence of heavy chain shown in SEQ ID NO:9 and SEQ ID NO:10
Chain-ordering or the anti-PD-L1 antibody or its antigen-binding fragment include sequence of heavy chain and SEQ ID shown in SEQ ID NO:1
Sequence of light chain shown in NO:2.
In specific embodiments, the present invention relates to polynucleotides, encode the anti-PD-L1 antibody or its antigen knot
Close segment.
In specific embodiments, the present invention relates to polynucleotides, encode polypeptide shown in SEQ ID NO:11,
Described in polypeptide be a part for specifically binding the antibody of PD-L1 molecule, and wherein the antibody also includes SEQ ID
Polypeptide shown in NO:12, it is preferable that the polynucleotide sequence is shown in SEQ ID NO:15.
In specific embodiments, the present invention relates to polynucleotides, encode polypeptide shown in SEQ ID NO:12,
Described in polypeptide be a part for specifically binding the antibody of PD-L1 molecule, and wherein the antibody also includes SEQ ID
Polypeptide shown in NO:11, it is preferable that the polynucleotide sequence is shown in SEQ ID NO:16.
In specific embodiments, the present invention relates to polynucleotides, encode polypeptide shown in SEQ ID NO:13,
Described in polypeptide be a part for specifically binding the antibody of PD-L1 molecule, and wherein the antibody also includes SEQ ID
Polypeptide shown in NO:14, it is preferable that the polynucleotide sequence is shown in SEQ ID NO:17.
In specific embodiments, the present invention relates to polynucleotides, encode polypeptide shown in SEQ ID NO:14,
Described in polypeptide be a part for specifically binding the antibody of PD-L1 molecule, and wherein the antibody also includes SEQ ID
Polypeptide shown in NO:13, it is preferable that the polynucleotide sequence is shown in SEQ ID NO:18.
The third aspect of the present invention is to provide the expression vector comprising the polynucleotides.
The fourth aspect of the present invention is to provide the host cell comprising above-mentioned expression vector.
The fifth aspect of the present invention is to provide the method for preparing the anti-PD-L1 antibody or its antigen-binding fragment, institute
The method of stating includes: 1) to cultivate the host cell；2) recycled from the host cell or culture medium the anti-PD-L1 antibody or
Its antigen-binding fragment.
The sixth aspect of the present invention is to provide a kind of drug containing the anti-PD-L1 antibody or its antigen-binding fragment
Composition also contains pharmaceutical carrier.
The seventh aspect of the present invention is to provide the anti-PD-L1 antibody or its antigen-binding fragment in preparation for improving
Purposes in the drug of T cell secretion of gamma-IFN level.
The eighth aspect of the present invention is to provide the anti-PD-L1 antibody or its antigen-binding fragment in preparation treating cancer
Purposes especially in the anti-tumor drug of non-small cell lung cancer etc..
Anti- PD-L1 antibody provided by the invention or its segment can specifically bind PD-L1 molecule, in conjunction with can hinder later
The combination of disconnected PD-L1 and PD-1, and a series of biological effects can be generated.These biological effects, which include, for example:, to be mentioned
The level of high tumor cases tumor specific T cells secretion of gamma-IFN is especially able to suppress mouse interior tumor growth.
In the present invention, statement " PD-L1 antibody " or " source of mouse PD-L1 antibody " is anti-for the source of mouse monoclonal for PD-L1
Body is in specific embodiments PD-L1.A antibody.Stating " humanization PD-L1 antibody " is on source of mouse PD-1 antibody basis
Upper humanization and be made, in specific embodiments, for PD-L1.A antibody carry out humanization and be made.
PD-1 molecule is the important member of CD28 family, which further includes CD28, CTLA-4, ICOS etc..PD-1 molecule
The B cell in activation, T cell and bone marrow cell etc. are expressed, the I type memebrane protein that molecular weight is about 55kDa has now been found that PD-1 has
Two ligands, are PD-L1 and PD-L2 respectively, and PD-1 inhibits T cell living by the interaction with (PD-L1 and/or PD-L2)
Property.PD-L1 can be expressed in kinds of tumor cells surface height, by the interaction of PD-1 and PD-L1, be able to suppress tumour leaching
The functional activity for moistening lymphocyte, including the TCR T cell proliferative capacity mediated and the secretion level of cell factor etc., therefore
The important mechanisms monitored by kinds of tumors using and as tumour cell escape immune system.And antibody specificity is utilized to block
The interaction of PD-1 and (PD-L1 and/or PD-L2), can activate the T cell in holddown, so that the function of T cell
It is released, restores the function of T cell, to reach using body immune system killing tumor cell and then carry out oncotherapy
Effect.
The present invention is made based on above-mentioned principle, and the anti-PD-L1 antibody or its antigen-binding fragment in the present invention are logical
It crosses in conjunction with PD-L1 molecular specific, blocks the combination of PD-L1 and PD-1, to make T cell activation, improve T cell and secrete IFN-
The level of γ.
The application includes the antibody or derivative with PD-L1 specific binding, also includes and original antibody display essence
The antigen-binding fragment of upper identical antigen-binding specificity." segment of antibody " or " antigen-binding fragment " refer to the anti-of antibody
Former binding fragment and antibody analog generally include the antigen binding domain or variable region of at least partly maternal antibody, such as one
A or multiple CDRs.The segment of antibody retains at least some of binding specificity of maternal antibody.Antigen-binding fragment includes being selected from
Fab、Fab′、Fab′-SH、Fv、scFv、F(ab′)2, double antibody, the peptide comprising CDRs etc..
" Fab segment " is made of the CH1 and variable region of a light chain and heavy chain.
Contain two heavy chain fragments of the CH1 and CH2 structural domain comprising antibody in the area " Fc ".Two heavy chain fragments by two or
Multiple disulfide bond are simultaneously kept together by the hydrophobic effect of CH3 structural domain.
" Fab ' segment " is containing a light chain and comprising between VH structural domain and CH1 structural domain and CH1 and CH2 structural domain
The part of one heavy chain in region forms interchain disulfide bond between two heavy chains of two Fab ' segments to form 2 points of F (ab ')
Son.
“F(ab′)2Part of the segment " containing two light chains and two comprising the constant region between CH1 and CH2 structural domain
Thus heavy chain forms interchain disulfide bond between two heavy chains.Therefore, F (ab ')2Segment is by passing through the disulfide bond between two heavy chains
The two Fab ' segments composition to keep together.
" area Fv " includes the variable region from both heavy chain and light chain, but lacks constant region.
" single-chain Fv antibody (scFv antibody) " refers to the antigen-binding fragment of the VH and VL structural domain comprising antibody, these knots
Structure domain is present in single polypeptide chain.In general, scFv polypeptide includes peptide linker, the connector between VH and VL structural domain
ScFv is enabled to form the required structure for antigen binding.
" double antibody " is that there are two the small antigen-binding fragments of antigen binding site for tool.The segment is included in identical more
The heavy-chain variable domains (VH) (VH-VL or VL-VH) being connect in peptide chain with light variable domains (VL).By using being as short as
The connector that cannot be matched between two structural domains of same chain, so that the complementary domain of the structural domain and another chain is matched
To and formed two antigen binding sites.
" humanization " form of non-human (such as mouse) antibody is to derive from non-human immune globulin containing minimal
The chimeric antibody of Bai Xulie.The major part of humanized antibody is human immunoglobulin(HIg), wherein some hypervariable region residues quilt of receptor antibody
With it is required specificity, affinity and ability non-human species hypervariable region residue displacement, non-human species for example have mouse,
Rat, rabbit or non-human primates.In some cases, the Fv framework region residue of human immunoglobulin(HIg) is residual by corresponding non-human
Base replaces.In addition, humanized antibody may include the not residue present in receptor antibody or donor antibody.Carry out these modification with
It is further improved antibody performance.
When referring to ligand/receptor, antibody/antigen or other combination clock synchronizations, " specificity " combine refer to albumen and/or its
The association reaction of the albumen such as PD-L1 is determined whether there is in the heterogeneous population of its biological reagent.Therefore, specified
Under the conditions of, specific ligand/antigen in conjunction with specific receptor/antibody, and not with present in significant quantity and sample its
Its protein binding.
The present invention also provides the pharmaceutical compositions containing PD-L1 antibody of the present invention or its antigen-binding fragment.In order to prepare
Pharmaceutical composition can be prepared into various institutes by mixing antibody or its antigen-binding fragment with pharmaceutical carrier or excipient
The dosage form needed.The type of dosage form as medical composition of the invention, such as the tablet as oral agents, powder can be enumerated
Agent, pill, powder, granule, granula subtilis, soft/hard capsule, film coating agent, pilule, sublingual tablet, paste etc., as non-
Oral agents can enumerate injection, suppository, percutaneous agent, ointment, emplastrum, liquid for external use etc., those skilled in the art's energy
It is enough that dosage form appropriate is selected according to administration route and administration object etc..
The dosage of the effective component of pharmaceutical composition of the invention, according to administration object, object internal organs, symptom, administration
Method etc. is different and has differences, it may be considered that type, medication, the age of patient and weight, the symptom of patient of dosage form
Deng being determined according to the judgement of doctor.
Pharmaceutical composition of the present invention can also contain other medicaments, including but not limited to cytotoxic agent, cell growth inhibition
Agent, anti-angiogenetic therapy drug or antimetabolite, target tumor drug, immunostimulant or immunomodulator or and cell toxicant
The antibody that agent, cytostatic agent or other drug toxicities combine.
Detailed description of the invention
Fig. 1 is the sieve chromatography and SDS-PAGE qualification figure for indicating the PD-L1 albumen of 293T cell expression system preparation.
Fig. 2 is the expression and purification for indicating PD-L1.A antibody protein and the figure of SDS-PAGE purity detecting result.
Fig. 3 is to indicate that PD-L1.A antibody can block the figure of the combination of PD-1 and PD-L1.
Fig. 4 is to indicate that PD-L1.A antibody is capable of the figure of activating T cell.
Fig. 5 is the expression and purification for indicating humanization PD-L1.A antibody protein and the figure of SDS-PAGE purity detecting result.
Fig. 6 is the affinity for indicating SPR detection PD-L1.A antibody and humanization PD-L1.A antibody combination PD-L1 albumen
Figure
Fig. 7 is the figure for indicating humanization PD-L1.A antibody NCG mouse HCC-827 tumor model inhibition assay result.
Specific embodiment
Hereinafter, carrying out more specific description to the present invention by embodiment.But it will be appreciated by those skilled in the art that
It is the purpose that embodiment below is intended to be merely illustrative of the present, and is not intended to limit the present invention.
The preparation of embodiment 1.PD-L1.A antibody
The building of 1.PD-L1 recombinant expression plasmid
It is reference with the cDNA (NM_001267706.1) of PD-L1, synthesizes the DNA sequence dna of PD-L1 (amino acid 1-238),
And restriction enzyme site EcoRI and BglII are introduced respectively, C-terminal introduces the label of 6 His amino acid, wherein EcoRI restriction enzyme site position
In 5 ' ends of sequence, the label and restriction enzyme site BglII of 6 His amino acid are sequentially located at 3 ' ends of sequence.Utilize restriction enzyme site
The DNA sequence dna of the PD-L1 of synthesis is cloned into expression vector pCAGGS (Addgene company) by EcoRI and BglII, establishes PD-L1
The eukaryotic expression recombinant plasmid of full-length proteins.
The expression and purification of 2.PD-L1 recombinant protein
1) transfect HEK293T cell: HEK293T cell (ATCC) is reached with 1: 3 to be continued to cultivate in culture dish；Take 7.5mL
DMEM (serum-free and antibiotic) (GIBCO) is added 300 μ L polyetherimide (PEI) 1.0 and mixes into 50mL pipe；75 μ are added
G PD-L1 recombinant plasmid dna mixes into mixing liquid and stands 30 minutes；Take 515 μ L into each culture dish in 37 DEG C respectively
5%CO2It is cultivated in incubator.
2) collect supernatant: after transfection 48 hours, supernatant is collected in 4 DEG C of centrifugations.
3) nickel affinity chromatography column purification: with 0.45 μM, supernatant after 33mm pvdf membrane filter filter centrifugation, under the conditions of 4 DEG C
It is combined 4 hours or more with nickel affinity chromatography column (GE Health)；Later with containing 10mM, 20mM, 50mM, 100mM, 200mM,
The eluent of the various concentrations imidazoles such as 300mM, 500mM is eluted, and changes liquid later to 20mM Tirs-HCl, 150mM NaCl
Buffer is spare, and SDS-PAGE identifies purity (Fig. 1).
The expression and purification of 3.PD-L1-mFc albumen
It is reference with the cDNA (NM_001267706.1) of PD-L1, synthesizes the DNA sequence dna of PD-L1 (amino acid 1-238),
And restriction enzyme site EcoRI and BglII are introduced respectively, C-terminal introduces the label of mouse Fc amino acid, and wherein EcoRI restriction enzyme site is located at
5 ' ends of sequence, Fc label and restriction enzyme site BglII are sequentially located at 3 ' ends of sequence.It will using restriction enzyme site EcoRI and BglII
The DNA sequence dna of the PD-L1 of synthesis is cloned into expression vector pCAGGS (Addgene company), establishes the recombination of PD-L1-mFc albumen
Eukaryon expression plasmid.
PD-L1-mFc protein expression:
1) transfect HEK293T cell: HEK293T cell (ATCC) is reached with 1: 3 to be continued to cultivate in culture dish；Take 7.5mL
DMEM (serum-free and antibiotic) is added 300 μ L polyetherimide (PEI) 1.0 and mixes into 50mL pipe；75 μ g PD- are added
L1-mFc recombinant plasmid dna mixes into mixing liquid and stands 30 minutes；Take 515 μ L into each culture dish in 37 DEG C 5% respectively
CO2It is cultivated in incubator.
2) collect supernatant: after transfection 48 hours, supernatant is collected in 4 DEG C of centrifugations.
3) Protein A affinity chromatography column purification: with 0.45 μM, supernatant after 33mm pvdf membrane filter filter centrifugation passes through
Centrifugation, which goes after precipitating isometric 20mM Na3PO4 (pH 7.0) is added thereto, to be uniformly mixed, then with 0.22 μm of filter membrane mistake
Filter combines 4 hours or more under the conditions of 4 DEG C with Protein A affinity column (GE Health)；0.1M Gly is used later
PH3.0 elution hangs over the antibody on pillar, and the about 0.8mL of Tris pH 9.0 (collected volume 3.2mL) of 1M, Zhi Houhuan is added
For liquid to 20mM Tirs-HCl, 150mM NaCl buffer is spare.
The preparation and screening of 4.PD-L1 source of mouse monoclonal antibody
Immunization method is injected intraperitoneally according to Freund's complete adjuvant, with the overall length PD-L1 recombinant protein for the purifying that recombination generates
(hereinafter referred to as PD-L1 antigen) is for exempting from B6/C57 mouse (Beijing Vital River Experimental Animals Technology Co., Ltd.)
Epidemic disease.The specific method is as follows:
1) animal immune: purified PD-L1 antigen is with complete Freund's adjuvant (Sigma) emulsification, using intraperitoneal injection
6-8 week old B6/C57 mouse is immunized in method, and immunizing dose is 50 μ g/, and progress is immune for the second time after two weeks at interval, with incomplete
Freund's adjuvant emulsification, immunizing dose are 50 μ g/.It is immune that tail blood is taken to measure serum titer with ELISA method gradient dilution afterwards twice；
Booster immunization is determined whether according to result, is chosen the highest mouse of antibody titer and is carried out cell fusion.
2) cell fusion: myeloma cell uses the sp2/0 in the source BALB/c, and logarithmic growth phase is in when fusion；It takes
Immune mouse spleen, is made lymphocyte single cell suspension；Mouse spleen lymphocyte is mixed with myeloma cell with 1: 5-1: 10,
37 DEG C 50%PEG (pH 8.0) 1mL is added dropwise, incomplete culture medium and remaining terminate liquid is added, HAT is added after abandoning supernatant in centrifugation
Culture medium, which suspends, to be mixed, and MC constant volume to 50mL is dispensed into 3.5cm culture dish, is put in wet box, is placed in 37 DEG C, 5%CO2It is permanent
It is cultivated in warm incubator.
3) screen and clone: fusion selected cell clone in 7-10 days, was carried out using the PD-L1 recombinant protein of purifying
The binding ability of ELISA test hybridoma supernatant and PD-L1 albumen.By PD-L1 recombinant protein according to the hole 300ng/, coating
To elisa plate, 4 DEG C are incubated overnight, and are cleaned 3 times with PBS buffer solution later, and the confining liquid of 5% skimmed milk power (Erie), room is added
25 DEG C of temperature are incubated for 2 hours；It is cleaned 3 times with PBS buffer solution later, the culture supernatant of cell clone is added, 25 DEG C of room temperature are incubated for 1
Hour；It is cleaned 3 times with PBS buffer solution later, the sheep anti-mouse igg antibody of horseradish peroxidase-labeled is added, 25 DEG C of room temperature incubate
It educates 1 hour；It is cleaned 3 times with PBS buffer solution later, TMB developing solution is added, terminate liquid is added after 15min, and be applicable in spectrophotometric
The absorption value of meter detection OD450.The cell clone for screening OD450 > 0.5 carries out subsequent screening.Mark cell strain number.To the positive
Hole cell carries out limiting dilution, 5-6 days measurement ELISA values after each limiting dilution, picking ELISA detection OD450 positive value compared with
High monoclonal hole carries out limiting dilution, until ELISA measures 96 orifice plates, hardened fruit is the positive entirely.The picking positive is worth high Dan Ke
Grand singling.It is PD-L1.A that it, which corresponds to fusion plate cell strain,.
4) preparation and purification of cell conditioned medium monoclonal antibody: the DMEM culture medium containing 15% serum is used to train cell strain PD-L1.A
It supports and is cultivated in 10cm culture dish, spread cultivation to about 4 × 107When a, 800rpm is centrifuged 5min, abandons supernatant and cell is transferred to 2L
In rolling bottle, serum free medium is added, making cell density is about 3 × 105A/mL.After continuing culture 1~2 week, work as cell death
(cell density is about 1-2 × 10 at this time when rate reaches 60%-70%6A/mL), collect cell suspension 6000rpm high speed from
Heart 20min takes supernatant, and affinity chromatography carries out supernatant purifying, corresponding column material is selected according to antibody die mould, using protein G
It is purified.Monoclonal antibody concentration mensuration after purification, is stored in 4-8 DEG C at packing (100uL/ pipe, concentration 1mg/mL).
5. the expression and purification of source of mouse PD-L1.A antibody
By 2 × 106Cell strain PD-L1.A cell by intraperitoneal injection 6-8 week old BALB/C mice (purchased from dimension tonneau
Hua company), mouse ascites are collected after 2-3 weeks, the ascites of acquisition is first passed through into centrifugation and goes after precipitating to be added thereto in equal volume
20mM Na3PO4(pH 7.0) is uniformly mixed, then with 0.22 μm of membrane filtration ascites, is mainly prevented other miscellaneous in ascites
Confrontation pillar causes to damage；Prepare loading purifying after the completion of filtering.
Protein G (5mL) HP affinity column (GE company) is connected to AKTA Purifier/Explorer/FPLC/
On START (GE company), following process is operated on machine: first 20% ethyl alcohol in column being gone out with water, then uses 20mM
Na3PO4, the buffer of pH 7.0 balances pillar, above-mentioned ascites passed through 5mL loop after show that conductance is 4.5% on instrument
The mode of ring loading is injected in conjunction with Protein G, flow velocity 1mL/min；After UV is steady, 1M is added in subsequent collecting pipe
The about 0.8mL of Tris pH 9.0 (collected volume 3.2mL), 100% 0.1M Gly pH 3.0 is then changed in program
Elution hangs over the antibody on pillar, collects the sample of elution, sample preparation, and gel electrophoresis identification judges that glue figure stripe size is correct, and
It is further purified by superdex200 sieve chromatography, SDS-PAGE identifies its purity (Fig. 2)；If above-mentioned identification nothing
Accidentally, the method for changing liquid using concentration, the antibody of concentration is constantly diluted with PBS, repeatedly by sample after 100 times of concentration and dilution or more
Packing, directly uses or is stored in -80 DEG C of refrigerators.
The screening of embodiment 2.PD-L1 blocking antibody and affinity analysis
Mouse immune is carried out by the PD-L1 albumen that 293T cells in vitro is expressed, the monoclonal antibody of acquisition carries out PD-1
The antibody of specific inhibition PD-1 and PD-L1 interaction is capable of in blocking experiment screening with PD-L1.
1.PD-1 total length expressed 293T cell preparation
In the present embodiment, by the way that the PD-1-GFP-p matter of PD-1 overall length (pEGFP-N1 carrier-GFP tagged-plasmid) will be contained
Grain (Clontech company) transfection 293T cell (ATCC) obtains the 293T cell of expression PD-1 overall length.Transfection first 1 day according to
0.5~2 × 105Cell per well is inoculated in 24 well culture plates, and the not antibiotic DMEM complete medium of 500 μ L is added
(GIBCO company), cell confluency is up to 70~80% when guaranteeing transfection.1 μ g PD-1-GFP-p plasmid is diluted in 50 μ L without blood
In the culture medium of cleer and peaceful antibiotic, mix gently.2gL PEI (Sigma) (4mg/m1) is diluted in 50 μ L without serum and is resisted
In the culture medium of raw element, mix gently.After five minutes, 50 μ L PEI dilutions are added drop-wise to the dilution of 50 μ L PD-1-GFP-p plasmids
It in liquid, mixes gently, is incubated at room temperature 20 minutes.100 μ L PEI/PD-1-GFP-p plasmid composites are added drop-wise in every hole simultaneously light
Jog is dynamic to mix it uniformly with fresh culture medium.After cell is put into 4~6h of incubator incubation, serum-containing medium is replaced
Remove compound.Cell is placed on 37 DEG C, CO2Incubator continues after being incubated for 24 hours, passes through stream type cell analyzer (BD
ARIA II) detection GFP expression, evaluate the expression of the total length expressed 293T cell of PD-1.
2. antibody blocking experiments
PD-L1.A antibody and PD-L1-mFc albumen (acquisition of embodiment 1) prepared by embodiment 1 is according to molar ratio 2: 1
Ratio mixing postposition is incubated for 1 hour on ice, is added to later containing 2 × 105The total length expressed 293T cell of PD-1 in, set and incubate on ice
It educates 30 minutes.It is negative control that Ebola virus GP protein specific antibody 4G7 (Mapp Biopharmaceutical), which is arranged,；
Twice, the anti-mouse IgG secondary antibody (BD) of APC label is added in PBS cleaning later, is cleaned twice after being incubated for 30 minutes with PBS buffer solution,
Flow cytometry is carried out after being finally resuspended with 300mL PBS solution.As a result as shown in Figure 3, the results showed that, PD-L1-mFc energy
It is enough to be significantly integrated on the total length expressed 293T cell of PD-1, and PD-1 and PD- can be completely inhibited after PD-L1.A antibody is added
The combination of L1, so that PD-L1-mFc not can be incorporated on the PD-1 albumen of 293T cell surface (Fig. 3).Therefore, PD-
L1.A antibody can significantly inhibit the combination of PD-1 and PD-L1 in cellular level.
Activated in Vitro ability of the embodiment 3.PD-L1 blocking antibody to T cell
The important application of PD-L1 blocking antibody first is that its antitumor action, the present embodiment acquire and have separated 3 health
A peripheral blood lymphocytes, the antitumor potential for the PD-L1 blocking antibody cell in vitro level that the evaluation present invention screens.
1. sample enters a group screening
The case being included in the present embodiment is healthy individuals.
2.PBMCs sorting
Vein peripheral blood of the lymphocyte used in the present invention from individual.Individual by screening is through clinician's physical examination
After qualification, the quantity of detailed programs process and required blood is informed by experimenter, informed consent form is agreed to and signed through volunteer,
By clinical worker, to volunteer, blood was collected.Using containing EDTA-K when blood sampling2Anticoagulant 9mL disposal vacuum heparin tube
(VACUETTE, Austrian Gray receive company), every volunteer take a blood sample about 20-25mL, overturn anti-hemostasis-coagulation after blood sampling immediately.
1) first by the peripheral blood of fresh acquisition with 121 DEG C of high pressure sterilization phosphate buffers after cooling (PBS, pH7.4)
It is (raw purchased from the ocean Tianjin Hao to be carefully added into preprepared 15mL lymphocyte separation medium by one times of dilution for diluted blood sample
Object Technology Co., Ltd.) in, when addition, is careful, is slowly added to, and avoids interface chaotic；
2) 20 points are centrifuged with horizontal centrifuge (SORVALL Stratos, Thermo company of the U.S.) 700g under the conditions of 25 DEG C
Clock, reduction of speed is adjusted to most slow when stopping；
Four layers of sample point after centrifugation, top layer's blood plasma is sucked out with Pasteur pipette, and the second layer is carefully sucked out later
Buffy coat is into new sterile centrifugation tube, as thick pure PBMCs cell；
3) thick pure PBMCs cell is diluted in equal volume with isometric phosphate buffer (PBS, pH7.4), later at 25 DEG C
Under the conditions of with centrifugal force 10 minutes of 800g；
Supernatant is discarded, is resuspended with 7mL serum-free RPMI1640 (GE company of U.S. Hyclone brand), the 500g at 25 DEG C
Centrifugation 5 minutes；
Supernatant is discarded, is resuspended, adding 7mL containing 10% fetal calf serum, (FBS, U.S. Fisher company brand Australia Thermo are come
Source) RPMI-1640 culture medium cleaning, at 25 DEG C 500g be centrifuged 5 minutes；
4) it discards the RPMI-1640 culture medium after supernatant with 3mL containing 10%FBS to be resuspended, takes appropriate gravity treatment liquid in hemocytometer
Cell count is carried out on number plates, and with containing the RPMI-1640 culture medium final adjustment of 10% serum to 2.5 × 106Cell/mL is close
It is spare to obtain PBMCs cell for degree；
5) ELISPOT detects the T cell activation level of antibody
A.ELISPOT detects cytositimulation culture
ELISPOT plate (Merck Millipore Corp.) shifts to an earlier date 12 hours or more to be resisted with phosphate buffer (pH7.4) is diluted
Human gamma-interferon monoclonal antibody (BD company) coating, 4 DEG C horizontal positioned.With containing 10% serum before stimulator antigen and cell is added
(HyClone) RPIM-1640 culture medium is closed 1 hour under room temperature.
PD-L1.A antibody and negative control antibody will be diluted with the RPMI-1640 culture medium of 10% serum (HyClone)
(EBOLA virus GP protein antibody 4G7 (Mapp Biopharmaceutical)), 100 μ L are added in every hole in ELISPOT plate hole,
Separately set non-stimulated blank control wells and phytolectin (PHA) (Sigma) stimulation Positive control wells.The control packet of this experimental setup
It includes:
The antibody being wherein added includes: PD-L1.A antibody and 4G7 negative control antibody antibody according to 10 μ g/mL final concentrations
It is added in 96 holes and carries out stimulation culture.
By the ELISPOT plate that 100 μ L and 100 μ l PBMCs dilutions are added in the PBMCs cell per well after dilution set 37 DEG C,
5%CO2It is incubated for 18 hours under the conditions of (carbon dioxide).
B.ELISPOT board-washing and result obtain
A) after being incubated for, hole inner cell liquid is discarded, is cleaned 2 times in the deionized water that 200 μ L room temperature are added in every hole rapidly,
The PBS (PBST) containing 5 ‰ Tween-20 is cleaned 3 times later.
B) washing lotion is removed, firmly buckles and does on blotting paper, the diluted detection antibody (BD) of 100 μ L is added in every hole, and room temperature is incubated
Educate 2h.
C) removal detection antibody-solutions, PBST are cleaned 3 times, and every hole is added the Streptavidin-HRP diluted and combines later
100 μ L of object (BD) is incubated at room temperature 1h.
D) develop the color: removal Streptavidin-HRP conjugate solution, PBST are cleaned 3 times, and PBS is cleaned 2 times later.It is absorbing water
It firmly buckles and does on paper, 100 μ L AEC substrate (BD) solution are added in every hole later, clear when seeing after incubation at room temperature 15~30 minutes
When clear spot, with distilled water flushing with stopped reaction.
E) 37 DEG C or dry at room temperature, later by ELISPOT plate with automatic plate reader (C.T.L) to anti-in the hole ELISPOT
The spot answered is counted, and adjusting parameter and is carried out quality control later, is provided end reaction result.
3. interpretation of result
By counting 105The specific spots number that generates after stimulating in cell is simultaneously analyzed, and evaluation PD-L1 is blocked
Influence of the antibody to T cell activation.
By the response situation to this 3 healthy individuals PBMCs to PD-L1 antibody, T cell is evaluated to PD-L1 antibody
Reaction effect.(Fig. 4) as the result is shown, the stimulation hole of PD-L1 antibody is added, and relative to negative control hole can to generate stronger T thin
Born of the same parents' immune response, while comparable levels of t cell immune response can be generated, and 4G7 negative control antibody is then compared with negative control
Hole is without significant difference.Therefore, PD-L1 antibody can the intracorporal T cell of effective activation healthy individuals under condition of culture in vitro, promote
IFN-γ is generated into it, to enhance its T cell immune function.
The humanization and humanized antibody and PD-1 affinity of 4. source of mouse PD-L1.A antibody of embodiment detect
According to the sequence homology of PD-L1.A antibody, the present invention is retaining the CDR region basis of antibody light chain and heavy chain
On, by replacing human antibody skeleton, obtain humanization PD-L1.A antibody (hu-PD-L1.A).
SEQ ID NO.:1:PD-L1.A source of mouse PD-L1.A heavy chain of antibody
SEQ ID NO.:2:PD-L1.A source of mouse PD-L1.A antibody light chain
SEQ ID NO.:3:PD-L1.A heavy chain CDR1
SEQ ID NO.:4:PD-L1.A heavy chain CDR2
SEQ ID NO.:5:PD-L1.A heavy chain CDR3
SEQ ID NO.:6:PD-L1.A light chain CDR1
SEQ ID NO.:7:PD-L1.A light chain CDR2
SEQ ID NO.:8:PD-L1.A light chain CDR3
SEQ ID NO.:9: humanization PD-L1.A heavy chain
SEQ ID NO.:10: humanization PD-L1.A light chain
SEQ ID NO:11: the heavy chain variable region of source of mouse PD-L1.A antibody
SEQ ID NO:12: the light chain variable region of source of mouse PD-L1.A antibody
SEQ ID NO:13: the heavy chain variable region of humanization PD-L1.A antibody
SEQ ID NO:14: the light chain variable region of humanization PD-L1.A antibody
SEQ ID NO:15: the coded sequence of the heavy chain variable region of source of mouse PD-L1.A antibody
SEQ ID NO:16: the coded sequence of the light chain variable region of source of mouse PD-L1.A antibody
SEQ ID NO:17: the coded sequence of the heavy chain variable region of humanization PD-L1.A antibody
SEQ ID NO:18: the coded sequence of the light chain variable region of humanization PD-L1.A antibody
SEQ ID NO:19: the coded sequence of humanization PD-L1.A heavy chain of antibody
SEQ ID NO:20: the coded sequence of humanization PD-L1.A antibody light chain
1) expression and purification of humanization PD-L1.A antibody:
Humanization PD-L1.A antibody passes through constructs the amino containing coding SEQ ID NO.:9 and SEQ ID NO.:10 respectively
The pCAGGS expression vector (Addgene company) of the polynucleotides (SEQ ID NO:19 and SEQ ID NO:20) of acid sequence, and
Respectively introduce restriction enzyme site EcoRI and BglII, wherein EcoRI restriction enzyme site be located at sequence 5 ' hold, restriction enzyme site BglII
In 3 ' ends of sequence.Using restriction enzyme site EcoRI and BglII by the hu-PD-L1.A light chain of synthesis or the DNA sequence dna gram of heavy chain
It is grand enter expression vector pCAGGS (Addgene company), establish the recombinant eukaryon expression matter of hu-PD-L1.A antibody light chain and heavy chain
Grain.By the expression plasmid of hu-PD-L1.A antibody light chain and heavy chain according to 2: 1 ratio, 293T cell is transfected jointly, expression
Antibody is purified through Protein G affinity column chromatography.It specifically includes:
A. the 14-16h before transfection divides the biggish cell of cell density to disk (a such as disk 100% is paved with the 10cm of cell
Culture dish is passed on 1: 3), after 14-16h, cell density reaches can be transfected on 70%.
B. by taking 10cm culture dish transfects adherent 293T cell as an example: transfect required plasmid amount be 20 μ g/ disks (light chain:
Heavy chain=1: 1, mass ratio), it is diluted in the HBS liquid of 100 μ L/ disks, is stood after mixing；With PEI (μ L): plasmid quality (μ g)=
The dosage of 1: 4 ratio-dependent PEI (1mg/mL) is diluted in the HBS liquid of 100 μ L/ disks, stands after mixing.Above-mentioned two solution
Mixing 5min is individually stood, the two is mixed continue to stand 20min later, be finally added to the cell culture fluid to be transfected
In.
C. after transfecting 4-6h, liquid is changed to the cell of transfection, first changes fresh nothing into again with after twice of the PBS rinse of 2-3mL
The DMEM culture medium (joined mycillin by 1: 1000) of serum, in 37 DEG C of constant temperature, 5%CO2Incubator in cultivate expression.
By the cell culture fluid after above-mentioned transfection, supernatant is collected after culture 3 days, changes liquid with DMEM culture medium, then to the
Seven days again by a supernatant.The supernatant that 2 times are collected mixes, and purifies destination protein, and purification process uses the above-mentioned step of the present embodiment
Protein G source of mouse PD-L1.A antibody purification process in rapid 5.Antibody purity after Protein G pillar affinity chromatography reaches
To 95% or more (such as Fig. 5).
2) the affinity verifying of humanization PD-L1.A antibody:
In the present embodiment, by surface plasma resonance technology (SPR) to PD-L1.A antibody and humanization PD-L1.A
Antibody carries out affinity identification.
The PD-L1 albumen prepared in embodiment 1 and humanization PD-L1.A antibody are changed into liquid (10mM into SPR buffer
HEPES-HCl, 150mM Na-Cl, 0.005%Tween-20, pH 7.4).Antibody protein is diluted to 20 μ g/mL to be fixed to
On CM5 chip (GE Health), the PD-L1 of gradient dilution is respectively flowed through into each channel of CM5 chip later, utilizes BIAcore
T100 analyzes binding kinetics parameter, and calculates affinity constant.
Pass through the detection to source of mouse PD-L1.A antibody and humanization PD-L1.A antibody and PD-L1 affinity, the results showed that,
The affinity of source of mouse PD-L1.A antibody and PD-L1 are 8.81nM, and the affinity of humanization PD-L1.A antibody and PD-1 are
7.77nM.Therefore, it can be seen that from SPR result and humanization PD-L1.A affinity of antibody is suitable with source of mouse PD-L1.A antibody,
Still keep the affinity (such as Fig. 6) of the nM order of magnitude.
The NCG Immune deficient mice HCC827 tumor suppression of embodiment 6.hu-PD-L1.A antibody is tested
The tumor suppression of the present embodiment application NCG immunodeficient mouse HCC827 tumor model evaluation hu-PD-L1.A antibody
Effect.
The NCG mouse tumor Inhibition test step of hu-PD-L1.A antibody includes:
1.NCG mouse source of people immune system is rebuild
NCG mouse derives from Nanjing University-Nanjing biological medicine research institute.Health is inoculated with first into every NCG mouse
The PBMCs cell in individual source establishes the mouse with source of people immune system in NCG Mice Body:
I. be inoculated with human PBMC s cell quantity: 1 × 107Cell/200 μ L/ is only；
Ii inoculation position: tail vein；
2.HCC-827 cell line tumor formation
(the Chinese Academy of Sciences typical case culture of HCC-827 non-small cell is inoculated with after inoculation PBMCs cell 3 days in every NCG mouse
Object preservation committee cell bank) lung cancer cell line:
A) inoculation HCC-827 cell quantity: 5 × 107Cell/200 μ L/ is only；
B) inoculation position: dorsal sc；
3. grouping and processing:
The mouse for selecting tumor formation more uniform after about 1 week after tumor cell injection is grouped, and carries out antibody abdominal cavity later
Injection.The present embodiment is negative right with Ebola virus specific antibody 13C6 (Mapp Biopharmaceutical) injection group
According to hu-PD-L1.A antibody is that processing group carries out parallel laboratory test, every group of 7 mouse.
| Mice group | Antibody injection and dosage | Mouse quantity |
| Negative antibody control group | 10mg/kg, 100 μ L | 7 |
| Hu-PD-L1.A antibody group | 10mg/kg, 100 μ L | 7 |
Antibody injection: after mouse tumor formation is obvious (7 days), point 4 injection of antibodies, 200 μ g/ only, are spaced two later for the first time
It, i.e., 200 μ g/ are injected only in third day second, later every three days or injection in four days (be 200 μ g/ only).
The tumor size of detection in every three to four days after tumor formation, last time continue observation two weeks after injecting.
4. therapeutic effect is observed:
1) tumor size detects:
A) diameter measurement, unit mm, calculation formula are carried out with slide calliper rule after antibody injection are as follows: v=1/2 × a × b × b
(a is major diameter, and b is minor axis)；
B) it tests and terminates after last time is observed, separation tumor tissues are directly taken pictures observation；
The result shows that the control group mice of injection 13C6 antibody equal fast-growth after the injection of 13C6 antibody.hu-PD-
L1.A antibody injection group tumour growth after antibody injection quickly enters plateau, has two mouse to inject 10 days in antibody respectively
Tachyauxesis after with 14 days, but still significantly less than 13C6 antibody control group.The present embodiment the result shows that, hu-PD-L1.A antibody energy
It is enough effectively to inhibit tumour growth, there is potential oncotherapy value (such as Fig. 7).
Claims (17)
1. anti-PD-L1 antibody or its antigen-binding fragment in conjunction with PD-L1 molecular specific, it includes such as SEQ ID NO:3,
Heavy chain CDRs shown in SEQ ID NO:4 and SEQ ID NO:5；And such as SEQ ID NO:6, SEQ ID NO:7 and SEQ ID
Light chain CDRs shown in NO:8.
2. anti-PD-L1 antibody as described in claim 1 or its antigen-binding fragment, wherein the antigen-binding fragment is selected from
Fab、Fab′、Fab′-SH、Fv、scFv、F(ab′)2, double antibody and the peptide comprising CDRs, the anti-PD-L1 antibody or its antigen
Binding fragment can block the combination of PD-L1 and PD-1.
3. anti-PD-L1 antibody as claimed in claim 1 or 2 or its antigen-binding fragment, wherein the anti-PD-L1 antibody includes
Sequence of light chain shown in sequence of heavy chain shown in SEQ ID NO:9 and SEQ ID NO:10.
4. anti-PD-L1 antibody as claimed in claim 1 or 2 or its antigen-binding fragment, wherein the anti-PD-L1 antibody includes
Sequence of light chain shown in sequence of heavy chain shown in SEQ ID NO:1 and SEQ ID NO:2.
5. anti-PD-L1 antibody as claimed in claim 1 or 2 or its antigen-binding fragment, wherein the anti-PD-1 antibody includes
Light-chain variable sequence shown in weight chain variabl area sequence shown in SEQ ID NO:11 and SEQ ID NO:12, or, including SEQ
Light-chain variable sequence shown in weight chain variabl area sequence shown in ID NO:13 and SEQ ID NO:14.
6. anti-PD-L1 antibody as claimed in claim 1 or 2 or its antigen-binding fragment are source of mouse or the anti-PD-L1 of humanization
Monoclonal antibody, the preferably described humanization PD-L1 monoclonal antibody include the region people Fc.
7. polypeptide, sequence is as shown in SEQ ID NO:11 or 13, wherein the polypeptide is to specifically bind resisting for PD-L1 molecule
A part of body, and wherein the antibody also includes polypeptide shown in SEQ ID NO:12 or 14.
8. polypeptide, sequence is as shown in SEQ ID NO:12 or 14, wherein the polypeptide is to specifically bind resisting for PD-L1 molecule
A part of body, and wherein the antibody also includes polypeptide shown in SEQ ID NO:11 or 13.
9. polynucleotides, the anti-PD-L1 antibody or its antigen-binding fragment of any one of coding claim 1-4.
10. polynucleotides encode polypeptide shown in SEQ ID NO:11 or 13, wherein the polypeptide is specific binding PD-
A part of the antibody of L1 molecule, and wherein the antibody also includes polypeptide shown in SEQ ID NO:12 or 14, it is preferable that
The polynucleotide sequence is shown in SEQ ID NO:15 or 16.
11. polynucleotides encode polypeptide shown in SEQ ID NO:12 or 14, wherein the polypeptide is specific binding PD-
A part of the antibody of L1 molecule, and wherein the antibody also includes polypeptide shown in SEQ ID NO:11 or 13, it is preferable that
The polynucleotide sequence is shown in SEQ ID NO:17 or 18.
12. the expression vector of the polynucleotides comprising any one of claim 9-11.
13. the host cell of the expression vector comprising claim 12.
14. the method for preparing the anti-PD-L1 antibody or its antigen-binding fragment of any one of claim 1-6, the method packet
It includes:
1) host cell as described in claim 13 is cultivated；
2) the anti-PD-L1 antibody or its antigen-binding fragment are recycled from the host cell or culture medium.
15. pharmaceutical composition contains anti-PD-L1 antibody of any of claims 1-6 or its antigen-binding fragment
And pharmaceutical carrier.
16. the anti-PD-L1 antibody or its antigen-binding fragment of any one of claim 1-6 are in preparation for improving T cell point
Secrete the purposes in the drug of IFN-γ level.
17. the anti-PD-L1 antibody or its antigen-binding fragment of any one of claim 1-6 include but not in preparation treating cancer
The purposes being limited in the anti-tumor drug of non-small cell lung cancer.
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| CN113372449A (en) * | 2020-03-10 | 2021-09-10 | 中国科学院微生物研究所 | Non-erythrocyte agglutination anti-PD-L1/CD 47 bispecific antibody and application thereof in anti-tumor treatment |
| CN116813783A (en) * | 2023-08-31 | 2023-09-29 | 苏州为度生物技术有限公司天津分公司 | Anti-human CD28 engineering antibody and application thereof |
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| CN116813783B (en) * | 2023-08-31 | 2023-10-27 | 苏州为度生物技术有限公司天津分公司 | Anti-human CD28 engineering antibody and application thereof |
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