CN104560884A - Monoclonal antibodies for antagonizing and inhibiting combination of programmed death-1 (PD-1) and ligands of PD-1, hybridoma cell line secreting monoclonal antibodies and application of monoclonal antibodies - Google Patents
Monoclonal antibodies for antagonizing and inhibiting combination of programmed death-1 (PD-1) and ligands of PD-1, hybridoma cell line secreting monoclonal antibodies and application of monoclonal antibodies Download PDFInfo
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Abstract
The invention discloses mice monoclonal antibodies for antagonizing and inhibiting combination of programmed death-1 (PD-1) and ligands of PD-1 and a hybridoma cell line secreting the monoclonal antibodies. The preservation number of the hybridoma cell line is CGMCC No. 8351. The invention further discloses an application of the monoclonal antibodies to detection of PD-1 protein.
Description
Technical field
The invention belongs to biotechnology-monoclonal antibody art.The present invention relates to a kind of antagonism and suppress PD-1(programmed death-1) monoclonal antibody of albumen and its ligand binding and secrete its hybridoma cell line and purposes.
Background technology
For a long time, biomedical boundary thinks that the immune functional state in the generation development of tumour and body is closely bound up always.In body, immunologic function plays the effect that the tumour cell of monitoring variation and Tumor suppression occur to develop in normal state.But when immune functional state in body occurs lowly or function is suppressed, the variation of tumour cell then can be accelerated with growth, more critical life time serious.Therefore a general objective in current international clinical therapy of tumor and means have been become (see summary: Topalian SL etc.: Cancer immunotherapy comes of age.JCO2011,29:4828-4836 by the immunotherapy (Immunotherapy) improved for the purpose of immunologic function; Pardoll D and Drake C:Immunotherapy earns its spot in the ranks of cancer therapy, J Exp Med2012,209:201-209).The recent breakthrough of immunotherapy and its widespread use then have benefited from amynologic basis progress and the development that is the modern biotechnology of representative with hybridoma/monoclonal antibody etc.
Immunology Today research shows that the T lymphocyte come by thymus development plays a part very crucial in identification with killing off tumor cells.T lymphocyte is functionally broadly divided into two large classes: namely to regulate and control the helper cell (T helper cells) of immunologic function and to participate in the cytotoxic T cell (cytotoxic T cells, CTL) identifying target antigen and kill and wound target cell directly.The activation of this two classes T cell and hyperplasia generally all need the synergy of two signal paths in body: wherein the 1st signal path be by T cell can receptor protein (the T-cell receptor of specific recognition antigen, TcR) with express at the target cells such as tumour or antigen presenting cell (antigen-presenting cells, APC) upper corresponding antigenic peptide-MHC (major histocompatibility complex) mixture combination and conduct, this signal path is antigen-specific; 2nd signal path is by the collaborative stimulation (co-stimulation) in T cell or collaborative suppress (co-inhibition) factor and the ligand binding of expressing on the target cells such as tumour or antigen presenting cell and conduct, and this signal path is antigen-non-specific.Just occur to activate and hyperplasia under the state that T lymphocyte only all exists at the 1st and the 2nd signal path.
The representative factor playing collaborative hormesis (i.e. forward or rise immunologic function) in the 2nd signal path is CD28 and its part B7-1, B7-2; The representative factor playing coordinate repression (i.e. negative sense or downward immunologic function) is then CTLA-4 and its part B7-1, B7-2, and programmed death acceptor (programmed death-1) PD-1 and its part PD-L1 and PD-L2; These collaborative stimulation/co-repressors are structurally all very similar, equal contactin member is (see summary: Chen LP:Co-inhibitory molecules of the B7-CD28family in the control of T cell immunity, Nature Immunol2004,336-347).
In theory, at least can reach by two different approach the object improving immunologic function in body:
Approach 1 directly reaches by the expression of the collaborative stimulating factors such as raising CD28 the effect raising immunologic function;
Approach 2 expresses by reducing or blocking the co-repressors such as CTLA-4 or PD-1/PD-L1, thus the immunosuppression that removal or reduction tumour target cell bring, and then indirectly reach the effect raising immunologic function.
Although the expression of the collaborative stimulating factors such as direct raising CD28 can reach the effect raising immunologic function, but due to 2006 Britain carry out one during to relate to code name be study clinical I phase of the anti-CD28 monoclonal antibody drug of TGN1412, namely 6 same day that health volunteers are accepting this antibody drug of injection there occurs extremely serious untoward reaction (Suntharalingam G etc.: Cytokine storm in a phase1trial of the anti-CD28monoclonal antibody TGN1412.NEJM2006,355:1018-1028), therefore scientific circles generally believe that excited type anti-CD28 antibody class medicine has very high security risk with uncertain in clinical application at present, also there is a lot of worry to the prospect of its following exploitation listing.Contrary, express and active antagonism type monoclonal antibody drug to reduce or to block the co-repressors such as CTLA-4 or PD-1/PD-L1, in recent years because it all shows obvious antitumor curative effect and acceptable security in the ongoing multiple early clinic research of the U.S., now become the research and development focus in international tumour medicine field on the contrary (see summary: Quezada SA and Peggs KS:Exploiting CTLA-4, PD-1and PD-L1to reactivate the host immune response against cancer.British Journal of Cancer2013，108：1560–1565；Flemming A.Cancer:PD-1makes waves in anticancer immunotherapy.Nat Rev Drug Discov.2012;11:601）。
PD-1 gene is found and clone in 1992 by Japanese Scientists Tasuku Honjo and colleague thereof the earliest, there is 1 GeIgVYang district its extracellular region, has homology (Ishida, the Y. of 23% with CTLA-4, Agata, Y., Shibahara, K.and Honjo, T.Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death.EMBO J.1992,11:3887; Summary is shown in: Okazaki T and Honjo T:PD-1and PD-1ligands:from discovery to clinical application.International Immunology2007,19:813-824).PD-1 is mainly expressed on the T lymphocyte of activation, bone-marrow-derived lymphocyte and monocyte (Internationl Immunology1996, the 8:675 such as Yasutoshi Agata).The acceptor of PD-1 or part there will be a known two: be respectively PD-L1(Freeman GJ etc., JEM2000,192:1027-1034), or also known as B7-H1(Dong H etc., Nature Medicine1999,5:1365-1369) with PD-L2(Latchman Y etc., Nat.Immunol.2001,2:261-268), or also known as B7-DC(Tseng SY etc., JEM2001,193:839-845).PD-L1 and PD-L2 mainly expresses (Thompson RH etc., Cancer Res2006 on the target cells such as tumour or antigen presenting cell; 66:3881-3885).These material facts that PD-1 participates in immunologic function in negative regulation body are observed in research PD-1 knock out mice by Tasuku Honjo and colleague thereof the earliest.They find that the mouse of PD-1 gene knockout is under C57BL/6 genetic background, there are lupoid acne glomerulonephritis and sacroiliitis (Nishimura H etc., Development of lupus-like autoimmune diseases by disruption of the PD-1gene encoding an ITIM motif-carrying immunoreceptor.Immunity1999,11:141); And under Balb/c genetic background, then produce the anti-cardiac muscular tissue antibody of high titre and cause serious autoimmune cardiomyopathy (Nishimura thus, H. etc., Autoimmune dilated cardiomyopathy in PD-1receptor-deficient mice.Science2001,291:319).
The expression of PD-L1 gene in normal tissue cell and the immunosuppression mediated or immunological tolerance; that its healthy tissues of endogenous protective avoids attacking and important mechanisms (the Keir M.E. etc. killed and wounded by surrounding Autologous lymphocyte; Tissue expression of PD-L1mediates peripheral T cell tolerance.J Exp Med2006,203:883-895).Tumour cell then can borrow expression by heightening PD-L1 or PD-L2 albumen and with the interaction of the PD-1 acceptor of its expression on lymphocyte and conduct suppression signal, immunity in body is made to occur suppressing or tolerating and escape lymphocytic identification and attack (Dong H etc., Nature Medicine2002,8:793-800; Azuma T etc., Blood2008,111:3635-3643); And as given PD-1 or PD-L1 antibody to block the interphase interaction of PD-1/PD-L1, then can break or eliminate the immunosuppression because tumour causes or immunological tolerance, recover the identification of T lymphocyte and the ability of attacking tumour target cell in body, and then reach good result (the Iwai Y etc. of neoplasm growth and transfer, PNAS2002,99:1229; Hirano F etc., Cancer Res 2005,65:1089-1096).Based on these fundamental research in early stage and preclinical animal studies result, have many pharmacy corporations in recent years in the world as U.S. Bristol-Myers Squibb(BMS)/Medarex, it is various by the antagonism antibody-like new drug blocked for the purpose of PD-1 and its ligand binding that the companies such as Merck company and Roche/Genentech are stepping up development, and applied for Patents for this reason.
Such as, Medarex company of the U.S. and Japanese Ono Pharmaceutical company are No8008449 U.S. patent Nos (contriver Korma AJ etc. in the patent No., denomination of invention: Human monoclonal antibodies to programmed death1 (PD-1) and methods for treating cancer using anti-PD-1antibodies alone or in combination with other immunotherapeutics) in disclose by transfection CHO cell and expression thereof containing total length or containing after birth outskirt people PD-1 albumen as hybrid antigen, the multiple anti-human hybridoma of PD-1 antibody that in cross immunity tool human normal immunoglobulin (Ig) genetically modified engineering mouse (HuMAb Moues), screening obtains and the antibody protein of secretion thereof, the nucleotide sequence of encoding antibody albumen, and this antibody is used for the application of the disease such as PD-1 Protein Detection and treatment tumour.
Merck Sharp & Dohme company is No8168757 U.S. patent Nos (contriver Finnefrock AC etc. in the patent No., denomination of invention PD-1binding proteins) in, also disclose it and screen hybridoma and antibody protein, its coding nucleotide sequence of the multiple anti-human PD-1 antibody of acquisition by containing after the DNA molecular immune mouse of PD-1 gene, and this antibody is used for the object being reached treatment tumour and infectious diseases by immunologic function in enhancing body.
Genentech company is U.S. patent Nos (the contriver Irving B etc. of No8217149 in the patent No., denomination of invention Anti-PD-L1antibodies, compositions and articles of manufacture) in, disclose the antibody protein fragment, its coding nucleotide sequence and uses thereof of showing multiple anti human PD-L 1 antibody that screening amplification obtains gene pool (phage-display) from phage antibody.
U.S. Weythe(Hui Shi) company and Medimmune(Medimmune Ltd.) state's number of patent application is 201010170022.4 wherein, denomination of invention is disclose in the patent document of " anti-PD-1 antibody and uses thereof " to show that the fragment of the anti-human PD-1 antibody obtained, its DNA sequences encoding and this antibody-like are screened as pharmaceutical cpd in storehouse from phagemid scFv gene, is used for the treatment of the application comprising the immunity system relative diseases such as autoimmune disease, transformation reactions, cancer.
U.S.'s Medarex(Mead arranges this) company's application number of then entering China at one International PCT/US2006/026046 is 200680028238.9, denomination of invention is the method disclosing its monoclonal antibody of screening the high-affinity specific combination human PD-L 1 obtained from immune mouse, its DNA sequences encoding in the patent document of " anti-programmed death ligand 1(PD-L1) human monoclonal antibodies " and be used for the treatment of the various diseases comprising cancer and transmissible disease with this antibody-like.
In PD-1/PD-L1 antibody drug research and development field, the anti-PD1 antibody Nivolumab(code name BMS-936558 of what current progress was the most forward surely belong to Bristol-Myers Squibb/Medarex company of the U.S., once code name MDX-1106 was used) and the anti-PD1 antibody lambrolizumab(code name MK-3475 of Merck company of the U.S.), these two kinds of antibody drugs have entered clinical II/III phase conceptual phase all in the U.S. at present.What be in progress next is the anti-PD-L1 antibody (code name MPDL3280A) of Roche/Genentech company, and this medicine has entered clinical I/II phase conceptual phase in the U.S. at present.These antibody drugs are mainly used in the multiple late tumors such as treatment lung cancer, melanoma, kidney, wherein early studies in man (I phase) result shows that anti-PD-1 or anti-PD-L1 antibody drug not only have Tumor suppression growth and extend the objective curative effect of survival of patients phase, and after its life-time service security also at tolerance interval (Brahmer JR etc., JCO2010,28:3167-3175; Topalian S etc., NEJM2012,366:2443-2454; Brahmer JR etc., NEJM2012,366:2455-2465; Hamid O etc., NEJM2013,369:134-144).As Topalian etc. reported in June, 2012: in an I phase clinical study having 296 routine patient with advanced cancer to participate in, the Nivolumab(code name by name giving BMS company of patient's intravenous injection U.S. for every 2 weeks is BMS-93655) anti-PD-1 antibody after, result has up to the melanoma patients of 28%, the renal cell carcinoma patients of 27% and unexpected high 18% nonsmall-cell lung cancer (NSCLC) patient, and suppressing or reducing appears in its tumour; In addition, this antibody drug also shows the feature of long-term sustainable on clinical efficacy: as in 31 patients following up a case by regular visits to more than 1 year, has 20 (64.5%) clinical signs still effectively (Topalian S etc., NEJM2012,366:2443-2454).
Similar, Hamid equals in July, 2013 report: in the I phase clinical study that another has 135 routine advanced melanoma patients to participate in, the Lambrolizumab(code name by name giving patient's intravenous injection Merck company every 2 to 3 weeks is MK-3475) anti-PD-1 antibody after, result have up to 38% patient tumors occur suppress or reduce; This antibody drug also shows the feature of long-term sustainable on clinical efficacy: as being in 52 tumour patients of 11 months following up a case by regular visits to median time, there are 42 (81%) still effective clinically, and also continuing to accept this pharmacological agent (Hamid O etc., N EJM2013,369:134-144).
In view of the combination between PD-1 and its acceptor PD-L1 or PD-L2 there is the various complexity in site, the amino acid number that relates to reaches tens with first-class feature, therefore infers the various anti-PD-1 monoclonal antibodies newly should prepared for different binding site or aminoacid sequence in theory.These monoclonal antibodies in conjunction with different loci should have new values and utilization: use as this antibody-like can merge with the anti-PD-1 being in clinical investigation phase above-mentioned at present or anti-PD-L1 antibody drug on the one hand, reach and strengthen immunologic function and better antitumor curative effect; On the other hand, this antibody-like is also expected to develop the Novel immune function enhancers or antitumor drug that become independent use.
Therefore, the more and anti-PD-1 monoclonal antibody that upgrades of research and development and the hybridoma cell line of secreting it just seem highly significant with necessity.
Summary of the invention
One of the technical problem to be solved in the present invention provides a kind of antagonism to suppress programmed death acceptor PD-1(programmed death-1) the mouse resource monoclonal antibody that combines with its part (PD-L1 and PD-L2).
Two of the technical problem to be solved in the present invention is to provide the mouse hybridoma cell system that this monoclonal antibody is produced in secretion; This hybridoma cell line code name is AB7, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (deposit number is CGMCC No.8351. preservation place: China, Beijing) on October 17th, 2013.
Three of the technical problem to be solved in the present invention is to provide the purposes of this antibody, comprises using above-mentioned antibody as detection reagent, for PD-1 albumen in quantitative and qualitative analysis biological sample.
In order to obtain anti-PD-1 monoclonal antibody that is more and that upgrade, the outer albumen of recombinant human PD-1 after birth choosing Mammals expression is in the present invention immunizing antigen, adopts traditional mouse hybridoma technology and recombination engineering method to obtain the monoclonal antibody of multiple anti-PD-1 newly.Wherein a code name is the hybridoma of AB7, monoclonal antibody of its secretion not only can with people PD-1 albumen specific combination, and the combination of PD-1 and its acceptor (PD-L1 and PD-L2) can be suppressed by antagonism.Antibody of the present invention can be used for the PD-1 albumen in quantitative and qualitative detection analysis different kind organism sample.Antibody of the present invention also can be used for preparation people-mouse mosaic gene engineered antibody and redundant organism thereof.
The present invention chooses the outer protein immunogen of recombinant human PD-1 after birth, by the mouse subcutaneous inoculation of repeated multiple times low dose, obtains the anti-PD-1 polyclonal antibody of secretion high-titer; Therefrom picking mouse again, gets its spleen cell, by the external hybridoma monoclonal cell merging with murine myeloma cell, establish many strains stably excreting anti-human VEGF antibody again through the step such as drug screening and subclone.Wherein a code name is AB7 hybridoma cell strain, through the qualification of the multiple method such as ELISA, immunoblotting, immunohistochemical methods, confirms that monoclonal antibody secreted by it can specific recognition in conjunction with people PD-1.In vitro study also proves that this monoclonal antibody can the combination of Competitive assays PD-1 and its part (i.e. PD-L1 and PD-L2).
In one aspect of the invention, there is provided a kind of can the hybridoma cell line of secrete monoclonal antibody, this hybridoma cell line code name is AB7, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as CGMCC) on October 17th, 2013, deposit number is CGMCC No.8351, preservation place: China, Beijing.
In another aspect of this invention, providing one can the antagonism monoclonal antibody that suppresses people PD-1 and its part (i.e. PD-L1 and PD-L2) to combine, be the hybridoma cell line secretion generation of CGMCC No.8351 by above-mentioned deposit number.
A third aspect of the present invention provides the purposes of this antibody, and namely this antibody is detecting the application in PD-1 albumen.As agent formulations, for the PD-1 albumen in quantitative or qualitative detection analytic sample.Adopt quantitatively and qualitative detection method be well-known in the art, comprise as, ELISA, immunoblotting, Western blot, immunofluorescence, immunoprecipitation, flow cytometer etc.
The term " monoclonal antibody (monoclonal antibody) " adopted herein refers to the immunoglobulin (Ig) obtained from a pure lines cell, has identical structure and chemical property, has specificity to single antigenic determinat.Monoclonal antibody is different from conventional polyclonal antibody preparation (normally having the different antibodies for different determinant), and each monoclonal antibody is for the single determinant on antigen.Except their specificity, the benefit of monoclonal antibody is also that they are obtained by hybridoma or recombined engineering cell cultures, can not mix and have other immunoglobulin (Ig).Modifier " mono-clonal " illustrates the characteristic of antibody, is to obtain from homogeneous antibody population, and this should not be construed as needing to produce antibody with any special methods.
The term " hybridoma cell line " (English name: hybridoma cell line) adopted herein, refers to the clone that screening and culturing goes out from hybridoma.
The term " Humanized monoclonal antibodies " adopted herein is except retaining complementary determining region (complementarity-determining regions by the aminoacid sequence of mouse resource monoclonal antibody, CDR) outward, other sequence (comprising the framework sequence in variable region) all or major part replace to the aminoacid sequence of human normal immunoglobulin, to be reached through the immunogenicity that genetic engineering means reduces mouse monoclonal antibody to greatest extent.
Term used herein " antibody " and " immunoglobulin (Ig) " have about 150000 of same structure feature daltonian different four glycan albumen, and it is made up of the heavy chain (H) that two identical light chains (L) are identical with two.Every bar light chain is connected with heavy chain by a covalent disulfide bonds, and the disulfide linkage number difference between the heavy chain of different Immunoglobulin Isotype.The intrachain disulfide bond at every bar heavy chain and light chain also regular interval.There is variable region (V one end of every bar heavy chain
h).Thereafter be multiple constant region.There is variable region (V one end of every bar light chain
l), the other end has constant region; The constant region of light chain is relative with first of heavy chain constant region, and the variable region of light chain is relative with the variable region of heavy chain.Special amino-acid residue forms interface between light chain and the variable region of heavy chain.
Term used herein " variable " represents that some part of variable region in antibody is different in sequence, which forms various specific antibodies to the combination of its specific antigen and specificity.But mutability is not evenly distributed in whole antibody variable region.It concentrates in light chain and variable region of heavy chain in three fragments become in complementary determining region (CDR) or hypervariable region.Part comparatively conservative in variable region is called framework region (Framework regions, FR).Each self-contained four FR districts in the variable region of heavy chain of antibody and light chain, they in beta sheet configuration, are connected by three CDR forming shack haply, in some cases can forming section β-pleated sheet structure structure.CDR in every bar chain is closely close together by FR district and together form the antigen-binding site (see Kabat etc., NIH Publ.No.91-3242, volume 1,647-669 page (1991)) of antibody with the CDR of another chain.Antibody constant region does not participate in the combination of antibody and antigen directly, but they show different effector functions, such as participate in cytotoxicity (the antibody-dependent cellular cytotoxicity depending on antibody of antibody, or complement-mediated toxicity (complemnt-dependent cytotoxicity, CDC) ADCC).
Suppress PD-1(programmed death-1 for obtaining a kind of antagonism) with the mouse resource monoclonal antibody of its ligand binding and the hybridoma cell line secreting it, the outer albumen of recombinant human PD-1 after birth that the present invention chooses Mammals expression is immunizing antigen, by the mouse subcutaneous inoculation of repeated multiple times low dose, obtain the polyclonal antibody of the anti-PD-1 albumen of secretion; Therefrom picking, containing the mouse of high-titer antibody, gets its spleen cell, by the external hybridoma monoclonal cell merging with murine myeloma cell, establish the antibody of the anti-human PD-1 albumen of many strains stably excreting again through the step such as drug screening and subclone again.Wherein a code name is the mouse hybridoma cell strain of AB7, through the multiple method qualification such as ELISA, immunoblotting, immunohistochemical methods, confirm that monoclonal antibody secreted by it not only can specific combination people PD-1 albumen, and the combination of suppression PD-1 albumen capable of blocking and its part (PD-L1, PD-L2).
Accompanying drawing explanation
Fig. 1 detects mouse hybridoma cell (AB7) culture supernatant protein bound result schematic diagram outer with the recombinant human PD-1 after birth be coated on 96-orifice plate with ELISA method in the embodiment of the present invention 1.The SP2/0 myeloma cell's culture supernatant wherein do not merged is negative control sample.
Fig. 2 be with the Chinese hamster ovary celI of antibody and PD-1 gene transfection and untransfected in flow cytomery AB7 Hybridoma Cell Culture supernatant liquor in embodiment 2 in conjunction with result schematic diagram.The SP2/0 myeloma cell's culture supernatant wherein do not merged is negative control; Wherein, Fig. 2 A represents the Chinese hamster ovary celI of PD-1 gene untransfected in SP2/0 negative control Supernatant samples, Fig. 2 B represents the Chinese hamster ovary celI of PD-1 gene untransfected in AB7 Hybridoma Cell Culture Supernatant samples, Fig. 2 A represents the Chinese hamster ovary celI of PD-1 gene transfection in SP2/0 negative control Supernatant samples, and Fig. 2 D represents the Chinese hamster ovary celI of PD-1 gene transfection in AB7 Hybridoma Cell Culture Supernatant samples.
Fig. 3 is with the result schematic diagram of SDS-PAGE Analysis and Identification antibody protein that affinity chromatography purification obtains from AB7 Hybridoma Cell Culture supernatant liquor in the embodiment of the present invention 3.Wherein swimming lane 1 is unreduced AB7 antibody protein samples, and swimming lane 2 is the AB7 antibody protein samples after DTT reduction; M is protein molecular weight mark.
Fig. 4 is that in embodiment 4, flow cytomery analyzes the protein bound result schematic diagram of PD-1 on people Jurkat T lymphocyte that mouse source AB7 monoclonal antibody and PHA activate.Wherein with irrelevant mouse antibodies IgG sample for negative control.Wherein, Fig. 4 A represents irrelevant mouse antibodies IgG negative control sample, and Fig. 4 B represents the sample of AB7 antibody.
Fig. 5 detects with external Inhibition ELISA in the embodiment of the present invention 5 to prove that AB7 antibody can block biotin labeled people PDL1-Fc albumen (Biotin-PDL1-Fc) and the result schematic diagram of combination being coated on people PD-1 albumen on 96-orifice plate by antagonism.AB7 & Biotin-PDL1-Fc group is wherein that AB7 antibody and Biotin-PDL1-Fc compete sample, MouseIg & Biotin-PDL1Fc group is that uncorrelated mouse antibodies and Biotin-PDL1-Fc compete sample, as negative control.
Fig. 6 detects with external Inhibition ELISA in the embodiment of the present invention 5 to prove that AB7 antibody can block biotin labeled people PDL2-Fc albumen (Biotin-PDL2-Fc) and the result schematic diagram of combination being coated on people PD-1 albumen on 96-orifice plate by antagonism.AB7 & Biotin-PDL2-Fc group is wherein that AB7 antibody and Biotin-PDL2-Fc compete sample, MouseIg & Biotin-PDL2Fc group is that uncorrelated mouse antibodies and Biotin-PDL2-Fc compete sample, as negative control.
Code name is that the mouse hybridoma cell strain (deriving from mouse Mus musculus) of AB7 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 17th, 2013 (deposit number is CGMCC No.8351; Preservation place: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica).
Embodiment
Further describe the present invention below in conjunction with embodiment, these embodiments just in order to play illustration, instead of are used for limiting the present invention.
Embodiment 1: foundation and the Screening and Identification of secreting the mouse hybridoma cell system of anti-PD-1 antibody
The acquisition of step 1. recombinant human PD-1 albumen (immunizing antigen) and animal immune
In embodiments of the present invention, the people PD-1 albumen for immunity is the outer albumen (Yi Qiao Divine Land, Beijing Products) of recombinant human PD-1 after birth of being expressed by Mammals.After this recombinant human PD-1 albumen mixes with Freund's complete adjuvant (Sigma Co., USA's product), in subcutaneous multi-point injection Balb/c mouse (100 μ l/ only, are total to 5-10 μ g PD-1 albumen).After first immunisation 2-3 week, mouse gives the mixture that subcutaneous multi-point injection contains PD-1 albumen and Freund's incomplete adjuvant (Sigma Co., USA's product) again, after booster immunization 2-3 time, take a morsel mice serum, with bag by the 96-plate of people PD-1 albumen tiring with anti-PD-1 antibody in ELISA method detection mice serum, get the cytogamy of splenocyte for next step of high person mouse of tiring.
Step 2, cytogamy
After final immunization 3 days, asepticly prepare mouse boosting cell suspension, with mouse SP2/0 myeloma cell (purchased from school of life and health sciences cyropreservation center, Chinese Academy of Sciences Shanghai), with the ratio of 5:1 at 50%PEG-1000(Sigma Co., USA product) the lower fusion of effect.Merge method (Kohler G.and Milstein C:Nature1975 routinely; 256:495-497), PEG consumption 1ml, slowly added in 1 minute.React after 90 seconds, with the RPMI-1640 substratum termination reaction of serum-free, the centrifugal 10min of 1000rpm, remove supernatant liquor, again cell concn is adjusted to 1 Х 10 with the RPMI1640-10% FCS substratum containing 10% HAT (H is xanthoglobulin, the amino dish purine of A, T thymidine, is Sigma Co., USA's product) by the cell under centrifugation
6/ ml, adds the 96 flat Tissue Culture Plates in hole (every hole 200 μ l), in 37 DEG C, cultivates 2-3 week in 5%CO2 incubator.
The mouse hybridoma cell that the secretion of step 3, enzyme linked immunosorbent assay (ELISA) screening antibodies is positive
With recombinant human PD-1 albumen (2 μ g/ml, pH9.6,0.1M NaHCO3 liquid) coated elisa plate, 37 DEG C of bags are spent the night by 2 hours or 4 DEG C; 2% bovine serum albumin (BSA) 4 DEG C is closed and is spent the night.After the washing of PBS-0.1%Tween20 liquid, add Hybridoma Cell Culture supernatant to be checked (with the thin culture supernatant of SP2/0 myelomatosis do not merged for negative control) 37 DEG C hatch 2 hours; After the washing of PBS-0.1%Tween20 liquid, add the goat anti-mouse igg (Sigma Co., USA's product) that horseradish peroxidase HRP-marks, hatch 1 hour for 37 DEG C; Again after PBS-0.1%Tween20 liquid fully washs, add O-Phenylene Diamine (OPD)-0.1%H
2o
2substrate solution colour developing 10-15min, with 0.1M HCl termination reaction.492nm place OD value is read in MK3-Multiskan microplate reader (U.S. Thermo Scientific Products).The OD492 value recorded is than negative control height 5-10 hybridoma cloning more doubly, and it is frozen to carry out amplification.
Subclone-the limiting dilution assay of step 4, positive hybridoma cell
The positive cell obtained by above-mentioned primary dcreening operation is diluted to every hole 1-10 cell with RPMI-1640-10%FCS substratum, is laid on 96-porocyte culture plate, in 37 DEG C, cultivates 2-3 week in 5%CO2 incubator.Treat that clone grows up to, get supernatant liquor with the secretion of the ELISA anti-PD-1 antibody of Testing and appraisal again.Identify after testing, obtain the strain of multiple antibody-secreting positive cell.Through subclone qualification, wherein a strain code name is the anti-PD-1 monoclonal antibody of AB7 hybridoma cell strain Absorbable organic halogens secreting, expressing.Fig. 1 detects with ELISA method to survey AB7 hybridoma supernatant and the protein bound result schematic diagram of recombinant human PD-1, and result proves the antibody of this hybridoma supernatant containing the anti-human PD-1 albumen of high-titer.This antibody is through being accredited as IgG class.This hybridoma cell strain is again through a large amount of amplification, and Long Term Passages is cultivated and is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (deposit number is CGMCC No.8351. preservation place: China, Beijing) on October 17th, 2013.
Embodiment 2 flow cytomery analyzes the combination of the Chinese hamster ovary celI of mouse source AB7 monoclonal antibody and people PD-1 gene transfection
In the present embodiment, with AB7 antibody for primary antibodie, be two to resist with the fluorescently-labeled goat anti-mouse igg of FITC, adopt flow cytomery to analyze AB7 antibody and be combined with the Chinese hamster ovary celI of people PD-1 gene transfection.For this reason, first from human peripheral lymphocyte (PBL), extract total mRNA, then obtain the cDNA fragment containing coding PD-1 full-length proteins (comprising outside born of the same parents and intracellular region) with Reverse transcription-polymerase chain reaction (RT-PCR) method amplification in vitro.Primer for RT-PCR is PD-1 forward primer: ATTAAGCTTGAGCAGTGGAGAAGGCGG, PD-1 reverse primer: AATTGGATCCCTCCTGAGGAAATGGGCTGA).Forgive the whole coding region of PD-1 albumen with this to primer amplification acquisition cDNA fragment, length is 989bp.This DNA after restriction enzyme ferment treatment, then is inserted into expression plasmid pCDNA3.1(American I nvitrogen company) in, thus obtain the recombinant plasmid pCDNA3.1-PD-1 containing PD-1cDNA.After being mixed with Fugen-6 liposome (Roche Products) by this expression plasmid subsequently, cotransfection enters Chinese hamster ovary celI (purchased from American Invitrogen company), transfection is after 2 days, cell is after centrifugation, heavily be dissolved in the solution containing mouse source AB7 mono-clonal supernatant (for test sample) or SP2/0 supernatant (for negative control) respectively, hatch 1 hour for 4 DEG C; After the washing of PBS-0.1%Tween20 liquid, add the goat anti-mouse igg (Sigma Products) of FITC-mark, hatch 1 hour for 4 DEG C; Again after the washing of PBS-0.1%Tween20 liquid, by sample loading to Cytomics FC500MCL) flow cytomery (Beckman Coulter company of the U.S.).
Fig. 2 is this flow cytomery result schematic diagram.As shown in Figure 2: in the sample of transfected with human PD-1 gene, have nearly 30% cell can with AB7 monoclonal antibody specific combination (Fig. 2 D); And in SP2/0 negative control Supernatant samples, no matter be that transfection (Fig. 2 C) or the Chinese hamster ovary celI of untransfected (Fig. 2 A) all combine without obvious; The Chinese hamster ovary celI of untransfected and AB7 antibody, also without obviously combining (Fig. 2 B), show that this flow cytomery result has higher specificity.
The external preparation in embodiment 3. mouse source anti-human PD-1 monoclonal antibody (AB7) and purifying
In the present embodiment, affinity chromatography separation and purification is adopted to obtain mouse-anti people PD-1 monoclonal antibody (AB7) albumen.
Its purification step is as follows:
After AB7 hybrid tumor cell amplification, be inoculated in 1640 substratum of 200-500ml serum-free, cultivate 5 days, collect culture supernatant subsequently for 37 DEG C, after 0.45 μm of membrane filtration, loading is to containing Protein G-Sepharose Fast Flow(purchased from GE company of General Electric) affinity chromatographic column; After chromatography column first removes foreign protein with the rinsing of PBS liquid, then with low pH (2.7-3.0) glycine (0.1M) liquid wash-out by the AB7 antibody protein adsorbed.Elutriant regulates pH to 7.0 with 1mol/L Tris (pH8.5-9.0), then to after the PBS liquid dialysis 12 ~ 16 of 10 times of volumes (period changes liquid 2-3 time), namely the sample after dialysis obtains the AB7 antibody of purifying again after 0.45 μm of membrane filtration.
The AB7 antibody of purifying is carried out according to a conventional method under DTT reduction and non-reductive condition through Polyacrylamide Gel Electrophoresis (SDS-PAGE) (separation gel is 10%, concentrated glue 5%).Fig. 3 is this electrophoretic analysis result collection of illustrative plates: wherein swimming lane 1 is unreduced complete AB7 antibody samples, and molecular weight is at about 150kD; Swimming lane 2 is the AB7 antibody samples after DTT reduction, and be separated into 2 master tapes, being wherein in molecular weight band on about 50kD is AB7 heavy chain of antibody, and being in molecular weight band below about 25kD is AB7 light chain of antibody.
Embodiment 4 mouse source AB7 monoclonal antibody analyzes the PD-1 albumen of expressing on human T lymphocyte for detecting
In the present embodiment, AB7 monoclonal antibody is as primary antibodie, and the fluorescently-labeled goat anti-mouse igg of FITC resists as two, analyzes the PD-1 albumen of expressing on human T lymphocyte for detecting.Human T lymphocyte is wherein Jurkat cell (purchased from school of life and health sciences cyropreservation center, Chinese Academy of Sciences Shanghai).For this reason, first Jurkat cell is placed in containing T lymphocyte activating factor (LAF) PHA(Sigma Co., USA product, PHA final concentration: 3 μ g/mL) RPMI-10%FCS nutrient solution in cultivate to activate and the expression of inducing PD-1 albumen.Activate and induce after 24-48 hour, cell is heavily dissolved in test sample AB7 antibody (whole solubility: 5 μ g/ml) containing purifying or irrelevant mouse IgG (MouseIgG respectively after centrifugation, negative control sample, whole solubility: 5 μ g/ml) solution in, hatch 1 hour for 4 DEG C; After the washing of PBS-0.1%Tween20 liquid, add the goat anti-mouse igg (Sigma Products, 1:200) of FITC-mark, hatch 1 hour for 4 DEG C; Again after the washing of PBS-0.1%Tween20 liquid, by sample to Cytomics FC500MCL flow cytomery (Beckman Coulter company of the U.S.).Flow cytometry analysis.
Fig. 4 is the result collection of illustrative plates that this flow cytomery is analyzed.As shown in Figure 4 B: the Jurkat cell that AB7 monoclonal antibody sample can activate with PHA is combined, with non-activated Jurkat cell without obvious association reaction.In irrelevant mouse IgG negative control sample (Fig. 4 A), no matter be activation or non-activated Jurkat cell, all without obvious association reaction.
Embodiment 5 AB7 monoclonal antibody biological activity determination: the combination of competitive ELISA method Testing and appraisal mouse source its acceptor of AB7 monoclonal antibody extracorporeal blocking PD-L1 and PD-L2 and PD-1 albumen
One of bioactive method of external qualification AB7 monoclonal antibody adopts competitive ELISA method to detect the combination whether this antibody can block PD-1 and its PD-1 part (PD-L1 and PD-L2).The ultimate principle of this competitive ELISA method from process is: first biotin labeled people PD-1 ligandin (as PDL1-Fc or PDL2-Fc fusion rotein) mixed with the monoclonal antibody of different solubility, again mixture is proceeded to the 96-orifice plate being coated with PD-1 albumen in advance afterwards, through hatching and after wash-out, adding the Avidin of Avidin(as horseradish peroxidase-labeled of enzyme labelling); Again through hatching and after wash-out, adding substrate and show and measure OD value.
The concrete steps that this competitive ELISA method detects are as follows:
1) wrap by 96-orifice plate (wrapping by solubility: 2 μ g/ml, 50 μ l/ holes) with the outer albumen (Yi Qiao Divine Land, Beijing Products) of recombinant human PD-1 after birth, 4 DEG C are spent the night;
2) after the rinsing of PBS liquid and 2%BSA (being diluted in PBS-0.1%tween20 liquid) room temperature are closed, add containing the biotin labeled PDL1-Fc albumen of fixing solubility or PDL2-Fc albumen (PDL1-Fc albumen and PDL2-Fc albumen are Yi Qiao Divine Land, Beijing Products) the AB7 antibody from different solubility respectively, or uncorrelated mouse antibodies (mouse IgG), hatch 2h for 37 DEG C;
3) after PBS-T wash-out, the Avidin(1:5000 of horseradish peroxidase-labeled is added), hatch 1h for 37 DEG C;
4) after PBS-T wash-out, add nitrite ion (O-Phenylene Diamine)-3% hydrogen peroxide, room temperature 10min is to colour developing;
5) add HCL termination reaction, measure the light absorption value in each hole, 492nm wavelength place with enzyme linked immunological instrument.
Fig. 5 is the representative result of AB7 antibody in vitro and biotin labeled PDL1-Fc albumen (Biotin-PDL1Fc) competition binding PD-1 albumen.As shown in Figure 5: adding in the sample that AB7 antibody and biotin labeled PDL1-Fc albumen (AB7 & Biotin-PDL1Fc) competes, each hole color reaction OD value and the AB7 antibody protein amount that adds be inversely: the amount of the AB7 antibody namely added is higher, and its OD value is lower.And adding in the sample that irrelevant mouse antibodies and biotin labeled PDL1-Fc albumen (MouseIgG & Biotin-PDL1Fc) competes, the antibody protein amount that each hole color reaction OD value is not added affects.This result clearly shows that AB7 antibody can be competed and blocks PD-L1(PDL1-Fc) with the combination of PD-1.
Fig. 6 is then the representative result of AB7 antibody in vitro and biotin labeled PDL2-Fc albumen (Biotin-PDL2Fc) competition binding PD-1 albumen.As shown in Figure 6: adding in the sample that AB7 antibody and biotin labeled PDL2-Fc albumen (AB7 & Biotin-PDL2Fc) competes, each hole color reaction OD value and the AB7 antibody protein amount that adds be inversely: the amount of the AB7 antibody namely added is higher, and its OD value is lower.And adding in the sample that irrelevant mouse antibodies and biotin labeled PDL2-Fc albumen (MouseIgG & Biotin-PDL2Fc) competes, the antibody protein amount that each hole color reaction OD value is not added affects.This result shows that AB7 antibody also can be competed and blocks PD-L2(PDL2-Fc) with the combination of PD-1.
Claims (3)
1. a hybridoma cell line for secrete monoclonal antibody, the deposit number of this hybridoma cell line is CGMCC No.8351.
2. antagonism suppresses a mouse resource monoclonal antibody for programmed death acceptor PD-1 and its ligand binding, is secreted produce by hybridoma cell line according to claim 1.
3. antibody according to claim 2 is detecting the application in PD-1 albumen.
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