CN104974253A - Anti-CTLA-4/PD-1 bispecific antibody as well as preparation method and application thereof - Google Patents
Anti-CTLA-4/PD-1 bispecific antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the biotechnology field, and more particularly discloses an anti-CTLA-4/PD-1 bispecific antibody as well as a preparation method and an application thereof. The anti-CTLA-4/PD-1 bispecific antibody disclosed by the invention can be combined with CTLA-4 and PD-1 and effectively enhances immune response by blocking the CTLA-4 and/or PD-1 signal transduction pathways, and therefore, the anti-CTLA-4/PD-1 bispecific antibody may have good anti-tumor immune response enhancement effect.
Description
Technical field
The invention belongs to biological technical field, more specifically, the invention discloses class bi-specific antibody and an its preparation method, and it prepares the application in antitumor drug.
Background technology
Two kinds of signals are at least needed to exist in the process that the activation startup of T cell and effect play, the first signal is antigen-specific, by major histocompatibility complex (Major histocompatibility complex, MHC) and with the antigen peptide of its combination by φt cell receptor (T cell receptor, TCR) identify and after combining, in T cell, conduct activation signal by TCR mixture; The second activation signal is antigen-non-specific, by antigen presenting cell (antigen presenting cell, APC) pairing and in T cell between multiple accessory molecule and interaction provide, a class T cell membranin of the second activation signal can be provided to be otherwise known as costimulatory molecules, wherein CD28 is the important costimulatory molecules of a kind of expression on T cell surface, plays a significant role in T cell activation.After TCR and MHC-antigenic peptide complexes combines, if costimulatory molecules lacks, by causing T cell to be incompetent state to special antigen expression, not only can not be activated, and the specific activation signals of antigen is not all replied; If costimulatory molecules as CD28 and B7 molecular juction merges activate after, can the activation of mediate T cell in multiple level.The T cell of activation the form of autocrine and paracrine can produce interleukin II (interleukin2; IL-2), act on the acceptor IL2R of cell surface, stimulate the propagation of T cell further, thus ready for playing effector function.
Cytotoxic T lymphocyte associated antigen-4 (cytotoxic T lymphocyte-associated antigen-4, CTLA-4) CD152 is had another name called, belong to leukocyte differentiation antigen, it is a kind of transmembrane receptor in T cell, jointly enjoy B7 molecule ligand (B7-1/CD80, B7-2/CD86) with CD28, and CTLA-4 and B7 molecule can bear the reactive behavior lowering T cell after combining, participating in immunoreactive negative regulation, is therefore a kind of immune Co inhibitor.The activity of combination to T cell of the B7 family molecule on APC and the aglucon CTLA4 in T cell plays important negative regulation effect, blocks this signal path, can promote the activation of T cell.The avidity of CTLA-4 and B7 is significantly higher than CD28, can with the B7 molecule on CD28 competition binding APC surface, block CD28 and B7 costimulatory signal conduction, the generation of suppressor T cell antigen recognition second signal, thus performance immunosuppressive effect.Under normal circumstances, be stored in the vesica in T cell kytoplasm after CTLA-4 synthesis, activation signal can induce CTLA-4 to insert on cytolemma, the CTLA-4 on film surface thus play restraining effect with CD28 competition binding respective ligand, and this refers to a kind of normal negative feedback mechanism.
Large quantity research shows, tumour is in forming process, and by mechanism and multiple, the supervision of escape from immune system, this process is referred to as immunologic escape or the immunoediting of tumor tissues by us.Tumour cell escapes immune attack by following approach: 1. the antigenic modulation of tumour antigen causes immunogenicity to reduce; 2. tumor cell surface MHC developed by molecule defect or expression amount reduce, as offered the defect of antigen peptide process and submission genes involved (as LMP/TAP gene); 3. tumour cell suppresses the lethal effect of NK cell and part cytotoxic T cell by non-classical human leucocyte antigen (humanleukocyte antigen, HLA) equimolecular (as HLA-G and HLA-E); 4. tumour cell can autocrine or some inhibitive ability of immunity cytokines of paracrine, and as IL-10, TGF-β etc., these factors can weaken the lethal effect of immunity system to tumour, also can the immunosuppressive action of activating regulatory T-cells; 5. tumour cell costimulatory molecules part and expression of adhesion molecule decline, or the part of tumour cell high expression level Co inhibitor; 6. tumour cell discharges tumour antigen molecule, becomes mixture with antibodies, by the Fc receptors bind of the Fc section of antibody and lymphocyte, NK cell, scavenger cell, thus closes ADCC effect; 7. the part of tumor cell secretion solubility activated receptor, as the part of NK cell activation receptors, lowers the expression of immunocyte surface active acceptor, immune effector cell function is lowered; 8. the coming off of tumor mortality acceptor; 9. the tolerance (being caused by host antigen presenting cells, medullary cell or regulatory T cells) of T cell tumour specific antigen.T cell is in core status in the process of tumour immunity, if can transfer the antipersonnel weapon in body, will be very effectively and the therapeutic strategy of safety.The Immune escaping mechanism of tumour and body are to there is very complicated relation between the immunne response of tumour.In the process of immunotherapy of tumors, the specific CD8 positive T cell of infantile tumour activates, and along with tumor growth loses the function of killing and wounding to the later stage, tumour produces resistibility.Generally speaking, self immune system response original should be activated in vivo, the reaction strengthen continuously transferred also will be allowed to be only the best strategy of tumor immunotherapy.
The activation of T cell is except needs provide the first signal by APC submission MHC-antigen peptide to T cells with antigenic specificity, a series of costimulatory molecules is also needed to provide second signal, and then T cell just can be made to reach physiological activation threshold value produce normal immunne response, this explains regulation mechanism accurate and delicate when immunity system produces immunological response to self and non-self antigen in theory better.If lack the second signal that costimulatory molecules provides, the anergy of T cell or specific immunologic tolerance will be caused even to enter apoptosis, therefore, the adjustment of positivity and negativity costimulatory signal and being equilibrated in the whole process of polar body immunne response between the two play important regulating effect.
PD-1(programmed death-1) be obtain in the T cell hybridoma of apoptosis at first, be named as programmed death-1 acceptor because it is relevant with apoptosis.At present, the part of PD-1 has been proved two, is PD-L1(B7-H1 respectively) and PD-L2(B7-DC).PD-L1(B7-H1) belong to B7 family, have IgV and IgC sample district, cross-film district and cytoplasmic domain afterbody, the acceptor PD-1 in PD-L1 and its T cell interacts, and plays an important role in the negative regulate of immunne response; This molecule has broad distribution expression pattern, and wide expression is in antigen presenting cell (APCs), activation T/B cell, scavenger cell, placental trophoblast, myocardium endothelium and thymic cortical epithelial cells.PD-L1 all can detect the expression of PD-L1 albumen in many mankind tumor tissues, and the PD-L1 expression level in many cancerous tissue compared with normal tissues obviously raises.Application immunohistochemical method, in the mankind tumor tissues such as mammary cancer, lung cancer, cancer of the stomach, intestinal cancer, the esophageal carcinoma, ovarian cancer, cervical cancer, kidney, bladder cancer, carcinoma of the pancreas, neurospongioma, melanoma, the expression of PD-L1 albumen successively detected, and the clinical and prognosis of the expression level of PD-L1 and patient is closely related.Much research all shows that PD-L1 is relevant to the Immune escaping mechanism of tumour.Research shows, the PD-L1 that the APC in tumour cell and tumor microenvironment expresses all through the activation of PD-1/PD-L1 signal path Tumor suppression T cells with antigenic specificity, can lower the tumor immune response of T cell mediation.Experimental results demonstrate, block the increment that PD-1/PD-L1 signal can promote specific for tumour antigen T cell, play the effect of killing tumor cell, effective Tumor suppression growth.Therefore, intervene PD-1/PD-L1 signal and be expected to the New Policy becoming immunotherapy of tumors.
In antineoplastic immune, the immunne response of the cytotoxic T cell of the CD8 positive plays central role, and its primary process can be divided into antigen recognition, cell activation/proliferation/differentiation and effect to kill and wound the stage.Wherein Co inhibitor CTLA-4 and PD-1 plays important negative sense immunoregulation effect in T cell activation/proliferation/differentiation stage and the effect stage of killing and wounding respectively.Under normal circumstances, play an important role in the maintenance that these two kinds of molecules tolerate at autoimmunity, the generation of autoimmune disease can be prevented; But in tumour patient body, these two kinds of molecules may play a part to suppress antineoplastic immune.Clinical study finds, anti-CTLA-4 antibody and the antibody combined application of anti-PD-1 are used alone than arbitrary antibody has better anti-tumor activity.Therefore, work out the bi-specific antibody that simultaneously can block CTLA-4 and PD-1 immune suppression function, thus more effectively strengthen antineoplastic immune, this kind of high efficiency anti-tumor medicine is those skilled in the art's problem anxious to be resolved always.
Summary of the invention
In order to solve the problem, the present inventor has carried out lot of experiments, and obtaining one can be combined with CTLA-4, the bi-specific antibody that can also combine with PD-1, thus complete the present invention i.e. " anti-CTLA-4/PD-1 bi-specific antibody " of the present invention.
The invention discloses:
1. an anti-CTLA-4/PD-1 bi-specific antibody, it can be combined with CTLA-4, can also combine with PD-1.
2. the anti-CTLA-4/PD-1 bi-specific antibody described in above-mentioned 1, it comprises anti-CTLA-4 variable region of mab and anti-PD-1 variable region of mab.
3. the anti-CTLA-4/PD-1 bi-specific antibody described in above-mentioned 1 or 2, its light-chain amino acid sequence is as shown in SEQID NO:4, and heavy chain amino acid sequence is for shown in SEQ ID NO:2.
4. the nucleic acid molecule be separated, the arbitrary described bi-specific antibody of its above-mentioned 1-3 that encodes, preferably, its light chain nucleotide sequence of described nucleic acid molecule is for shown in SEQ ID NO:3, and heavy chain nucleotide sequence is for shown in SEQ ID NO:1.
5. an expression vector, containing the expression regulation sequence that above-mentioned 4 or 5 arbitrary described nucleic acid molecule are connected with the sequence being operational of described nucleic acid molecule, preferably, described carrier can be pCHO1.0, pBudCE4.1 or pCGS3 carrier, most preferably is pCHO1.0 carrier.
6. a host cell, it contains the carrier described in above-mentioned 5.Preferably, described host cell is mammalian cell, and more preferably, described host cell is COS, CHO, NS0; Most preferably, described host cell is
or
gS
-/-cell.
7. prepare a method for above-mentioned 1 ~ 3 arbitrary described anti-CTLA-4/PD-1 bi-specific antibody, the method comprises:
A) under expression condition, cultivate the host cell described in above-mentioned 6, express anti-CTLA-4/PD-1 bi-specific antibody;
B) isolated or purified a) described in anti-CTLA-4/PD-1 bi-specific antibody.
Preferably, described preparation method comprises the following steps:
A) heavy chain fusion gene and the light chain fusion gene of anti-CTLA-4/PD-1 bi-specific antibody is built;
B) by above-mentioned a) fusion gene cloning to expression vector pCHO1.0, construction of expression vector pCHO1.0(DVD-CTLA-4/PD-1);
C) by above-mentioned b) expression vector pCHO1.0(DVD-CTLA-4/PD-1) transfection CHO cell, screening, carries out subclone, carries out enlarged culturing by screening the high-expression clone obtained;
D) isolated or purified c) the anti-CTLA-4/PD-1 bi-specific antibody that obtains.
8. a composition, containing above-mentioned 1 ~ 3 arbitrary described anti-CTLA-4/PD-1 bi-specific antibody and pharmaceutically acceptable carrier.
9. above-mentioned 1 ~ 3 arbitrary described anti-CTLA-4/PD-1 bi-specific antibody or the composition described in above-mentioned 8 are preparing the purposes in antitumor drug.
10. the purposes described in above-mentioned 9, it also comprises the antitumor drug conbined usage with other.
The suitable DNA of any coding anti-CTLA-4 antibody variable region and anti-PD-1 antibody variable region is suitable for the present invention.
In the present invention, any suitable carrier can use, and can be one of pCHO1.0, pBudCE4.1 or pCGS3, preferred pCHO1.0, and expression vector comprises and is connected with the suitable fusion dna sequence with translational regulation sequence of transcribing.
The expression of Mammals or insect host cell culture systems fusion rotein used in the present invention, the zooblasts such as COS, CHO, NS0, sf9 and sf21 are all applicable to the present invention, as being
or
gS
-/-cell.
One of available host cell also can be the prokaryotic cell prokaryocyte containing above-mentioned carrier, can be DH5 α, BL21(DE3) and TG1.
Disclosed in the present invention, the preparation method of anti-CTLA-4/PD-1 bi-specific antibody is cultivate above-mentioned host cell under expression condition, thus expresses bi-specific antibody, and then the bi-specific antibody described in isolated or purified.
The method of affinity chromatography can be utilized to carry out separation and purification to bi-specific antibody disclosed by the invention, according to the characteristic of utilized affinity column, the conventional method such as fusion rotein polypeptide of method elution of bound on affinity column such as high-salt buffer, change PH can be used.
Utilize aforesaid method, can be substantially homogeneous material by fusion protein purification, such as, on SDS-PAGE electrophoresis, be shown as single band.
Above-mentioned anti-CTLA-4/PD-1 bi-specific antibody disclosed by the invention obtains by the following method: full-length gene (light chain and the heavy chain) sequence of the anti-CTLA-4 monoclonal antibody in total man source is (see the aminoacid sequence that patent US8318916B2 announces, variable region of light chain SEQ ID NO7, variable region of heavy chain SEQ ID NO17, constant region of light chain SEQ ID NO39, CH SEQ ID NO40) synthesized by Suzhou Jin Weizhi biotechnology Services Co., Ltd full genome, full-length gene (light chain and the heavy chain) sequence of the anti-PD-1 monoclonal antibody in total man source is (see aminoacid sequence disclosed in patent US20130133091A1, variable region of light chain SEQ ID NO8, variable region of heavy chain SEQ ID NO3, light chain is identical with SEQ IDNO39-40 described in the above-mentioned US8318916B2 of CH) also synthesized by Suzhou Jin Weizhi biotechnology Services Co., Ltd full genome.Recombinate in rear clone to pCHO1.0 carrier with over-lap PCR method with the heavy chain of anti-PD-1 monoclonal antibody and light chain gene respectively and are built into carrier for expression of eukaryon pCHO1.0(DVD-CTLA-4/PD-1 in the heavy chain of anti-CTLA-4 monoclonal antibody and variable region of light chain), above-mentioned plasmid liposome transfection CHO-S cell, and with screening the cell clone of the anti-CTLA-4/PD-1 bi-specific antibody of stably express containing the methotrexate (MTX) of proper concn and the Selective agar medium of tetracycline.Utilize the anti-CTLA-4/PD-1 bi-specific antibody of Protein A post affinity chromatography purifying from cell culture supernatant.Gained anti-CTLA-4/PD-1 bi-specific antibody variable region comprises above-mentioned anti-CTLA-4 monoclonal antibody variable region and above-mentioned anti-PD-1 monoclonal antibody variable region, its constant region is above-mentioned anti-PD-1 monoclonal antibody constant region, anti-its light-chain amino acid sequence of CTLA-4/PD-1 bi-specific antibody is as shown in SEQ ID NO:4, and heavy chain amino acid sequence is for shown in SEQ ID NO:2.
Applicant of the present invention carries out avidity detection, mixed lymphocytes proliferation experiment, the experiment of protein drug dynamic metabolism to above-mentioned anti-CTLA-4/PD-1 bi-specific antibody, experimental result shows, anti-CTLA-4/PD-1 bi-specific antibody disclosed by the invention both can combine with one of CTLA-4 or PD-1, also can be combined with CTLA-4 and PD-1 simultaneously, block its intracellular signaling function.In addition, this anti-CTLA-4/PD-1 bi-specific antibody has excellent pharmacokinetic property.Experiment shows, under equal dose, anti-CTLA-4/PD-1 bi-specific antibody has than the independent anti-CTLA-4 antibody of application and the better antitumous effect of anti-PD-1 antibody, reaches object of the present invention.The present invention discloses above-mentioned anti-CTLA-4/PD-1 bi-specific antibody; can pharmaceutical preparations composition be formed thus more play consistently curative effect together with pharmaceutically acceptable auxiliary material; these preparations can ensure the Conformational Integrity of fusion receptors amino acid core sequence disclosed by the invention, also want the polyfunctional group of protected protein matter to prevent its degraded (including but not limited to cohesion, deamination or oxidation) simultaneously.Generally, for liquid preparation, usually can stablize under 2 DEG C of-8 DEG C of conditions and preserve at least one year; For freeze-dried preparation, within least six months, keep stable at 30 DEG C.Here preparation can be the conventional preparation such as suspendible, liquid drugs injection, freeze-drying of pharmacy field, preferred liquid drugs injection or freeze-dried preparation.For liquid drugs injection or the freeze-dried preparation of above-mentioned anti-CTLA-4/PD-1 bi-specific antibody disclosed by the invention, pharmaceutically acceptable auxiliary material includes but not limited to tensio-active agent, solution stabilizer, isotonic regulator and damping fluid one or a combination set of, wherein tensio-active agent includes but not limited to: nonionic surface active agent is as Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80), poloxamer(is as poloxamer188), Triton, sodium lauryl sulphate (SDS), Sulfuric acid,monododecyl ester, sodium salt, tetradecyl, sub-oil base or octadecyl sarkosine, Pluronics, MONAQUAT
tMdeng, its add-on should make the granulating trend of anti-CTLA-4/PD-1 bi-specific antibody minimum, solution stabilizer can be carbohydrate, amino acids or alcohols, wherein carbohydrate can be reducing sugar and nonreducing sugar, amino acids comprises msg powder type or Histidine, alcohols comprises trivalent alcohol, senior sugar alcohol, propylene glycol, polyoxyethylene glycol one or a combination set of, keeps steady state in the time that the add-on of solution stabilizer should make last preparation those skilled in the art formed think to reach stable, isotonic regulator can be one of sodium-chlor, N.F,USP MANNITOL, damping fluid can be one of Tris, histidine buffering liquid, phosphate buffered saline buffer.
Above-mentioned preparation is the composition comprising anti-CTLA-4/PD-1 bi-specific antibody, and after to the animals administer comprising people, antitumous effect is obvious.Specifically, preventing and/or treating effectively tumour, can use as antitumor drug.
The tumour of indication of the present invention, include but not limited to: gland cancer, leukemia, lymphoma, melanoma, sarcoma, the source of tumor tissues includes but not limited to suprarenal gland, gall-bladder, bone, marrow, brain, mammary gland, bile duct, gi tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate gland, skin, sialisterium, spleen, testis, thymus gland, Tiroidina and uterus.Except above-mentioned tumour, also can be used for the tumour of central nervous system as spongiocyte diversity knurl, astrocytoma etc., in addition the tumour of eye comprises rodent cancer, squamous cell carcinoma, melanoma etc., also comprise endocrine disrupting effects, neuroendocrine system tumour, gi tract pancreatic endocrine system tumor, genital system tumor and tumor of head and neck etc.Here will not enumerate.
Antitumor drug alleged by the present invention, refer to that there is the medicine suppressing and/or treat tumour, can postpone with the related indication development of tumor growth and/or the severity reducing these symptoms, it can also alleviate the already present symptom that accompanies with tumor growth further and prevent the appearance of other symptoms, also reduces or prevents transfer.
In the present invention, anti-CTLA-4/PD-1 bi-specific antibody and composition thereof are to when comprising the animals administer of people, dosage is because of age of patient and body weight, disease traits and seriousness, and route of administration and different, can with reference to zooperal result and all situations, total dosage can not exceed certain limit.Specifically intravenous dosage is 1 ~ 1800mg/ days.
Anti-CTLA-4/PD-1 bi-specific antibody disclosed by the invention and composition thereof can also with other antitumour drug Combined Preparation to reach the object of more effectively treating tumour, and these antitumour drugs include but not limited to: 1, cytotoxic drug: as (1) acts on the medicine of nucleic acid chemistry structure: alkylating agent is as nitrogen mustards, nitrosourea, methanesulfonate ester class; Platinum compound is as cis-platinum (Cisplatin), carboplatin (Carboplatin) and RP-54780 (Oxaliplatin) etc.; Antibiotics is as Zorubicin (Adriamycin/Doxorubicin), dactinomycin (Dactinomycin D), daunorubicin (Daunorubicin), pidorubicin (Epirubicin), aclacinomycin (Aclarubicin), Plicamycin (Mithramycin) etc.; (2) medicine of nucleic acid metabolism is affected: dihydrofolate reductase inhibitor is as Rheumatrex (MTX) and pemetrexed (Pemetrexed) etc.; Thymus nucleoside synthetase inhibitors is as fluorouracil (5-FU, capecitabine) etc.; Purine nucleoside synthetase inhibitors is as Ismipur etc.; Ribonucleotide reductase inhibitor is as hydroxyurea (Hydroxycarbamide) etc.; DNA polymerase inhibitor is as cytosine arabinoside (Cytosine arabinoside) and gemcitabine (Gemcitabine) etc.; (3) medicine of tubulin is acted on: docetaxel (Docetaxel), vincaleucoblastine (Vincristine), vinorelbine (Vinorelbine), Podophyllum emodi var chinense alkali class, homoharringtonine etc.; 2, hormone medicine: estrogen antagonist is as tamoxifen (Tamoxifen), droloxifene (Droloxifene), Exemestane (Exemestane) etc.; Arimedex is as aminoglutethimide (Aminoglutethimide), Formestane (Formestane), letrozole (Letrozle), Anastrozole (Anastrozole) etc.; Androgen antagonist: its ammonia RH-LH agonist/antagonist of fluorine: Zoladex, enatone etc.; 3, biological response modifier class medicine: this type of medicine mainly through conditioner body immunity function with to antineoplastic effect, as interferons (Interferon); Interleukin II (Interleukin-2); Thymic (Thymosins) etc.; 4, monoclonal antibodies medicine: bent appropriate former times monoclonal antibody (Trastuzumab), Rituximab (Rituximab), Cetuximab (Cetuximab), rhuMAb-VEGF (Bevacizumab) etc.; 5, other series antineoplastic medicaments: comprise some current mechanism and be still not clear, need the medicine etc. of further research.Anti-CTLA-4/PD-1 bi-specific antibody disclosed by the invention and composition thereof can with above-mentioned antitumor drug one or a combination set of drug combination.
Accompanying drawing explanation
Fig. 1. anti-CTLA-4/PD-1 bi-specific antibody and PD-1 bonding force are tested;
Fig. 2. anti-CTLA-4/PD-1 bi-specific antibody and CTLA-4 bonding force are tested;
Fig. 3. the mixed lymphocytes proliferation experiment of anti-CTLA-4/PD-1 bi-specific antibody
Fig. 4. anti-CTLA-4/PD-1 bi-specific antibody time front of blood concentration
Embodiment
Following examples, experimental example further illustrate of the present invention, should not be construed as limitation of the present invention.Embodiment does not comprise the detailed description to ordinary method, as the method that those draw for carrier construction and matter, the gene of proteins encoded is inserted into the method that such carrier and matter are drawn or method plasmid being introduced host cell.Such method is well-known to person having ordinary skill in the art, and described by having in many publications, such as: Sambrook, J., Fritsch, E.F.and Maniais, T.(1989) MolecularCloning:A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.
The structure of embodiment 1. anti-CTLA-4/PD-1 bi-specific antibody expression vector
Full-length gene (light chain and the heavy chain) sequence of the anti-CTLA-4 monoclonal antibody in total man source is (see the aminoacid sequence disclosed in patent US8318916B2, variable region of light chain SEQ ID NO7, variable region of heavy chain SEQ ID NO17, constant region of light chain SEQ ID NO39, CH SEQ ID NO40) synthesized by Suzhou Jin Weizhi biotechnology Services Co., Ltd full genome, full-length gene (light chain and the heavy chain) sequence of the anti-PD-1 monoclonal antibody in total man source is (see aminoacid sequence disclosed in patent US20130133091A1, variable region of light chain SEQ ID NO8, variable region of heavy chain SEQ ID NO3, light chain is identical with SEQ IDNO39-40 described in above-mentioned US8318916B2 with CH) also synthesized by Suzhou Jin Weizhi biotechnology Services Co., Ltd full genome.Design the light chain of the anti-CTLA-4 monoclonal antibody of primer polymerase chain reaction (PCR) amplification respectively according to DNA sequence dna and reconnect variable region, and the light chain of anti-PD-1 monoclonal antibody and reconnecting; The complementary overhangs district of certain length is introduced, so that on the light chain variable region gene of anti-CTLA-4 monoclonal antibody being connected to anti-PD-1 monoclonal antibody by recombinant PCR method and heavy chain gene segment during design primer; , there is certain space operation degree of freedom the link zone (aminoacid sequence is: GGGGSGGGGS) of complementary overhangs district encoding exogenous in order to make two adjacent Variable domain.
PCR all adopts high-fidelity DNA polymerase (purchased from Takara company
hS DNAPolymerase).The reaction conditions of anti-PD-1 monoclonal antibody weight chain full-length gene and the anti-CTLA-4 weight chain variable region fragment of increasing rationally is arranged according to archaeal dna polymerase manufacturer specification sheets: 95 DEG C 20 seconds; 55 DEG C 10 seconds, 72 DEG C of 1 point/kb; 30 circulations.Above PCR primer is for the template of recombinant PCR after agarose gel electrophoresis purifying reclaims, and recombinant PCR reaction conditions is: 95 DEG C 20 seconds; 55 DEG C 10 seconds, 72 DEG C 2 points; Corresponding primer is added again after 6 circulations; 95 DEG C 20 seconds; 55 DEG C 10 seconds, 72 DEG C of 1 point/kb.PCR primer reclaims through agarose gel electrophoresis purifying and is cloned in pCHO1.0 carrier (purchased from Life Technologies company), confirms to obtain correct clone after sequence verification.SEQ ID NO:1 and SEQ ID NO:2 respectively illustrates Nucleotide and the aminoacid sequence of anti-CTLA-4/PD-1 bi-specific antibody heavy chain; SEQ ID NO:3 and SEQ ID NO:4 respectively illustrates Nucleotide and the aminoacid sequence of anti-CTLA-4/PD-1 bi-specific antibody light chain.Correct clone in this example is denoted as pCHO1.0(DVD-CTLA-4/PD-1).
The expression of the anti-CTLA-4/PD-1 bi-specific antibody of embodiment 2. in Chinese hamster ovary cell (CHO)
In 125ml cell cultures shaking flask, inoculate the CHO-S cell of 30ml density 5 × 105, second day cell density increase to 1 × 106 and Cell viability higher than 95% time carry out transfection: the plasmid pCHO1.0(DVD-CTLA-4/PD-1 by 50 μ l concentration being the coding anti-CTLA-4/PD-1 bi-specific antibody weight chain of 1 μ g/ μ l) be diluted to 1.45mlOptiPRO
tMsFM(is purchased from Life Technologies company, as follows) in, mix gently; By 50 μ l transfection reagent FreeStyle
tMmAX(is purchased from Life Technologies company, as follows) be diluted to 1.45mlOptiPRO
tMin SFM, mix gently; Immediately by the FreeStyle after dilution
tMmAX solution slowly joins in the DNA solution after dilution, adds fashionable rifle head and to be dipped under liquid level and slowly expel liquid; Turning upside down immediately makes solution mix for several times, and incubated at room temperature about 10 minutes is formed to make DNA-liposome complex; By 3mlDNA-FreeStyle
tMmAX complexes drop-wise joins in the shaking flask of above-mentioned culturing cell, limit edged jog culturing bottle; Cell culture after transfection be placed in 37 DEG C, 8% CO2, rotating speed 130rpm cell culture table on cultivate.Transfection is after 48 hours, centrifugal collecting cell, nutrient solution changed into the resistance clone of the Selective agar medium screening stable transfection containing 20 μ g/ml tetracyclines (Puromycin) and 200nM Rheumatrex (MTX); After about two weeks, when Cell viability and density recover, centrifugal collecting cell, changes the Selective agar medium containing 50 μ g/ml tetracyclines (Puromycin) and 1000nM Rheumatrex (MTX) into by nutrient solution; After about one week, when Cell viability and density recover, centrifugal collecting cell, changes into not containing antibiotic substratum by nutrient solution, be placed in 37 DEG C, 8%CO2, rotating speed 130rpm shaking table on suspension culture 10 days, period added glucose at the 3rd, 5,7,9 day with the final concentration of 4g/L; After 10 days, cell culture after low-speed centrifugal (300g) removes most cells and cell debris in 5 minutes, then removes the solid substance still suspended, then suction filtration (filter sizes 0 in 10 minutes through high speed centrifugation (10000g).45 μm), finally use the anti-CTLA-4/PD-1 bi-specific antibody of Protein A affinity column (purchased from GE company) separation and purification from the clarified liq obtained.Purified product is through order-checking, and consistent with SEQ ID NO:2, SEQ IDNO:4 sequence, being dialysed by purified fusion acceptor PBS, is finally standard substance BCA(Bicinchoninic acid with human normal immunoglobulin) method carries out quantitatively.
Embodiment 3. anti-CTLA-4/PD-1 bi-specific antibody avidity test experience-ELISA method
Experimental procedure: recombinant protein c TLA-4(is purchased from R & D company, as follows) and PD-1(purchased from R & D company, as follows) be diluted to 2 μ g/ml with 0.05mol/L carbonate buffer solution (pH9.6), the protein solution after the above-mentioned dilution of 100 μ l is added in the 96 every holes of hole enzyme plate, and 4 DEG C are spent the night in wet box; Next day, discard solution in hole, wash 3 times with lavation buffer solution (phosphate buffered saline buffer containing 0.05%Tween-20), each 3 minutes; Dilution for difference anti-CTLA-4 antibody (to be prepared according to embodiment 1 and embodiment 2 method according to CTLA-4 antibody sequence anti-described in embodiment 1, as follows), anti-PD-1 antibody (to prepare according to embodiment 1 and embodiment 2 method according to PD-1 antibody sequence anti-described in embodiment 1, as follows) and anti-CTLA-4/PD-1 bi-specific antibody 100 μ l be added on above-mentioned bag in the reacting hole of quilt, put 37 DEG C and hatch 1 hour; Then with lavation buffer solution washing, goat anti-human igg 1 antibody (Fc is special, available from Sigma) adding horseradish peroxidase-labeled, in each reacting hole, hatches 1 hour for 37 DEG C, washing; The tmb substrate solution 0.1ml adding Extemporaneous in each reacting hole develops the color, and places 10 ~ 30 minutes for 37 DEG C; 2mol/L sulfuric acid 0.05ml termination reaction is added in each reacting hole; Put (SpectraMax i3Multi-Mode Platform, BIO-TEK company of the U.S.) in microplate reader and measure 450nm wavelength place mensuration light absorption value.
Experimental result as depicted in figs. 1 and 2.Result shows: anti-CTLA-4/PD-1 bi-specific antibody both can in conjunction with CTLA-4, also can in conjunction with PD-1, and compare with anti-PD-1 antibody with anti-CTLA-4 antibody, anti-CTLA-4/PD-1 bi-specific antibody does not weaken because of the transformation of structure the bonding force of two kinds of target spots.
The anti-CTLA-4/PD-1 bi-specific antibody of embodiment 4. stimulates the experiment of mixed lymphocyte reacion
Mixed lymphocyte reacion (mixed lymphocyte reaction, MLC) be that the lymphocyte of two independent individuals of the same race is mixed cultivation, because the histocompatibility antigen on the two Lymphocyte Membrane is different, can stimulate mutually, cause the lymphocytic cell division of the other side, propagation and differentiation, the lymphoblast of final generation shows as cell volume and increases, and in nuclear dna and endochylema, RNA increases.By the lymphocyte percentage of morphological method counting differentiation, also by measuring the precursor of the lymphocyte picked-up DNA synthesis activated
3h mark thymidine (
3h-TdR) number of incorporation judges.Isotope-labelling method is comparatively objective, reproducible and result is more accurate.Lymphproliferation response intensity is directly proportional to the difference degree of both sides' histocompatibility antigen.Both consistencies are poorer, react stronger.This law have two-way and unidirectional MLR point, in two-way MLR, the lymphocyte of both sides stimulate mutually and hyperplasia, differentiation, not only the lymphocyte of both sides is irritation cell but also reacting cells; In unidirectional MLC, by the lymphocyte roentgen radiation x of a side or use ametycin process, it is made to lose proliferative response ability and still retain its antigenic stimulation effect, MLR now only has side's lymphocyte generation proliferative response, therefore can understand Different Individual LS intensity and proliferative response intensity.Human peripheral monokaryon/lymphocyte (Peripheral Blood Mononuclear Cells, PBMC) prepare with density gradient centrifugation: the anticoagulant heparin peripheral blood getting two Different Individual A and B, monocyte is isolated with human peripheral lymphocyte parting liquid (purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), (include through 56 DEG C, the calf serum 10% of 30min deactivation with RPMI-1640 nutrient solution after washing, the penicillin of suitable concentration, Streptomycin sulphate, use saturated NaHCO
3solution adjust pH to 7.2 ~ 7.4) adjustment cell density be 1 × 10
6/ ml.
MLC implements by following experimental procedure: 1, by A, B bis-the lymphocyte suspension of individuality mark respectively, using the reacting cells as MLR of individual A, the irritation cell as MLR of individual B; 2, irritation cell process: the lymphocyte suspension 0.9ml getting individual B, add ametycin (mitomycin C) 0.1ml of people 400 μ g/mL, its final concentration is made to be 40 μ g/m, cell suspension puts 37 DEG C of water-bath 30min, centrifugally abandon supernatant, sedimentation cell nutrient solution is washed once, adjustment cell concn to 1 × 10
6/ ml, the lymphocyte of the individual B of ametycin process is denoted as Bm; 3, respectively get 0.1mL reacting cells (A) and irritation cell (Bm) (A+Bm) in the hole of a round bottom Tissue Culture Plate with micro sample adding appliance, put 37 DEG C, 5%CO2 incubator interior cultivation 5d; 4, stop front 15 ~ 16h in cultivation, add in every hole with isotropic substance micro sample adding appliance
3h-TdR1 μ L(is containing 0.5 μ Ci), now blank group does not add isotropic substance; 5, sample preparation: with cell collector collecting cell in glass fiber filter paper, wash away free with physiological saline
3h-TdR, puts filter disc in 60 DEG C of baking ovens and dries, and after cooling, filter disc is put into the measuring cup containing scintillation solution, and detect intensity of radioactivity with beta scintillator survey meter, blank adds isotropic substance before sample preparation, the same experimental group for the treatment of process; 6, experimental result calculates: result is with per minute umber of pulse (counts per minute, cpm) represent, all samples all should deduct the cpm value of blank group before data processing, MLR response intensity is usually with the cpm net value of sample or stimulation index (stimulation index, SI) represent, cpm net value equals experimental group cpm value and deducts control group cpm, and SI equals experimental group cpm divided by control group cpm.
Experimental result as shown in Figure 3.Result shows: compared with anti-CTLA-4 antibody or anti-PD-1 antibody, anti-CTLA-4/PD-1 bi-specific antibody has the lymphopoietic effect of stronger stimulation.
The pharmacokinetic studies of the anti-CTLA-4/PD-1 bi-specific antibody of experimental example 5.
Experimental procedure: 10 week age, no-special pathogen (Specific-pathogen free, SPF) Thirty male rats (purchased from Shanghai Slac Experimental Animal Co., Ltd.), body weight 350-400g, be divided into eight groups at random, often organize four, give drug solvent, anti-CTLA-4 antibody, anti-PD-1 antibody and anti-CTLA-4/PD-1 bi-specific antibody respectively by intravenous injection, the dosage of antibody is set to 4mg/kg.All rats get blood (angular vein gets blood) after 0.5h, 1h, 2h, 6h, 12h, 24h, 48h, 5d, 10d, 15d, 20d, 25d, 30d after injection, and every rat extracting blood about 100 μ l, adds 0.The heparin mixing of 1%, the centrifugal 20min of 4000rpm, gets supernatant, abandons precipitation.The concentration detecting antibody in blood plasma adopts ELISA method: recombinant protein c TLA-4 and PD-1 0.05mol/L carbonate buffer solution (pH9.6) are diluted to 2 μ g/ml, the protein solution after the above-mentioned dilution of 100 μ l is added in the 96 every holes of hole enzyme plate, and 4 DEG C are spent the night in wet box; Next day, discard solution in hole, wash 3 times with lavation buffer solution (phosphate buffered saline buffer containing 0.05%Tween-20), each 3 minutes; Dilution for difference rat plasma is added on above-mentioned bag in the reacting hole of quilt, puts 37 DEG C and hatch 1 hour; Then with lavation buffer solution washing, the rat anti-human IgG1 antibody (Fc is special, Sigma Products) adding horseradish peroxidase-labeled, in each reacting hole, hatches 1 hour for 37 DEG C, washing; The tmb substrate solution 0.1ml adding Extemporaneous in each reacting hole develops the color, and places 10 ~ 30 minutes for 37 DEG C; 2mol/L sulfuric acid 0.05ml termination reaction is added in each reacting hole; Put (SpectraMax i3Multi-Mode Platform, BIO-TEK company of the U.S.) in microplate reader and measure 450nm wavelength place mensuration light absorption value.As shown in Figure 4, the Half-life in vivo of anti-CTLA-4 antibody, anti-PD-1 antibody and anti-CTLA-4/PD-1 bi-specific antibody is all longer, does not have significant difference for detected result.
Claims (10)
1. an anti-CTLA-4/PD-1 bi-specific antibody, is characterized in that, described antibody can be combined with CTLA-4, can also combine with PD-1.
2. anti-CTLA-4/PD-1 bi-specific antibody according to claim 1, is characterized in that, described antibody comprises anti-CTLA-4 variable region of mab and anti-PD-1 variable region of mab.
3. anti-CTLA-4/PD-1 bi-specific antibody according to claim 1 or 2, is characterized in that, described light chain of antibody aminoacid sequence is as shown in SEQ ID NO:4, and heavy chain amino acid sequence is as shown in SEQ ID NO:2.
4. a nucleic acid molecule, the arbitrary described anti-CTLA-4/PD-1 bi-specific antibody of its coding claims 1 to 3, its light chain nucleotide sequence is as shown in SEQ ID NO:3, and heavy chain nucleotide sequence is as shown in SEQ ID NO:1.
5. an expression vector, containing the expression regulation sequence that nucleic acid molecule according to claim 4 is connected with the sequence being operational of described nucleic acid molecule, described carrier can be pCHO1.0, pBudCE4.1 or pCGS3 carrier.
6. a host cell, it contains above-mentioned 5 ~ 6 arbitrary described carriers, and described cell can be COS, CHO or NS0 cell.
7. prepare a method for the arbitrary described anti-CTLA-4/PD-1 bi-specific antibody of claims 1 to 3, the method comprises:
A) under expression condition, cultivate host cell according to claim 6, express anti-CTLA-4/PD-1 bi-specific antibody;
B) isolated or purified a) described in anti-CTLA-4/PD-1 bi-specific antibody.
8. a composition, containing the arbitrary described anti-CTLA-4/PD-1 bi-specific antibody of claims 1 to 3 and pharmaceutically acceptable carrier.
9. the arbitrary described anti-CTLA-4/PD-1 bi-specific antibody of claims 1 to 3 or the composition described in the claims 8 are preparing the purposes in antitumor drug.
10. the purposes described in the claims 9, also comprises the antitumor drug conbined usage with other.
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