Antibody that binds to OX40 and use thereof
A technology of antibody and monoclonal antibody, which is applied in the field of cancer and infectious diseases, and the treatment of OX40-related diseases, can solve the problem of reducing Treg activity and achieve good anti-tumor effect in vivo
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[0122] Preparation of monoclonal antibody of the present invention
[0123] The monoclonal antibodies of the present invention can be prepared using the somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See, eg, US Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.
[0124] Generation of Transfectomas to Produce Monoclonal Antibodies of the Invention
[0125] Antibodies of the invention can also be produced in host cell transfectomas using, for example, recombinant DNA techniques combined with gene transfection methods (eg Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is insert...
Embodiment 1
[0167] Example 1 Construction of HEK293A cell strain stably expressing human, monkey or mouse OX40
[0168] cDNA sequences encoding human, monkey or mouse OX40 (SEQ ID NOs: 31, 33 and 35, respectively encoding amino acid sequences SEQ ID NOs: 32, 34 and 36) were synthesized and cloned into pLV-EGFP(2A) -Puro vector (Beijing Yingmao Shengye Biotechnology Co., Ltd., China). The resulting pLV-EGFP(2A)-Puro-OX40, psPAX and pMD2.G plasmids were transfected into HEK293T cells (Nanjing Kebai Company, China) by lipofection to produce lentiviruses. The method is completely consistent with the instructions of Lipofectamine 3000 (Thermo Fisher Scientific, USA). Three days after transfection, lentivirus was harvested from the cell culture medium (DMEM medium (Cat#: SH30022.01, Gibco) supplemented with 10% FBS (Cat#: FND500, Excell)) of HEK293T cells. Then HEK293A cells (Nanjing Kebai Company, China) were transfected with lentivirus to obtain HEK293A cells stably expressing human, monk...
Embodiment 2
[0169] Example 2 Preparation of hybridoma cell lines secreting human OX40 antibody
[0170] Mouse anti-human OX40 monoclonal antibody was obtained by conventional hybridoma fusion technique with slight modification of the protocol.
[0171] inoculation
[0172] Twenty BALB / C mice (Victoria, China) were cross-injected with recombinant human OX40(ECD)-hFc protein (SinoBiological, CN, Cat#: 10481-H08H) and monkey OX40-hFc protein (Cat#: 90846 -C08H, Yiqiao Shenzhou, China) for immunization, the specific immunization process is shown in Table 2. Human OX40(ECD)-hFc protein and monkey OX40-hFc protein were treated with equal volume of complete Freund's adjuvant (Cat#: F5881-10*10ML, Sigma, USA) or incomplete Freund's adjuvant (Cat#: F5506-6* 10ML, Sigma, USA) or PBS phacoemulsification.
[0173] Table 2. Immunization schedule
[0174]
[0175] One week after each booster immunization, 50 μl serum was taken from each mouse, and the titer was detected by ELISA, using recom...
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