anti-b7-h3 antibody
A B7-H3, antibody technology, applied in the direction of antibodies, antibody medical components, anti-tumor drugs, etc., can solve problems such as changes in antibody performance
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[0181] 2. Preparation of Anti-B7-H3 Antibody
[0182] Anti-B7-H3 antibodies of the present invention can be obtained by immunizing animals with B7-H3 or any polypeptide selected from the amino acid sequence of B7-H3 according to conventional methods, and collecting and purifying the antibodies produced in vivo. The biological species of B7-H3 used as an antigen is not limited to humans, and animals can be immunized with B7-H3 derived from animals other than humans, such as mice or rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity of the antibodies with the resulting binding of heterologous B7-H3 and human B7-H3.
[0183] Furthermore, according to known methods (for example, Kohler and Milstein, Nature, (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibody, pp. 365-367, Prenum Press, N.Y. (1980)) , Monoclonal antibodies can be obtained by fusing antibody-producing cells that produce anti-B7-H3 antibodies with mye...
Embodiment 1
[0350] Example 1. Preparation of plasmids
[0351] 1)-1 Preparation of human B7-H3 expression vector
[0352] 1)-1-1 Preparation of expression vector of full-length human B7-H3 variant 1
[0353] Using cDNA synthesized from total RNA of LNCaP cells (American Type Culture Collection (ATCC)) as a template, and also using the following primer set, a PCR reaction was performed to amplify the cDNA encoding human B7-H3 variant 1 :
[0354] Primer 1:
[0355] 5'-ctatagggagacccaagctggctagcatgctgcgtcggcggggcag-3' (SEQ ID NO: 1 in the Sequence Listing); and
[0356] Primer 2:
[0357] 5'-aacgggccctctagactcgagcggccgctcaggctatttcttgtccatcatcttctttgctgtcag-3' (SEQ ID NO: 2 in the sequence listing).
[0358] Subsequently, the PCR product thus obtained was purified using MagExtractor PCR & Gel cleanup (TOYOBO, Co., Ltd.). Then, the PCR product was digested with restriction enzymes (NheI and NotI), followed by purification using MagExtractor PCR & Gel cleanup (TOYOBO, Co., Ltd.). The p...
Embodiment 2
[0419] Example 2. Preparation of monoclonal antibodies and screening of antibodies
[0420] 2)-1 Immunization
[0421] 4-6 week-old BALB / cAnNCrlCrlj mice (Charles River Laboratories Japan, Inc.), FcgRII KO mice (Taconic, Inc., IBL Co., Ltd.) or GANP mice (Transgenic, Inc.) were used. On days 0, 7, 15 and 24, at 5 x 10 6 LNCaP cells, MCF7 cells (ATCC) or AsPC1 cells (ATCC) detached with ethylenediaminetetraacetic acid (Invitrogen Corporation) were administered subcutaneously to the dorsal region of each mouse at a dose of 2 cells / mouse. On day 31, with 5 x 10 6 A dose of each cell was administered intravenously to each mouse. On day 34, spleens were excised from each mouse and used to prepare hybridomas.
[0422] 2)-2 Preparation of hybridomas
[0423] Using PEG 4000 (manufactured by IBL Co., Ltd.), spleen cells and mouse myeloma P3X63Ag8U.1 cells (ATCC) were subjected to cell fusion, whereby hybridomas were prepared.
[0424] As a result, the following clones were establ...
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