Pharmaceutical composition for treating and/or preventing cancer
A technology of chimeric antibody and antibody, which is applied in the direction of drug combination, medical preparations containing active ingredients, anti-tumor drugs, etc.
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[0046] The preparation of the antigen, the preparation of the antibody, and the pharmaceutical composition related to the present invention will be described below.
[0047]
[0048] The protein or its fragment used as a sensitizing antigen for obtaining the antibody against CAPRIN-1 used in the present invention can be derived from humans, dogs, cows, horses, mice, rats, chickens, etc. The types of animals are not limited. However, the protein or fragment thereof is preferably selected in consideration of suitability for mother cells used in cell fusion, and generally, mammalian-derived proteins are preferred, and human-derived proteins are particularly preferred. For example, when CAPRIN-1 is human CAPRIN-1, human CAPRIN-1 protein and / or partial peptides thereof, cells expressing human CAPRIN-1, etc. can be used.
[0049] The base sequence and amino acid sequence of human CAPRIN-1 and its homologs can be obtained, for example, by accessing GenBank (US NCBI), using algorit...
Embodiment 1
[0180] Example 1 Identification of a new cancer antigen protein using the SEREX method
[0181] (1) Preparation of cDNA library
[0182] Total RNA was extracted from testicular tissues of healthy dogs by the Acidguanidium-Phenol-Chloroform method (Acidguanidium-Phenol-Chloroform method), and poly-ARNA was purified using the Oligotex-dT30 mRNA Purification Kit (manufactured by Takara Shuzo Co., Ltd.) according to the instructions attached to the kit .
[0183] The thus obtained mRNA (5 µg) was used to synthesize a dog testis cDNA phage library. The cDNA phage library was prepared using the cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit (manufactured by STRATAGENE), and the library was prepared according to the instructions attached to the kits. The size of the cDNA phage library produced was 7.73×10 5 pfu / ml.
[0184] (2) Screening of cDNA library using serum
[0185] The dog testis cDNA phage library prepared above was used for immu...
Embodiment 2
[0213] Example 2 Preparation of Human CAPRIN-1
[0214] (1) Preparation of recombinant protein
[0215] Based on the gene of sequence number 1 obtained in Example 1, the recombinant protein of the human homologous gene was prepared by the following method. PCR was carried out as follows: 1 μl of cDNA whose expression could be confirmed by RT-PCR using various tissues and cell cDNAs produced in Example 1, and two primers (sequence numbers 38 and 39) containing SacI and XhoI restriction enzyme cleavage sequences. ) each of 0.4 μM, 0.2 mM dNTP, and 1.25 U PrimeSTARHS polymerase (manufactured by Takara Shuzo Co., Ltd.)) were added to make the total amount 50 μl of each reagent and accompanying buffer solution, and the thermal cycler (manufactured by BIORAD Co., Ltd.) was used to cycle 98° C. for 10 seconds, The cycle of 68°C - 2.5 minutes was repeated 30 times. In addition, the above-mentioned two types of primers are primers for amplifying the region encoding the entire length ...
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