Use for preventing and treating myeloid-derived suppressor cell-related diseases
A source, immunosuppressive technology, applied in the field of improvement or treatment of MDSC-related diseases, prevention, and can solve research puzzles and other issues
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Embodiment 1
[0132] Example 1: Preparation of anti-CD66c chimeric antibody
[0133] 1.1. Gene sequence cloning of anti-CD66c antibody
[0134] The 8F5 antibody gene was cloned using a mouse Ig primer set (Millipore, catalog number: 69831). RNA isolated from the 8F5 hybridoma was subjected to PCR using a mouse Ig primer set, inserted into the pGem-T vector (Promega, catalog number: A3600), sequenced to confirm the DNA sequence, and passed through the IMGT site (www.imgt. org) identified mouse antibody genes. The heavy chain variable region sequence and the light chain variable region sequence of the analyzed 8F5 antibody are as follows.
[0135] [Table 4]
[0136]
[0137]
[0138] 1-2. Production of chimeric antibodies
[0139] Based on the amino acid sequence of the constructed anti-CD66c mouse antibody 8F5, an anti-CD66c chimeric antibody was prepared.
[0140] 1-2-1. Plasmid production
[0141] For expression of the anti-CD66c chimeric antibody, heavy chain expression ...
Embodiment 2
[0146] Example 2: Preparation of humanized anti-CD66c monoclonal antibody
[0147] 2.1 Selection of recombinant antibody sequences by in silico humanization
[0148] By maintaining the mouse anti-CD66c antibody as much as possible, 8F5 (heavy chain amino acid sequence: SEQ ID NO: 7, heavy chain encoding DNA: SEQ ID NO: 62; light chain amino acid sequence: SEQ ID NO: 8, heavy chain encoding DNA: CDRH1: ASGYSFTDYTMN) SEQ ID NO: 1, CDRH2: SEQ ID NO: 2 (LINPFHGGGTVSNQRFKV); CDRH3: SEQ ID NO: 3 (VRGDPVRHYYALAY); CDRL1: SEQ ID NO: 4 (GASENVYGTL); CDRL2: SEQ ID NO: 5 (GATNLAD); CDRL3: SEQ ID NO: 6 (VATYYCQNVLSAPYT) CDRs are as similar as possible, if antigen binding capacity is equal or higher, based on the gene encoding the human antibody The germline sequence of the humanized antibody sequence obtained by recombining the site sequence of the framework region was selected by computer simulation. As shown in Table 5, the human antibody germline genes used as the backbone of the r...
Embodiment 3
[0199] CHO cell expression and analysis of DNP002
[0200] The humanized anti-CD66c antibody DNP002 was expressed and analyzed in CHO cells used to express most of the therapeutic antibodies. To test the functional difference according to the subtype of the DNP002 antibody, IgG1 type and IgG2 type antibodies were prepared.
[0201] After codon-optimizing the DNA sequences of the light chain variable region and the heavy chain variable region used to construct the humanized recombinant antibody, they were synthesized and ligated with the constant region of human IgG1 or IgG2 by overlapping PCR method, and The XhoI and EcoRI gene fragments were cloned into the pcDNA3.4 vector (Life Technology Company).
[0202] [Table 15]
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[0204]
[0205] In order to prepare afucosylated DNP002 humanized antibody, when expressing DNP002 IgG1 type antibody, 2F-PF (2F-peracetyl-fucose; Merck company, catalog number: 344827) was added at 50uM to culture medium, and then purified...
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