Antibody binding to chondroitin sulfate proteoglycan-5
A chondroitin sulfate, proteoglycan technology, applied in the direction of antibodies, immunoglobulins, anti-animal/human immunoglobulins, etc., can solve problems such as short half-life
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Embodiment 1
[0548] [Example 1] Obtaining of anti-CSPG5 antibody
[0549] (1) Obtaining antibodies using human antibody phage library
[0550] VH gene fragments and VL gene fragments were amplified by PCR from cDNA derived from human PBMCs. The VH gene fragment and VL gene fragment were respectively inserted into phagemid vector pCANTAB 5E (manufactured by Amersham Pharmacia Biotech, Inc.), and plasmids were obtained by transforming Escherichia coli TG1 (manufactured by Lucigen Corporation). The resulting plasmid was infected with M13KO7 helper phage (manufactured by Invitrogen, Inc.), whereby a human antibody M13 phage library of VH gene and VL gene was obtained.
[0551] In addition, a synthetic human antibody M13 phage library incorporating random mutations in CDR3 was generated in the same manner.
[0552] Using the human antibody M13 phage library, the anti-CSPG5 monoclonal antibody was obtained using the following phage display method. The human CSPG5-FLAG_Fc or mouse CS...
Embodiment 2
[0560] [Example 2] Production of antibody
[0561] (1) Construction of CSPG5 scFv-hG4PE (R409K) expression vector
[0562] An expression vector for producing scFv-Fc antibodies in which each anti-CSPG5 scFv antibody was linked to a human IgG4 antibody comprising amino acid residue substitutions according to EU numbering S228P, L235E, and R409K (sometimes hereinafter) was constructed. referred to simply as the "IgG4 variant") Fc region.
[0563] Using the phagemid vector pCANTAB_CSPG5115 as a template, the gene fragment of the scFv region was amplified by PCR. The gene fragment for the hinge-CH2-CH3 region was amplified by PCR using a synthetic gene for the heavy chain constant region as a template. The obtained gene fragment was inserted into a pCI vector (manufactured by Promega, Inc.), thereby generating a pCI_CSPG5115scFv-hG4PE(R409K) vector.
[0564] Antibody expression vectors into which gene fragments of the scFv regions of each of the various types of anti-...
Embodiment 3
[0578] [Example 3] Analysis of reactivity with CSPG5 expressing cells
[0579] The nucleotide sequence encoding human CSPG5 is represented by SEQ ID NO: 159, the amino acid sequence deduced from the nucleotide sequence is represented by SEQ ID NO: 160, the nucleotide sequence encoding mouse CSPG5 is represented by SEQ ID NO: 161 represents, the amino acid sequence deduced from the nucleotide sequence is represented by SEQ ID NO: 162, the nucleotide sequence encoding monkey CSPG5 is represented by SEQ ID NO: 163, and the amino acid sequence deduced from the nucleotide sequence is represented by SEQ ID NO: 162 ID NO: 164 said.
[0580] The full-length gene sequences of human CSPG5, mouse CSPG5, and monkey CSPG5 were synthesized, and the gene sequences were respectively inserted into the BamHI-NotI sites of the pEF6 / V5-His (manufactured by Thermo Fisher Scientific, Inc.) vector, The following plasmid vectors for membrane expression of the corresponding types of CSPG5 were ...
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