Antibody which binds to myelin oligodendrocyte glycoprotein
a technology of myelin oligodendrocytes and antibodies, applied in the field of antibodies, can solve the problems of difficult drug development, side effects such as the production of neutralizing antibodies, and the inability to obtain effective concentration of drug administration, and achieve the effect of improving the property of the binding molecule itsel
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example 1
[Example 1] Acquisition of Anti-MOG Antibodies
(l) Acquisition of Antibodies Using Human Antibody Phage Libraries
[0569]A VH gene fragment and a VL gene fragment were amplified from human PBMC-derived cDNA by PCR. The VH gene fragment and the VL gene fragment were inserted to a phagemid vector pCANTAB 5E (manufactured by Amersham Pharmacia Biotech), and a plasmid was obtained by transforming Escherichia coli TG1 (manufactured by Lucigen).
[0570]The obtained plasmid was infected with M13K07 Helper Phage (manufactured by Invitrogen), and a human antibody M13 phage library of the VH gene and the VL gene was thus obtained.
[0571]Using the human antibody M13 phage libraries, anti-rat MOG (rMOG) monoclonal antibodies were obtained using the phage display method described below. rMOG-FLAG_Fc of Example 4 described below was immobilized on a MAXISORP STARTUBE (manufactured by NUNC), and the sites to which rMOG-FLAG_Fc was not bound were blocked using SuperBlock Blocking Buffer (manufactured by ...
example 2
[Example 2] Construction of Antibody Expression Vectors
(l) Construction of Anti-MOG Antibody Expression Vectors
[0591]To produce anti-MOG antibodies of human IgG type, expression vectors for anti-MOG antibodies in which the DNA sequences encoding the amino acid sequences of the variable regions of the human antibody phage library-derived anti-MOG scFv antibodies obtained in Example 1 were incorporated into a nucleotide sequence encoding the amino acid sequence of a constant region of human IgG antibody were produced by the method described below.
[0592]A nucleotide sequence encoding the lambda chain constant region of human IgG was synthesized and inserted to the BglII-EcoRI site of N5KG4PE vector (described in International Publication No. 2002 / 088186), and N5LG4PE vector was thus produced.
[0593]Expression vectors obtained by inserting nucleotide sequences encoding the amino acid sequences of VH and VL of MOG01 antibody and MOG09 antibody into N5LG4PE were named N5LG4PE_MOG01 and N5L...
example 3
[Example 3] Construction of Bispecific Antibody Expression Vectors
(l) Production of Vector Expressing Bispecific Antibody Binding to Her2 and MOG
[0610]A vector expressing a bispecific antibody binding to HER2 and MOG, pCI-Trastuzumab-hKG4PE(R409K)_MOG01scFv was produced by the following method. In the bispecific antibody, scFv of an anti-MOG antibody is fused to the C-terminuses of the two H chains of IgG of an anti-HER2 antibody.
[0611]Using a synthetic gene of the heavy chain constant region as a template and using primer 17 (SEQ ID NO: 59) and primer 18 (SEQ ID NO: 60) and KOD plus DNA Polymerase (manufactured by Toyobo Co., Ltd.), a gene fragment of the CH1-Hinge-CH2-CH3-linker region was amplified by PCR.
[0612]In the PCR, 30 cycles of reaction of 30 seconds at 94° C., 30 seconds at 58° C. and two minutes at 68° C. were conducted. Using a phagemid vector pCANTAB_MOG01 as a template and using primer 19 (SEQ ID NO: 61) and primer 20 (SEQ ID NO: 62) and KOD plus DNA Polymerase (manu...
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