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Example 3
Measurement of Dissociation Constant of KLHa505 and KLHb1501 for Binding to rhVEGF
With the use of BIAcore-3000, Kd of KLHa505 and Kd of KLHb1501 for binding to VEGF were determined. rhVEGF was immobilized on a CMS chip, serial dilution was performed twice with HBS-EP buffer, and then each antibody was injected at a flow rate of 30 μL/min. Kd is Koff/Kon.
As a result, it was demonstrated that the dissociation constant for binding of KLHa505 to rhVEGF was 4.23 pmol/L, and the dissociation constant for binding of KLHb1501 to rhVEGF was 0.60 pmol/L.
When compared with the dissociation constant for an existing antibody (for example, the dissociation constant for Avastin (bevacizumab) is 4.9 nmol/L), the dissociation constants for the antibodies of the present invention were each 1/1000 or less that for the existing antibody. Hence, the binding activity of the antibodies that bind to VEGF is significantly high.
That is, in this Example, the antibodies (high-affinity anti-VEGF antibodies) having extremely high affinity for VEGF were obtained.