Abstract
Recombinant Fv derivative of the high affinity murine anti-fluorescein monoclonal antibody 4-4-20 was constructed and expressed in high yields, relative to the single chain antibody (SCA) derivative (2 3-fold), in Escherichia coli. Both variable heavy (VH) and variable light (VL) domains, that accumulated as insoluble inclusion bodies, were isolated, denatured, mixed, refolded, and affinity-purified to yield active Fv 4-4-20. Affinity-purified Fv 4-4-20 showed identical ligand binding properties compared with the SCA construct, both were slightly lower than the affinities expressed by Fab or IgG 4-4-20. Proper protein folding was shown to be domain-independent by in vitro mixing of individually refolded variable domains to yield functional Fv protein. In solid phase and solution phase assays, Fv 4-4-20 closely approximated the SCA derivative in terms of both idiotype and metatype, confirming identical active site structures and conformations. The equilibrium dissociation constant (Kd) for the VL/VH association (1.43 x 10(-7) M), which was determined using the change in fluorescein spectral properties upon ligand binding, was relatively low considering the high affinity displayed by the Fv protein for fluorescein (Kd, 2.9 x 10(-10) M). Thus, domain-domain stability in the Fv and SCA 4-4-20 proteins cannot be the sole cause of reduced affinity (2-3-fold) for fluorescein as compared with the Fab or IgG form of 4-4-20. With their identical ligand binding and structural properties, the decreased SCA or Fv affinity for fluorescein must be an ultimate consequence of deletion of the CH1 and CL constant domains. Collectively, these results verify the importance of constant domain interactions in antibody variable domain structure-function analyses and future antibody engineering endeavors.
Highlights
The equilibrium dissociation constant (Kd) for the variable light (VL)/variable heavy (VH) association (1.43 ؋ 10؊7 M), which was determined using the change in fluorescein spectral properties upon ligand binding, was relatively low considering the high affinity displayed by the Fv protein for fluorescein (Kd, 2.9 ؋ 10؊10 M)
If the initial decrease in Fv affinity for antigen was due to decreased domain-domain interactions, the properties governing stable variable domain association in relation to antigen binding must be identified
As such antibody proteins continue to be modified and applied to different systems, the nature of this affinity decrease, including how VL/VH affinity correlates with antigen binding affinity, FIG. 7
Summary
Being covalently coupled by an interdomain linker, SCA proteins have been suggested to possess greater interdomain stability than their Fv counterparts due to the favorable entropic effect of domain coupling [6, 8] This would, in turn, suggest that in the appropriate Fv molecule (one with high affinity for antigen), interdomain associative properties would dictate the overall affinity displayed for antigen because only associated VL/VH proteins would bind antigen. To study the relation between interdomain association and affinity for antigen, an antibody protein must be available in many derivative forms (e.g. with and without constant domain, with and without an intervariable domain linker) To satisfy these criteria, the Fv derivative of mAb 4-4-20, a high affinity murine anti-fluorescein antibody, has been synthesized. Results indicated that the reduced affinity of Fv and SCA 4-4-20 did not correlate with reduced variable domain association, but with the absence of antibody constant domains, emphasizing their role in antibody/ antigen interactions
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