Comparative properties of the single chain antibody and Fv derivatives of mAb 4-4-20 - Relationship between interdomain interactions and the high affinity for fluorescein ligand

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 (V-H) and variable light (V-L) 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 (K-d) for the V-L/V-H 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 (K-d, 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 C(H)1 and C-L constant domains. Collectively, these results verify the importance of constant domain interactions in antibody variable domain structure-function analyses and future antibody engineering endeavors.