FcgammaRIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific antibodies (bsAbs) targeting FcgammaRIII represent a powerful alternative to the recruitment of the receptor via the Fc fragment, but are not efficiently produced. Single-domain antibodies (sdAbs) endowed with many valuable structural features might help to bypass this problem. In the present work, we have isolated anti-FcgammaRIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with FcgammaRIIIB extra-cellular domains. These sdAbs bind FcgammaRIIIA+ NK cells and FcgammaRIIIB+ polymorphonuclear cells, but not FcgammaRI+ or FcgammaRII+ cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different FcgammaRIII epitopes, with C21 recognizing a linear and C28 a conformational epitope of the receptor. Surface plasmon resonance experiments showed that C21 and C28 sdAbs bind FcgammaRIII with a K(D) in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of FcgammaRIIIA expressed by transfected Jurkat T cells or by NK cells derived from peripheral blood induced a strong IL-2 and IFN-gamma production, respectively. These anti-FcgammaRIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit FcgammaRIII killer cells to target and destroy tumor cells.
Production of recombinant antibody fragments in bacterial expression systems results in intentional or fortuitous differences compared to the original products prepared by hybridoma technology. These differences may have significant effects not only on antigen-binding properties but also on pharmacokinetic and tumor-seeking properties. Our major goal was to investigate some of these possible differences. We produced in Escherichia coli an rFab fragment containing only 1 cysteine residue in the hinge region; the fragment was derived from a mouse MAb (F6) specific for CEA. The rFab had a slightly lower m.w. and a higher isoelectric point relative to the corresponding nonrecombinant fragment (pFab ). This was explained by the absence of N-glycosylation on the V domain of rFab . V glycosylation had no significant effect on antibody-binding affinity and kinetics. However, rFab was eliminated from the circulation much faster than pFab , and the maximal dose accumulated in the tumor was reduced relative to pFab . Thus, glycosylation appears to modify the targeting efficiency of antibody fragments. rF(ab ) 2 fragments were obtained either spontaneously from the culture supernatant of E. coli or by chemical cross-linking [rcF(ab ) 2 ]. We observed improved tumor targeting with rcF(ab ) 2 compared to rF(ab ) 2 , which could be explained by the greater stability of the thioether compared to the disulfide linkage. These results demonstrate that a single cysteine residue in the hinge region of rFab is particularly well suited to prepare stable, chemically coupled, bivalent or bispecific antibodies, avoiding intrahinge disulfide bonding and thus achieving higher production yields.
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