Influence of affinity and antigen density on antibody localization in a modifiable tumor targeting model

A persistent, question in the field of antibody imaging and therapy is whet her increased affinity is advantageous for the targeting of tumors. We have addressed this issue by using a manipulatabte model system to investigate the impact of affinity and antigen density on antibody localization. In vit ro enzyme-linked immunosorbent assays and bead-binding assays were carried out using BSA conjugated with high and low densities (HD and LD, respective ly) of the chemical hapten rho -azophenyl-arsonate as an antigen. Isotype-m atched monoclonal antibodies (mAbs) 36-65 and 36-71, with identical epitope specificity but 200-fold differences in affinity, were chosen as targeting agents. The relative in vitro binding of 36-65 and 36-71 was compared with an artificial "tumor'' model in vivo using antigen-substituted beads s.c. implanted into SCID mice. Nonsubstituted BSA beads were implanted in the co ntralateral groin as a nonspecific control. The efficacy of the targeting o f [I-125]-labeled antibodies was assessed by the imaging of animals on a ga mma-scintillation camera using quantitative region-of-interest image analys is over the course of 2 weeks and by postmortem tissue counting. In vitro, both antibodies bound well to the HD antigen, whereas only the high-affinit y mAb 36-71 bound effectively to the LD antigen. In vivo, high-affinity mAb 36-71 bound appreciably to both LD and HD beads. In contrast, there was no specific localization of low-affinity mAb 36-65 to LD antigen beads, altho ugh the antibody did bind to the beads with the HD antigen. Whereas the hig h-affinity mAb 36-71 increased its binding to HD beads throughout the 14 da ys of observation, binding of the high affinity antibody to LD beads and of the low affinity antibody to HD beads plateaued between 10-14 days. These in vitro and in vivo findings demonstrate that the need for a high-affinity antibody is dependent on the density of the target antigen. High-affinity antibodies bind effectively even with a single antigen-Fab interaction, irr espective of the antigen density. In contrast, low-affinity antibodies, bec ause of weak individual antigen-Fab interactions, require the avidity confe rred by divalent binding for effective attachment, which can only occur if antigen density is above a certain threshold, An understanding of the diffe rential behavior of high- and low-affinity antibodies and the impact of avi dity is useful in predicting the binding of monovalent antibody fragments a nd engineered antibody constructs and underlies the trend toward developmen t of multivalent immunological moieties. Consideration of the relative dens ity of the antigen on the tumor and the background tissues may enable and e ven favor targeting with low-affinity antibodies in selected situations.