Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins

The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen bindin g affinity: internalization rate, intracellular processing and intrinsic to xin domain potency. This study examines the influence of antigen binding af finity on potency in various immunotoxin fusion proteins where target antig en binding is mediated by single chain antibody variable region fragments ( scFv). Firstly, the relationship between affinity and potency was examined in a panel of four scFv immunotoxins generated from different anti-CD3 mono clonal antibodies fused to the 38 kDa fragment of Pseudomonas aeruginosa ex otoxin A (PE38). Of these four scFv-PE38 immunotoxins, the one derived from the anti-CD3 monoclonal antibody UCHT1 has highest cell killing potency. A nalysis of these four scFv-PE38 immunotoxins indicated a correlation betwee n antigen binding affinity and immunotoxin potency in the cell killing assa y with the exception of the scFvPE38 immunotoxin derived from the antibody BC3. However this scFv appeared to suffer a greater drop in affinity ( simi lar to 100 x), relative to the parent Mab than did the other threo scFvs us ed in this study (2 - 10 x). secondly, the scFv(UCHT1)PE38 immunotoxin was then compared with a further panel of scFv(UCHT1)-derived immunotoxins incl uding a divalent PE38 version and both monovalent and divalent Corynehacter ium diphtheriae toxin (DT389) fusion proteins. When the scFv-UCHT1 domain w as amino-terminally positioned relative to the toxin, as in the scFv(UCHT1) -PE38, an approximately 10-fold higher antigen-binding affinity was observe d than with the C-terminal fusion, used in the DT389-scFv(UCHT1) molecule. Despite this lower antigen-binding activity, the DT389-scFv immunotoxin had a 60-fold higher potency in the T-cell-killing assay. Thirdly, a divalent form of the DT389-scFv construct. containing tandem scFv domains, had a 10- fold higher binding activity, which was exactly reflected in a 10-fold incr ease in potency. Therefore. when comparing immunotoxins in which scFvs from different antibodies are fused to the same toxin domain (DT or PE) a broad correlation appears to exist between binding affinity and immunotoxin pote ncy. However. no correlation between affinity and potency appears to exist when different toxin domains are combined with the same scFv antibody domai n. (C) 2001 Elsevier science Ltd. All rights reserved.