G. Pavlinkova et al., Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-TAG-72 single-chain Fvs, INT J CANC, 94(5), 2001, pp. 717-726
One major constraint in the clinical application of murine monoclonal antib
odies (MAbs) is the development of a human antimurine antibody response. Th
e immunogenicity of MAbs can be minimized by replacing nonhuman regions wit
h corresponding human sequences. The studies reported in our article were u
ndertaken to analyze the immunoreactivity and the immunogenicity of the CC4
9 single-chain antibody fragments (scFvs): (i) an scFv construct comprised
of mouse CC49 V-L and V-H (m/m scFv), (ii) a light chain shuffled scFv with
human V-L (Hum4 V-L) and mouse CC49 V-H (h/m scFv), and (iii) a humanized
scFv assembled from Hum4 VL and CC49 V-H complementary determining regions
(CDRs) grafted onto a V-H framework of MAb 21/28' CL (h/CDR scFv). The CC49
scFvs competed for an antigen binding site with CC49 IgG in a similar fash
ion in a competition radioimmunoassay and were able to inhibit the binding
of CC49 IgG to the antigen completely. The immunogenicity of CC49 scFvs was
tested using sera with antiidiotypic antibodies to MAb CC49 obtained from
patients treated by CC49 IgG in clinical trials. All tested sera exhibited
the highest reactivity to the m/m scFv. However, the sera demonstrated diff
erential reactivities to h/CDR scFv and h/m scFv. Replacement of the mouse
chain in h/m scFv and h/CDR scFv decreased or completely averted serum reac
tivity. Our studies Compared for the first time the antigen binding and imm
unogenicity of different scFv constructs containing the mouse, CDR grafted
or human variable chains. These results indicate that the humanized CC49 sc
Fv is potentially an important agent for imaging and therapeutic applicatio
ns with TAG-72-positive tumors. (C) 2001 Wiley-Liss, Inc.