Structural Correlates of an Anticarcinoma Antibody: Identification of Specificity-Determining Residues (SDRs) and Development of a Minimally Immunogenic Antibody Variant by Retention of SDRs Only

Abstract

Clinical utility of murine mAbs is limited because many elicit Abs to murine Ig constant and variable regions in patients. An Ab humanized by the current procedure of grafting all the complementarity determining regions (CDRs) of a murine Ab onto the human Ab frameworks is likely to be less immunogenic, except that its murine CDRs could still evoke an anti-variable region response. Previous studies with anticarcinoma mAb CC49 showed that light chain LCDR1 and LCDR2 of humanized CC49 could be replaced with the corresponding CDRs of a human Ab with minimal loss of Ag-binding activity. The studies reported in this paper were undertaken to dissect the CC49 Ag-binding site to identify 1) specificity determining residues (SDRs), the residues of the hypervariable region that are most critical in Ag-Ab interaction, and 2) those residues that contribute to the idiotopes that are potential targets of patients’ immune responses. A panel of variants generated by genetic manipulation of the murine CC49 hypervariable regions were evaluated for their relative Ag-binding affinity and reactivity to sera from several patients who had been immunized with murine CC49. One variant, designated HuCC49V10, retained only the SDRs of CC49 and does not react with the anti-variable region Abs of the sera from the murine CC49-treated patients. These studies thus demonstrate that the genetic manipulation of Ab variable regions can be accomplished by grafting only the SDRs of a xenogeneic Ab onto human Ab frameworks. This approach may reduce the immunogenicity of Abs to a minimum.

Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-tag-72 single-chain Fvs

“…It was evident that the pattern of the anti-V region response differed from patient to patient. Similar findings were reported by Tamura et al, 6 who studied the serum reactivity of the genetically modified humanized CC49 IgG variants. Some sera responded to certain idiotypes more than other patients' sera.…”

“…Some sera responded to certain idiotypes more than other patients' sera. 6 Furthermore, current studies suggest that CDR grafting can remove existing epitopes, but it can also generate new ones. 6,9 This was observed in our studies with serum #2; there was higher binding of h/CDR scFv in comparison with h/m scFv.…”

“…32,52 The murine CDRs can also represent the idiotopes that are recognized as targets by the human immune system, and at the same time they can be crucial for antigen binding. 6,9,53 An analysis of the known structures of the antigen-antibody complexes suggests that only 20 -33% of CDR residues are involved in the antigen contact. 54 These specificity-determining residues (SDRs), which may be the most critical in the antigenantibody interaction, can be identified by genetic manipulations or by X-ray crystallography.…”

“…Humanization by grafting only SDRs could further reduce the immunogenic potential of murine antibodies with a minimal decrease in their immunoreactivity. 6 The development of single-chain Fv constructs opened new possibilities for manipulation of antibody properties. The molecular structure of the single-chain antibodies has been modified to study the antigen binding, kinetics, penetration into tissues, metabolism, specificity, pharmacokinetics and stability.…”

“…6 -9 Furthermore, CDR grafting could also generate new immunogenic epitopes. 6,9 The murine MAb CC49 recognizes tumor-associated glycoprotein (TAG-72), which is expressed by most human adenocarcinomas of the pancreas, colon, ovary, prostate, lung and esophagus 10 and is absent in most normal tissues. 11 Radiolabeled CC49 IgG has shown excellent tumor localization in several clinical trials.…”