Humanization of Predicted T-Cell Epitopes Reduces the Immunogenicity of Chimeric Antibodies: New Evidence Supporting a Simple Method

Abstract

Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies. We described previously a new method based on the humanization of the linear epitopes presented to T cells. In brief, potential immunogenic epitopes in the variable region were identified and subjected to point mutations to make them human and/or to modify amphipatic motifs. The resulting recombinant antibody retained its antigen binding affinity and was less immunogenic in monkeys than their murine or chimeric predecessors are. The present study provides two new examples of this T-cell epitope humanization approach: ior-t1A murine monoclonal antibody (mMAb), which recognizes the human-CD6 molecule, and ior-C5 mMAb, which recognizes a novel glycoprotein expressed on the surface of malignant colorectal cells. Seven amino acids were substituted in ior-C5 and eleven residues in ior-t1A, by the corresponding residues from the highest homologous human sequences. Surprisingly, the homology between re-shaped chimeric antibody variable region frameworks and human sequences was 80-90%. Experiments in monkeys showed that T1AhT and C5hT "detopes" antibodies were less immunogenic than their chimeric analogues while they retained 30-50% of antigen binding affinities. The proposed method might be of general applicability to reduce immunogenicity of chimeric antibodies with therapeutic potential.

Immunological evaluation of rheumatoid arthritis patients treated with itolizumab

“…35 Itolizumab F(ab) 2 was used to coat ELISA plates. Sera from Cercopitecus aethiops monkeys containing anti-idiotypic antibodies against itolizumab 67 were used as positive control. Absorbance values of at least 2-fold the signal with the pre-immune sera were considered positive for anti-idiotypic antibodies.…”

Immunopotentiating properties of a multispecific α-anti-idiotype antibody

“…The chimeric antibodies (ChAbs; human IgG1, κ) B7Y33, 16 and VHB7Y33/VκP3 (hybrid antibody), 16 and the anti-mucin C5, 23 were purified from transfectoma culture supernatants by Protein-A affinity Chromatography (Amersham Biosciences, 17-5280-02) and analyzed by SDS-PAGE under reducing conditions.…”

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

“…There are several potential advantages of using scFv fragments over whole antibodies or larger fragments for liposome targeting. These include: i) slower clearance than mAb-targeted liposomes, as Fc-mediated clearance is eliminated [55]; ii) theoretically lower production cost for scFv fragments generated from bacterial cultures relative to whole antibodies generated from animal ascites or cell culture [56,57]; iii) the ability to select scFv with the desired affinity and specificity using phage display [58]; iv) the option of engineering tags into scFv constructs, which can aid in their identification and purification [59]; and v) the ability to engineer fully human fragments or fragments with low levels of non-human content, which will reduce the risk of immunogenic reactions [60,61].…”