Removal of Amphipathic Epitopes from Genetically Engineered Antibodies: Production of Modified Immunoglobulins with Reduced Immunogenicity

Abstract

Several approaches have been developed to reduce the human immune response to nonhuman antibodies. However, chimeric antibodies and humanized antibodies often have decreased binding affinity. We described a new approach for reducing the immunogenicity of chimeric antibodies while maintaining the affinity. This approach seeks to prevent the recognition of murine immunogenic peptides from the antibody variable region by human lymphocytes. Putative immunogenic epitopes in the variable region are identified and subjected to site directed mutagenesis to make them human and/or to break the amphipathic motifs. The R3 antibody, which blocks the epidermal growth factor (EGF) receptor, was used as a model system to test this approach. Four segments containing possible amphipathic epitopes were found in the heavy variable domain using the program AMPHI. Six amino acids within two of these segments were substituted by the corresponding residues from a homologous human sequence. No mutations were made in the murine light variable domain. Experiments in monkeys suggested that the "detope" R3 antibody was less immunogenic than its chimeric analogue. A search for possible amphipathic epitopes in the Kabat database revealed the presence of conserved patterns in the different families of variable region sequences, suggesting that the proposed method may be of general applicability.

Estimating design space available for polyepitopes through consideration of major histocompatibility complex binding motifs

“…The approach for mitigation of immunogenicity of existing proteins is much as for elimination of junctionals from polyepitopes, involving sequence changes to violate MHC binding motif spacing/amino acid content requirements. The immunological consequences of eliminating all class II MHC binding motifs from an immunogenic protein are not entirely certain, but the possibility is that proteins so engineered might be much less immunogenic (or entirely non-immunogenic) in individuals with MHC alleles targeted in the protein design (Mateo et al 2000;Roque-Navarro et al 2003). This approach to mitgated immunogenicity is historically precedented: the discovery of random co-polymers of amino acids which triggered no immune responses in certain host backgrounds provided a vital clue to the nature of the role of the MHC in governing and mediating specific immunity (Katz et al 1973).…”

A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates

“…Chimeric antibodies, which consist of murine antigen binding variable regions bound genetically to human antibody constant regions, were an early attempt to reduce this undesirable effect (Morrison et al 1984;Wright et al 1992), but that still evoke a strong HAMA in many cases (Hwang and Foote 2005). At present, fully human antibodies (Green 1999) or humanized molecules generated by complementary determining regions (CDRs) grafting of a murine antibody into the corresponding regions of a human immunoglobulin (Riechmann et al 1988), or by any other methodology (Mateo et al 2000;Almagro and Fransson 2008), are the preferred choice for proving the therapeutic potential of a new mAb in the clinics (Liu et al 2008).…”

Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity

“…We also evaluated the correlation between exterior fluorescence intensity and antibody productivity using quantitative enzyme-linked immunosorbent assay (ELISA). The hum-C2 mabs were engineered to have lower immunogenicity compared to its precursor which was developed in the mouse through hybridoma technology (Mateo et al 2000;Yazaki et al 2004). The hum-C2 mAb is of immunoglobulin isotype G subclass 1(IgG1) isotype and it is highly specific for a colorectal tumor associated antigen (C2), a novel 145-190 kilo dalton (kDa) glycoprotein preferentially expressed on the surface of malignant colorectal cells (Iznaga-Escobar et al 2004).…”