The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions

Abstract

Monoclonal antibodies represent an attractive therapeutic tool as they are highly specific for their targets, convey effector functions and enjoy robust manufacturing procedures. Humanization of murine monoclonal antibodies has vastly improved their in vivo tolerability. Humanization, the replacement of mouse constant regions and V framework regions for human sequences, results in a significantly less immunogenic product. However, some humanized and even fully human sequence-derived antibody molecules still carry immunological risk. To more fully understand the immunologic potential of humanized and human antibodies, we analyzed CD4(+) helper T cell epitopes in a set of eight humanized antibodies. The antibodies studied represented a number of different VH and VL family members carrying unique CDR regions. In spite of these differences, CD4(+) T cell epitopes were found only in CDR-sequence containing regions. We were able to incorporate up to two amino acid modifications in a single epitope that reduced the immunogenic potential while retaining full biologic function. We propose that immunogenicity will always be present in some antibody molecules due to the nature of the antigen-specific combining sites. A consequence of this result is modifications to reduce immunogenicity will be centered on the affinity-determining regions. Modifications to CDR regions can be designed that reduce the immunogenic potential while maintaining the bioactivity of the antibody molecule.

Figure 1

CD4⁺ T cell responses to peptides derived from cetuximab (black squares) and humanized cetuximab (open circles) V regions. 15-mer peptides overlapping by 12 amino acids describing the VH and VL regions of cetuximab were tested for their ability to induce proliferative responses with CD4⁺ Tcells and dendritic cells from 87 community donors. peptides derived from humanized cetuximab were tested with 106 community donors.

Figure 2

CD4⁺ T cell responses to framework and CDR-sequence-containing peptides derived from cetuximab (A) and humanized cetuximab (B) V regions. (A) 15-mer peptides overlapping by 12 amino acids describing the VH and VL regions of cetuximab were tested for their ability to induce proliferative responses with CD4⁺ cells and dendritic cells from 87 community donors. (B) peptides derived from humanized cetuximab were tested with 106 community donors. Framework sequence peptides are depicted by the open circles. CDR sequence-containing peptides are depicted by the black squares.

Figure 3

CD4⁺ T cell epitope responses to eight humanized and fully human antibodies. (A) Framework (circle; n = 188) and CDR region-containing (square; n = 364) peptide responses. (B) Average ± standard deviation for all framework and CDR region containing peptides tested.

Figure 4

Reduction and exacerbation of in vitro immunogenicity by epitope amino acid sequence modifications. All proliferative responses rates to epitope variants were standardized to the unmodified parent epitope peptide response. (A) Distribution of standardized response rates of the variant peptides. (B) Distribution of standardized average stimulation index of the variant peptides.

Figure 5

Two amino acid modifications in epitope sequences result in more potential deimmunized peptides. (A) Distribution of response rates to single point mutations. (B) Distribution of response rates to double mutations. (C) Box and whiskers plot standardized to the unmodified parent epitope peptide response. Arrow indicates the approximate location of the median.