Murinization and H Chain Isotype Matching of the Anti-GITR Antibody DTA-1 Reduces Immunogenicity-Mediated Anaphylaxis in C57BL/6 Mice.

Recent advances in immuno-oncology have shown that the immune system can be activated to induce long-term, durable antitumor responses. For immuno-oncology drug development, immune activation is often explored using rat Abs in immunocompetent mouse models. Although these models can be used to show efficacy, antidrug immune responses to experimental protein-based therapeutics can arise. Immunogenicity of surrogate Abs may therefore represent an important obstacle to the evaluation of the antitumor efficacy of immunomodulator Abs in syngeneic models. A recent publication has shown that anti-glucocorticoid-induced TNFR family-related protein agonistic Ab DTA-1 (rat or murinized IgG2a) can induce the development of anaphylaxis in C57BL/6 mice upon repeated i.p. dosing because of an anti-idiotypic anti-drug Ab immune response. This study was undertaken to address the impact of the immunogenicity derived from the Fc and variable domains. To this end, chimerized (rat V domains/mouse constant regions) and murinized (95% mouse sequence) DTA-1-based surrogate Abs with a murine IgG2c H chain isotype were created. Chimerization and murinization of DTA-1 did not affect receptor binding and glucocorticoid-induced TNFR family-related protein-induced T cell agonistic properties. Similar in vivo antitumor efficacy and intratumoral CD8+/regulatory T cells were also observed. Finally, treatment of C57BL/6 mice with the chimerized and murinized DTA-1 Abs on a C57BL/6-matched IgG2c isotype resulted in reduced development and severity of anaphylaxis as measured by decline of body temperature, behavioral effects, serum IL-4, IgE, and anti-drug Ab levels. These results suggest that careful murinization and selection of a strain-matched H chain isotype are critical to generate ideal surrogate Abs for testing immuno-oncology mechanisms in vivo.

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

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.

Enhanced immunogenicity of a functional enzyme by T cell epitope modification.

BACKGROUND: T helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines. RESULTS: Hartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone. CONCLUSIONS: With a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzyme's original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics.