Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes.

A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mAb induce anti-mAb responses. To analyze CD4 T cell reactions against IgG in various physical states, we developed an adoptive transfer model using CD4+ T cells specific for a Vκ region-derived peptide in the hapten-specific IgG mAb 36-71. We found that heat-aggregated or immune complexes (IC) of mAb 36-71 elicited anti-idiotypic (anti-Id) antibodies, while the deaggregated form was tolerogenic. All 3 forms of mAb 36-71 induced proliferation of cognate CD4+ T cells, but the aggregated and immune complex forms drove more division cycles and induced T follicular helper cells (TFH) development more effectively than did the deaggregated form. These responses occurred despite no adjuvant and no or only trace levels of endotoxin in the preparations. Physical analyses revealed large differences in micron- and nanometer-sized particles between the aggregated and IC forms. These differences may be functionally relevant, as CD4+ T cell proliferation to aggregated, but not IC mAb 36-71, was nearly ablated upon peritoneal injection of B cell-depleting antibody. Our results imply that, in addition to denatured aggregates, immune complexes formed in vivo between therapeutic mAb and their intended targets can be immunogenic.

The Role of Protein Excipient in Driving Antibody Responses to Erythropoietin.

Human serum albumin (HSA) is an excipient present in formulations of several recombinant protein products that are approved for clinical use. We investigated the relative contributions of HSA and HSA particles to the generation of antibody responses against recombinant human erythropoietin (rhEPO) and the excipient HSA itself. Protein samples were characterized before injection for quantities of monomeric proteins, soluble protein aggregates, and nano- and micron-sized particles. rhEPO, containing various concentrations of HSA particles, were injected three times a week for 8 weeks into mice. Hematocrits and the production of anti-rhEPO and anti-HSA antibodies were determined at various time points. Levels of antibodies against rhEPO in mice injected with HSA-containing rhEPO were higher than those in mice treated with HSA-free rhEPO. Mice injected with formulations that contained particles of HSA produced strong anti-HSA antibody responses; whereas these responses were greatly reduced when particle-free formulations were administered. In contrast, anti-rhEPO antibody responses were not affected by the presence of particles.

Recombinant murine growth hormone particles are more immunogenic with intravenous than subcutaneous administration.

Evaluation and mitigation of the risk of immunogenicity to protein aggregates and particles in therapeutic protein products remains a primary concern for drug developers and regulatory agencies. To investigate how the presence of protein particles and the route of administration influence the immunogenicity of a model therapeutic protein, we measured the immune response in mice to injections of formulations of recombinant murine growth hormone (rmGH) that contained controlled levels of protein particles. Mice were injected twice over 6 weeks with rmGH formulations via the subcutaneous, intraperitoneal, or intravenous (i.v.) routes. In addition to soluble, monomeric rmGH, the samples prepared contained either nanoparticles of rmGH or both nano- and microparticles of rmGH.The appearance of anti-rmGH IgG1, IgG2a, IgG2b, IgG2c, and IgG3 titers following the second injection of both preparations implies that multiple mechanisms contributed to the immune response. No dependence of the immune response on particle size and distribution was observed. The immune response measured after the second injection was most pronounced when i.v. administration was used. Despite producing high anti-rmGH titers mice appeared to retain the ability to properly regulate and use endogenous growth hormone.