Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated biotherapeutics.

We describe a novel human immunoglobulin G2 (IgG2 )-tolerant and immune-competent heterozygous mouse model (Xeno-het) developed by crossbreeding a human Ig-tolerized XenoMouse® with a C57BL/6J wild-type mouse. The Xeno-het mouse expresses both mouse and human immunoglobulin G (IgG) genes, resulting in B-cells expressing human and mouse IgG, and secretion of human and mouse Ig into serum. This model was utilized to evaluate the immunogenicity risk of aggregated and chemically modified human antibodies. The mice were tested for their ability to break tolerance to self-tolerant monomeric antibodies. Aggregates made by mechanical stirring elicited an anti-drug antibody (ADA) response, but did not induce a robust and long-term memory B and T-cell response. Chemically modified antibodies made by oxidation were only weak and transient inducers of an immune response, as measured by a lack of both an ADA response and a B-cell antigen-specific response. Aggregate size was an important characteristic, as specific-sized protein-coated beads were able to elicit an immune response. We propose the use of this model to identify risk factors such as aggregation during manufacturing at early development for an increased potential immunogenicity risk.

Kinetic analysis of unpurified native antigens available in very low quantities and concentrations.

Affinity measurements of antigen-antibody interactions are generally performed using known concentrations of purified or recombinant materials. In addition, many technologies that measure affinity require the interacting components to be present in at least microgram quantities. Specifically, if the antigen is either available only in low quantities or unable to be purified, or if the quantity is unknown, then the measurement of affinity can be very difficult. Using the Kinetic Exclusion Assay (KinExA) technology, here we describe a method that overcomes the requirement for large amounts of purified and known quantities of antigen. We used this method to precisely measure the affinity of fully human anti-human interleukin 13 (IL13) monoclonal antibodies to IL13 produced in native form from primary T cells derived from a variety of species, including human. These antigens were available only in the limited quantities present in the conditioned cell culture medium, and the affinity was measured directly without further purification.