Mice lacking the p110δ catalytic subunit of phosphatidylinositol 3-kinase have reduced numbers of B1 and marginal zone B cells, reduced levels of serum immunoglobulins, respond poorly to immunization with type II thymus-independent antigen, and are defective in their primary and secondary responses to thymus-dependent antigen. p110δ−/− B cells proliferate poorly in response to B cell receptor (BCR) or CD40 signals in vitro, fail to activate protein kinase B, and are prone to apoptosis. p110δ function is required for BCR-mediated calcium flux, activation of phosphlipaseCγ2, and Bruton's tyrosine kinase. Thus, p110δ plays a critical role in B cell homeostasis and function.

The immunogenicity of protein therapeutics has so far proven to be difficult to predict in patients, with many biologics inducing undesirable immune responses directed towards the therapeutic resulting in reduced efficacy, anaphylaxis and occasionally life threatening autoimmunity. The most common effect of administrating an immunogenic protein therapeutic is the development of a high affinity anti-therapeutic antibody response. Furthermore, it is clear from clinical studies that protein therapeutics derived from endogenous human proteins are capable of stimulating undesirable immune responses in patients, and as a consequence, the prediction and reduction of immunogenicity has been the focus of intense research. This review will outline the principle causes of the immunogenicity in protein therapeutics, and describe the development of pre-clinical models that can be used to aid in the prediction of the immunogenic potential of novel protein therapeutics prior to administration in man.

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.

RhoG is a low-molecular-weight GTPase highly expressed in lymphocytes that activates gene transcription and promotes cytoskeletal reorganization in vitro. To study the in vivo function of RhoG, we generated mice homozygous for a targeted disruption of the RhoG gene. Despite the absence of RhoG, the development of B and T lymphocytes was unaffected. However, there was an increase in the level of serum immunoglobulin G1 (IgG1) and IgG2b as well as a mild increase of the humoral immune response to thymus-dependent antigens. In addition, B-and T-cell proliferation in response to antigen receptor cross-linking was slightly increased. Although RhoG deficiency produces a mild phenotype, our experiments suggest that RhoG may contribute to the negative regulation of immune responses. The lack of a strong phenotype could indicate a functional redundancy of RhoG with other Rac proteins in lymphocytes.

B cells from phospholipase C (PLC)γ2‐deficient mice express reduced levels of the pro‐survival protein Bcl‐2 and show a defect in the development of transitional T3 and marginal zone (MZ) B cells that reflects reduced B cell survival. Introduction of a bcl‐2 transgene restored the numbers of MZ, T3 and follicular B cells in PLCγ2–/– mice. Restricting the B cell repertoire in PLCγ2‐deficient mice by the introduction of a BCR transgene resulted in a striking reduction in the number of IgM‐positive B cells and a paucity of IgD‐expressing cells in the spleen which was also rescued by the bcl‐2 transgene. BCR‐stimulated ERK and IκBα phosphorylation were PLCγ2 dependent, while calcium flux was reduced, but not abrogated, in the absence of PLCγ2, suggesting an ancillary role for PLCγ1. The bcl‐2 transgene rescued development of PLCγ2–/– B cells and serum IgM levels but did not restore BCR‐mediated signaling, proliferation or serum IgG3 levels. These data suggest that PLCγ2 performs a critical role in B cell development through regulation of survival rather than differentiation.