Comprehensive mechanism-based antibody pharmacokinetic modeling.

Pharmacokinetic models of antibody distribution and dynamics are useful for predicting and optimizing therapeutic behavior. Targeted antigens are produced and distributed in various tissues in specific patterns in disease phenotypes. Existing models leave out significant mechanistic detail which would enable an understanding of how to modify therapeutics in an optimal manner to allow appropriate tissue penetration in either a healthy or diseased state. The model presented here incorporates additional complexity such as diffusion through endothelial barriers, differential transcytosis properties, FcRn-mediated recycling, and incorporates these properties in an organ-specific manner. This creates a platform which can be expanded upon to include understanding of the effect of target on therapeutic distribution and clearance, differences in dynamics during a diseased versus healthy state, differential dose strategies, and mechanistic translation between animal models and human disease state. This model represents a superior alternative to typical and potentially over-simplified scaling strategies utilized in most existing physiologically-based pharmacokinetic models. Ultimately, this will enable better therapeutic design and greater pharmacological effects.

Impaired SHP2-mediated extracellular signal-regulated kinase activation contributes to gefitinib sensitivity of lung cancer cells with epidermal growth factor receptor-activating mutations.

Most non-small cell lung cancers (NSCLC) display elevated expression of epidermal growth factor receptor (EGFR), but response to EGFR kinase inhibitors is predominantly limited to NSCLC harboring EGFR-activating mutations. These mutations are associated with increased activity of survival pathways, including phosphatidylinositol 3-kinase/AKT and signal transducer and activator of transcription 3/5. We report that EGFR-activating mutations also surprisingly lead to decreased ability to activate extracellular signal-regulated kinase (ERK) compared with wild-type EGFR. In NSCLC cells and mouse embryonic fibroblasts expressing mutant EGFR, this effect on ERK correlates with decreased EGFR internalization and reduced phosphorylation of SHP2, a tyrosine phosphatase required for the full activation of ERK. We further show that ERK activation levels affect cellular response to gefitinib. NSCLC cells with EGFR mutation display reduced gefitinib sensitivity when ERK activation is augmented by expression of constitutively active mutants of mitogen-activated protein kinase/ERK kinase (MEK). Conversely, in a NSCLC cell line expressing wild-type EGFR, gefitinib treatment along with or following MEK inhibition increases death response compared with treatment with gefitinib alone. Our results show that EGFR-activating mutations may promote some survival pathways but simultaneously impair others. This multivariate alteration of the network governing cellular response to gefitinib, which we term "oncogene imbalance," portends a potentially broader ability to treat gefitinib-resistant NSCLC.

Input-output behavior of ErbB signaling pathways as revealed by a mass action model trained against dynamic data.

The ErbB signaling pathways, which regulate diverse physiological responses such as cell survival, proliferation and motility, have been subjected to extensive molecular analysis. Nonetheless, it remains poorly understood how different ligands induce different responses and how this is affected by oncogenic mutations. To quantify signal flow through ErbB-activated pathways we have constructed, trained and analyzed a mass action model of immediate-early signaling involving ErbB1-4 receptors (EGFR, HER2/Neu2, ErbB3 and ErbB4), and the MAPK and PI3K/Akt cascades. We find that parameter sensitivity is strongly dependent on the feature (e.g. ERK or Akt activation) or condition (e.g. EGF or heregulin stimulation) under examination and that this context dependence is informative with respect to mechanisms of signal propagation. Modeling predicts log-linear amplification so that significant ERK and Akt activation is observed at ligand concentrations far below the K(d) for receptor binding. However, MAPK and Akt modules isolated from the ErbB model continue to exhibit switch-like responses. Thus, key system-wide features of ErbB signaling arise from nonlinear interaction among signaling elements, the properties of which appear quite different in context and in isolation.

B lymphocyte deficiency in IgH-transgenic rabbits.

We developed IgH-transgenic rabbits carrying a productive VDJ-Cmu Tg and found the rabbits were B cell-deficient, with a 50-100% reduction in serum IgM and IgG levels. The bone marrow of newborn Tg rabbits contained severely reduced levels of preB cells and almost no B cells. The few preB cells present in the bone marrow were large, cycling cells that expressed the VDJ-Cmu Tg, indicating that the block in B cell development likely occurred at or before the transition from large (early) preB to small (late) preB cells. By immunoprecipitation, the Tg mu-chain paired with VpreB and lambda5, suggesting that the B cell deficiency is not due to an inability to form a preB cell receptor. Despite the block in B cell development, a few B cells, expressing predominantly endogenous mu-chains, began the second stage of development in GALT. B cells were localized in and beneath the follicle-associated epithelium of GALT prior to B cell follicle formation, suggesting to us that B cell follicle formation is initiated near the follicle-associated epithelium, possibly through contact with intestinal microbiota. These IgH-Tg rabbits should provide a useful model for studies of B cell development both in bone marrow and in GALT.

Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express V(H)n allotype immunoglobulin (Ig)M. Within weeks, the number of V(H)n B cells decreases, whereas V(H)a allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from V(H)n to V(H)a B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the V(H)n to V(H)a repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of V(H)a B cells. By comparing amino acid sequences of V(H)n and V(H)a Ig, we identified a putative V(H) ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with V(H)a B cells results in their selective expansion.

B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis.

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.