Effects of Upstream and Downstream Processes

Beyond the interaction of the drug with its target, variations in downstream effector pathways such as signaling proteins and transcription factors may influence the overall action of the treatment. We recently published a comprehensive review of Pgx studies of mechanistically related candidate markers in drug target pathways for protein therapeutics (15). A few examples are provided to illustrate the effects of downstream processes on drug responses.

One example illustrating this effect involves the presence of somatic mutations of KRAS in certain tumors. Activating KRAS mutations have been associated with lack of response to panitumumab, which targets EGFR upstream of this GTPase (11,12,94). Mutations in KRAS may subvert the mechanism of EGFR inhibitors, since downstream signaling is no longer dependent on signals through EGFR. In a similar fashion, loss of function of PTEN has also been associated with poor response to trastuzumab and cetuximab therapy (98-100). The mutation status of downstream signaling molecules in drug target pathways is especially important to consider with oncology agents, since tumor escape mechanisms generally involve coincident or sequential alteration of several growth-regulatory pathways (21).

The suppressors of SOCS system, which modulates certain cell surface receptors by inhibiting downstream signal transduction pathways and targeting receptors for degradation through the proteosome, may have effects upstream or downstream of the therapeutic target (see sect. "Target-Mediated Elimination Pathways"). Specific SOCS family proteins have been shown to regulate signaling by IFN-a, insulin, leptin, G-CSF, and somatotropin (31,32,39-41). Several Pgx studies have found significant associations between SOCS1 and SOCS3 genotypes or pretreatment mRNA levels and response to IFN-a treatment in chronic hepatitis C patients (35-38).

By far, the largest literature on Pgx of downstream effectors has described the effects of coding polymorphisms in the FcR genes. While FcR-dependent activities of phagocytic cells and the reticuloendothelial system can indirectly mediate clearance of some therapeutic proteins, most studies have focused on the efficacy side of the equation. Pharmacogenomic studies have repeatedly demonstrated FcR associations with responses to selected therapeutic mAb drugs. mAbs that bind cellular targets may activate FcR on phagocytic cells and NK cells, stimulating the destruction of the targeted cell by antibody-mediated cellular cytotoxicity (ADCC) or by phagocytosis of the opsonized cell.

Several publications describe the association of coding polymorphisms in FCGR3A with responses to rituximab in oncology and autoimmunity trials (101105). Similar findings have been reported for FCGR2A, although it may be that these associations result from linkage within the genetic region encoding several FcRs (103). In vitro studies have shown that the FCGR3A variant 158V has higher affinity for IgG than the 158F variant, effectively broadening the concentration range in which Fc engagement activates ADCC in NK cells (106,107). Likewise, it has been observed that the anti-TNF mAb drugs may act as cell-targeting agents, since TNF-a is produced in a transmembrane form before release from the cell surface by cleavage (108). Recent studies of FCGR3A and infliximab therapy demonstrated that patients homozygous for the high-affinity 158V allele had a greater decrease in C-reactive protein, a PD marker in Crohn's disease, compared with 158F carriers. A follow-up study demonstrated that variations in the CRP gene itself did not account for this association. While the association of FCGR3A genotype with biological response in serum CRP levels to infliximab was confirmed, no association was shown with clinical response in the two cohorts studied (109-111).

Fewer studies have investigated associations between FcR variants and erbB-targeting mAbs, perhaps because endocytosis of the mAb-receptor complex reduces the window of opportunity for immune effector cell recognition. However, a role for ADCC in trastuzumab and cetuximab responses has been postulated, and recently, several studies have found associations between FcR variants and/or NK cell activity with clinical response to trastuzumab in breast cancer patients (112-115).

Nonfucosylated mAb constructs have been designed to increase cellmediated killing of tumor cells by binding with higher affinity to FcR and overcoming the variability associated with coding polymorphisms in FCGR3A and FCGR2A (116). Interestingly, the phagocytic activity of polymorphonuclear cells appears to favor high fucose content of IgG Fc, whereas the ADCC performed by mononuclear effector cells such as NK is enhanced by low fucose content (116,117).

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