Identification of a Diagnostic Gene Signature for SGN-40, Anti-CD40 Monoclonal Antibody, in Pre-Clinical NHL Models and the Role of FAS in SGN-40 Mediated Apoptosis.

SGN-40, an anti-CD40 monoclonal antibody, is a humanized IgG1 antibody that binds to CD40, mediates effector cell functions (ADCC/ADCP) and activates downstream signaling pathways. SGN-40 has shown activity in a phase I single agent multi-dose trial in non Hodgkin’s lymphoma, with greatest activity in diffuse large B-cell lymphoma (Advani et al. 2008, ICML). Previous in vitro studies implicated down-regulation of the germinal center expressed protein Bcl-6, upregulation of p53 family member TAp63a, and FAS death receptor induction as potential mechanisms leading to lymphoma cell death (Lewis et al. 2007, AACR, ASH). In order to further define the apoptosis signaling mechanism, we assessed the ability of SGN-40 to inhibit proliferation and promote apoptosis across a large panel of non-Hodgkin’s lymphoma cell lines. SGN-40 reduced cell viability in 58% (18/31) of cell lines tested. To identify the genes that may be promoting apoptosis and/or inhibiting proliferation, we compared gene expression levels before and after SGN-40 exposure in both sensitive and resistant cell lines, as well as in normal B-cells. SGN-40 strikingly and specifically upregulated FAS on the cell surface of sensitive cell lines. Furthermore, the addition of soluble FAS-Fc dampened SGN-40-induced apoptosis in a subset of sensitive cell lines, suggesting a dependence on a FAS-FASL interaction. These data imply that FAS-dependent apoptosis may directly contribute to the anti-tumor effect of SGN-40. Our data also demonstrate that SGN-40 sensitivity is dependent on the point in B-cell development at which the NHL cell lines were transformed. Sensitive cell lines had a gene signature characteristic of minimal activation of CD40 signaling prior to SGN-40 exposure, whereas resistant cell lines had a signature consistent with prior constitutive signaling downstream of CD40. Thus, SGN-40 appears to elicit its apoptotic properties through activation of CD40 signaling in NHL cell lines not previously exposed to CD40L signaling in the germinal center environment at the time of lymphocyte transformation (GCB lymphomas). In order to develop a clinically feasible assay from FFPE tissue, we developed a 14-gene signature by Stepwise Linear Modeling, utilizing genes from the CD40 pathway activation and GCB gene sets; the classifier gave >96% accuracy (30/31) on the ‘training’ set of cell lines and 75% (3/4) accuracy on a ‘test’ set of xenografts. Overall, our data provides unique insights into SGN-40 mechanisms of action, provides a testable hypothesis of the clinical mechanism of action, and a potential diagnostic test to identify patients more likely to benefit from SGN-40. Efforts are currently underway to test the clinical relevance of these findings.