Tetraspanin CD37 Directly Mediates Transduction of Survival and Apoptotic Signals

Abstract

SUMMARY Tetraspanins are commonly believed to act only as “molecular facilitators”, with no direct role in signal transduction. We herein demonstrate that upon ligation, CD37, a tetraspanin molecule expressed on mature normal and transformed B-cells, becomes tyrosine phosphorylated, associates with proximal signaling molecules, and initiates a cascade of events leading to apoptosis. Moreover, we have identified two tyrosine residues with opposing regulatory functions, one lies in the N-terminal domain of CD37 in a predicted “ITIM-like” motif and mediates SHP1-dependent death whereas the second lies in a predicted “ITAM motif” in the C-terminal domain of CD37 and counteracts death signals by mediating phosphatidylinositol 3-kinase-dependent survival.

Glycovariant anti-CD37 monospecific protein therapeutic exhibits enhanced effector cell-mediated cytotoxicity against chronic and acute B cell malignancies

“…2C). In particular, western blot analysis of cellular lysates with an anti-phosphotyrosine antibody revealed a predominant protein at ~65 kD in cells treated with SMIP-016 or SMIP-016 GV , an observation previously described by our group 8,21 and determined to be SHP-1 and Lyn phosphorylation. The identical binding affinity to the target antigen CD37 along with the equivalent amount of cytotoxicity against CLL cells via identical phosphorylation of SMIP-016 GV mediates direct cytotoxicity in primary CLL B cells with goat anti-human Fc crosslinker in comparable levels to those seen with the parent (unmodified) protein ( Fig.…”

“…SMIP-016-mediated direct cytotoxicity has been shown to be dependent on antigen density. 8,21 We wanted to test whether antigen density played a role in ADCC with the SMIP-016 and whether the glycovariant could overcome this. To test this, we created a model system by retrovirally transducing 697 cells (CD37 negative cell line) with a pBABE-CD37 vector construct and isolated clones with differing levels of surface CD37 by limiting dilution cloning.…”

“…The trend in cytotoxicity for SMIP-016 GV downstream proteins collectively suggests that the parent compound SMIP-016 and the glycovariant SMIP-016 GV are working through similar molecular pathways in CLL cells to cause direct cytotoxicity, as previously described. 21 Next, we tested if SMIP-016 GV had any complement initiating capabilities. We have shown that the SMIP-016 is unable to mediate complement-dependent cytotoxicity.…”

“…8 This was subsequently shown to involve SHP1 and a complex array of signaling via the ITIM motif of CD37. 21 To test whether the SMIP-016 GV was signaling within the cells via the same molecular pathways, we stimulated CLL cells with SMIP-016 or SMIP-016 GV along with anti-human Fc crosslinker. Both SMIP-016 and SMIP-016 GV show similar patterns of increased tyrosine phosphorylation after 10 min of in vitro stimulation of CLL cells (Fig.…”

“…In this paper, we demonstrate that the production process yielded functional afucosylated SMIP-016, which suggests that it may also enhance effector cell function modification of the molecule does not affect its binding affinity to CD37 and subsequent direct cytotoxicity, nor does it alter SMIP-016 GV from signaling through mechanisms previously described by our group. 8,21 From these data, we hypothesized that SMIP-016 GV 's direct cytotoxicity is dependent on surface levels of CD37 on CLL cells and is irrespective of prognostic factors such as IgVH mutational status, Rai stage and common cytogenetic abnormalities, as described with SMIP-016. 21 SMIP-016 GV , however, shows enhanced NK cell-mediated ADCC against primary CLL B cells compared with its parent compound SMIP-016.…”