Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1–CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.

Human monocytes undergo spontaneous apoptosis upon culture in vitro; removal of serum from the media dramatically increases the rate of this process. Monocyte apoptosis can be significantly abrogated by the addition of growth factors or proinflammatory mediators. We have evaluated the role of the endogenous Fas–Fas ligand (FasL) interaction in the induction of this spontaneous apoptosis and found that a Fas–immunoglobulin (Ig) fusion protein, an antagonistic anti-Fas monoclonal antibody and a rabbit anti-FasL antibody all greatly reduced the onset of apoptosis. The results indicate that spontaneous death of monocytes is mediated via an autocrine or paracrine pathway. Treatment of the cells with growth factors or cytokines that prevented spontaneous apoptosis had no major effects on the expression of Fas or FasL. Additionally, monocyte-derived macrophages were found to express both Fas and FasL but did not undergo spontaneous apoptosis and were not sensitive to stimulation by an agonistic anti-Fas IgM. These results indicate that protective mechanisms in these cells exist at a site downstream of the receptor–ligand interaction.

No abstract

Elotuzumab is a humanized monoclonal antibody specific for signaling lymphocytic activation molecule-F7 (SLAMF7, also known as CS1, CD319, or CRACC) that enhances natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) of SLAMF7-expressing myeloma cells. This study explored the mechanisms underlying enhanced myeloma cell killing with elotuzumab as a single agent and in combination with lenalidomide, to support ongoing phase III trials in patients with relapsed/refractory or newly-diagnosed multiple myeloma (MM). An in vitro peripheral blood lymphocyte (PBL)/myeloma cell co-culture model was developed to evaluate the combination of elotuzumab and lenalidomide. Expression of activation markers and adhesion receptors was evaluated by flow cytometry, cytokine expression by Luminex and ELISPOT assays, and cytotoxicity by myeloma cell counts. Elotuzumab activated NK cells and promoted myeloma cell death in PBL/myeloma cell co-cultures. The combination of elotuzumab plus lenalidomide demonstrated superior anti-myeloma activity on established MM xenografts in vivo and in PBL/myeloma cell co-cultures in vitro than either agent alone. The combination enhanced myeloma cell killing by modulating NK cell function that coincided with the upregulation of adhesion and activation markers, including interleukin (IL)-2Rα expression, IL-2 production by CD3+CD56+ lymphocytes, and tumor necrosis factor (TNF)-α production. In co-culture assays, TNF-α directly increased NK cell activation and myeloma cell death with elotuzumab or elotuzumab plus lenalidomide, and neutralizing TNF-α decreased NK cell activation and myeloma cell death with elotuzumab. These results demonstrate that elotuzumab activates NK cells and induces myeloma cell death via NK cell-mediated ADCC, which is further enhanced when combined with lenalidomide.

No abstract

Members of the TNF superfamily, including Fas, Fas ligand, and CD40, have been shown to be expressed on tumor cells. In the studies described in this work, we report that another family member, the ligand for 4-1BB (CD137), is expressed on various human carcinoma cell lines, on cells of solid tumors derived from these cell lines, and cells obtained from human tumors. Expression of 4-1BB ligand (4-1BBL) mRNA was detected by both RT-PCR and Northern blot analysis, and expression of 4-1BBL protein was detected by Western blot analysis of whole cell lysates and by FACS analysis of tumor cells and cell lines. Incubation of tumor cells with a 4-1BB-Ig fusion protein led to the production of IL-8 by the cells, demonstrating that the 4-1BBL is functionally active and signals back into the tumor cells. Furthermore, 4-1BBL expressed on the carcinoma cells functioned as a costimulatory molecule for the production of cytokines (most notably IFN-γ) in cocultures of T cells and tumor cells. These findings suggest that 4-1BBL expressed on carcinoma cells may significantly influence the outcome of a T cell-tumor cell interaction.

Binding studies with a CD6 immunoglobulin fusion protein (CD6 Rg) resulted in the identification and cloning of a CD6 ligand. This ligand was found to be a member of the immunoglobulin supergene family and was named ALCAM (activated leukocyte cell adhesion molecule). Cell adhesion assays showed that CD6-ALCAM interactions mediate thymocyte-thymic epithelium cell binding. ALCAM is also expressed by activated leukocytes and neurons and may be involved in interactions between T cells and activated leukocytes and between cells of the immune and nervous systems, respectively. Herein we describe the preparation of domain-specific murine CD6 Rg fusion proteins and show that the membrane-proximal SRCR (scavenger receptor cysteine-rich) domain of CD6 contains the ALCAM binding site. We also show that mAbs which bind to this domain preferentially block CD6-ALCAM binding. These results demonstrate that the membrane-proximal SRCR domain of CD6 is necessary for CD6 binding to ALCAM and provide the first direct evidence for the interaction of an SRCR domain with a ligand.

Maximal T lymphocyte responses require presentation of antigen by major histocompatibility complex molecules and delivery of one or more co-stimulatory signals. Interaction of the CD28 molecule on T lymphocytes with its ligands on antigen-presenting cells (APC) initiates a critical co-stimulatory pathway inducing T lymphocyte proliferation and cytokine secretion. Dendritic cells (DC) are potent APC for a primary T lymphocyte response and potential CD28/CTLA-4 ligands on DC are, therefore, of particular functional relevance. In these experiments, the expression and function of the CD28/CTLA-4 ligands B7.1 (CD80) and B7.2 (CD86) were examined on human blood DC. Resting DC populations directly isolated by immunodepletion of lineage marker-positive cells lacked cell membrane expression of CD80 and expressed little or no CD86, although CD86, but not CD80 mRNA was detected by reverse transcription-polymerase chain reaction analysis. In contrast, low-density DC isolated after culture in vitro strongly expressed CD86 surface protein, but expressed limited or no CD80, although mRNA for both molecules were detected. Short-term culture of directly isolated DC up-regulated both CD80 and CD86 expression. Analysis of the kinetics of CD28/CTLA-4 ligand induction showed that surface CD86 was present within 8 h, whereas CD80 antigen was first detected after 24 h of culture. The functional importance of CD28/CTLA-4 ligand up-regulation on DC during T lymphocyte interactions was demonstrated by the ability of both CTLA-4Ig and CD86 monoclonal antibodies (mAb), but not CD80 mAb, to block an allogeneic mixed lymphocyte reaction stimulated by DC populations initially negative for CD80 and CD86. These results demonstrate that CD86 is both the earliest and functionally the predominant co-stimulatory CD28/CTLA-4 ligand on DC.