ALBERT

Description: Key Points An anti-CD37 antibody-drug conjugate can kill B-lymphoma cells via direct inhibition, effector function, and payload delivery. Targeting CD37 with an antibody-drug conjugate results in selective depletion of malignant human B cells.

Description: Abstract 1862 Poster Board I-887 Background: Biotest AG (Dreieich, Germany) is developing the immunoconjugate BT062, which comprises the anti-CD138 chimerized MAb (nBT062) and the cytotoxic agent maytansinoid (DM4). Once bound to CD138 on a target cell, the conjugate is internalized and releases DM4. At present, CD138 represents one of the most reliable target antigens for identification of multiple myeloma (MM) cells and has been reported to be a highly sensitive and specific diagnostic marker of MM. Preclinical investigations demonstrated significant in vitro and in vivo anti-MM activity of BT062, providing the rationale for the conduct of clinical trials (Ikeda et al., 2009). Objectives: To determine the maximum tolerated dose (MTD), the dose-limiting toxicities (DLTs), pharmacokinetics (PK) and anti-MM activity of increasing doses of BT062 on a repeated single dose schedule once every three weeks in relapsed or relapsed/refractory MM. Clinical response was assessed as per the international working group criteria (Durie et al., 2006). Methods: This is a prospective, open label, dose-escalation multicenter study. Patients aged ≥ 18 years with relapsed or relapsed/refractory MM who have failed previous treatments including an immunomodulating agent and a proteasome inhibitor were eligible to participate. Patients with clinical response (or no evidence of progressive disease) and without unacceptable toxicities were eligible for further treatment cycles. Patients are enrolled in cohorts of 3 at each dose level, with DLT in the first cycle triggering cohort expansion. Results: To date 20 patients have been treated with BT062 at 7 dose levels ranging from 10 mg/m2 to 200 mg/m2. Maximum administered dose has not been defined to date with continued enrollment at 200 mg/m2 dose. None of the patients treated experienced serious hypersensitivity reactions or humoral responses (HAHA) against BT062. The most frequently reported adverse events to date cover primarily events expected for the underlying disease. Nevertheless, a few adverse events have also been observed involving skin and mucosa (tissues of epithelial origin with CD138 expressing cells). No grade 4 toxicity has been reported. Preliminary PK results indicate an unusual rapid clearance from plasma in the early elimination phase, followed by a generally normal terminal elimination phase at dose levels up to 120 mg/m2, whereas a more typical clearance profile was observed for all 3 patients at the 160 mg/m2 dose. Interestingly, even in phase I study decreased urine M-Protein or serum FLC levels have been observed in 2 patients. One of these patients showed a decrease in urine M-Protein by more than 50% after administration of 8 repeated low doses. At a high dose level another patient without detectable M-Protein levels, showed a decrease of serum FLC by more than 50% after having received the second dose of BT062. Furthermore, evidence of clinical benefit has been observed in at least 6 patients with early stabilization of M-protein levels (and light-chain burden) in serum and /or urine. Conclusion: Development of a monoclonal antibody in MM remains an important therapeutic option and BT062 is an exciting possibility. Preliminary data from this phase I study, demonstrate an acceptable toxicity profile of BT062 in the clinics. Even in phase I study, evidence of clinical activity is observed. These encouraging results and the unique PK observed support investigation of a more frequent dosing regimen for optimizing anti-MM responses. Updated data on safety, PK and efficacy of BT062 from this clinical trial will be presented at the meeting. Disclosures: Jagannath: Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Avigan:Genzyme: Consultancy, Research Funding; Celgene: Research Funding. Lutz:Immunogen, Inc.: Employment. Haeder:Biotest AG: Employment. Ruehle:Biotest AG: Employment. Uherek:Biotest AG: Employment. Wartenberg-Demand:Biotest AG: Employment. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

Description: Abstract 3008 Background: BT062 represents a CD138 specific antibody-drug conjugate comprising a chimerized anti-CD138 antibody conjugated to maytansinoid (DM4), an inhibitor of tubulin polymerization. CD138 (syndecan-1) which is highly upregulated on neoplastic plasma cells, is currently used as a standard identification marker for multiple myeloma (MM) and provides therefore a promising target for therapeutic intervention in MM. We have previously reported that BT062 exerts high selective in vitro and in vivo cytotoxic activity against CD138 positive MM cells (Ikeda et al., Clin Cancer Res. 2009; 15(12)). Based on these results, a Phase I clinical trial has been conducted in relapsed/refractory MM patients, which demonstrated an acceptable toxicity profile after repeated administration of single doses up to 160 mg/m2, as well as first signs of clinical activity in heavily pretreated patients (Chanan-Khan et al., ASH 2009). Here, we investigated the anti-myeloma efficacy of BT062 when combined with clinically approved myeloma drugs in vitro and in experimental animals in vivo. Methods and Results: In vitro drug combination studies on different myeloma cell lines indicated that BT062 exerts synergistic cytotoxic activity when combined for example with lenalidomide on RPMI8226 myeloma cells. None of the myeloma drug combinations with BT062 resulted in a strong antagonistic effect. Lenalidomide and bortezomib, representing novel established anti-myeloma drugs, were further tested for combination with BT062 in SCID mice bearing MOLP-8 MM xenografts. A single intravenous injection of BT062 was active against these tumors (400 μg/kg: T/C = 7%, Log cell kill (LCK) = 2.7) without signs of toxicity (no body weight loss). Monotherapy of bortezomib (1 mg/kg, twice weekly for 2 weeks) was inactive against the MOLP-8 xenografts. However, when combined with BT062, a superior anti-tumor effect compared to BT062 monotherapy was observed: Single injection of BT062 at different concentrations (100 μg/kg, 200 μg/kg, 400 μg/kg) followed by i.v. administration of bortezomib (1 mg/kg, twice weekly for 2 weeks) resulted in a dose dependent inhibition of tumor cell growth (at 200 μg/kg: LCK = 2.0; T/C = 7%, at 400 μg/kg LCK = 3.2; T/C = 7%). No increased in toxicity have been observed in the combination treatment compared to each monotherapy. BT062 was further evaluated for combination with lenalidomide which was administered intraperitoneally, daily for 5 days (2 weeks in total): BT062 administered alone at concentrations of 400 μg/kg was highly active against the MOLP-8 tumors reflected by a LCK of 2.5 (T/C = 13%). A lower anti-tumor effect has been observed by lenalidomide monotherapy (LCK = 0.8, T/C = 38%). Combination of both drugs demonstrated superior anti-tumor activity compared to each monotherapy (e.g. 400 μg/kg BT062 + lenalidomide: LCK = 3.5; T/C = 8%). Remarkably, neither body weight loss nor symptoms of illness have been observed confirming that this combination is also well tolerated. Conclusion: These data demonstrate that BT062 is not only effective in MM monotherapy but represents further a promising candidate for combination with currently used anti-MM drugs such as bortezomib or lenalidomide. No increased toxicities have been observed in all in vivo xenograft studies indicating that BT062 combined with other cytotoxic compounds might be well tolerated in MM therapy. Interestingly, although BT062 has been administered as a single dose, the anti-tumor effect in combination with either bortezomib or lenalidomide was significantly enhanced. These data provide a rationale to further investigate BT062 in clinical combination trials. Disclosures: Zuber: Biotest AG: Employment. Daelken:Biotest AG: Employment. Aigner:Biotest AG: Employment. Haeder:Biotest AG: Employment. Ab:ImmunoGen, Inc.: Employment. Whiteman:ImmunoGen, Inc.: Employment. Lutz:ImmunoGen, Inc.: Employment. Osterroth:Biotest AG: Employment. Uherek:Biotest AG: Employment.

Description: CD38 is a promising target for antibody therapeutics for the treatment of various hematological malignancies, including multiple myeloma as well as non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, and chronic myeloid leukemia. CD38 is a type II transmembrane glycoprotein with ectozyme activity that has been implicated in Ca2+ mobilization. CD38 expression correlates with the poor disease prognosis in some hematological malignancies. It has been proposed that rituximab, a well-established anti-CD20 antibody for the treatment of non-Hodgkin’s lymphoma, works by several mechanisms, including antibodydependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis. A panel of murine anti-CD38 antibodies was first screened for their ability to induce apoptosis, as measured by Annexin V staining, in various lymphoma, leukemia, and multiple myeloma cell lines. Chimeric human IgG1 versions of the selected murine antibodies with potent apopototic activity were then produced and screened for ADCC and CDC activities. Among the screened antibodies, a chimeric version of SAR650984, a humanized anti-CD38 antibody, had the best overall activities. SAR650984 induced potent apoptosis in Daudi, Raji, Ramos, and SU-DHL-8 lymphoma cells, as well as MOLP-8 multiple myeloma (MM) cells and DND-41 acute T lymphocytic leukemia (T-ALL) cells, which like most, if not, all MM and T-ALL cells, express CD38, but not CD20. SAR650984 also induced potent ADCC and CDC in these tumor cells. When the activities of SAR650984 and rituximab were directly compared in Daudi and Raji lymphoma cells that express similar levels of CD38 and CD20, SAR650984 had similar ADCC and CDC activities as rituximab, and, in addition, SAR650984 induced a greater percentage of lymphoma cells to undergo apoptosis than did rituximab. SAR650984 and rituximab showed similar efficacy in a SCID disseminated survival model with Daudi lymphoma cells. Groups of 10 mice were intravenously injected with 5 × 106 Daudi cells on day 0 and then treated with 40 mg/kg SAR650984, 40 mg/kg rituximab, or PBS control on days 7, 11, 14, 18, 21 and 25. The median survival (range) was 28 days (26–30 days) for the PBS-treated group, 47 days (40–51 days) for the SAR650984-treated group and 42.5 days (40–51 days) for the rituximab-treated group. SAR650984 also showed strong anti-tumor activity in a subcutaneous xenograft SCID model with CD38+ CD20− NCI-H929 multiple myeloma cells. Groups of 10 mice were subcutaneously injected with 106 NCI-H929 cells on day 0, and then treated with 40 mg/kg SAR650984, 40 mg/kg rituximab (non-binding IgG1 control), or PBS control twice weekly for three weeks starting on day 6 when the tumors were palpable. A mean tumor volume of 1000 mm3 was reached by PBS-treated group on day 89 and rituximab-treated group on day 84 (70–80% tumor take rate for NCI-H929). SAR650984 treatment completely prevented the tumor growth in all 10 mice (tumor free at the end of study on day 128). In summary, the humanized anti-CD38 antibody, SAR650984, has potent ADCC, CDC, and apoptotic activities in vitro and anti-tumor activity in vivo. SAR650984 is a promising therapeutic antibody candidate for various hematological malignancies, especially in diseases such as multiple myeloma, where rituximab is inactive.

Description: Erythrocytes have a defined lifespan in vivo, and the signals that maintain their survival in circulation or trigger their death are unknown. Here, we investigated the control of erythrocyte survival and death in an in vitro culture system where erythrocytes survived for 10 days in serum-free medium in the presence or absence of bovine serum. Death of the cells in culture was correlated with increased exposure of phosphatidylserine and increased levels of intracellular calcium. Cell death could be suppressed by supplementing the medium with human plasma or serum, resulting in a doubling of the lifespan to 20 days. Freshly isolated erythrocytes and cultured erythrocytes were both found to express Bcl-XL and, to a lesser extent, Bak in membrane protein extracts. Treatment of the cells with a Bak-derived BH3 peptide fused to the internalization sequence of the antennapedia protein, which has previously been shown to enter cells by diffusion and antagonize Bcl-XL, resulted in substantial cell death in erythrocyte cultures. BH3-induced death was accompanied by an immediate increase in accumulation of intracellular calcium and could be suppressed by plasma, but not by the caspase inhibitor zVAD. A BH3 peptide mutated at amino acid 78 of full-length Bak required for heterodimerization with Bcl-XL had no effect on cell viability or calcium levels. We conclude that the BH3 peptide accelerates erythrocyte death through antagonization of Bcl-XL. The data suggest that erythrocyte survival is promoted by survival factors in plasma and by membrane-associated Bcl-XL.