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Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo.Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model).Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G2-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model.Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.

Translation on this Article from The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Selective Cytotoxicity Against CD138-Positive Multiple Myeloma Cells In vitro and In vivo

Supplementary Data from The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Selective Cytotoxicity Against CD138-Positive Multiple Myeloma Cells In vitro and In vivo

CD4(+)CD25(+) regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.

Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G2-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.

Objectives: Individual immunoglobulins expressed by B-cell lymphomas represent tumor-specific antigens (‘idiotypes’). Immunization with idiotype in follicular lymphoma patients may induce specific immune responses, sustained progression-free survival, and disappearance of minimal residual disease. Manufacturing of idiotype vaccines has mostly relied on heterohybridomas established from viable lymphoma cells. This paper describes the feasibility of production of GMP-grade idiotype vaccines as recombinant Fab fragments in Escherichia coli. Methods: IgH and IgL transcripts were analyzed by anchored PCR from 106 lymphoma and nine control biopsies. Lymphoma-derived V segments were inserted into prokaryotic expression plasmids. Recombinant idiotype Fab fragments were expressed in E. coli in a fermentation system. Results: Idiotype IgH and IgL transcripts were identified in 95% of 106 lymphoma biopsies according to stringent clonality criteria. Large-scale idiotype expression was successful in 69 of 78 cases (89%) and yielded a median of 17 mg (range: 1.2–250 mg) recombinant Fab protein. After affinity chromatography, median vaccine purity was 99% heterodimeric Fab protein (range: 72–100%). Bacterial protein contamination was detectable in one vaccine only. Fab proteins with IgL lambda chains had a tendency for inferior yield and lesser purity than κ-type Fabs. Among other structural idiotype features (isotype, V family usage, somatic hypermutation pattern, novel glycosylation sites, CDR III net charge), no consistent influences on Fab yield or purity were detected. Conclusions: Anchored PCR cloning and subsequent protein expression in E. coli provides a reliable technological basis for clinical idiotype vaccination trials.

Malignant lymphomas are clonal neoplasms of lymphoid origin. By definition, all cells of the malignant clone have undergone the same rearrangement of antigen receptor genes and express identical antigen receptor molecules (immunoglobulin for B cell lymphomas, T cell receptor for T cell lymphomas). The hypervariable stretches within the variable regions of these receptors are considered true tumor-specific antigens (‘idiotypes’). In several animal models, protective humoral or cellular immunity can be induced against the malignant lymphoma by vaccination with the tumor-derived idiotype. Successful experimental immunization strategies in animals include idiotype protein vaccines combined with various adjuvants, genetically or immunologically modified lymphoma cells, idiotype-presenting dendritic cells, idiotype-encoding viral vectors, and DNA immunization. Firm evidence for the induction of lymphoma-specific immunity has also been obtained from human idiotype vaccination trials. Furthermore, some trials have provided strong but hitherto formally unproven evidence for clinical benefit of idiotype-vaccinated patients. Alternative vaccination approaches are based on immunologically modified tumor cells. Current research efforts concentrate on the identification of the most efficacious vaccination route, on definitive proof of clinical efficacy, and on the development of convenient methods to manufacture individual idiotype vaccines.

By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmo- nella phage P22 have been identified. Temperature- sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect ther- mal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel b-helix. We modified tailspike by rational single amino acid replace- ments at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) muta- tions in a similar structural context as two of the four known su mutations, which have been sug- gested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the b-helix core. The high-resolution crystal struc- ture of mutant T326F proves that structural rear- rangements only take place in the remarkably plas- tic lumen of the b-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the sur- face of the protein unaffected. This supports the notion that changes in the stability of an intermedi- ate, in which the b-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mu- tants, on the other hand, appears to be more diffi- cult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interac- tions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statisti- cal analysis of the distribution of backbone dihedral angles in known three-dimensional protein struc- tures that the adoption of f/c angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89 -101.

An expression system for rapid and standardized production of human recombinant immunoglobulin Fab fragments in E. coli was developed. Functional folding of the Fab fragments was accomplished by the dicistronic expression vector pFab.gammakappa containing specialized leader sequences to direct the immunoglobulin heavy and light chains to the periplasmic bacterial space. A C-terminal hexahistidine tag of the Fd chain facilitated metal affinity chromatography and purification to homogeneity as assessed by SDS PAGE and silver staining. Specific antigen recognition by a hybridoma-derived Fab fragment was indistinguishable from that of the corresponding monoclonal antibody. This protocol may be useful for analysis of the antigen specificity of human B cells and for convenient production of lymphoma-derived idiotype protein for vaccination strategies. To obtain unmodified immunoglobulin cDNA sequences from small human biopsies for insertion into pFab.gammakappa, oligo(dG)-tailed cDNA was amplified with an oligo(dC)- and nested mu or kappa constant region-specific primers. Using single sets of primers for each class of immunoglobulin transcripts, the products of this anchored PCR reflected the relative abundance of the starting cell population and permitted reliable identification of clonal, lymphoma-derived sequences for subsequent expression cloning.

CD138 (Syndecan1) is highly expressed on multiple myeloma (MM) cells. In this study, we examined the anti-MM effect of murine/human chimeric CD138-specific monoclonal antibody (mAb) nBT062 conjugated with highly cytotoxic maytansinoid derivatives in vitro and in vivo. These agents significantly inhibited growth of CD138-positive MM cell lines and primary tumor cells from MM patients, without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G2/M cell cycle arrest followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Non-conjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells (BMSCs). Co-culture of MM cells with BMSCs, which protects against dexamethasone-induced death, had no impact on the cytotoxicity of the immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo in both human multiple myeloma xenograft mouse models and in SCID-human bone grafts (SCID-hu mouse model). These studies provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.

Abstract 4042 Background: CD138 (Syndecan-1) is highly overexpressed in various solid tumors and hematological malignancies, and represents one of the most specific target antigens for identification of multiple myeloma (MM) cells. BT062 (Biotest AG Dreieich, Germany) is an antibody-drug conjugate, comprised of the anti-CD138 chimerized MAb (nBT062) and the cytotoxic agent DM4. Once bound to CD138 on a target cell, the conjugate is internalized and releases DM4, leading to target cell death. Data from the first in human study (969) of BT062 demonstrated an acceptable toxicity profile and evidence of clinical activity in heavily pretreated relapsed and/or refractory MM patients(1). Based on these data, a Phase I/IIa study in MM (975) was initiated to further evaluate the safety and efficacy of BT062 when given more frequently. Objectives: To determine the maximum tolerated dose (MTD), the dose-limiting toxicities (DLTs), pharmacokinetics (PK), and anti-MM activity of increasing doses of BT062, when administered on a repeated, multiple dose schedule (Days 1, 8, and 15, every 4 weeks) in patients with relapsed and/or refractory MM. Methods: This is a prospective, open label, dose-escalation, multicenter Phase I/IIa study. The Phase I part includes dose escalation, and the Phase IIa the expansion of the MTD or Recommended Phase II Dose (RPTD) cohort. Patients aged ≥18 years with relapsed or relapsed/refractory MM who have failed previous treatment, including an immunomodulatory 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 were enrolled in cohorts of at least 3 at each dose level; DLT in the first cycle triggered cohort expansion. Toxicities were assessed by CTCAE v4 and clinical response was assessed according to the international myeloma working group criteria. Results: A total of 29 patients have been treated with BT062, receiving 1 of the first 8 dose levels ranging from 40 mg/m2 to 160 mg/m2. One out of 6 patients treated at 140 mg/m2 experienced a palmar-plantar erythrodysesthesia syndrome that was assessed as DLT (SAE, CTC grade 3). Dose was escalated to 160 mg/m2, and 1 of the 2 patients treated experienced an elevation of liver enzymes that was reported as DLT (non-serious, CTC grade 3). Maximum administered dose has not been reached and recruitment into the 160 mg/m2 cohort is ongoing. About 90% of the reported Adverse Events (AE) are grades 1–2. The most frequently reported AEs are anemia, diarrhea, and fatigue. Among the 23 patients evaluable for efficacy, 1 patient achieved a partial response (PR) and has been on study treatment for more than 1 year. Stable disease (SD) for at least 3 months was noted in an additional 11 patients, with median progression free survival of 112 (90–245) days. Thus disease control (PR + SD) was noted in more than 50% (12/23) of patients. The most common reason for discontinuation was disease progression. Preliminary pharmacokinetic results indicate rapid early clearance of BT062 from plasma consistent with rapid delivery and binding of BT062 to the MM cells. No significant accumulation of BT062 was noted, even after the end of the 2nd and 3rd infusions within a cycle. Conclusion: Preliminary data from this ongoing study indicate that BT062 is well tolerated even in this multiple dose schedule and provide further evidence of its clinical activity. Dose escalation is ongoing (now at 160 mg/m2) to define the MTD/RPTD, with cohort expansion then planned to further evaluate the safety and efficacy of BT062 at this dose. Based on the favorable safety profile, a Phase I/IIa study (983) has been initiated to evaluate the safety and efficacy of BT062 in combination with lenalidomide and dexamethasone. Disclosures: Heffner: Millennium: Research Funding. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: 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: Honoraria, Membership on an entity's Board of Directors or advisory committees. Zimmerman:Novartis: Consultancy; Millennium: Honoraria; Celgene: Honoraria. Lonial:Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Merck and Co: Consultancy. Lutz:ImmunoGen, Inc.: Employment. Czeloth:Biotest AG: Employment. Osterroth:Biotest AG: Employment. Ruehle:Biotest AG: Employment. Beelitz:Biotest Pharmaceuticals Corporation: Employment. Wartenberg-Demand:Biotest AG: Employment. Haeder:Biotest AG: Employment. Anderson:Celgene: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees; Oncopep: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Consultancy. Munshi:Oncopep: Patents & Royalties; Merck: Consultancy; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract 305 Background: CD138 (Syndecan-1) is highly overexpressed in various solid tumors and hematological malignancies and represents one of the most specific target antigens for identification of multiple myeloma (MM) cells. BT062 (Biotest AG Dreieich, Germany) is an antibody-drug conjugate, comprised of the anti-CD138 chimerized MAb (nBT062) and the cytotoxic agent DM4. Once bound to CD138 on a target cell, the conjugate is internalized and releases DM4, leading to target cell death. We performed the first in man study (969) to investigate safety and efficacy of BT062 in MM. 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 patients with relapsed and/or refractory MM. Methods: This is a prospective, open label, dose-escalation, multicenter phase I study. Patients aged ≥18 years with relapsed or relapsed/refractory MM who have failed previous treatments including an immunomodulatory 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 were enrolled in cohorts of 3 at each dose level, with DLT in the first cycle triggering cohort expansion. Toxicities were assessed by CTCAE v3 and clinical response was assessed according to the international myeloma working group criteria. Results: A total of 32 patients have been treated with BT062, receiving one of 7 dose levels ranging from 10 mg/m2 to 200 mg/m2. Maximum administered dose has been defined at 200 mg/m2, with mucositis as the dose limiting toxicity (CTC grade III in 2 of the 3 patients in this cohort). Thirteen of 32 patients have been treated in an expanded MTD-cohort at 160 mg/m2. The most frequently reported adverse events to date are mild to moderate and cover primarily events expected for the underlying disease and patient group. A few adverse events have also been observed involving skin and/or mucosa (tissues of epithelial origin with CD138 expressing cells), as well as the eye. CTC grade II/III toxicity involving skin and/or mucosa (e.g. mucositis, stomatitis, hand/foot syndrome) has been observed mainly at the dose levels 160 mg/m2 or higher. Adverse events involving the eye (e.g. blurred vision, dry eye) have also been reported mainly in patients at the dose levels 160 mg/m2 or higher, all restricted to CTC grade I/II. Among the 27 evaluable patients, 3 patients responded including 1 partial response and 2 minor responses, with one patient (minor response) remaining on treatment for more than a year. Stabilization of disease was noted in an additional 11 patients, receiving a median of 5 cycles of therapy (range of 4–10). Thus stable disease or better was noted in 52% of patients. Most patients came off study due to disease progression. Conclusion: Preliminary data from this study demonstrate an acceptable toxicity profile of BT062. Even in this phase I patient population, evidence of clinical activity was observed. Based on the favourable safety profile, the pharmacokinetic data and early signs of clinical activity, a Phase I/IIa study in MM (975) is initiated to further evaluate the safety and anti-MM efficacy of BT062 in a more frequent dosing regimen. To date 13 patients have been treated with BT062 on the intensified multi-dose regimen, receiving one of the first four dose levels. Updated results on safety, PK and anti-MM efficacy of BT062 will be presented. Disclosures: Jagannath: Celgene: Honoraria; Millennium/Takeda Pharma: Honoraria; J&J Family: Honoraria; Onyx: Honoraria; Merck: Honoraria. Heffner:Millennium: Research Funding. Avigan:Genzyme: Consultancy, Research Funding; Celgene: Research Funding; Curetec: Research Funding. Lutz:ImmunoGen, Inc.: Employment. Engling:Biotest AG: Employment. Uherek:Biotest AG: Employment. Osterroth:Biotest AG: Employment. Ruehle:Biotest AG: Employment. Beelitz:Biotest Pharmaceuticals Corporation: Employment. Niemann:Biotest AG: Employment. Wartenberg-Demand:Biotest AG: Employment. Haeder:Biotest AG: Employment. Anderson:Merck: Consultancy; Onyx: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. 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.

Abstract 3060 Background: 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. BT062 (Biotest AG Dreieich, Germany) is an antibody-drug conjugate, comprised of the anti-CD138 chimerized MAb (nBT062) and the cytotoxic agent DM4. Once bound to CD138 on a target cell, the conjugate is internalized and releases DM4, leading to targeted cell death. Preclinical investigations demonstrated strong 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 patients with relapsed and/or refractory MM. Toxicities were assessed by CTCAE v3 and clinical response was assessed according to the international working group criteria. 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 immunomodulatory 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 were enrolled in cohorts of 3 at each dose level, with DLT in the first cycle triggering cohort expansion. Results: A total of 32 patients have been treated with BT062, receiving one of 7 dose levels ranging from 10 mg/m2 to 200 mg/m2. Maximum administered dose has been defined at 200 mg/m2, with mucositis as the dose limiting toxicity (CTC grade III in 2 of the 3 patients in this cohort). Therefore, the MTD was defined at 160 mg/m2. Thirteen of 32 patients have been treated in an expanded MTD-cohort. No CTC grade 4 toxicity has been reported. The most frequently reported adverse events to date cover primarily events expected for the underlying disease and patient group. Most of the reported adverse events are CTC grade I to II. Nevertheless, a few adverse events have also been observed involving skin and/or mucosa (tissues of epithelial origin with CD138 expressing cells), as well as the eye. Severe events involving skin and/or mucosa (e.g. mucositis, hand/foot syndrome) have only been observed at the dose levels 160 mg/m2 or higher. Adverse events involving the eye (e.g. blurred vision, dry eye) have been reported in only 3 patients overall at the dose levels 160 mg/m2 or higher, all CTC grade I to II. At dose levels up to 120 mg/m2, preliminary PK results indicate an unusual rapid clearance from plasma in the early elimination phase, followed by a generally normal terminal elimination phase. A more typical clearance profile was observed for all patients at the 160 mg/m2 and 200 mg/m2 dose. To date, one patient showed a decrease in urine M-Protein by >50% after 8 repeated low doses of 20 mg/m2 each. At a high dose level of 160 mg/m2, another patient showed a >50% decrease of serum FLC after two doses of BT062. In total, stabilization of disease was noted in 13 patients. Patients with stable disease received a median of 5 cycles of therapy (range of 3–10). Most patients came off study due to disease progression. Conclusion: Preliminary data from this phase I study demonstrate an acceptable toxicity profile of BT062. Even in this phase I patient population, evidence of clinical activity was observed. Based on the favourable safety profile, the pharmacokinetic data and early signs of clinical activity, a Phase I/II study in MM is initiated to further evaluate the safety and anti-MM efficacy of BT062 in a more frequent dosing regimen. Updated results on safety, PK and efficacy of BT062 will be presented. Disclosures: Jagannath: Celgene: Honoraria; Millenium/Takeda Pharma: Honoraria; J&J Family: Honoraria; Onyx: Honoraria; Merck: Honoraria. Heffner:Millennium: Research Funding. Avigan:Genzyme: Consultancy, Research Funding; Celgene: Research Funding; Curetec: Research Funding. Lutz:ImmunoGen, Inc.: Employment. Uherek:Biotest AG: Employment. Osterroth:Biotest AG: Employment. Ruehle:Biotest AG: Employment. Haeder:Biotest AG: Employment. Niemann: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:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Acetylon: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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.

CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

Because of their hypervariable regions and somatic mutations, the antigen receptor molecules of lymphomas (idiotypes) are tumor-specific antigens and attractive targets for antilymphoma immunotherapy. For the optimal induction of human idiotype-specific cytotoxic T cells (CTL), idiotype was presented to CD8(+) peripheral blood mononuclear cells by monocyte-derived autologous dendritic cells (DC) after the endocytosis of idiotype protein or by idiotype-expressing DC. Recombinant idiotype was obtained as a functionally folded Fab fragment by periplasmic expression in Escherichia coli. Idiotype-expressing DC were generated by transduction with recombinant Semliki forest virus vectors encompassing heavy- or light-chain idiotype genes. Autologous lymphoblastoid cell lines stably transfected with Epstein-Barr virus-based idiotype expression vectors were used as target cells to detect idiotype-specific lysis. CTL stimulated with idiotype-loaded DC showed strong specific, CD8-mediated, and major histocompatibility complex (MHC) class I-restricted cytotoxicity against autologous heavy- and light-chain idiotype. In contrast, stimulation with idiotype-transduced DC resulted in only moderate natural killer cell activity. These data confirm the existence of idiotype-specific CTL in patients with lymphoma, define a "good manufacturing practice"-compatible protocol for the generation of these cells without the requirement of viable lymphoma cells, and favor the processing of exogenous antigen over DC transduction for the induction of MHC I-restricted CTL against idiotypes with unknown antigenicity. (Blood. 2000;95:1342-1349)