Abstract: Background: CD19 and CD20 are B-cell lineage-specific antigens expressed on the cell surface of most B-cell lymphomas. While CD20 is acquired during late stages of B-cell lymphogenesis and is then lost upon differentiation into plasma cells, CD19 expression covers the entire spectrum of early B-cell genesis and maturation. CD20-targeting agents have been broadly integrated into the therapeutic armamentarium for B-cell lymphomas. More recently, CD19-targeting agents have emerged as promising alternatives with demonstrated therapeutic value. Given the imminent availability of both CD19 and CD20 targeted therapies, and potential for combinational approaches, we studied the surface expression of these antigens at the single-cell level on lymphoma cells and benign background lymphoid subsets from biopsy specimens. Methods: Flow cytometric analysis (seven-color) was performed on biopsy specimens from 47 patients with newly diagnosed B-cell lymphomas, including diffuse large B-cell lymphoma (n=15), follicular lymphoma (n=15), marginal zone lymphoma (n=9), mantle cell lymphoma (n=9), Burkitt lymphoma (n=2), and unclassifiable low-grade B-cell lymphoma (n=2). Small lymphocytic lymphoma was intentionally excluded, given its well-described loss of CD20 expression. Biopsies from eight additional patients with persistent/recurrent B-cell lymphomas after anti-CD20 therapy (greater than 6 months after last dose) were also evaluated. Thresholds for CD19 or CD20 antigen positivity were defined for each case, based on the 95th percentile fluorescence intensity of the respective marker on tumor-infiltrating T-cells (internal negative control). In addition, CD19 or CD20 fluorescence intensities of tumor cells were normalized to background benign B-cells (internal positive control) using the median fluorescence ratio (MFR). Results: Both CD19 and CD20 were highly expressed on CD20 treatment naïve tumor cells, with a slightly higher median percentage of positive tumor cells for CD19 (98%) compared with CD20 (93%) (p=0.003), and one case lacking CD20 expression. When compared with background benign B-cells, CD20 was frequently overexpressed on tumor cells (mean MFR=1.8), while CD19 expression was overall similar to background benign B-cells (mean MFR=0.9) (p=0.001). As the surface density of CD20 on benign B-cells is reportedly higher than CD19 (~100,000 vs ~20,000 molecules per cell, respectively), these findings are consistent with a higher density of surface CD20 than CD19 on most B-cell lymphomas. However, CD20 expression was more heterogeneous (within individual patient samples and across patients), with a higher median percentage of CD20-negative tumor events (median=0.5%, range=0-98%) compared with CD19-negative events (median=0%, range=0-28%) (p=0.003). Interestingly, expression of CD19 within the CD20-negative tumor subsets was largely preserved (mean % CD19-positive events=97.86%, min=40%). In addition, percentages of CD19/CD20 double-negative tumor events were very small (median=0%, range=0-4.9%), and only detectable in 15 cases (32%). Of eight additional cases studied post-anti-CD20 immunotherapy (6-84 months after last dose), the percentage of antigen-positive events by tumor cells was largely preserved for both CD19 (median=99.6%, range=93.5-100%) and CD20 (median=95.7%, range=55.6-100%), similar to the pre-therapy cohort. Conclusions: CD19 and CD20 are both highly and consistently expressed in B-cell lymphomas. While CD20 has a higher average density of surface molecules per tumor cell, CD19 expression is more homogenous and is preserved in small CD20-negative tumor subsets and after anti-CD20 targeted therapy. These findings support the clinical evaluation of anti-CD19 immunotherapies and combinational therapies targeting both surface antigens. Disclosures Horna: MorphoSys AG: Research Funding. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Curis: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; MorphoSys: Consultancy, Research Funding; NanoString: Research Funding. Endell:MorphoSys AG: Employment, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties.

Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. Approximately 60% of DLBCL patients can be cured using standard chemotherapy, including an anti-CD20 antibody (R-CHOP; rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). However, 40% of DLBCL patients have refractory disease or experience relapse. This highlights the need for more effective therapies for this subset of relapsing or refractory patients. Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal anti-CD19 antibody, currently in clinical development in patients with relapsed or refractory (r/r) DLBCL in combination with either the immunomodulatory drug lenalidomide (LEN) (L-MIND study) or the chemotherapeutic agent bendamustine (B-MIND study). The B-lymphocyte antigen CD19 is broadly and homogeneously expressed throughout B-cell development and across different B-cell malignancies, including DLBCL. CD19 has been reported to enhance B-cell receptor signaling, which is assumed important for B-cell survival. This makes CD19 a promising therapeutic target in B-cell malignancies. Macrophages are abundant in the bone marrow stroma and lymph nodes of lymphoma patients. These lymphoma-associated macrophages (LAMs) support proliferation, survival, and drug resistance of lymphoma cells; therefore, serving a tumor-promoting function (Farinha et al, Blood, 2005). However, macrophages can also act as immune effector cells for antitumor effects exerted by therapeutic monoclonal antibodies, like tafasitamab. Given their abundance in DLBCL, an attractive therapeutic approach would be to stimulate their tumoricidal activity in order to promote antitumor immunity. Consequently, the immunomodulatory agent LEN has the potential to synergize with tafasitamab by enhancing antibody dependent cellular phagocytosis (ADCP). Here, we demonstrate that monocyte-derived macrophages and isolated LAMs from DLBCL patients can kill lymphoma cell lines and autologous lymphoma cells by tafasitamab-mediated phagocytosis. Tafasitamab-mediated ADCP activity was correlated with FcγRIII/FcγRI levels and inversely correlated with SIRPα expression on macrophage/effector cells (Spearman r between 0.51 and 0.62). Analyzing immune checkpoint receptor expression on LAMs of DLBCL patients, we found increased expression of VISTA, SLAMF7 and SIRPα compared with normal macrophages from healthy volunteers (VISTA: 2.8-fold, p=0.0006; SLAMF7: 2.9-fold, p=0.03; SIRPα: 1.8-fold, p=0.04). In contrast, we found no significant difference in FcγR expression (CD16: 1.4-fold, p=0.12; CD32: 1.3-fold, p=0.32; CD64: 1.2-fold, p=0.12). Moreover, pretreatment with lenalidomide showed a 3 to 5-fold enhanced tafasitamab-mediated cytotoxicity by macrophages and isolated LAMs (Figure 1A). Of note, the increased cytotoxicity was tafasitamab-specific and neither attributable to direct cytotoxic effects of LEN, nor increased CD19 expression on target cells. In addition, LEN treatment decreased the expression of SIRPα on macrophages (control vs. LEN: 3,117±652 MFI [mean fluorescent intensity] vs 2,327±610 MFI) (Figure 1B). This suggests that the CD47-SIRPα pathway may be suppressed by downregulation of SIRPα by LEN, contributing to the observed ADCP enhancement. These results suggest that tafasitamab leverages the presence of tumor-promoting LAMs as effector cells. The inherent ADCP activity of tafasitamab can be further improved by the immunomodulatory agent LEN, potentially via reduction of SIRPα levels on macrophages. Finally, our results provide a rationale for targeting the CD47-SIRPα axis that should be further explored in preclinical and clinical studies. Figure 1 Disclosures Endell: MorphoSys AG: Employment, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Bruns:Morphosys AG: Research Funding.

Abstract: Introduction In the era of CD19-targeting cancer therapies, e.g. CD19 CAR-T cell therapy, it is key to understand if CD19 expression on cancer cells is maintained after the previous line of therapy. In this context, we evaluated data from a Phase I trial of the CD19-targeting antibody tafasitamab (MOR208; XmAb®5574) in chronic lymphocytic leukemia (CLL). The trial was designed to evaluate the safety, tolerability, and pharmacokinetics (PK) of tafasitamab in patients with relapsed or refractory CLL and enrolled 27 patients (NCT01161511). The trial consisted of a dose escalation, expansion and follow-up phase and results were published by Woyach JA, et al. Blood 2014;124:3553-60. Methods Enumeration of B cells was performed by flow cytometry using the CD24 antigen expression as an alternative pan B cell marker in CLL. Routinely used B cell marker i.e. CD20 and CD19 were precluded as useful pan B cell marker in this trial: CD20 antigen, in particular after a prior anti-CD20 antibody containing therapy, is potentially dim or undetectable in CLL. CD19 antigen becomes undetectable on B cells following administration of tafasitamab, as commercial anti-CD19 reagent antibodies are cross-blocking with tafasitamab. Irrespective of the obvious limitation i.e. blockade of CD19 binding by the already bound tafasitamab during therapy and, for a certain time period, after the last tafasitamab administration, CD19 antigen quantification on CD24-positive B cells was performed with a phycoerythrin (PE) conjugated tafasitamab in conjunction with Quantum Simply Cellular microspheres (Bangs Laboratories, INC). This method allowed an accurate determination of CD19 antigen level at baseline (Cycle 1 Day 1, pre-treatment). In contrast, validity of determined CD19 antigen level after the first administrations of tafatsitamab was uncertain due to the above mentioned limitation of the applied method. Results We observed a significant discrepancy between CD24-positive and CD24/CD19 double-positive B-cell counts (the latter was near to zero counts) immediately after the first administration of tafasitamab. This finding is a clear indicator for interference of tafasitamab with accurate CD19 detection. Contrary, covergence of CD24-positive and CD24/CD19 double-positive B-cell counts should serve as an indicator for data validity. A pharmacokinetic/pharmacodynamic analysis of CD24-positive compared with CD24/CD19 double-positive B-cell counts at remaining tafasitamab serum concentrations after treatment end revealed an estimated threshold of ≤7 µg/mL tafasitamab serum concentration to allow for an evaluable CD19 detection using the tafasitamab-PE detection antibody. In case where a patient had more than one evaluable CD19 data point after tafasitamab treatment, mean CD19 expression levels were used for comparison to baseline. Applying the identified tafasitamab serum concentration threshold for detection, blood samples taken at a median of 84 days (range: 34-114 days) after the last tafasitamab dose were deemed evaluable. As a result, 14 of 27 enrolled patients had evaluable CD19 data. For these 14 patients, the median absolute CD19 antigen expression level at baseline was 27,500 antibodies bound per cell (ABC) (range: 13,000-35,300) (Figure 1). All of these patients maintained CD19 antigen expression level on the surface of CD24-positive B cells after tafasitamab treatment, with a relative median CD19 expression level of 109% (range: 71-166%) when compared with the CD19 expression level at baseline. Conclusion CD19 expression data observed in this Phase I study in CLL before and after cessation of tafasitamab treatment (median 84 days) indicate that CD19-targeted antibody treatment does not induce a loss of CD19 expression on CLL cells. Further studies are warranted to substantiate these findings. Figure Disclosures Boxhammer: MorphoSys AG: Employment, Patents & Royalties. Striebel:MorphoSys: Employment. Baumgartner:MorphoSys: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties.

Abstract: Introduction:CD38 is a type II transmembrane glycoprotein widely expressed in many hematological malignancies including multiple myeloma (MM). MOR202, a HuCAL-derived, human IgG1 CD38 monoclonal antibody, induces antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). MOR202 does not induce complement-dependent cytotoxicity, which is suspected to be a major contributor to infusion-related reactions (IRRs). Preclinical models of MM demonstrate high single-agent antitumor activity of MOR202 and synergy in combination with immunomodulatory drugs (IMiDs), lenalidomide (LEN) or pomalidomide (POM). Methods: This is an interim analysis of a multicenter, dose-escalation phase I/IIa study of MOR202 in relapsed or refractory (RR)MM. Preliminary safety and efficacy data from 3 patient cohorts treated with clinically relevant doses of MOR202 (administered as an IV 2-hour infusion), alone or in combination with an IMiD are presented: MOR202 4, 8 and 16 mg/kg weekly; MOR202 8 or 16 mg/kg weekly with either LEN or POM. All patients in these cohorts also received low dose dexamethasone. Primary objectives were to evaluate the safety, maximum tolerated dose (MTD) and recommended phase II dose of MOR202. Secondary objectives included an assessment of overall response rate, duration of response and progression-free survival. Results: As of July 12, 2016, a total of 66 patients had been treated; 31 in clinically relevant cohorts, including 18 patients receiving MOR202 alone, 8 receiving MOR202 + LEN and 5 receiving MOR202 + POM. Patients treated with MOR202 alone and MOR202 + POM had both received a median of 4 prior lines of therapy; 78% and 100% had been refractory to last prior treatment, respectively. Patients treated with MOR202 + LEN had received a median of 2 prior lines of therapy and 50% had been refractory to last prior treatment. Most of the patients had received bortezomib, LEN, cyclophosphamide, and melphalan alone or in combination with autologous stem cell transplant as part of their prior regimens. In this trial the MTD has not been reached yet. MOR202 alone or in combination with an IMiD was well tolerated, with mainly hematological toxicity reported. A 2-hour MOR202 infusion was feasible in all patients. In the clinically relevant cohorts only 1 patient discontinued due to an adverse event considered to be related to MOR202 (platelet count decreased) and no deaths related to any of the study drugs occurred. IRRs were seen in only 3/31 (10%) patients, all occurring during the first infusion. All IRRS were ≤ grade 2. So far, 28 patients were evaluable for response in the MOR202 clinically relevant cohorts. Of 16 evaluable patients in the MOR202 alone cohort, 3 partial responses (19%) and 2 very good partial responses (13%) were reported. In the MOR202 + LEN cohort 5/7 partial responses were seen, and 3/5 patients responded to MOR202 + POM treatment including 2 complete responses. Median time to response was 4 weeks, with responses tending to deepen over time. Most responses (10/13) are ongoing with the longest duration of response currently being 48 weeks. Preliminary analysis in 5 patients revealed preservation of high CD38 levels on MM cells under MOR202 therapy, with a mean decrease of only 10% from baseline to day 1 cycle 2 (4 weeks). Conclusions: In this analysis, a 2-hour infusion of MOR202 (up to 16 mg/kg) alone, or in combination with POM or LEN showed a very good safety profile, particularly an excellent infusion tolerability in heavily pretreated patients with RRMM. Promising preliminary efficacy and long-lasting tumor control was seen for MOR202 +/- IMiDs. The data suggest that CD38 expression on patient MM cells is preserved during treatment. Disclosures Raab: Novartis: Consultancy, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Amgen: Consultancy, Research Funding. Goldschmidt:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Honoraria, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Agis:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Einsele:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau. Engelhardt:Amgen: Research Funding; Janssen: Research Funding; MSD: Research Funding; Celgene: Research Funding. Ferstl:Novartis: Other: Case report presentation; Bristol Myers Squibb: Other: Advisory Board. Weisel:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Onyx: Consultancy; BMS: Consultancy, Honoraria; Novartis: Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Jarutat:MorphoSys AG: Employment. Weinelt:MorphoSys: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Peschel:MophoSys: Honoraria.

Abstract: Background: MOR202 is a fully human anti-CD38 antibody currently being tested in a Phase I/IIa clinical trial in multiple myeloma (MM). It mediates antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in MM cells with high potency (EC50 ~200 pM) representing a possible promising new therapy for MM patients. In this in vitro study, we evaluated the synergistic potential of MOR202 and pomalidomide (POM), a newly approved IMiD® immunomodulatory agent in MM therapy. Methods: Using flow cytometry analysis, POM was evaluated in relation to its effects on several parameters anticipated to be relevant for anti-tumor activity when combined with MOR202. This included the induction of direct cytotoxicity and CD38 upregulation in several MM cell lines, as well as the activation of human immune effector cells derived from peripheral blood mononuclear cells of healthy donors. On a functional level the interaction of MOR202 and POM was assessed using FACS-based ADCC assays. Different incubation schemes prior to the ADCC assays were evaluated in order to distinguish the influence of POM on ADCC activity when pre-incubated for 72 hours either on target or effector cells or on both in parallel. The observed combination effects were analyzed for synergistic potential. Experiments were carried out in triplicate and mean values (±SEM) were calculated. Results: POM as a single agent showed cytotoxic effects on MM cell lines with high potency (EC50 ~150 nM) and additionally induced an up to 2.7-fold upregulation of CD38 (EC50 ~20 nM) on CD38-expressing MM cell lines. Both effects were maximal at the last tested time point of 72 hours and strongest on cell lines with comparably lower CD38 expression levels. In combination with the observed activation of effector cells these POM-mediated mechanisms lead to a synergistically enhanced cytotoxic activity of MOR202. This synergistic benefit ranged between 1.2-fold and 3.1-fold above theoretical additivity depending on the cell line used and was most pronounced in the case of strong CD38 upregulation. Figure 1 Exemplary ADCC dose-response curves for the comparably lower CD38 expressing cell line AMO-1 after POM pre-treatment of effector cells, target cells or both. Figure 1. Exemplary ADCC dose-response curves for the comparably lower CD38 expressing cell line AMO-1 after POM pre-treatment of effector cells, target cells or both. Conclusions: The cytotoxic activity of MOR202 on MM cells was enhanced and synergized when combined with the immunomodulator agent POM via multiple mechanisms, namely direct cytotoxicity, CD38 upregulation and activation of effector cells. These results provide a mechanistic rationale for combining MOR202 and POM and warrant further evaluation in the clinical setting. Disclosures Endell: MorphoSys AG: Employment, Patents & Royalties. Boxhammer:Morphosys AG: Employment, Patents & Royalties. Steidl:MorphoSys AG: Employment, Patents & Royalties.

Abstract: Abstract 4018 Background: MOR202, a HuCAL-derived fully human anti-CD38 antibody has proven to be highly effective in preclinical models of multiple myeloma (MM) and is currently evaluated in a phase I/II clinical trial. Having previously identified ADCC as potent effector mechanism of MOR202, the capability to induce killing of MM patient cells via antibody dependent cellular phagocytosis (ADCP) was assessed. Furthermore, the immunomodulatory agent lenalidomide (LEN), a commonly used agent in MM therapy was investigated for its potential to enhance the cytotoxicity of MOR202 in vitro and in vivo. Methods: Drug effects in presence or absence of LEN were characterized in vitro in a FACS-based phagocytosis assay using macrophages derived from M-CSF stimulated monocytes and a panel of CD38+ MM target cells. The therapeutic potential of MOR202 in vivo as a single agent and in combination with LEN was studied in a disseminated survival model of iv injected Ramos lymphoma cells. Synergy was determined using the fractional product concept. Results: MOR202 induced ADCP with high potency showing EC50 values between 40 and 130pM. Maximal killing was donor and cell line dependent ranging from 11 to 72%. The combination with LEN induced additive to synergistic enhancement of MOR202 activity which was mediated by several mechanisms identified to be direct cytotoxicity and increased CD38 expression levels on MM cells. In vivo, MOR202 at 1mg/kg increased median survival (MS) by 142% compared to vehicle control (MS day 48.5 vs. day 20), while LEN had no distinct effect at any of the tested doses between 25 and 100mg/kg (MS day 21–22). Intriguingly, despite the apparent lack of efficacy by LEN alone co-administration of 1mg/kg MOR202 and 100mg/kg LEN resulted in a superior increase of MS by 225% (MS day 65), thus demonstrating strong synergism. Of note, while no mouse survived beyond day 65 in any other group, 3/8 animals in the combo group were completely free of tumor until study termination on day 98. Conclusions: We here demonstrate ADCP to be an additional potent effector mechanism of MOR202. The combination of MOR202 and LEN enhanced ADCP-mediated killing in vitro and increased median survival in vivo in a synergistic manner. These results suggest a mechanistic rationale for MOR202 combinations with LEN including in particular LEN non-responders or relapse/refractory patients, thus warranting further evaluation in future clinical trials. Disclosures: Endell: MorphoSys AG: Employment. Boxhammer:MorphoSys AG: Employment. Wurzenberger:MorphoSys AG: Employment. Ness:MorphoSys AG: Employment. Steidl:MorphoSys AG: Employment.