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Despite the recent advancements in treatment options with the introduction of anti-CD20 monoclonal antibody therapy, approximately 50% of patients with non-Hodgkin's lymphomas (NHL) will not sustain a durable response to standard of care (SOC) treatment. Thus, there remains a continuous need for safer and more effective anti-cancer therapies in this indication. T-cell bispecific antibodies (TCBs) represent a new class of disease targeting agents shown to promote the activation of a patient's own T cells to attack and kill cancer cells. CD20 TCB is a new bispecific antibody with IgG-like pharmacokinetic properties whose unique "2:1" structure leads to increased tumor antigen avidity, T cell activation, and tumor cell killing, as compared to other T cell engaging bispecific antibody molecular formats. The molecule comprises two CD20 binding Fabs (derived from the Type II CD20 IgG1 obinutuzumab), one CD3e binding Fab (fused to one of the CD20 Fabs via a short flexible linker), and an engineered, heterodimeric Fc region with completely abolished binding to FcgRs and C1q. In vitro, CD20 TCB was shown to dose-dependently induce tumor lysis with EC50 values in the range of 0.05 - 3.1 pM. The "2:1"format of CD20 TCB was shown to confer superior potency (up to 10 - 1000x) when compared to CD20 TCBs having the conventional "1:1" IgG-based format (i.e., one binding domain for CD20 and one for CD3). CD20 TCB-mediated tumor lysis resulted in T-cell activation, proliferation and cytokine release with up-regulation of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis upon tumor lysis. CD20 TCB also demonstrated potent ex vivo activity in whole bone marrow aspirate samples of NHL and CLL patients (n=17). Such primary tumor samples preserve the native tumor microenvironment and bear low effector to target cell ratios ranging in this study from 0.02 to 0.8 (average value 0.3). CD20 TCB activity was consistently superior to that of the "1:1" CD20 TCB and demonstrated faster, more profound and more potent B cell depletion with EC50 values ranging from 0.002 to 2.7 nM. In vivo, CD20 TCB displayed potent anti-tumor activity in xenograft models in stem cell humanized mice and induced regression of large, aggressive WSU-DLCL2 lymphoma tumors (0.5 mg/kg, weekly administration). In addition to tumor regression, CD20 TCB treatment led to fast and complete elimination of peripheral blood B cells within 24 h after the first administration (0.05, 0.15 and 0.5 mg/kg, weekly administration) and to a complete elimination of B cells in spleen, bone marrow and lymph nodes after two administrations. B cell depletion was paralleled by transient decrease of T-cell counts in the peripheral blood and by the peak of cytokine release 24 h after the first administration, followed by rapid recovery and return to baseline levels at 72 h post treatment. Tumor growth inhibition mediated by CD20 TCB was accompanied by increase in intra-tumor T-cell infiltration, up-regulation of PD-1 receptor on T cells and PD-L1 in the tumor. Combination studies of CD20 TCB with PD-L1 blocking antibody led to more profound and faster tumor growth inhibition. Taken together, the preclinical data show that CD20 TCB is a novel differentiated CD20-targeting T cell bispecific antibody with promising anti-tumor activity and the ability to modify the tumor microenvironment. CD20 TCB consistently demonstrated superior potency compared to other CD20 TCBs with a conventional "1:1" IgG format. This translated into superior efficacy in vitro, ex-vivo and in vivo, which could not be matched by increasing doses of the "1:1" TCBs. The molecule is now scheduled to start clinical trial by December 2016. Disclosures Bacac: Roche: Employment, Equity Ownership, Patents & Royalties. Umaña:Roche: Employment, Equity Ownership, Patents & Royalties. Herter:Roche: Employment, Patents & Royalties. Colombetti:Roche: Employment. Sam:Roche: Employment. Le Clech:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Richard:Roche: Employment. Nicolini:Roche: Employment. Gerdes:Roche: Employment. Lariviere:Roche: Employment. Neumann:Roche: Employment. Klein:Roche: Employment, Patents & Royalties.

Background & Aim: T-cell bispecific antibodies (TCBs) binding to a target on tumor cells and CD3 on T cells induce potent T-cell mediated killing of cells carrying the target. In contrast to targets like e.g. CD38 or CD138, B-cell maturation antigen (BCMA) is suggested to be only expressed on plasma cells (PCs) and multiple myeloma (MM) PCs. Therefore, a BCMA-TCB should specifically act on these cell types. We report on a new class of BCMA-TCBs designed for effective and convenient therapy of MM. Molecular structure & its rationale (fig. 1A): The new class of BCMA-TCBs are asymmetric two-arm IgG-based human antibodies, bivalent to BCMA, monovalent to CD3 and comprising an engineered Fc. To achieve tumor specificity of the BCMA-TCBs and avoid unspecific T-cell activation, monovalent binding to CD3 was fixed at an affinity of KD= 70 nM, whereas various BCMA-TCBs with binding affinities to BCMA ranging from KD= 50 pM to 10 nM have been investigated (measurement of binding affinities by surface plasmon resonance and flow cytometry (FC)). For long elimination half-life, an Fc was introduced to enable once a week intravenous or subcutaneous administration. Fc is engineered to abolish binding to FcgR and C1q to minimize risk of infusion reactions without interfering with FcRn binding. The BCMA-TCBs can be well-manufactured and have no tendency to aggregation. In vitro profile: Potency of BCMA-TCBs to activate T cells and to induce killing of human MM cell lines was measured in a 24h co-culture assay with human PBMCs and MM cells in a ratio of 10:1. Results: (i) NCI-H929 cells expressing on cell surface up to 100 times more BCMA than primary patient MM cells were killed in presence of BCMA-TCBs with EC50 ranging from 5 to 50 pM, but not in presence of a control-TCB binding to CD3 only. (ii) As next, RPMI-8226 cells were used as target cells because surface BCMA expression was found to be only slightly higher than on primary patient MM cells (specific antigen binding capacity SABC as measured by FC between 1165 and 5461 per RPMI-8226 cell compared to 116 to 4479 per primary patient MM cell). Effective killing of RPMI-8226 MM cells was observed; the killing potency was higher respectively EC50 lower with the BCMA-TCBs having binding affinities below 1 nM (fig. 1B; EC50 from 50 pM to 1000 pM). (iii) Killing of U266 and L363 human MM cell lines was also observed with BCMA-TCBs. (iv) T-cell activation and increased T-cell function go in parallel with lysis of MM target cells as observed by an upregulation of CD69 and CD25 expression, release of granzyme B (>20 ng/mL at 3 nM vs. 20 pg/mL for control) and proinflammatory cytokines e.g. IFN-g, TNF-a, IL-2, IL-6, and IL-10. (v) At 120h incubation, BCMA-TCBs induced concentration dependant CD4 and CD8 T-cell proliferation as observed by CFSE dilution. A small exploratory study in whole bone marrow (BM) aspirates from MM patients (n=3) suggested a concentration dependent killing of MM PCs induced by BCMA-TCBs in presence of autologous T cells, thus justifying for a much larger trial to investigate one of the BCMA-TCB of this new class to induce killing and T-cell activation/function in MM patient BM aspirates performed by two clinical groups (Abstract also submitted to ASH). In vivo profile: New BCMA-TCBs bind to BCMA as well as CD3 of cynomolgus monkeys (cyno). Single dose pharmacokinetics (PK) and pharmacodynamics of a BCMA-TCB was studied in cyno at 0.003, 0.03, 0.3 mg/kg intravenously. Dose linear PK was observed. Mean serum concentrations in the first 7 days after 0.03 mg/kg were ≈1 to 2 nM, BM aspirates collected at 96h showed also concentrations of ≈1 to 2 nM. Elimination between 24 h and 504 h was found to be first order with a half-life of approximately 6 to 8 days. These data suggest a convenient clinical dosing schedule, e.g. once a week. Peripheral blood T-cell redistribution was observed 24h after dosing. Reduction of PCs could be observed by FC while total B cells and other cell types were unaffected. Summary: Effective and specific killing of MM cells was demonstrated with the BCMA-TCBs. Killing goes in parallel with T-cell activation and increased function. TCB with binding affinity to BCMA below 1 nM have higher potency/lower EC50 than those with affinity above 1 nM. TCBs could be produced with high purity and were stable with no tendency to aggregation. In cynomolgus monkeys, a PK profile has been found, suitable for once a week administration. This new class of BCMA-TCB has promises for clinical development. Disclosures Vu: EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Moser:Roche: Employment. Delon:Roche: Employment. Latzko:Roche: Employment. Gianotti:Roche: Employment. Lüoend:Roche: Employment. Friang:Roche: Employment. Murr:Roche: Employment. Duerner:Roche: Employment. Weinzierl:Roche: Employment. Fauti:Roche: Employment. Bacac:Roche: Employment. Ast:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Rodriguez Diaz:Roche: Employment. Zielonka:Roche: Employment. van Puijenbroek:Roche: Employment. Hosse:Roche: Employment. Bruenker:Roche: Employment. Mössner:Roche: Employment. Klein:Roche: Employment. Umaña:Roche: Employment. Strein:EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BB Biotech AG: Membership on an entity's Board of Directors or advisory committees; Novimmune SA: Membership on an entity's Board of Directors or advisory committees.

Introduction IL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the known IL-2-based immunocytokines has yet progressed to pivotal clinical trials due to various constraints in their design; in particular, the fusion of two wild-type IL-2 moieties to the antibody and retained FcgR binding of IgG-based immunocytokines. This design results in 1) high affinity binding with pM affinity to IL-2Raβγ on immune cells compromising tumor targeting and inducing rapid systemic clearance and short half-life; 2) high affinity for CD25 (IL-2Ra) expressed on pulmonary vascular endothelium contributing to pulmonary toxicity; and 3) preferential activation of Tregs over immune effectors. Here we describe a novel class of monomeric tumor-targeted immunocytokines where a single, engineered IL-2 variant (IL2v) with abolished CD25 binding is fused to the C-terminus of an antibody with a heterodimeric Fc-part. FcγR and C1q binding is completely abolished by a novel Fc mutation. For tumor targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA-IL2v recognizes a membrane proximal epitope of human carcinoembryonic antigen (CEA) and binds preferentially to membrane-bound CEA, but not shed CEA. Methods CEA- and FAP-IL2v were produced as recombinant proteins and their activity tested oneffector cells by assessing the activation of P-STAT5, cell proliferation, sensitivity to Fas-induced apoptosis, expression of activation markers and cytokine release upon treatment. Safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were analyzed in SCID and fully immunocompetent C57Bl/6 mice as single agent and in combination with trastuzumab and cetuximab. Tumor targeting was investigated in the orthotopic syngeneic Renca renal cell cancer tumor model in Balb/c mice by SPECT imaging. Results FAP- and CEA-IL2v completely lack binding to CD25, but retain IL-Rβγ binding, and show pM binding affinity to respective antigens, FAP on fibroblasts and CEA on tumor cells. As consequence of abolished binding to CD25 these molecules do not preferentially activate Tregs. The treatment of effector cells with IL2v reduces their sensitivity for Fas-mediated apoptosis (also known as activation induced cell death) as compared to wild-type IL-2 based immunocytokine. IL-2Rβγ bioactivity was retained and FAP- and CEA-IL2v activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers, cell proliferation and cytokine release. Furthermore, CEA-IL2v and FAP-IL2v enhanced the cytotoxic activity of NK cells when combined with ADCC-competent antibodies. Mechanism of action studies in fully immunocompetent mice showed that the molecules strongly expand and activate NK, CD8+ T cells and gd T cells (up to 100-fold) and skew the CD4:CD8 ratio strongly towards CD8+ T cells in the peripheral blood, lymphoid tissues, and in the tumor. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite a higher exposure and circulatory half-life than the analogous IL-2 based immunocytokine. MicroSPECT/CT imaging with radioactively labeled FAP-IL2v revealed good FAP-mediated tumor targeting in the orthotopic syngeneic Renca model with low normal tissue uptake and low accumulation in lymphoid tissues, contrary to analogous IL-2 based immunocytokine that showed preferential targeting to lymphoid tissue. Studies in tumor-bearing mice showed dose-dependent anti-tumor efficacy of FAP-IL2v and CEA-IL2v in syngeneic models. Additional studies in xenograft models in SCID mice transgenic for human CD16A showed that CEA-IL2v strongly enhances the antitumor efficacy and/or survival mediated by ADCC-competentantibodies, including trastuzumab and cetuximab. Conclusion CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 binding, monovalency and high-affinity tumor-targeting as compared to classical IL-2-based immunocytokines. They retain capacity to activate and expand NK and CD8+ effector T cells through IL-2Rβγ in the periphery and the tumor microenvironment. These data support their further nonclinical and clinical investigation for immunotherapy of cancer. Clinical trials with CEA-IL2v are foreseen in 2014. Disclosures: Klein: Roche Glycart AG: Employment. Waldhauer:Roche: Employment. Nicolini:Roche: Employment. Dunn:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Danny:Roche: Research Funding. Boerman:Roche: Research Funding. Nayak:Roche: Employment. Herter:Roche: Employment. Van Puijenbroek:Roche: Employment. Ast:Roche: Employment. Hofer:Roche: Employment. Hosse:Roche: Employment. Lang:Roche: Employment. Neumann:Roche: Employment. Kettenberger:Roche: Employment. Neubauer:Roche: Employment. Gorr:Roche: Employment. Tuerck:Roche: Employment. Evers:Roche: Employment. Gerdes:Roche: Employment. Levitsky:Roche: Employment. Bacac:Roche: Employment. Moessner:Roche: Employment. Umana:Roche: Employment, Equity Ownership.