ALBERT

Description: 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.

Description: Antibody-based immunotherapy represents a promising strategy to target chemo-resistant leukemic cells. However, current antibody-based approaches are restricted to cell lineage surface antigens. Targeting intracellular antigens enables to enlarge the number of suitable tumor-associated target antigens with a more restricted expression profile. In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody for immunotherapy of acute myeloid leukemia (AML). The T cell receptor (TCR)-like TCB targets the intracellular tumor antigen Wilms tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A*02 (HLA-A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR-specificity. We further analyzed enhancement of TCB-mediated T cell cytotoxicity through combination with the immune-modulatory drug lenalidomide. WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real-time PCR, western blot and immunohistochemical staining. WT1-TCB-mediated cytotoxicity was analyzed by co-cultivation of WT1-expressing HLA-A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCB-mediated cytotoxicity against primary AML cells and combination with 10 μM lenalidomide was evaluated in our pre-established feeder layer-based ex vivo long-term culture system. For in vivo testing, NSG mice (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) were humanized with human HLA-A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1-expressing HLA-A2+ SKM-1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1-TCB. In accordance with previous reports, we observed WT1 expression in 79% (n=38) of cancer cell lines and in 92% (n=65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n=26), whereas WT1 was expressed again at time of relapse (n=21). WT1-TCBs elicited antibody-mediated T cell cytotoxicity against peptide-pulsed T2 cells and AML cell lines in a WT1 and HLA-restricted manner. Equally, TCR downstream signaling was observed in a WT1-restrictive manner by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18). Correspondingly, up-regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3±1.5, PD-1: 5.1±0.7, TIM-3: 4.7±0.6; ±SEM; n=22). WT1-TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38±13% after 13-14 days; ±SEM; n=5). In comparison with WT1RMF-specific T cells, only bivalent binding by WT1-TCB induced efficient lysis of primary AML cells. Interestingly, combination of WT1-TCB with lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 32±10% vs 59±9%; p=0.0017; ±SEM; n=13). This was accompanied by an increased secretion of the proinflammatory cytokines IL-2, IFN-γ and TNF-α and promoted the differentiation of naïve T cells towards a memory phenotype characterized by a downregulation of CD45RA. Furthermore, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control. TCR-like TCBs targeting intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide-MHC complex on the cell surface validating WT1-TCB as a promising therapeutic agent for the treatment of AML. Our results further indicate that the combinatorial approach with lenalidomide leads to increased TCB-mediated T cell cytotoxicity. Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Xu:Roche: Employment, Equity Ownership, Patents & Royalties. Heitmüller:Roche: Employment. Hanisch:Roche: Employment, Equity Ownership, Patents & Royalties. Sam:Roche: Employment, Equity Ownership, Patents & Royalties. Pulko:Roche: Employment, Equity Ownership, Patents & Royalties. Schönle:Roche: Employment, Equity Ownership, Patents & Royalties. Challier:Roche: Employment, Equity Ownership, Patents & Royalties. Carpy:Roche: Employment, Equity Ownership, Patents & Royalties. Lichtenegger:Roche: Employment. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Subklewe:Roche: Consultancy, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Morphosys: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy.

Description: Synthetic T cell redirecting therapies, using chimeric antigen receptor (CAR)-T cells or CD3-bispecific antibodies targeting B-cell surface antigens such as CD19 and CD20, currently in clinical development, are emerging as promising, potential therapeutic approaches for the treatment of non-Hodgkin lymphomas (NHL). CD3-bispecific antibodies and first generation CAR-T cells only provide T cell receptor stimulation, so-called "signal 1", to the redirected T cells, but lack costimulatory, so-called "signal 2", support of those T cells. Agonism of costimulatory receptors on T cells, such as CD28 and/or 4-1BB, can increase the strength and durability of a T cell-mediated response via multiple mechanisms. Co-stimulation can enhance T cell specific cytotoxicity, proliferation, secretion of Th1-polarizing cytokines, recruitment of additional T cells via increased chemokine secretion, T cell metabolic fitness, and resistance to T-cell exhaustion and to activation-induced T-cell death. Indeed, 2nd generation CAR-T cells that incorporate CD28 or 4-1BB co-stimulation have replaced 1st generation ones in clinical development. However, complex manufacturing logistics and the need of specialized clinical centers for the administration of CAR-T cells significantly limit their broad application. In order to provide an off-the-shelf, synthetic T cell redirection approach delivering both signals 1 and 2 to T cells, CD3-bispecific antibodies would need combination with systemically administered T-cell costimulatory agonists. Yet, clinical development of 1st generation costimulatory agonists has not been successful to date due to on-target, off-tumor immune-mediated toxicity, such as hepatotoxicity. To overcome this limitation, we have generated a novel 4-1BB costimulatory agonist, CD19-targeted 4-1BBL (CD19-4-1BBL, RG6076, RO7227166), and are developing it in combination with a potent CD20xCD3 T cell bispecific antibody, CD20-TCB (RG6026 or glofitamab). CD19-4-1BBL consists of a trimeric, human 4-1BBL fused to a monovalent CD19-targeting IgG1 antibody with an engineered Fc region devoid of FcgR binding. As effective agonism of 4-1BB receptor requires crosslinking of more than three receptor units on a T cell, CD19-4-1BBL is systemically inactive unless it binds to CD19 and clusters on the surface of targeted B-cells to hyper-crosslink multiple 4-1BB receptors on redirected T cells. In our off-the-shelf, combination approach, glofitamab binds to CD20 on B-cells and engages CD3 on redirected T cells, providing signal 1 and inducing the expression of 4-1BB on those T cells. CD19-4-1BBL can then target those activated T cells and provide them with signal 2. In preclinical experiments, we show that CD19-4-1BBL can boost glofitamab-mediated cytokine release by activated T cells in healthy donor as well as DLBCL patient-derived PBMCs. Using a human diffuse large B cell lymphoma (DLBCL) tumor-bearing (WSU-DLCL2) fully humanized mouse model, we observed a CD19-4-1BBL dose-dependent, synergistic combination effect with glofitamab, leading to strongly increased T cell accumulation in tumors, tumor growth inhibition and regression. Importantly, CD19-4-1BBL was also able to prevent tumor escape to glofitamab monotherapy at late treatment time points in a fully humanized mouse model bearing large OCI-Ly18 human DLBCL tumors. Glofitamab monotherapy has recently demonstrated encouraging activity in relapsed/refractory NHL patients with reported complete response rates in DLBCL in the same range as those of 2nd generation CAR-T cells that already incorporate both T cell signals 1 and 2. The preclinical data we report here provide a strong rationale for adding CD19-4-1BBL-mediated T cell signal 2 to glofitamab in the clinic to further boost treatment efficacy and deliver an off-the-shelf, enhanced T cell redirection approach alternative to CAR-T cell therapy. CD19-4-1BBL is currently in clinical trials (NCT04077723). Disclosures Herter: Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.Sam:Roche Glycart AG:Current Employment.Ferrara Koller:Roche Glycart AG:Current Employment.Diggelmann:Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company.Bommer:Roche Glycart AG:Current Employment.Schönle:Roche Glycart AG:Current Employment.Claus:Roche Glycart AG:Current Employment.Bacac:Roche Glycart AG:Current Employment, Patents & Royalties.Klein:Roche:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.Umana:Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Description: Traditional drug safety assessment often fails to predict complications in humans, especially when the drug targets the immune system. Here, we show the unprecedented capability of two human Organs-on-Chips to evaluate the safety profile of T-cell bispecific antibodies (TCBs) targeting tumor antigens. Although promising for cancer immunotherapy, TCBs are associated with an on-target, off-tumor risk due to low levels of expression of tumor antigens in healthy tissues. We leveraged in vivo target expression and toxicity data of TCBs targeting folate receptor 1 (FOLR1) or carcinoembryonic antigen (CEA) to design and validate human immunocompetent Organs-on-Chips safety platforms. We discovered that the Lung-Chip and Intestine-Chip could reproduce and predict target-dependent TCB safety liabilities, based on sensitivity to key determinants thereof, such as target expression and antibody affinity. These novel tools broaden the research options available for mechanistic understandings of engineered therapeutic antibodies and assessing safety in tissues susceptible to adverse events.