ALBERT

Description: Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.

Description: Introduction Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcgRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in CLL, indolent NHL and DLCBL. ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and MM. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl-2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. Experimental Methods The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2, SU-DHL4 DLBCL and Z138 MCL cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. Results GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC-0199 and GA101 induced a greater than additive anti-tumor effects in the SU-DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Moreover, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 model. Conclusions Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to the combination of rituximab with GDC-0199. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively the preclinical data presented here supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently in a phase Ib clinical trial (clinical trial.gov identifier NCT01685892). Disclosures: Sampath: Genentech: Employment, Equity Ownership. Herter:Roche: Employment. Herting:Roche: Employment. Ingalla:Genentech: Employment. Nannini:Genentech: Employment. Bacac:Roche: Employment. Fairbrother:Genentech: Employment, Equity Ownership. Klein:Roche Glycart AG: Employment.

Description: Introduction: Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody that strongly induces direct cell death. As a result of glycoengineering, obinutuzumab has increased affinity for FcgRIII on innate immune effector cells resulting in enhanced induction of ADCC and ADCP. Obinutuzumab has been approved for first line treatment of CLL patients in combination with chlorambucil in the US and Europe and is currently in pivotal clinical trials in indolent NHL and DLCBL. RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound are novel, orally bioavailable, selective MDM2 antagonists which reactivate p53 and thereby mediate cell cycle arrest and subsequent apoptotic cell death in solid and hematologic tumors. RG7388 is currently in clinical trials for the treatment of AML and prostate cancer. Based on the fact that the majority of B lymphoid malignancies including NHL and CLL bear wildtype p53, and the complementary mechanisms of action involving increased apoptosis (MDM2 antagonist) or direct cell death (obinutuzumab), the combination of both compounds has the potential for superior efficacy in treating B lymphoid malignancies. Experimental methods: The combination of obinutuzumab and RG7388 (or obinutuzumab and rituximab with RG7112, the frontrunner compound with identical mode of action) was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (Annexin V/PI positivity) on p53 wildtype Z138 Mantle cell lymphoma (MCL) and DoHH-2 Diffuse large B-Cell lymphoma (DLBCL) cells by FACS and the impact of MDM2 inhibition on ADCC induction and whole blood B cell depletion. In vivo efficacy of the combination of obinutuzumab or rituximab with RG7122 and RG7388 was evaluated in the s.c. Z138 MCL xenograft model in immunodeficient SCID beige mice. Results: RG7338 induced concentration-dependent cell death of Z-138 and DOHH-2 cell lines. At concentrations 〉 10-100 nM RG7388 resulted in enhanced cell death induction of DOHH-2 and Z-138 cells in combination with obinutuzumab. Notably, RG7388 did not influence obinutuzumab mediated ADCC during 4 h up to concentrations of 1000 nM and did not affect obinutuzumab mediated NK cell activation (CD16 downregulation, CD107a upregulation). Similarly, addition of RG7388 did not interfere with obinutuzumab mediated B cell depletion in healthy human whole blood at concentrations up to 1000 nM. In the Z-138 xenograft model, the combination of suboptimal doses of 0.5 mg/kg obinutuzumab or 1 mg/kg rituximab with 150 mg/kg RG7112 (three times a week p.o. for 3 weeks) resulted in superior tumor growth inhibition by obinutuzumab as compared to rituximab including the induction of complete tumor remission. In a second Z-138 study, the combination of the suboptimal dose of 0.5 mg/kg obinutuzumab with 80 mg/kg RG7388 yielded similar anti-tumor activity. In summary, the combination of either obinutuzumab or rituximab with RG7112 or the combination of obinutuzumab with RG7388 showed superior in vivo efficacy with no clinical signs of toxicity. Conclusions: The combination of obinutuzumab with MDM2 antagonists results in enhanced cell death of p53 wildtype NHL tumor cells while not affecting obinutuzumab mediated ADCC of tumor cells or B cell depletion in whole blood from healthy donors. In vivo the combination of obinutuzumab with MDM2 inhibitors RG7112 and RG7388 results in robust combined anti-tumor efficacy in xenograft models. Taken together, these preclinical data strongly support the investigation of obinutuzumab and RG7388 combination therapy in clinical trials. Disclosures Herting: Roche: Employment, Patents & Royalties. Herter:Roche: Employment. Friess:Roche: Employment, Patents & Royalties. Lehmann:Roche: Employment. Bacac:Roche: Employment. Dangl:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Description: Introduction: Previous preclinical studies have shown that the CD20 antibody obinutuzumab achieved greater anti-tumor efficacy together with the Bcl-2 inhibitor venetoclax (GDC-199) (Sampath et al, Blood 122 (21), 4412), and the MDM2 inhibitor idasanutlin (RG7388) (Herting et al., Blood 124 (21), 1780). Clinical combination studies combining obinutuzumab with venetoclax (NCT01685892) or idasanutlin (NCT02624986) are currently ongoing. Based on the data we investigated whether the triple combination of obinutuzumab with venetoclax and idasanutlin can further improve outcome in two preclinical human wildtype p53 NHLtumor xenograft models. Experimental Methods: The in vivo antitumor efficacy of the triple combination and the respective double combinations and monotherapies was evaluated in two different CD20 positive p53 wildtype xenograft models in female SCID beige mice bearing established s.c. human DoHH-2 diffuse large B cell lymphoma (DLBCL) or human Z-138 mantle cell lymphoma (MCL) tumors. In the DoHH-2 model, mice were treated when tumors reached 200 mm3 with vehicle control, obinutuzumab (ip, 10 mg/kg, q7d, days 13, 21, 27), idasanutlin (po, 30 mg/kg, days 13-17, 20-24, 27-29) or venetoclax (po, 100 mg/kg, days 13-29). One study group received the triple combination at the indicated days. In the Z-138 model, mice were treated when the tumors reached 500 mm3 with vehicle control, a sub-optimal dose of obinutuzumab (ip, 0.5 mg/kg, q7d, days 18, 25, 32), idasanutlin (po, 100 mg/kg, days 18-22, 80 mg/kg days 25-36) or venetoclax (po, 100 mg/kg, daily, days 18-36). In the combination groups, obinutuzumab, idasanutlin and venetoclax were administered at the same dosages and on the same days. Results: In the DoHH-2 model all monotherapies resulted in significant anti-tumor efficacy with 56% tumor growth inhibition (TGI) for idasanutlin (npTCR 0.48, CI 0.33-0.68), 60% TGI for venetoclax (npTCR 0.43, CI 0.27-0.67) and a TGI of 90% for obinutuzumab (npTCR 0.17, CI 0.08-2.24). Superior efficacy compared to the respective monotherapies was observed for the triple combination group which induced tumor regression in 90% of the animals with 30% reaching complete tumor remission (npTCR 0.009, CI 0.00-0.02). In the Z-138 model monotherapy treatment using obinutuzumab, venetoclax or idasanutlin resulted in TGI of 47 % (npTCR 0.56, CI 0.42 - 0.76), 53% (npTCR 0.50, CI 0.40 - 0.64) or 67% (npTCR 0.43, CI 0.32 - 0.55), respectively. Combination of obinutuzumab with venetoclax or idasanutlin yielded a TGI of 85% (npTCR 0.26, CI 0.20 - 0.34) or 86% (npTCR 0.26, CI 0.19 - 0.35), respectively. Combination of idasanutlin with venetoclax and the triple combination showed tumor regression (TGI〉100%) on day 32. To elucidate the long term effects a time-to-event (TTE) analysis was performed until study termination on day 125. Whereas, monotherapy with obinutuzumab at the suboptimal dose of 0.5 mg/kg resulted in 2/10 and the combination of idasanutlin and venetoclax in 5/10 tumor-free animals, the triple combination resulted in a complete tumor remission in 10/10 animals until study termination on day 125. Conclusions: These preclinical data demonstrate a strong anti-tumoral efficacy of combining the CD20 antibody obinutuzumab with the Bcl-2 inhibitor venetoclax and the MDM2 inhibitor idasanutlin, in particular regarding complete tumor remissions and long term response, and support the clinical investigation of this triple combination for the treatment of B cell malignancies. Disclosures Herting: Roche: Employment, Equity Ownership, Patents & Royalties: Roche. Friess:Roche: Employment, Equity Ownership, Patents & Royalties: Roche. Umaña:Roche: Employment, Equity Ownership, Patents & Royalties. Steven:Roche: Employment, Equity Ownership. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Description: Abstract 3925 GA101 is Type II, glycoengineered CD20 monoclonal antibody currently in PhII/III clinical trials. We have previously shown that GA101 mediates superior in vitro and in vivo activity compared to the Type I CD20 antibody rituximab. By epitope mapping and crystallography we have shown that GA101 recognizes CD20 in a unique way that is different from Type I CD20 antibodies and have proposed that this may be the basis for the Type II character of GA101. Here we compare for the first time GA101 with rituximab, the standard of care in various clinical settings in NHL and B-CLL in combination with chemotherapy, as well as with the Type I CD20 antibody ofatumumab, which was recently approved for treatment of B-CLL patients refractory to fludarabine and alemtuzumab. The following assays were used to compare the three anti-CD20 antibodies: i) Binding to NHL cell lines Z138 (MCL, ca. 60.000 CD20 binding sites per cell) and SU-DHL4 (DLBCL, ca. 1 Mio CD20 binding sites per cell) assessed by FACS, ii) Cell death induction, detected by AxV/PI staining and FACS, on a panel of NHL cell lines, iii) Antibody dependent cellular cytotoxicity mediated by PBMNCs as effector and Z138, SU-DHL4 as target cells (ADCC, LDH release assay); iv) Complement dependent cytotoxicity with Z138, SU-DHL4 as target cells (CDC, LDH release assay) and v) B-cell depletion (assessed by FACS) in whole blood from healthy donors. Dose-dependent anti-tumoral activity was assessed in a s.c. SU-DHL4 NHL xenograft model in Scid beige mice. Survival experiments in a disseminated Z138 MCL model are ongoing and an update on the results will be included as part of the poster presentation. Ofatumumab (“Arzerra”) was purchased from a local pharmacy, GA101 and rituximab were obtained from Hoffmann La Roche AG, Basel. First, binding studies confirmed that GA101 shows half-maximal binding to NHL cells relative to rituximab and ofatumumab, a known property of Type II CD20 antibodies. EC50 values of binding were comparable indicating that GA101, rituximab and ofatumumab have apparent binding affinities in the low nanomolar range on NHL cells independent of the level of CD20 expression. Second, the three CD20 antibodies were compared for their induction of direct cell death as measured by AxV/PI staining. Overall, GA101 mediated superior direct cell death induction compared to rituximab and ofatumumab utilizing a panel of NHL cell lines of different origins. Immune effector-related mechanisms of action were subsequently compared by ADCC and CDC assays. GA101, a glycoengineered antibody with enhanced affinity for FcgRIIIa, was found to exhibit up to 100-fold higher ADCC potency than rituximab and ofatumumab on Z138 and SU-DHL4 cells. CDC, as expected for a Type II CD20 antibody was ca. 10 to 1,000 less potent compared to the Type I antibodies rituximab and ofatumumab. In order to integrate the different mechanisms of action (direct cell death, ADCC, CDC), autologous ex vivo B-cell depletion assays with whole blood from healthy donors containing natural immune effector cells, human complement and physiological concentrations of human immunoglobulins were performed. These studies showed that GA101 was more potent in terms of EC50 values and more efficacious in terms of absolute B-cell depletion when compared to rituximab and ofatumumab. Finally, the dose-dependent effects of the three CD20 antibodies was studied on the growth of s.c. SU-DHL4 DLBCL xenografts in SCID beige mice. GA101 induced a dose-dependent anti-tumoral effect including complete tumor remission and was superior to the Type I antibodies rituximab and ofatumumab at saturating antibody doses. In summary, the preclinical data presented herein demonstrate that the Type II, glycoengineered CD20 antibody GA101 is differentiated from the Type I CD20 antibodies rituximab and ofatumumab by its superior overall activity supporting its further clinical investigation. Of note, in contrast to previous publications, in this series of assays no superior preclinical activity of ofatumumab was observed when compared to rituximab. Disclosures: Herter: Roche: Employment, Patents & Royalties. Waldhauer:Roche: Employment. Otz:Roche: Employment. Herting:Roche: Employment, Patents & Royalties. Lang:Roche: Employment. Nicolini:Roche: Employment. Römmele:Roche: Employment. Friess:Roche: Employment, Patents & Royalties. Van Puijenbroek:Roche: Employment. Bacac:Roche: Employment. Weidner:Roche: Employment, Equity Ownership. Gerdes:Roche: Employment, Equity Ownership, Patents & Royalties. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Description: Abstract 3915 GA101 is type II, glycoengineered CD20 antibody currently in PhII/III clinical trials. GA101 mediates enhanced direct cell death with a concomitant reduction of CDC; and high ADCC induction due to increased affinity for FcgRIIIa. We have shown that GA101 compared to rituximab mediates superior efficacy in NHL xenograft models including the induction of complete tumor remission. In clinical practice the combination of rituximab with chemotherapy e.g. CHOP, CVP, bendamustine, fludarabine or FC results in a substantial clinical benefit. To assess the potential of GA101 for combination with bendamustine or fludarabine, in vivo combination studies in s.c. Z138 (MCL) xenografts in Scid beige mice were devised. GA101 and rituximab at sub-optimal doses of 1 mg/kg (once weekly) were combined with 3 mg/kg bendamustine (days 19, 20, 21, 22); or with 40 mg/kg fludarabine (days 22, 23, 24) and compared to the corresponding monotherapy arms. GA101 in combination with bendamustine mediated statistically superior efficacy in terms of tumor growth inhibition (TGI) compared to the combination of rituximab and bendamustine: TGI values on day 33 were 29% for rituximab, 42% for rituximab + bendamustine, 47% for GA101 and 72% for GA101 + bendamustine. Treatment with bendamustine did not show significant antitumor activity. Statistical evaluation based on sAUC showed a more than additive and significant effect on tumor growth for the combination of GA101 with bendamustine compared to the corresponding monotherapy arms. GA101 in combination with fludarabine demonstrated statistically superior efficacy in terms of TGI and yielded a significant difference compared to the combination of rituximab and fludarabine or GA101 as monotherpy. TGI values on day 36 were 50% for fludarabine, 60% for rituximab, 85% for rituximab + fludarabine, 86% for GA101 and 〉100% for GA101 + fludarabine. Furthermore, the superiority of the GA101-fludarabine combination was demonstrated by the observation of 3 tumor-free animals at the end of the study. ABT-263 (navitoclax) is a Bcl-2 family inhibitor that is currently in Phase I/II clinical trials for lymphoid malignancies. To provide evidence that GA101 can be combined with ABT-263 and the experimental Bcl-2 family inhibitor ABT-737, in vivo combination studies in s.c. SU-DHL4 (DLBCL) xenografts in Scid beige mice were devised. 10 mg/kg GA101 and rituximab (once weekly) were combined with 50 mg/kg ABT-737 (i.p., days 19, 22, 24, 26, 29, 31, 33). In a second study, 3 mg/kg GA101 or 10 mg/kg rituximab (once weekly) were combined with 100 mg/kg ABT-263 (orally, once daily). GA101 at a sub-optimal dose of 10 mg/kg demonstrated statistically superior efficacy in combination with ABT-737 in terms of tumor growth inhibition compared to GA101 alone or the combination of 10 mg/kg rituximab and ABT-737. Both combination treatments were statistically significant compared to the corresponding monotherapy arms. TGI values based on means on day 36 were 20% for ABT-737, 45% for rituximab, 92% for rituximab + ABT-737, 96% for GA101 and 〉100% for GA101 + ABT-737. The superiority of the combination of GA101 and ABT-737 was supported by complete tumor regression in all animals whereas none was observed with the combination of rituximab and ABT-737. GA101 at a sub-optimal dose of 3 mg/kg or rituximab at a dose of 10 mg/kg mediated statistically superior efficacy in terms of tumor growth inhibition in combination with ABT-263 compared to the respective monotherapy arms. TGI values based on means on day 43 were 15% for ABT-263, 89% for rituximab, 〉100% for rituximab + ABT-263, 80% for GA101 (at the sub-optimal dose) and 〉100% for GA101 (sub-optimal dose) + ABT-263. Taken together i) GA101 as single agent was at least as efficacious as the combination of rituximab with bendamustine, fludarabine or ABT-737; ii) the combination of GA101 with bendamustine or fludarabine was superior to the respective monotherapy arms and resulted in an enhanced, at least additive effect of the combination; iii) the combination of GA101 with Bcl-2 family inhibitors ABT-737 and ABT-263 was superior to the respective monotherapy arms and resulted in an enhanced effect of the combination including the induction of tumor remission. These data strongly support the further clinical investigation of GA101 in combination with Fludarabine, Bendamustine or Bcl-2 family inhibitors. Disclosures: Herting: Roche: Employment. Bader:Roche: Employment. Umana:Roche: Employment, Equity Ownership. Klein:Roche: Employment, Equity Ownership.