ALBERT

Description: Abstract 4304 Background: VEGF blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis. Several publications described that VEGF and Ang-2 are up-regulated in several hematological tumors like B-CLL, AML or multiple myeloma. In particular, Ang-2 may qualify as a novel prognostic marker in AML and CLL. We have generated TAvi6, a novel bispecific antibody targeting VEGF-A and Ang-2 and tested its anti-tumor and anti-angiogenic activity. TAvi6 is a tetravalent IgG-like bispecific antibody based on bevacizumab and targets Ang-2 with 2 disulfide-stabilized scFvs (LC06) fused to the C-terminus of the heavy chain. Material and Methods: TAvi 6 was profiled in biochemical and cellular (angiogenesis) assays. Antitumoral and anti-angiogenic efficacy was assessed in established s.c. Colo205, s.c. Calu-3 and orthotopic i.m.f.p. KPL-4 xenografts in SCID beige mice. Mice were treated with bevacizumab or antibody LC06 (10 mg/kg), the respective combination (each 10 mg/kg) and TAvi6 (13.3 mg/kg). In addition, TAvi6 was evaluated for inhibition of metastasis to the lung quantified by Alu-PCR. Tumors were explanted for histological analysis of angiogenesis and inhibition of angiogenesis was assessed in the cornea angiogenesis assay. Results: In biochemical assays (affinity, Tie2-Ang-2 interaction) and cellular assays (Tie2 phosphorylation, HUVEC proliferation, tube formation) TAvi6 shows properties identical to the parental antibodies bevacizumab and LC06. In the orthotopic KPL-4-003 xenograft tumor growth inhibition was 79% for bevacizumab; 39% for LC06; 90% for the combination and 91% for TAvi6. In the s.c. Colo205-009 xenograft TGI was 66% for bevacizumab; 50% for LC06; 78% for the combination and 87% for TAvi6. TAvi6 was able to suppress growth of tumors refractory to 1st-line treatment with bevacizumab (Colo205 xenograft) and suppressed metastasis to the lung significantly (KPL-4 xenograft). In the s.c. Calu-3 xenograft we observed a strong inhibition of angiogenesis by in vivo and ex vivo imaging and an advantage of TAvi6 compared to the antibody combination. Furthermore, TAvi6 potently inhibited VEGF-induced angiogenesis in the mouse corneal angiogenesis assay. Conclusions: We have generated a novel tetravalent IgG-like bispecific antibody targeting VEGF-A and Ang-2 simultaneously. TAvi6 shows identical properties compared to the respective parental antibodies. In particular, TAvi6 blocks angiogenesis in vivo efficacously and mediates strong tumor growth inhibition in various xenograft models with a slight advantage of TAvi6 over the combination of the respective single agents bevacizumab and LC06 in several models. Further studies are foreseen to assess the application of bispecific antibodies targeting Ang-2 and VEGF as novel therapeutic approach for the treatment of hematological cancers. Disclosures: Weidner: Roche: Employment, Equity Ownership. Scheuer:Roche: Employment, Patents & Royalties. Thomas:Roche: Employment, Patents & Royalties. Baehner:Roche: Employment, Patents & Royalties. Seeber:Roche: Employment, Patents & Royalties. Kettenberger:Roche: Employment, Patents & Royalties. Schanzer:Roche: Employment, Patents & Royalties. Brinkmann:Roche: Employment, Patents & Royalties. Regula:Roche: Employment, Patents & Royalties. 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 3721 The anti-CD20 antibody rituximab has become central to the treatment of B-cell malignancies over the last decade. Recently, it has been shown that anti-CD20 antibodies can be divided into two types based on their mechanisms of action on B cells. Rituximab is a type I antibody that redistributes CD20 into lipid rafts and promotes complement-dependent cytotoxicity (CDC), while the type II, glycoengineered antibody GA101 has lower CDC activity but higher antibody-dependent cellular cytotoxicity and direct cell death activity. In preclinical studies GA101 was superior to rituximab in B-cell killing in vitro, depletion of B cells from whole blood, and inhibition of tumour cell growth in lymphoma xenograft models. GA101 is currently being evaluated in Phase II/III trials, including comparative studies with rituximab. To investigate the differences in direct effects of GA101 and rituximab on B-cell lymphoma signaling, we have analysed the effects of antibody binding on gene expression in different B-cell lines using a GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Rituximab and GA101 rapidly induced gene expression changes in SUDHL4 and Z138 cells, including regulation of genes associated with B-cell-receptor activation such as EGR2, BCL2A1, RGS1 and NAB2. The effects on gene expression differed markedly between different cell lines and between the two antibodies. SUDHL4 cells showed pronounced changes in the gene expression pattern to rituximab treatment, while Z138 cells, which represent a different B-cell stage, showed less pronounced changes in gene expression. The reverse was true for GA101, suggesting not only that the signaling mediated by CD20 differs in different cell lines, but also that in a given cell line the two types of antibodies bind CD20 molecules with different signaling capacity. For each cell line, gene expression induced by other type I antibodies (LT20, 2H7, MEM97) was more like rituximab and that induced by other type II antibodies (H299/B1, BH20) was more like GA101 in terms of the number of genes regulated and the magnitude of changes in expression. Unbiased hierarchical clustering analysis of gene expression in SUDHL4 could discriminate type I from type II antibodies, confirming that the two classes of antibody recognised CD20 complexes with inherently different signalling capacities. By confocal and time-lapse microscopy using different fluorophores, rituximab and GA101 localised to different compartments on the membrane of lymphoma cells. GA101/CD20 complexes were relatively static and predominantly associated with sites of cell–cell contact, while rituximab/CD20 complexes were highly dynamic and predominantly outside areas of contact. These findings suggest that type II antibodies such as GA101 bind distinct subpopulations of CD20 compared with type I antibodies such as rituximab, accounting for the differences in mechanisms of action and anti-tumour activity between these antibodies. Disclosures: Niederfellner: Roche: Employment. Mundigl:Roche: Employment. Lifke:Roche: Employment. Franke:Roche: Employment. Baer:Roche: Employment. Burtscher:Roche: Employment. Maisel:Roche: Employment. Belousov:Roche: Employment. Weidner:Roche: Employment. Umana:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.