Thomas Friess

Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.

Strongly Enhanced Antitumor Activity of Trastuzumab and Pertuzumab Combination Treatment on HER2-Positive Human Xenograft Tumor Models

The human epidermal growth factor receptor (HER) family plays an important role in cell survival and proliferation, and is implicated in oncogenesis. Overexpression of HER2 is associated with aggressive disease and poor prognosis. Trastuzumab is a humanized monoclonal antibody targeting HER2 and has proven survival benefit for women with HER2-positive early and metastatic breast cancer. Pertuzumab, another monoclonal antibody, is a HER2 dimerization inhibitor that binds to a different epitope on HER2 than trastuzumab and inhibits HER2 dimer formation with other HER family members such as HER3 and HER1. We investigated the antitumor activity of these agents alone and in combination in HER2-positive breast and non-small cell lung cancer xenografts. Our data show that the combination of trastuzumab and pertuzumab has a strongly enhanced antitumor effect and induces tumor regression in both xenograft models, something that cannot be achieved by either monotherapy. The enhanced efficacy of the combination was also observed after tumor progression during trastuzumab monotherapy. Near-IR fluorescence imaging experiments confirm that pertuzumab binding to tumors is not impaired by trastuzumab pretreatment. Furthermore, we show by in vitro assay that both trastuzumab and pertuzumab potently activate antibody-dependent cellular cytotoxicity. However, our data suggest that the strongly enhanced antitumor activity is mainly due to the differing but complementary mechanisms of action of trastuzumab and pertuzumab, namely inhibition of HER2 dimerization and prevention of p95HER2 formation.

Preclinical Activity of the Type II CD20 Antibody GA101 (Obinutuzumab) Compared with Rituximab and Ofatumumab In Vitro and in Xenograft Models

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. Mol Cancer Ther; 12(10); 2031–42. ©2013 AACR.

Combination Treatment with Erlotinib and Pertuzumab against Human Tumor Xenografts Is Superior to Monotherapy

In many solid tumors, overexpression of human epidermal growth factor receptors (e.g., HER1/EGFR and HER2) correlates with poor prognosis. Erlotinib (Tarceva) is a potent HER1/EGFR tyrosine kinase inhibitor. Pertuzumab (Omnitarg), a novel HER2-specific, recombinant, humanized monoclonal antibody, prevents heterodimerization of HER2 with other HERs. Both mechanisms disrupt signaling pathways, resulting in tumor growth inhibition. We evaluated whether inhibition of both mechanisms is superior to monotherapy in tumor cell lines expressing different HER levels. Human non–small cell lung cancer (NSCLC) cells (Calu-3: HER1/EGFR 0+, HER2 3+; QG56: HER1/EGFR 2-3+, HER2 0+) and breast cancer cells (KPL-4: HER1/EGFR 2-3+, HER2 3+) were implanted into BALB/c nu/nu mice and severe combined immunodeficient beige mice, respectively. Tumor-bearing mice (n = 12 or 15 per group) were treated with vehicle (Captisol or buffer), erlotinib (orally, 50 mg/kg/d), pertuzumab (i.p. 6 mg/kg/wk with a 2-fold loading dose), or erlotinib and pertuzumab for 20 (QG56), 27 (KPL-4), or 49 (Calu-3) days. Drug monotherapy had antitumor activity in all models. Tumor volume treatment-to-control ratios (TCR) with erlotinib were 0.36 (Calu-3), 0.79 (QG56), and 0.51 (KPL-4). Pertuzumab TCR values were 0.42, 0.51, and 0.64 in Calu-3, QG56, and KPL-4 models, respectively. Combination treatment resulted in additive (QG56: TCR 0.39; KPL-4: TCR 0.38) or greater than additive (Calu-3: TCR 0.12) antitumor activity. Serum tumor markers for NSCLC (Cyfra 21.1) and breast cancer (soluble HER2) were markedly inhibited by combination treatment (80-97% in Calu-3 and QG56; 92% in KPL-4), correlating with decreased tumor volume. Overall, erlotinib and pertuzumab are active against various human xenograft models, independently of HER1/EGFR or HER2 expression. A combination of these HER-targeted agents resulted in additive or greater than additive antitumor activity.

Preclinical Studies on the Mechanism of Action and the Anti-Lymphoma Activity of the Novel Anti-CD20 Antibody GA101

GA101 is a novel glycoengineered Type II CD20 monoclonal antibody. When compared with rituximab, it mediates less complement-dependent cytotoxicity (CDC). As expected for a Type II antibody, GA101 appears not to act through CDC and is more potent than the Type I antibody rituximab in inducing cell death via nonclassical induction of apoptosis cytotoxicity, with more direct cytotoxicity and more antibody-dependent cell-mediated cytotoxicity. We evaluated the antitumor activity of GA101 against the human-transformed follicular lymphoma RL model in vivo in severe combined immunodeficient mice (SCID) mice. GA101 induced stronger inhibition of tumor growth than rituximab. Combination of GA101 with cyclophosphamide in vivo confirmed the superiority of GA101 over rituximab. Neutralizing the complement system with cobra venom factor partially impaired the antitumor activity of rituximab, but had no impact on the efficacy of GA101. In vitro GA101 more potently induced cell death of RL cells than rituximab. The expression of a limited number of genes was found to be induced by both antibodies after exposure in vitro. Among these, early growth response 1 and activation transcription factor 3 were confirmed to be increased at the protein level, suggesting a possible role of these proteins in the apoptotic signalling of anti-CD20 antibodies. These data imply that GA101 is superior to rituximab not only as a single agent, but also in combination with chemotherapy. These data suggest the presence of novel signalization pathways activated after exposure to anti-CD20 antibodies. Mol Cancer Ther; 10(1); 178–85. ©2011 AACR.

GA201 (RG7160): A Novel, Humanized, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior In Vivo Efficacy Compared with Cetuximab

Purpose: Anti-EGF receptor (EGFR) antibodies and small-molecule tyrosine kinase inhibitors have shown activity in epithelial tumors; however, agents that work by blocking the EGFR growth signal are ineffective when the oncogenic stimulus arises downstream, such as in tumors with KRAS mutations. Antibodies of the IgG1 subclass can also kill tumor cells directly through antibody-dependent cell-mediated cytotoxicity (ADCC), and the efficacy of this is determined by the interaction of the Fc portion of the target cell–bound antibody and Fc receptors present on immune effector cells. Experimental Design: We report the development of GA201, a novel anti-EGFR monoclonal antibody with enhanced ADCC properties. GA201 was derived by humanization of the rat ICR62 antibody. The Fc region of GA201 was glycoengineered to contain bisected, afucosylated carbohydrates for enhanced binding to FcγRIIIA. Results: In vitro binding of GA201 to EGFR inhibited EGF ligand binding, EGFR/HER2 heterodimerization, downstream signaling, and cell proliferation to a similar extent as cetuximab. However, GA201 exhibited superior binding to both the low- and high-affinity variants of FcγRIIIA. This resulted in significantly enhanced induction of ADCC compared with cetuximab against both KRAS-wild-type and -mutant tumor cells lines. This enhanced ADCC translated into superior in vivo efficacy in a series of mouse xenograft models. Efficacy of GA201 was further increased when administered in combination with chemotherapy (irinotecan). Conclusions: These data suggest that GA201 may be more effective than cetuximab in patients with EGFR-positive solid tumors and may also represent a first-in-class treatment of patients with KRAS-mutated tumors. Clin Cancer Res; 19(5); 1126–38. ©2012 AACR.

RG7116, a therapeutic antibody that binds the inactive HER3 receptor and is optimized for immune effector activation

The EGF receptor (EGFR) HER3 is emerging as an attractive cancer therapeutic target due to its central position in the HER receptor signaling network. HER3 amplifies phosphoinositide 3-kinase (PI3K)-driven tumorigenesis and its upregulation in response to other anti-HER therapies has been implicated in resistance to them. Here, we report the development and characterization of RG7116, a novel anti-HER3 monoclonal antibody (mAb) designed to block HER3 activation, downregulate HER3, and mediate enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) via glycoengineering of the Fc moiety. Biochemical studies and X-ray crystallography revealed that RG7116 bound potently and selectively to domain 1 of human HER3. Heregulin binding was prevented by RG7116 at concentrations more than 1 nmol/L as was nearly complete inhibition of HER3 heterodimerization and phosphorylation, thereby preventing downstream AKT phosphorylation. In vivo RG7116 treatment inhibited xenograft tumor growth up to 90% relative to controls in a manner accompanied by downregulation of cell surface HER3. RG7116 efficacy was further enhanced in combination with anti-EGFR (RG7160) or anti-HER2 (pertuzumab) mAbs. Furthermore, the ADCC potency of RG7116 was enhanced compared with the nonglycoengineered parental antibody, both in vitro and in orthotopic tumor xenograft models, where an increased median survival was documented. ADCC degree achieved in vitro correlated with HER3 expression levels on tumor cells. In summary, the combination of strong signaling inhibition and enhanced ADCC capability rendered RG7116 a highly potent HER3-targeting agent suitable for clinical development.

Enhanced anti-tumor activity of the glycoengineered type II CD20 antibody obinutuzumab (GA101) in combination with chemotherapy in xenograft models of human lymphoma

Abstract Obinutuzumab (GA101) is a novel glycoengineered type II CD20 antibody in development for non-Hodgkin lymphoma. We compared the anti-tumor activity of obinutuzumab and rituximab in preclinical studies using subcutaneous Z138 and WSU-DLCL2 xenograft mouse models. Obinutuzumab and rituximab were assessed alone and in combination with bendamustine, fludarabine, chlorambucil, doxorubicin and cyclophosphamide/vincristine. Owing to strong single-agent efficacy in these models, suboptimal doses of obinutuzumab were applied to demonstrate a combination effect. Obinutuzumab plus bendamustine achieved superior tumor growth inhibition versus rituximab plus bendamustine and showed a statistically significant effect versus the respective single treatments. Combinations of obinutuzumab with fludarabine, chlorambucil or cyclophosphamide/vincristine demonstrated significantly superior activity to rituximab-based treatment. Obinutuzumab monotherapy was at least as effective as rituximab plus chemotherapy in vivo, and obinutuzumab plus chemotherapy was superior to the respective monotherapies. These data support further clinical investigation of obinutuzumab plus chemotherapy.

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (89Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of 89Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of 89Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg 89Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent 89Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of 89Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of 89Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, 89Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.

RG7386, a novel tetravalent FAP-DR5 antibody, effectively triggers FAP-dependent, avidity-driven DR5 hyperclustering and tumor cell apoptosis

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946–57. ©2016 AACR.