Valeria Nicolini

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.

Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines

ABSTRACT We developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rβγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using 89Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo, CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8+ T cells, skewing the CD8+:CD4+ ratio toward CD8+ T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies.

CD20-TCB with Obinutuzumab Pretreatment as Next-Generation Treatment of Hematologic Malignancies

Purpose: Despite promising clinical activity, T-cell–engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug. Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug. Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell–binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD. Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785–97. ©2018 AACR. See related commentary by Prakash and Diefenbach, p. 4631

Abstract 486: Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer.

IL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the described IL-2-based immunocytokines has progressed beyond Phase II trials due to various constraints in their design: 1) pM affinity for IL-2Rαβγ on immune cells and pulmonary vascular endothelium compromising tumor targeting due to the fusion of two wildtype IL-2 moieties to the antibody, together with FcγR binding on the same cells; 2) Rapid systemic clearance and short half-life due to high affinity IL-2Rαβγ binding; 3) Preferential activation of Tregs over immune effectors by wt IL-2. Here, we describe a novel monomeric tumor-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Rα (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 targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA- and FAP-IL2v were recombinantly produced and induction of P-STAT5, proliferation, activation induced cell death (AICD), activation markers and cytokines were determined on effector cells. Safety, pharmacokinetics (PK), tumor targeting, pharmacodynamics and anti-tumor efficacy were analyzed in SCID and immunocompetent C57Bl/6 mice. FAP- and CEA-IL-2v completely lack binding to CD25, but retain IL-Rβγ binding. They do not bind to CD25 or preferentially activate Tregs, and induce lower degree of AICD. However, IL-2Rβγ bioactivity is retained and they activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers and proliferation. In particular, CEA- and FAP-IL2v expand and activate NK cells and skew the CD4:CD8 ratio towards CD8+ T cells in vivo. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite of higher exposure and circulatory half-life than the corresponding wt IL-2 immunocytokine. MicroSPECT/CT imaging revealed FAP-mediated tumor targeting of FAP-IL2v with low normal tissue uptake with FAP-IL2v tumor targeting being similar to the parental FAP antibody with low accumulation in lymphoid tissues and clearly superior to an FAP-targeted wt IL-2 immunocytokine that shows preferential spleen targeting. Studies in tumor-bearing mice showed dose dependent anti-tumor efficacy of CEA- and FAP-IL2v in established xenograft and syngeneic mouse models. Thus, CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 and FcγR binding, monovalency and high-affinity tumor-targeting as compared to classical immunocytokines. They retain capacity to activate NK and T‐effector cells through IL‐2Rβγ; in particular once targeted to the tumor microenvironment. These data support their investigation for the immunotherapy of CEA/FAP-positive tumors. Citation Format: Christian Klein, Inja Waldhauer, Valeria Nicolini, Claire Dunn, Anne Freimoser-Grundschober, Sylvia Herter, Edwin Geven, Otto Boerman, Tapan Nayak, Erwin van Puijenbroek, David Wittig, Samuel Moser, Oliver Ast, Peter Bruenker, Ralf Hosse, Sabine Lang, Sebastian Neumann, Hubert Kettenberger, Adelbert Grossmann, Ingo Gorr, Stefan Evers, Pavel Pisa, Jennifer Fretland, Victor Levitsky, Christian Gerdes, Marina Bacac, Ekkehard Moessner, Pablo Umana. Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 486. doi:10.1158/1538-7445.AM2013-486

Abstract 2481: CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors

T cell bispecific antibodies (TCBs) are potent molecules that upon simultaneous binding to tumor cells and T cells trigger strong T cell activation resulting in the killing of tumor cells. CEA TCB (RG7813) is a novel bispecific antibody targeting carcinoembryonic antigen (CEA), often overexpressed on solid tumors (e.g. colorectal, gastric, pancreatic, lung carcinoma etc.), and the CD3 epsilon chain present on T cells. CEA TCB bears several innovative technological features that distinguish it from other bispecific antibodies currently in (pre-)clinical development: (a) bivalency for tumor antigen translating into higher avidity, superior potency and better differentiation between high and low antigen-expressing cells; (b) head-to-tail fusion geometry for anti-tumor and CD3-binding domains, resulting in higher potency compared to conventional IgG-based TCBs; (c) extended half-life compared to non-Fc-based TCBs; (d) fully silent Fc ensuring lower risk of FcgR-mediated infusion reactions; and (e) robust production using standard manufacturing processes (enabled by “CrossMAb” and knob-into-hole bispecific antibody technologies). In vitro, CEA TCB mediates potent target-dependent T cell cytotoxicity, T cell activation, proliferation, and cytokine release in killing assays, exclusively in the presence of CEA-expressing target-cells. CEA TCB activity correlates with CEA expression level, showing higher potency against tumor cells with high expression of CEA. In vivo, CEA TCB induces dose- and time-dependent regression of CEA-expressing tumors with variable amounts of immune cell infiltrate. In fully humanized NOG mice, CEA TCB is efficacious in poorly-infiltrated tumors and converts non-inflamed into highly-inflamed tumors. Histological and FACS analyses revealed that CEA TCB recruits new T cells into tumors and/or expands pre-existing ones and is able to induce T cell re-localization from the tumor periphery into the tumor bed. Surprisingly, CEA TCB treatment also qualitatively alters the composition of intratumoral T cells resulting in an increased frequency of activated (CD69, CD25), proliferating (Ki67) and differentiated T cells (having effector memory phenotype) that are ready to kill (express high levels of Granzyme B). Taken together, these preclinical data show that CEA TCB is a novel tumor-targeted T cell bispecific antibody with promising anti-tumor activity and the novel ability to modify the tumor microenvironment. Phase 1 clinical trials with CEA TCB are currently ongoing. Future studies will focus on identification of combination partners that inhibit T cell suppression and unleash the full potential of T cell activity. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Johannes Sam, Valeria Nicolini, Nathalie Steinhoff, Oliver Ast, Peter Bruenker, Ralf Hosse, Thomas Hofer, Ekkehard Moessner, Christiane Jaeger, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2481. doi:10.1158/1538-7445.AM2015-2481

Abstract 2579: Combination with the novel tumor-targeted CEA-IL2v immunocytokine enhances the activity of ADCC-competent and glycoengineered antibodies in vitro and in vivo

We recently described a novel monomeric tumor-targeted CEA-IL2v immunocytokine. CEA-IL2v comprises a single IL-2 variant (IL2v) with abolished CD25 binding that is fused to the C-terminus of a humanized CEA antibody with a heterodimeric Fc devoid of FcγR and C1q binding. CEA-IL2v recognizes a membrane-proximal epitope of CEA. Here we show that CEA-IL-2v is able to enhance the activity of ADCC competent antibodies and glycoengineered IgG1 antibodies in vitro and in vivo. The combination of CEA-IL2v with ADCC-competent IgG1 antibodies was investigated in vitro in ADCC assays and in vivo in xenografts in hCD16a transgenic SCID mice that express hFcγRIIIα on NK cells and the homologous muFcγRIV on macrophages/monocytes. Antibodies tested include trastuzumab, cetuximab and the ADCC-enhanced glycoengineered EGFR antibody GA201. In the spontaneous genetically engineered BALB-neuT breast cancer mouse model a surrogate tumor stroma Tenascin C A2 (TNCA2)-targeted IL2v immunocytokine was used in combination with a ratHER2 antibody. PBMCs stimulated by CEA-IL2v in co-cultures with tumor cells showed dose-dependent elimination of tumor cells and activation of NK cells. Addition of trastuzumab or cetuximab resulted in induction of activation markers and enhanced killing of tumor cells. As a single agent, CEA-IL2v showed minor tumor growth inhibition in orthotopic i.m.f.p. KPL4 BC and s.c. N87 GC xenografts. The combination of CEA-IL2v (1 mg/kg, q7d×3) with trastuzumab (10 mg/kg in KPL4 and 25 mg/kg in N87, q7d×3) resulted in superior inhibition of tumor growth compared to the respective single-agent treatments, combination therapy induced 6/9 complete tumor remissions in N87 and 3/5 in KPL4. In the i.v. A549 lung cancer and i.s. LS174T CRC xenografts, single-agent CEA-IL2v showed a moderate effect on median survival, whereas the combination of CEA-IL2v (1 mg/kg, q7d×3) with cetuximab (25 mg/kg, q7d×3) increased median/overall survival. Similarly, in the i.v. A549 NSCLC and i.s. LS174T CRC xenografts, the combination of CEA-IL2v (1 mg/kg for A549 and 2 mg/kg for LS174T, q7d×3) with GA201 (25 mg/kg, q7d×3) resulted in superior median survival compared to the respective single-agent treatments with long term survival of 10/10 animals in the A549 model and survival of 2/8 animals in the LS174T model. Finally in the spontaneous GEM BALB-neuT breast cancer model the combination of TNCA2-IL2v with a ratHER2 IgG1 antibody resulted in a strong and significant reduction of tumor formation in 6/7 animals as compared to the respective single agents. Combining CEA-IL2v or TNCA2-IL2v with ADCC competent/enhanced IgG1 antibodies results in enhanced anti-tumor efficacy in xenografts and in a spontaneous immunocompetent genetically engineered BALB-neuT mouse model. These data support the clinical investigation of CEA-IL2v with ADCC competent/enhanced IgG1 antibodies. Citation Format: Valeria Nicolini, Inja Waldhauer, Anne Freimoser, Sara Colombetti, Federica Cavallo, Marina Bacac, Christian Gerdes, Pablo Umana, Christian Klein. Combination with the novel tumor-targeted CEA-IL2v immunocytokine enhances the activity of ADCC-competent and glycoengineered antibodies in vitro and in vivo . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2014-2579

Abstract 1494: Combination of CEA TCB, a novel T-cell bispecific antibody for the treatment of solid tumors, with PD-L1 checkpoint blockade

Recent results from clinical trials have shown that immune therapies, particularly immune checkpoint inhibitors, can extend the overall survival of cancer patients and lead to durable responses. Despite these promising results, current immune-based therapies are only effective in a proportion of patients and combination strategies are needed to improve therapeutic benefit. Programmed death-ligand 1(PD-L1) is found on the surface of immune and tumor cells and its expression is induced by interferon gamma (IFNg). It prevents the immune system from destroying cancer cells by interacting with the inhibitory programmed death-1 (PD-1) and B7.1 receptors on activated T cells, which results in a T-cell inhibitory signal. CEA TCB (RG7802, RO6958688) is a novel T cell bispecific antibody targeting the carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells, currently being investigated as single agent in a Phase I study in patients with advanced and/or metastatic CEA-expressing tumors. CEA TCB-mediated killing of tumor cells led to T cell activation, IFNg secretion and subsequent up-regulation of the PD-1/PD-L1 immune suppressive pathway in vitro and in vivo, similarly to what happens during tumor adaptive immune resistance mechanisms where upon recognition of tumor antigens, TILs produce IFNg, which drives PD-L1 expression in the tumor microenvironment and delivers a suppressive signal to T cells upon binding to PD-1. Incubation of the high-CEA expressing gastric carcinoma cell line (MKN45) with human PBMCs (E:T 10:1) and increasing concentrations of CEA TCB, led to a dose-dependent up-regulation of PD-1 receptor on both CD4+ or CD8+ T cell subsets as well as of PD-L1 on surviving tumor cells as early as 24 h following treatment. PD-1 expression was specific as it did not occur in the absence of CEA-expressing tumor cells or upon treatment with un-targeted control TCB. In vivo studies performed using MKN45 tumor xenografts (MKN45) in fully humanized NOG mice demonstrated that CEA TCB treatment led to increased frequency of intra-tumoral T cells expressing PD-1 and to a strong induction of PD-L1 expression in tumors. Combination of CEA TCB with a PD-L1 blocking antibody showed significant increase of anti-tumor activity as compared to the respective single agents. These preclinical data indicate that CEA TCB treatment leads to up-regulation of the PD-1/PD-L1 immune suppressive pathway and that the combination of CEA TCB with PD-L1 blocking agents results in enhanced anti-tumor activity. Phase Ib clinical trials investigating the combination of CEA TCB and atezolizumab are currently ongoing. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Linda Fahrni, Valeria Nicolini, Christian Gerdes, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. Combination of CEA TCB, a novel T-cell bispecific antibody for the treatment of solid tumors, with PD-L1 checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1494.

Abstract PR8: Novel tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer.

Introduction: IL-2 therapy can lead to durable responses in a modest proportion of cancer patients, but the treatment is associated with significant toxicity. Over the last decades, various IL-2-based immunocytokines have been generated by fusing IL-2 to tumor-targeting antibodies. However, none of these molecules have progressed beyond Phase II trials and they are hampered by various liabilities: 1) High functional affinity (low pM) for IL-2Rabg on immune cells and on pulmonary vascular endothelium (Krieg et al., PNAS, 2010) compromising preferential tumor targeting due to fusion of two IL-2 moieties to the antibody. This is further compounded when the immunocytokine binds to FcgRs on the same cells. 2) Rapid systemic clearance and short half-life due to high affinity IL-2Rabg binding. 3) Preferential activation of Tregs over immune effectors due to use of wildtype IL-2. Here, we describe a novel monomeric tumor-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Ra (CD25) binding is fused to the C-terminus of a tumor-specific hIgG1 antibody with a heterodimeric Fc-part. FcgR and C1q binding is completely abolished by a novel Fc mutation. For targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were chosen. Experimental procedures: CEA- and FAP-IL2v were recombinantly produced and characterized by surface plasmon resonance. Induction of P-STAT5, proliferation, activation induced cell death (AICD), various activation markers and cytokine release were determined on effector cells. Safety, pharmacokinetics (PK), tumor targeting by imaging, immune-pharmacodynamics and anti-tumor efficacy were analyzed in immunocompromised Scid and immunocompetent C57BL/6 mice. Results: IL-2v completely lacks binding to CD25, but retains IL-Rbg binding. In line with this, FAP- and CEA-IL2v do not bind to CD25 or preferentially activate Tregs, and do not cause AICD. However, IL-2Rbg bioactivity is retained and they are still able to activate NK, CD4 and CD8 T cells as shown by concentration dependent increase in activation markers and induction of proliferation. In particular, CEA- and FAP-IL2v expand and activate NK cells and skew the CD4:CD8 ratio towards activated CD8 T cells in vivo. In C57BL/6 mice CEA- and FAP-IL2v demonstrate improved safety despite of ca. 2-fold higher exposure and t1/2 than a wildtype IL-2-based IgG immunocytokine. SPECT/CT imaging revealed FAP-mediated tumor targeting and accumulation of FAP-IL2v with low normal tissue uptake. Notably, FAP-IL2v tumor targeting was similar to the parental FAP antibody with low accumulation in lymphoid tissues; clearly superior to an FAP-targeted wt IL-2 immunocytokine that showed preferential homing to the spleen. Studies in tumor bearing mice showed dose dependent efficacy of CEA- and FAP-IL2v in established xenograft and immunocompetent syngeneic mouse models in terms of survival. Conclusion: CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 and FcgR binding, monovalency and high-affinity tumor-targeting as compared to conventional immunocytokines. They retain the capacity to activate NK and T effector cells through IL 2Rbg; in particular once targeted and immobilized in the tumor microenvironment. These preclinical properties support further investigation for the immunotherapy of CEA/FAP-positive tumors. This abstract is also presented as Poster A23. Citation Format: Christian Klein, Waldhauer Inja, Valeria Nicolini, Dunn Claire, Anne Freimoser, Anne Freimoser, Sylvia Herter, Edwin Geven, Otto Boerman, Erwin van Puijenbroek, David Wittig, Samuel Moser, Oliver Ast, Ralf Hosse, Sabine Lang, Sebastian Neumann, Adelbert Grossmann, Ingo Gorr, Stefan Evers, Pavel Pisa, Jennifer Fretland, Victor Levitsky, Christian Gerdes, Marina Bacac, Ekkehard Moessner, Ekkehard Moessner, Pablo Umana. Novel tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR8.

Abstract LB-236: M4-3-ML2, a novel glycoengineered humanized IgG1 antibody, targeting a membrane-proximal epitope of MCSP/CSPG4 exhibits potent ADCC induction in vitro and in vivo anti-tumoral efficacy in disseminated melanoma models

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL MCSP/CSPG4 is a large transmembrane proteoglycan identified in melanomas as HMW-MAA. In the mouse it is known as neurite growth factor 2 (NG2), a marker of pericyte recruitment. MCSP has been used as a target for clinical imaging of (uveal) melanomas by immunoscintigraphy. MCSP shows uniform and abundant expression in ca. 60-80% of melanoma, and was described in lobular breast carcinoma, glioblastoma, osteo- & chondrosarcoma, and basal cell carcinoma. It is present at high levels on pericytes of tumor neovasculature, but down-regulated as vessels mature. Normal tissue expression is low and it is not detected on PBMCs. We have generated human/Cynomolgus cross-reactive antibodies against a membrane-proximal MCSP epitope by mouse immunization with a linear peptide derived from the membrane proximal D3 domain followed by boosting with melanoma cells. The mouse antibody LC007 was selected for humanization due to its potent induction of ADCC as a chimeric antibody, compared to antibodies to membrane distal epitopes of MCSP. LC007 as chimeric IgG1 and its humanized IgG1 derivative M4-3-ML2 are characterized by the following properties: i) Specific binding to the native epitope on MCSP+ melanoma cells, but no induction of internalization; ii) Specific IHC staining of MCSP+ cells in FFPET samples; iii) ca 10 nM monovalent affinity for hMCSP D3 domain. Moreover, glycoengineering of LC007 and M4-3-ML2 antibodies using GlycoMab technology resulted in increased binding affinity for hFcgRIIIa and enhanced ADCC potency and absolute killing of melanoma cell lines. As expected, neither up to 10 ug/mL wildtype, nor glycoengineered M4-3-ML2 induced relevant cytokine (IL-6, TNF-α, IFN-γ) release in human whole blood supporting that MCSP is not expressed there. Subsequently, we studied anti-tumoral efficacy of the chimeric antibody LC007 and the humanized antibody M4-3-ML2 in disseminated models of MV3 and MDA-MB435 melanoma after i.v. injection of tumor cells in hCD16 transgenic Scid mice, which express the functional human high affinity FcgRIIIa receptor on NK cells. Both, glycoengineered LC007 and M4-3-ML2 mediated efficacy in terms of enhanced median and overall survival in both disseminated xenograft models, and were superior to the respective non-glycoengineered antibodies. Taken together, our studies support MCSP/CSPG4 as an attractive target for antibody-based cancer immunotherapy. Further studies investigating the anti-angiogenic effect of MCSP antibodies via their action on pericytes/vascular smooth muscle cells are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-236. doi:1538-7445.AM2012-LB-236

Abstract A156: GA201 (RG7160) pretreatments and combination therapies improve efficacy without negatively affecting antitumoral ADCC.

GA201 is a humanized and glycoengineered IgG1 antibody which targets EGFR with boosted antibody dependent cell cytotoxicity (ADCC) in phase 2 clinical trials. Pretreatment with corticosteroids and/or anti-histaminics are of normal practice for monoclonal antibody therapy to prevent potential infusion reactions. GA201 follows a premedication regimen of anti-histaminics and hydrocortisone. Due to the dual GA201 mode of action, evaluation on the impact of these drugs on ADCC is critical and was previously assessed preclinically for potential negative impacts on immune effector cells and their capacity to mediate tumor cell killing. Combination phase II studies with GA201 + chemotherapy regimens are currently under investigation in NSCLC as first-line treatment (chemotherapy selection based on histology Cisplatin + gemcitabine for squamous and cisplatin + pemetrexed for non squamous) and in CRC as second-line treatment (FOLFIRI for KRAS-mutant CRC or cetuximab + FOLFIRI for KRAS-wildtype CRC). These combinations and others where tested prior use in the clinics to evaluate preclinical efficacy and their impact on the immune effectors in vitro, and in vivo. Chemotherapy combos showed additive or synergistc effect in anti-tumor efficacy as assessed in orthotopic xenograft models and all pre-medications and chemotherapies tested showed no negative impact on ADCC supporting their combination with GA201 in the clinics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A156.