Klaus-Peter Kuenkele

Development of tetravalent IgG1 dual targeting IGF-1R–EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.

Abstract 1755: Sensitization of radiotherapy and chemotherapy by adjunction of R1507 (anti-IGF-1R monoclonal antibody) in small cell lung cancer cell lines: Opportunity of translating the triple combination R1507-Cisplatin-radiotherapy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Insulin like growth factor receptor-1 (IGF-1R) inhibition could be a pertinent therapeutic approach in small cell lung cancer (SCLC), given the recognized activation of an IGF-1R autocrine loop. Materials and methods: We evaluated the importance of IGF-1R axis in SCLC by assessing IGF-1R expression and Akt activation in 83 human SCLC tissue specimens. In parallel, we evaluated in vitro the efficacy of R1507, an IgG1 fully human monoclonal antibody directed to IGF-1R. R1507 effects on IGF-1R and downstream pathways were assessed using three SCLC cell lines: H69, H146 and H526. The cytotoxicity of R1507, Cisplatin and ionizing radiation (IR) was evaluated by WST-1 cell proliferation assays and clonogenic survival assays. In vivo, the efficacy of R1507, Cisplatin and IR was assessed on H526 and H146 xenografts in nude mice. Results: IGF1R was overexpressed and Akt was activated in 38 (46%) and 31 (37%) of 83 SCLC tumors, respectively. In vitro, R1507 down-regulates IGF-1R receptor and disrupts downstream signaling through Akt and MAPK pathways in a dose dependent manner in selected small cell lung cancer cell lines. R1507 inhibits cell proliferation and colony forming capacity in H526 and H146 cells but not in H69 cells. The inhibition of PI3K-Akt pathway correlated with treatment response. The combination of R1507 (200 nM) and CDDP (3 μM) exhibited a synergistic inhibitory effect on the colony forming capacity of H146 cells and an additive inhibitory effect on H526 cells. R1507 showed synergistic effects with IR (2 Gy) in H146 cells and an additive inhibitory effect in H526 cells. R1507 potentiates the IR effects and induces a prolonged increase of the proportion of sub G1 cells in H526 but not in H146 cell line. The triple combination R1507-Cisplatin-IR potentiates the antitumoral effect of Cisplatin-IR (current standard treatment) in H146 and in H526 cell lines. In vivo, R1507 administered in monotherapy leads to a non-significant delay of tumor growth in H526 xenografts but sensitizes to both Cisplatin and IR. The triple combination R1507-Cisplatin-IR achieves a long lasting delay in tumor growth as compared with the current standard treatment Cisplatin-IR. Conclusion: R1507 has a single agent activity and remarkable chemo- and radiosensitizing effect in defined SCLC cell lines in vitro. Efficacy is dose-dependent and related to the capacity to inhibit PI3K-Akt signaling pathway. In vivo, it sensitizes to both IR and Cisplatin effects. The triple combination R1507-Cisplatin-IR exhibits a long lasting tumor growth delay as compared with the current standard treatment Cisplatin-IR, the potential of this combination should be evaluated in further early clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1755. doi:10.1158/1538-7445.AM2011-1755

Abstract LB-212: XGFR, an Fc-engineered dual signaling inhibitor targeting IGF-1R and EGFR

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background : Elevated signaling via the receptor tyrosine kinases IGF-1R and EGFR has been identified as common characteristic of multiple cancer type. IGF-1R and EGFR signal predominantly through the PI3K and MAPK signaling pathways and thereby mediate growth and survival signals crucial for the development and progression of cancer. There is strong cross talk on multiple levels between IGF-1R and EGFR dependent signaling pathways. Therefore, targeting IGF-1R and EGFR simultaneously is an attractive way to achieve maximal inhibition of signal transduction and to avoid resistance formation. Methods : Bispecific IGF1R-EGFR antibodies were engineered by linking scFv domains of an EGFR Mab (GA201) via Serine-Glycine linkers to an IgG1 IGF-1R Mab (RG1507). The functional properties of the bispecific antibodies were evaluated in cellular in vitro assays (IGF-1R/EGFR phosphorylation, downregulation, 3D proliferation and ADCC assays) and in in vivo xenograft models for tumor growth inhibition and survival. Results : Bispecific IGF-1R-EGFR antibodies (XGFR2, XGFR3, XGFR4) were successfully generated with yields and stability comparable to conventional IgG1 antibodies. XGFR antibodies effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation in H322M tumor cells and induced strong downmodulation of IGF-1R and enhanced EGFR downmodulation compared to the parental EGFR antibody GA201. XGFR antibodies showed strong anti-tumor efficacy comparable to the combination of monospecific IGF-1R and EGFR Mabs in the BxPC3 and H322M xenograft models. To enhance the ADCC properties of XGFR, afucosylated, glycoengineered bispecific antibodies with enhanced affinity for FcγRIIIA were generated using the GlycoMab technology. Glycoengineered bispecific antibodies were shown to have superior ADCC properties in in vitro ADCC assays and XGFR4 significantly prolonged median and overall survival of mice in an ADCC competent in vivo model (A549 i.v.). Conclusions : Bispecific IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components of multiple cancer types (IGF-1R and EGFR), resulting in effective inhibition of the PI3K and MAPK signaling pathway and to avoid the formation of resistance to therapy. Having overcome issues of stability and productivity, bispecific antibodies may become an advantageous way to reduce costs and infusion times in cancer therapy, while at the same time, achieving maximal anti-tumor effects through inhibition of multiple targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-212. doi:10.1158/1538-7445.AM2011-LB-212

Abstract LB-397: Functional characterization of IGF-1R antibodies and possible implications for clinical safety and efficacy

Background This is the first study performing a head to head comparison of monoclonal IGF-1R antibodies (mAb) based on published sequences for therapeutic mAbs from Pfizer (CP751,871, IgG2 kappa), Amgen (AMG479, IgG1 lambda), Merck (h7C10, cloned to R1507 backbone), Imclone (IMC-A12, IgG1 lambda) and Roche (R1507, IgG1). Methods In this study, sequence information for IGF-1R mAbs was extracted from patents and used to clone and transiently express IgGs (*=re-synthesized) in HEK293F cells. In vitro assays for ligand binding, IGF-1R auto-phosphorylation, IGF-1R downregulation, IR co-downregulation, and affinity analyses (Biacore) were used. In vivo comparison was done in BxPC3 xenograft mouse model. Results All antibodies inhibit IGF-1 binding and signaling at low nanomolar levels. While IMC-A12* and R1507 also prevent IGF-2 binding to IGF-1R and subsequent receptor activation, CP751,871* and h7C10* do not inhibit IGF-2 binding and have only limited impact (55%/30% inhibition) on IGF-2 signaling. Analysis of IGF-1R phosphorylation in 0.5 % FCS medium revealed that all antibodies except R1507 exert agonistic activity. Interestingly, Fab fragments of agonistic mAbs became antagonistic, indicating that bivalent binding is necessary for agonistic effects. All mAb (200nM, 24h treatment of MCF-7 cells) with the exception of AMG479* efficiently downregulated 78–82% the IGF-1R. Analysis of the same cell lysates revealed however striking differences in IR co-downregulation, a mechanism discussed as possible cause of clinical hyperglycemia. R1507 had the least side effects on Insulin co-downregulation (9%) compared to h7C10* (15%), CP751,871* (23%) and IMC-A12* (46%). Differences were also seen in the binding kinetics. Both AMG479* and R1507 showed faster k off rates resulting in shorter retention times at the receptor. Since k on /k off rates are discussed to influence tumor penetration (1), we compared downregulation of IGF-1R by R1507 and CP751,871* in xenograft tumors. Although both mAbs downregulate IGF-1R in vitro to the same extent, R1507 was significantly more effective in the in-vivo setting. Conclusion The head to head comparison of IGF-1R mAbs revealed differences in regard to binding properties, tumor penetration, IR codownregulation, and inhibition of signaling via the ligand IGF-2. Reference List 1. Adams, G. P., Schier, R., McCall, A. M., Simmons, H. H., Horak, E. M., Alpaugh, R. K., Marks, J. D., and Weiner, L. M. (2001) Cancer Res. 61 , 4750–4755 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-397. doi:10.1158/1538-7445.AM2011-LB-397