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.

Croasdale R ，Wartha K ，Schanzer JM ，Kuenkele KP ，Ries C ，Mayer K ，Gassner C ，Wagner M ，Dimoudis N ，Herter S ，Jaeger C ，Ferrara C ，Hoffmann E ，Kling L ，Lau W ，Staack RF ，Heinrich J ，Scheuer W ，Stracke J ，Gerdes C ，Brinkmann U ，Umana P ，Klein C ... -  《-》

A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor type I (IGF-1R) demonstrating unique molecular properties.

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

Schanzer JM ，Wartha K ，Croasdale R ，Moser S ，Künkele KP ，Ries C ，Scheuer W ，Duerr H ，Pompiati S ，Pollman J ，Stracke J ，Lau W ，Ries S ，Brinkmann U ，Klein C ，Umana P ... -  《-》

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity.

Dong J ，Sereno A ，Aivazian D ，Langley E ，Miller BR ，Snyder WB ，Chan E ，Cantele M ，Morena R ，Joseph IB ，Boccia A ，Virata C ，Gamez J ，Yco G ，Favis M ，Wu X ，Graff CP ，Wang Q ，Rohde E ，Rennard R ，Berquist L ，Huang F ，Zhang Y ，Gao SX ，Ho SN ，Demarest SJ ，Reff ME ，Hariharan K ，Glaser SM ... -  《-》

XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer.

Schanzer JM ，Wartha K ，Moessner E ，Hosse RJ ，Moser S ，Croasdale R ，Trochanowska H ，Shao C ，Wang P ，Shi L ，Weinzierl T ，Rieder N ，Bacac M ，Ries CH ，Kettenberger H ，Schlothauer T ，Friess T ，Umana P ，Klein C ... -  《-》

Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics.

Asano R ，Shimomura I ，Konno S ，Ito A ，Masakari Y ，Orimo R ，Taki S ，Arai K ，Ogata H ，Okada M ，Furumoto S ，Onitsuka M ，Omasa T ，Hayashi H ，Katayose Y ，Unno M ，Kudo T ，Umetsu M ，Kumagai I ... -  《-》