Abstract C022: Heterodimeric bispecific antibodies for highly specific treatment of EGFR(+)HER2(+) pancreatic ductal adenocarcinoma
Authors: Long Alan W. , Santich Brian , Guo Hongfen , Cheung Nai-Kong 
Published: November 2022

Abstract
Background: EGFR and HER2 are highly expressed in many pancreatic cancers which strongly correlate with poorer prognoses. We tested homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors. Methods: We used the 2+2 IgG(L)-scFv format to create BsAbs, previously shown to have exceptional anti-tumor properties compared to other structural platforms. These BsAbs utilize two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. Anti-EGFR was based on the Cetuximab and anti-HER2 on the Trastuzumab amino acid sequences. Using a ‘knob-into-hole’ strategy, we generated an EGFRxHER2 heterodimeric BsAb bearing one Fab specific for EGFR and one for HER2. T cells were tagged with luciferase for tracking by bioluminescence for in vivo trafficking studies. Results: The EGFRxEGFR, HER2xHER2, and EGFRxHER2 BsAbs demonstrate high avidity (KD: 2pM to 300 pM) and T cell-mediated cytotoxicity against several human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They mediate potent T cell-mediated antitumor effects against subcutaneous PDAC xenografts and were highly efficient in driving human polyclonal T cells into tumors as measured by bioluminescence. To examine the importance of anti-tumor valency in antitumor effects, EGFR and HER2 BsAbs were heterodimerized with a CD33 control BsAb to generate EGFRxCD33 and HER2xCD33 BsAbs. Both EGFRxCD33 and HER2xCD33 BsAbs showed modest reductions in target avidity by surface plasmon resonance (2.7-fold and 12.6-fold, respectively), cell binding strength by FACS (8.0-fold and 63.6-fold) and cytotoxicity (7.7-fold and 47.2-fold) compared to their homodimeric counterparts and, remarkably, showed no therapeutic efficacy against SW1990 cell line-derived and patient-derived PDAC xenografts, correlating with reduced tumor-infiltrating bioluminescent T cells, when compared to EGFRxHER2 BsAb. When tested against SW1990 with CRISPR-mediated target KOs, only HER2xHER2 BsAb treatment could eliminate EGFR-KO tumors, while EGFRxEGFR and EGFRxHER2 BsAbs were ineffective. Conversely, in HER2-KO SW1990 tumors, while EGFRxEGFR BsAb demonstrated strong tumor control, HER2xHER2 and EGFRxHER2 BsAbs demonstrated only weak activity. Conclusion: EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. HER2 was overall more effective as a target when compared to EGFR for pancreatic cancer in vivo. Bivalent Fab binding to tumor was critical for robust anti-tumor effect in vivo even when in vitro differences of monovalent BsAb were less obvious. EGFRxHER2 heterodimeric BsAb exhibited potent antitumor efficacy against EGFR(+)HER2(+) double positive PDAC xenografts, but not single-positive tumors, and could offer a strategy to spare healthy single-positive tissues to reduce ‘on target/off tumor’ side effects.
Citation Format: Alan W. Long, Brian Santich, Hongfen Guo, Nai-Kong Cheung. Heterodimeric bispecific antibodies for highly specific treatment of EGFR(+)HER2(+) pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C022.