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N.-K.V.C. also heterodimerized with a CD33 control T-BsAb to generate tumor-monovalent EGFRxCD33 and HER2xCD33 T-BsAbs. T-BsAb avidity for tumor cells was studied by flow cytometry, cytotoxicity by T-cell mediated 51Chromium release, and in vivo efficacy against cell line-derived xenografts (CDX) or patient-derived xenografts (PDX). Tumor infiltration by T cells transduced with luciferase reporter was quantified by bioluminescence. Results The EGFRxEGFR, HER2xHER2, and EGFRxHER2 T-BsAbs demonstrated high avidity and T cell-mediated cytotoxicity against human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They were highly efficient in driving human polyclonal T cells into subcutaneous PDAC xenografts and mediated potent T cell-mediated anti-tumor effects. Both EGFRxCD33 and HER2xCD33 tumor-monovalent T-BsAbs displayed substantially reduced avidity by SPR when compared to homodimeric EGFRxEGFR or HER2xHER2 T-BsAbs (150-fold SB-423557 and 6000-fold respectively), tumor binding by FACS (8.0-fold and 63.6-fold), and T-cell mediated cytotoxicity (7.7-fold and 47.2-fold), while showing no efficacy against CDX or PDX. However, if either EGFR or HER2 was removed from SW1990 by CRISPR-mediated knockout, the in vivo efficacy of heterodimeric EGFRxHER2 T-BsAb was lost. Conclusion EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. Two arm Fab binding to either one or both targets was critical for robust anti-tumor effect in vivo. By engaging SB-423557 both targets, EGFRxHER2 heterodimeric T-BsAb exhibited Rabbit Polyclonal to ZNF387 potent anti-tumor effects if CDX or PDX were EGFR+HER2+ double-positive with the potential to spare single-positive normal tissue. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-024-01538-5. Keywords: EGFR, HER2, Bispecific antibody, Pancreatic ductal adenocarcinoma Background Pancreatic cancer remains one of the most lethal forms of cancer despite considerable therapeutic efforts [1]. As of 2020, pancreatic cancer represents the 3rd leading cause of cancer-related deaths despite representing only 3% of all new cancer diagnoses [2]. With a high stroma to tumor ratio, poor immune infiltration, and an aggressive propensity for metastasis, treatment strategies for pancreatic cancer have seen modest advancement since the establishment of gemcitabine as the standard of care more than 20 years ago [3, 4]. The NIHs predicted 5-year survival rate for patients with pancreatic cancer is a discouraging 11.5%, a statistic that has remained largely unchanged for decades, loudly begging for more effective strategies [2]. EGFR and HER2 are highly expressed in many pancreatic ductal adenocarcinomas (PDAC) and correlate with poor prognoses [5, 6]. Tumors with both EGFR and HER2 over-expression can be accompanied by c-myc overexpression [7, 8], and AEG-1 expression [9, 10], SB-423557 associated with a more invasive phenotype. EGFR- and HER2-directed monoclonal antibodies have been well-tolerated but failed to exhibit meaningful clinical efficacy in the treatment of PDAC [11, 12]. A recent phase I clinical trial exploring the safety and efficacy of EGFR-directed chimeric antigen receptor T (CAR T) cells in metastatic pancreatic carcinoma reported partial response (PR) or stable disease (SD) in 12 out of 14 evaluable patients (4 PR and 8 SD), although the median progression-free survival was only 3 months [13]. Several patients experienced grade??3 adverse effects, including pleural effusion and pulmonary interstitial exudation toxicities [13], a possible side effect of EGFR-directed on-target/off-tumor responses [14]. Similarly, 6 out of 11 pancreatic cancer patients in a phase I trial responded to HER2-specific CAR T cell therapy (1 PR, 5 SD), though progression-free survival was similarly brief (4.8 months) [15]. As with the previous study, patients experienced adverse effects, though most were mild or moderate [15]. Both studies underscore a potential for EGFR- and HER2-directed T cell-based therapies in pancreatic cancer. Though antibody therapies directed at single SB-423557 receptors have been largely ineffective, the consistent expression of EGFR and HER2 in PDAC could be exploited to improve efficacy while reducing on-target/off-tumor dose limiting complications. We have previously described several T cell-engaging bispecific antibodies (T-BsAbs) [16C19], including a HER2-specific T-BsAb that demonstrated exceptionally strong anti-tumor activity in vitro and in vivo [20C22]. We have also elucidated the significance of interdomain spacing and spatial configuration of the.