Abstract Metastatic colorectal cancer (mCRC) remains one of the leading causes of cancer-related deaths worldwide with only a minority of patients responding to immune checkpoint targeting therapies. To effectively treat mCRC, we have developed a novel B7-H6/CD3 IgG-like T cell Engager (ITE) that mediates cytolytic synapse formation between T cells and tumor cells, re-directs their cytolytic activity selectively to B7-H6 (B7 homolog 6, NCR3LG1) expressing CRC cells, and increases infiltration of immune cells into the tumor tissue. B7-H6 is a member of the B7 family of immune receptors. We have identified B7-H6 as a novel antigen which is expressed in several solid tumor indications. In CRC tissues B7-H6 is expressed in >95 % of cases, while no to very little expression could be detected in normal tissues, thus making B7-H6 a promising antigen to re-direct cytolytic T cells to B7-H6-expressing CRC cells. The bispecific monovalent B7-H6/CD3 ITE is designed to bind simultaneously to CD3 on T cells and B7-H6 on CRC cells resulting in formation of a cytolytic synapse without interfering with the natural function of B7-H6 and has sustained serum exposure. In vitro, unstimulated T cells were co-cultured with B7-H6 expressing CRC cell lines and increasing concentrations of the B7-H6/CD3 ITE. The B7-H6/CD3 ITE induced dose-dependent lysis of CRC cell lines with EC50 values ranging from 0.9 to 25 ng/mL, whereas viability of B7-H6-negative cells was not affected, demonstrating the selectivity of the B7-H6/CD3 ITE for the B7-H6 antigen. In addition, the B7-H6/CD3 ITE induced B7-H6-dependent activation and proliferation of T cells, and granzyme B and cytokine secretion. In vivo, the B7-H6/CD3 ITE induced dose-dependent anti-tumor activity in subcutaneous CRC (NCI-H716, HCT-15, HT-29) xenograft tumor bearing immunodeficient mice which were reconstituted with human PBMCs or T cells. Tumor regressions were observed with single doses of < 0.5 mg/kg of B7-H6/CD3 ITE administered as i.v. injection, and anti-tumor activity could be further enhanced by q7d dosing. Consistent with the mode-of-action, the B7-H6/CD3 ITE led to a profound infiltration of both CD4+ and CD8+ T cell subsets into the tumor, which correlated with cleaved caspase 3 expression on tumor cells and tumor shrinkage. The inflammatory tumor microenvironment that was created by treatment with the B7-H6/CD3 ITE also led to an increase of PD-1 on T cells and PD-L1 on the tumor cells. The B7-H6/CD3 ITE shows cross-reactivity to both B7-H6 and CD3 of human and non-human primate origin respectively, and demonstrates antibody-like pharmacokinetic properties in a single dose study in cynomolgus monkeys. Our pre-clinical data demonstrate that the B7-H6/CD3 ITE is a highly B7-H6-selective and efficacious T cell engaging antibody with the ability to convert a non-inflamed into an inflamed tumor environment which supports clinical development. Citation Format: Susanne Hipp, Wei Zhang, Xiaoyun Liao, Aurelie Auguste, Annika Osswald, Kathrin Bauer, Christian Walterskirchen, Farzaneh H. Sayedian, Manuela Kellner, Craig Giragossian, Vladimir Voynov, Andrew E. Nixon, Paul J. Adam. A novel B7-H6/CD3 bispecific IgG-like T cell engaging antibody for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 56.