Abstract LB-232: BiTE antibody constructs can mediate bystander tumor cell killing
Authors: Ross Sandra L. , Mulen Marika , McElroy Patricia L. , Lofgren Julie , Moody Gordon , Baeuerle Patrick A. , Coxon Angela , Arvedson Tara L. 
Published: August 2015

Abstract
Recent clinical data demonstrate the significance of T cells in anti-tumor activity. For instance, the CD19/CD3 bispecific T cell engager (BiTE) blinatumomab is a proven means of harnessing T cells for cancer treatment. BiTE antibody constructs comprise an anti-CD3 scFv (single chain variable fragment) linked to an scFv binding a tumor-associated antigen (TAA). One potential challenge for TAA-targeted therapeutics is that treatment may only eliminate TAA-expressing tumor cells and heterogeneity of TAA expression becomes a potential means of resistance. To prevent escape of TAA-negative tumor cells, a treatment modality with a bystander effect on TAA-negative cells may be desirable.
To evaluate the potential of BiTE antibodies to mediate bystander cell killing, mixtures of TAA-positive and -negative (bystander) cells were co-cultured with human T cells and the effect of BiTE antibodies tested. Lysis of TAA-expressing and bystander cells was evaluated using both imaging and viability assays. For this study, we used BiTE antibodies recognizing either epidermal growth factor receptor (EGFR) or CD33. In the presence of TAA-positive cells, T cells were activated and bystander cells lysed. In the absence of TAA-positive cells, bystander cells were not killed. Bystander cell lysis was also observed in a xenograft mouse model with subcutaneous tumors comprising EGFR-positive and -negative cancer cells, and human T-cells.
The mechanism of BiTE-mediated bystander killing was further investigated. In the presence of TAA-positive cells, T cells released many cytokines, including IFN-γ and TNFα. However, exposure of bystander cells to just the soluble factors released by T cells did not induce their lysis, suggesting that a direct interaction between BiTE-activated T cells and bystander cells was required. BiTE treatment induced the expression on bystander cells of intercellular adhesion molecule 1 (ICAM-1), a protein involved in formation of cytolytic T cell synapses with target cells. ICAM-1 upregulation on bystander cells was also observed following exposure to recombinant IFN-γ and TNFα. These findings suggest that exposure of bystander cells to cytokines secreted by BiTE-activated T cells caused ICAM-1 expression on bystander cells leading to their improved attachment and cytolytic synapse formation. Blockade of ICAM-1 by an antibody partially protected bystander cells from lysis.
Our data suggest a model where BiTE-activated T cells secrete cytokines that cause upregulation of ICAM-1 on TAA-negative cells. This can then lead to T cell binding and T cell-induced bystander cell lysis. This mechanism is not expected to cause systemic cell death because only those cells proximal to the activated T cell in the tumor environment would be exposed to sufficiently high concentrations of ICAM-1-inducing cytokines. However, this locally confined bystander cell lysis may be sufficient to enable effective treatment of tumors that are heterogeneous for TAA expression.
Citation Format: Sandra L. Ross, Marika Mulen, Patricia L. McElroy, Julie Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara L. Arvedson. BiTE antibody constructs can mediate bystander tumor cell killing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-232. doi:10.1158/1538-7445.AM2015-LB-232