Bispecific T cell engagers have demonstrated clinical efficacy; however, their use can be accompanied by severe toxicity. Mechanistic understanding of these toxicities is limited by a lack of suitable immunocompetent preclinical models. In this study, we describe an immunocompetent mouse tumor model that exhibits bispecific T cell engager-induced toxicity and recapitulates key features similar to those in human cytokine release syndrome. In this study, toxicity occurred between the second and fourth injections of an NK Group 2D bispecific T cell engager protein. Symptoms were transient, peaking 3-4 h after treatment and resolving by 8 h. Mice developed weight loss, elevated plasma cytokines, a significant reduction in spleen white pulp, and lymphocyte infiltration in the liver. Systemic cellular immune changes also occurred; notably, an increase in CD8 + T cell activation, an increase in myeloid cells in the blood, and a population of Ly-6C int monocytes (CD11b + Ly-6G 2 F4/80 2 ) emerged in the liver and spleens of bispecific protein-treated mice. IFN-g was primarily produced by CD8 + T cells in the spleen and was required for the observed changes in both T cell and myeloid populations. Rag deficiency, IFN-g deficiency, or depletion of either CD4 + or CD8 + T cells prevented toxicity, whereas perforin deficiency, GM-CSF deficiency, or modulation of the myeloid population through clodronate-mediated depletion showed a partial abrogation of toxicity. Together, these findings reveal that T cell activation by a bispecific T cell engager leads to changes in the host myeloid cell population, both of which contribute to treatment induced toxicity in immunocompetent mice.
Chimeric antigen receptor (CAR) T cells and bispecific T cell engagers have demonstrated clinical efficacy, however are often accompanied by severe toxicity including cytokine release syndrome (CRS), neurotoxicity or macrophage activation syndrome (MAS). Mechanistic understanding of the development of these toxicities and testing of therapeutic interventions is limited by a lack of suitable immunocompetent pre-clinical models. We have developed both human and murine versions of a bispecific molecule with specific activity against target expressing tumor cells in vitro and in vivo. These bispecific proteins are constructed from the extracellular portion of either mouse or human NKG2D receptor and target cells expressing the cognate ligands by redirecting T cells through their CD3 binding arm. Using the murine bispecific protein, mNKG2DxCD3, we have developed an immunocompetent mouse tumor model which exhibits treatment induced toxicity and recapitulates key features similar to those observed in human CRS. Toxicity was observed between the 2nd and 4th bispecific protein injections but was not observed following subsequent injections. Monitoring of toxicity kinetics showed transient symptoms peaking 3-4 hours following bispecific protein injection with complete resolution after 8 hours. Weight loss and elevated plasma cytokines including IFNγ, IL6, TNFα, IL2 and IL10 were also associated with toxicity. Pathology analysis showed a reduction in white pulp in the mouse spleens, however, other organs evaluated (brain, kidney, lung and heart) were normal. Using mouse genetic knockout models, we were able to show that T cells, IFNγ and Perforin were required for development of this toxicity. Additionally, In vivo depletion of either CD4+ or CD8+ T cells was sufficient to prevent the onset of toxicity. These findings highlight the importance of T cell activation and function and shed light on the mechanistic underpinnings of bispecific T cell engager protein induced toxicity. Citation Format: Claire Godbersen-Palmer, Tiffany Coupet, Zakaria Grada, Charles Sentman. Bispecific T cell engager-induced toxicity in an immunocompetent solid tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1542.
Bispecific T cell Engagers are molecules composed of two single chain variable fragments where one arm binds CD3 on T cells and redirects them to a tumor antigen with the other arm. This causes immune synapse formation between the T cell and tumor cell resulting in T cell activation and destruction of tumor cells. Bispecific T cell engagers have been used successfully in the clinic, however, it is unclear which activities of the host immune system are required for efficacy. This study investigates the mechanisms of action of a Bispecific protein specific for the B7H6 tumor antigen in an immune intact tumor model of lymphoma. In this lymphoma model the median survival for the B7H6xCD3 bispecific treated group was greater than 80 days while the median survival of the control bispecific treated group was 20 days. Monitoring of cellular changes in the bone marrow, blood, spleen and lymph nodes by flow cytometry 24h after bispecific protein treatment revealed that most cellular changes occurred in the inguinal lymph nodes were increases in CD8+ T cell and NK cells while CD4+ T cells decreased. Additionally, intracellular staining of cytokines IFNγ, Mip1α and TNFα in mouse T cells cultured in vitro with target tumor cells and bispecific protein showed that CD8+ T cells had greater polyfunctionality than CD4+ T cells. We have shown that IFNγ is essential for efficacy against lymphoma in vivo. To study the relative contribution of CD4 and CD8 T cells in vivo, lymphoma bearing mice that were depleted of CD4+ T cells or were deficient in CD8+ T cells were monitored for survival after bispecific protein treatment. While both CD4 or CD8 deficient mice had a greater survival when treated with B7H6xCD3 bispecific compared to control bispecific protein, the loss of either cell type resulted in much lower survival compared to WT mice treated with B7H6xCD3. All WT mice receiving B7H6xCD3 bispecific protein survived at least 65 days compared to CD4-depleted mice (median survival 20 days) and CD8-deficient mice (median survival 30 days) receiving the same treatment. In conclusion, both CD4+ and CD8+ T cells play a critical role in bispecific therapy. Understanding the mechanisms of action of bispecific proteins in vivo will help to develop more effective ways to use these powerful immune activating proteins to treat cancer. Citation Format: Tiffany A. Coupet, Claire Godbersen-Palmer, Charles L. Sentman. Mechanisms of action of bispecific T cell engager-mediated immune response in a lymphoma tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1539.
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