Abstract B93: Tumor infiltrating lymphocytes as a biomarker of response for CTLA-4 targeting therapies
Authors: HILLAIRET DE BOISFERON MARC , BICHAT FRANCIS , MIGNARD CAROLINE , TIZON XAVIER , FRANCE DAMIEN , MAUBANT SYLVIE , Mirjolet Jean-Francois 
Published: May 2016

Abstract
Immunotherapy based on monoclonal antibodies (mAbs) targeting cancer cells is now developed as a valid approach to treat cancer. Suppressive mechanisms in immune responses normally play a critical role in maintaining immune homeostasis. However, these suppressive mechanisms are also considered as one of the main reasons for the failure of cancer immunotherapies because they induce peripheral tolerance of tumor-specific immune responses and allow tumor growth. Regulatory T cells have been revealed as the most important population of immune suppressors, and their depletion has been reported to enhance antitumor immune responses.
CTLA-4 was reported as a critical target for regulatory T cell function and thus blockade of CTLA 4 mediated signals has been suggested as a possible strategy to treat cancers. The first anti-CTLA-4 human mAb, ipilimumab, was approved in 2011 by the FDA for use in metastatic melanoma. Success for ipilimumab was reported in a large phase III clinical trial involving patients with metastatic melanoma, who had undergone previous failed treatment.
Mice obtained from Charles River (France) were orthotopically or subcutaneously injected with syngenic tumor cell lines. They received repeated intraperitoneal injections of antibody directed against CTLA-4. During the course of the experiment, animals were terminated under anesthesia when they displayed significant signs of physiological changes. Animal housing and experimental procedures were performed according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals. Animal facility is authorized by the French authorities (Agreement N°B21231011EA).
The tumor and tumor draining lymph nodes were collected for subsequent FACS analysis to study the immune response in mice. Cells suspensions were prepared from tissues either by mechanistic dissociation or by enzymatic digestion. The antigens associated antibodies used for FACS analyses were CD45, CD3 and CD8 for effector T cell lymphocytes, and were CD45, CD3, CD4, FoxP3 and CD25 for regulatory T cell lymphocytes. The stained cells were analyzed with a LSR II flow cytometer (BD Biosciences) equipped with 3 excitation lasers at wavelengths 405, 488 and 633nm.
In CT26 and EMT6 models, an increase in T effector vs T regulator infiltrating immune cells ratio was observed for responding mice treated with CTLA-4 mAb. In 4T1 model, no ratio change was observed for mice treated with CTLA-4 mAb.
Pending new generation of humanized mouse models, the growing interest in immunology as a cancer therapy shows the limitation of conventional xenograft models in immunodeficient animals. A more effective approach is the use of syngeneic mouse models.
We here report on syngenic tumor models our capacity to identify biomarkers of response to CTLA-4 targeting therapies using detailed analysis of tumor immune infiltrating cells.
Citation Format: MARC HILLAIRET DE BOISFERON, FRANCIS BICHAT, CAROLINE MIGNARD, XAVIER TIZON, DAMIEN FRANCE, SYLVIE MAUBANT, Jean-Francois Mirjolet. Tumor infiltrating lymphocytes as a biomarker of response for CTLA-4 targeting therapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B93.