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TGFβ may act as a tumor suppressor early in tumorigenesis; however, TGFβ is also known to support tumor growth via multiple mechanisms, including promotion of epithelial-mesenchymal transition, fibroblast recruitment, ECM deposition, neovascularization, and immunosuppression of both adaptive and innate immune cells. SAR439459 is pan-neutralizing TGFβ antibody that is being developed for the treatment of solid tumors. In this study, we investigated the effect of SAR439459 on the immune system in primary human immune cells in vitro and in murine syngeneic tumor models in vivo. Exogenous TGFβ inhibited proliferation of CD8pos primary human T cells and suppressed the production of inflammatory cytokines such as IFNγ. The addition of SAR439459 blocked these effects. Additionally, TGFβ enhanced the development of inducible Tregs by approximately 4-fold, while the inclusion of SAR439459 abrogated this effect. The effects of exogenous TGFβ and SAR439459 were next examined in the context of T Cell Receptor signaling in a co-culture system of Jurkat cells expressing luciferase reporter under the control of NFAT-responsive promoter and PD-L1pos APCs. The addition of anti-PD1 stimulated TCR signaling in this system, while the addition of TGFβ inhibited anti-PD1-mediated response. SAR439459 restored the ability of anti-PD1 to stimulate the TCR signaling. These data support the notion that TGFβ neutralization could positively impact T effector cells and inhibit Tregs development. Therefore, combining a TGFβ-neutralizing antibody with anti-PD1 could enhance T cell responsiveness. Compared to checkpoint inhibitors, which are suggested to cause the reactivation and de-repression of exhausted T cells, the effects of TGFβ on the immune system are thought to be distinct. To test this, the combination of anti-PD1 and SAR439459 was examined in syngeneic mouse models. Established MC38 tumors were treated with anti-PD1, SAR439459 or the combination of both antibodies. In tumor-bearing mice treated with either anti-PD1 or SAR439459 alone, neither Ab resulted in significant tumor growth inhibition. However, the combination of anti-PD-1 and SAR439459 led to tumor regression in the majority of tumor-bearing mice. These results provide the rationale for combining TGF-β neutralization with checkpoint inhibitors, specifically anti-PD1, for the treatment of human cancers. Citation Format: Richard C. Gregory, Rita Greco, Hongjing Qu, Natalia Malkova, Mikhail Levit, Keli Perron, Waldemar Racki, Fangxian Sun, Gary Shapiro, Christopher Winter, Dmitri Wiederschain, Tun Tun Lin, Jack Pollard. The anti-TGFβ neutralizing antibody, SAR439459, blocks the immunosuppressive effects of TGFβ and inhibits the growth of syngeneic tumors in combination with anti-PD1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2790.

TGFβ is a potent immunosuppressive cytokine, acting on multiple cell types of both the innate and adaptive immune systems. Emerging preclinical and clinical data implicate TGFβ in tumor immune evasion, poor prognosis and resistance to PD1/PDL1 directed checkpoint therapy. Thus, neutralization of TGFβ is suggested to relieve immunosuppression via mechanisms that are distinct yet complementary to checkpoint inhibitors such as PD1. SAR439459 is a potent human pan-neutralizing anti-TGFβ antibody that has entered first-in-human studies in advanced solid tumors (NCT03192345*). I. Biomarkers for indication selection: A gene expression signature of TGFβ pathway activation was derived from analysis of TGFβ-stimulated versus naïve cancer cell lines. Queries of the signature against The Cancer Genome Atlas (TCGA) revealed that primary head and neck, ovarian, and colorectal cancers are enriched for activation. II. Biomarkers for patient selection: An analysis of the patients enriched for activation revealed that mesenchymal tumors predominate. Machine learning methods were applied to panels of gene expression data for colorectal (CRC), ovarian serous and head and neck squamous cell carcinoma to derive a two-gene biomarker for selection of patients with mesenchymal tumors. Implementation in a logistic regression model achieves good for prediction in CRC, and similar performance in other indications. Patient cohorts are enriched over three-fold for the mesenchymal subtype and false-negative rates are acceptable. Implementation of the two-gene biomarker on a low complexity platform and its validation on an independent panel of samples is described. III. Biomarkers for assessment of the tumor microenvironment: In partnership with NeoGenomics, the immune contexture of patient tumors was evaluated using MultiOmyx, a multiplex IHC assay, on CRC and melanoma. Multiplexing was conducted with 12 biomarkers (jointly describing 22 immune cell types) on single FFPE section from each tumor sample. Pilot studies included a range of inflammation to evaluate how well the analytics assess each tumor type and correlate to possible treatment effects. Statistical methods were developed to assess differences at the cell population level, including replicate concordance, volcano plots for analyses of variance, and correlation matrices. The MultiOmyx assay demonstrated excellent technical reproducibility and precision, a favorable dynamic range, inflammation status differences in select immune cells and regions of interest, and included both positive and negative correlations between cell populations. * https://clinicaltrials.gov/ct2/show/NCT03192345 Citation Format: Joachim Theilhaber, Jean Cavallo, Stephen L. Madden, Charlene Manning, Sherry Cao, Parminder Mankoo, Robert Pomponio, Hongjing Qu, Natalia Malkova, Gary Shapiro, Christopher Winter, Dmitri Wiederschain, Michele Sanicola-Nadel, Fangxian Sun, Tun Tun Lin, Richard C. Gregory, Jack Pollard. Translational biomarkers for SAR439459, an anti-TGFβ antibody for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5550.