Abstract
BackgroundClinical targeting of TNFR family of receptors (CD40, CD134 and CD137) with immunostimulatory monoclonal antibodies has been successful in cancer immunotherapy. However, targeting of CD27 with a mAb is a relatively new approach to provide costimulation of immune cells undergoing activation. Thus, activation of human CD27 (TNFRSF7) with a monoclonal antibody (varlilumab) has previously been demonstrated to result in T cell activation and anti-tumor activity in preclinical models, and is currently in early phase clinical trials in patients with advanced malignancies. In this study we used an in vitro system using human peripheral blood T cells to characterize the varlilumab-mediated costimulatory effects in combination with TCR stimulation in terms of phenotypic, transcriptional and functionality changes.MethodsT cells were isolated from normal volunteer PBMCs using magnetic bead isolation kits and stimulated in vitro with plate bound anti-CD3 Ab (OKT3) and varlilumab or control Ab for 72 h. Activation profiles were monitored by ELISA or Luminex-based testing cytokine/chemokine releases, cell surface phenotyping for costimulatory and coinhibitory markers and CFSE dye dilution by proliferating T cells and Tregs. Changes in gene expression and transcriptome analysis of varlilumab-stimulated T cells was carried on Agilent Human whole genome microarray datasets using a suite of statistical and bioinformatic software tools.ResultsCostimulation of T cells with varlilumab required continuous TCR signaling as pre-activated T cells were unable to produce cytokines with CD27 signaling alone. Analysis of T cell subsets further revealed that memory CD4+ and CD8+ T cells were specifically activated with a bias toward CD8+ T lymphocyte proliferation. Activation was accompanied by upregulated cell surface expression of costimulatory [4-1BB, OX40, GITR and ICOS] and coinhibitory [PD-1] molecules. Importantly, varlilumab costimulation did not activate purified Tregs as measured by cytokine production, proliferation and suppression of dividing non-Treg T cells. Analysis of changes in gene expression during varlilumab stimulation of T cells revealed modulation of pro-inflammatory signatures consistent with cellular activation and proliferation, with the IL-2 pathway showing the highest frequency of gene modulation.ConclusionsAltogether, the data reveal the requirements and T cell subtype-specific effects of CD27 costimulation, and helps select relevant biomarkers for studying the effects of varlilumab in patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0080-2) contains supplementary material, which is available to authorized users.
Highlights
Clinical targeting of TNFR family of receptors (CD40, CD134 and CD137) with immunostimulatory monoclonal antibodies has been successful in cancer immunotherapy
We initially showed that this method provides effective costimulation through different TNFRSF members- CD27, 4-1BB, OX40 and GITR (Fig. 1a)
We investigated the kinetics of costimulation for cytokine production under these conditions, and found that 72 h was required to observe a high response to costimulation, and that these kinetics were similar for each cytokine analyzed (Fig. 1b)
Summary
Clinical targeting of TNFR family of receptors (CD40, CD134 and CD137) with immunostimulatory monoclonal antibodies has been successful in cancer immunotherapy. Activation of human CD27 (TNFRSF7) with a monoclonal antibody (varlilumab) has previously been demonstrated to result in T cell activation and anti-tumor activity in preclinical models, and is currently in early phase clinical trials in patients with advanced malignancies. CD27, a member of the TNFR superfamily (TNFRSF7), is constitutively expressed as a costimulatory molecule on naïve, activated and memory T cells, NK and NKT cells, Treg and B lymphocytes. Consistent with its role in T cell activation and proliferation, CD27 costimulation improves the function of T cells genetically engineered to express chimeric antigen receptors (CARs) [10]. The role of CD27 signaling in T cells is complex as it is unclear whether the different lymphocyte subsets that express this molecule are under the control of different transcription factors or in a state of activation or differentiation
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