CC-122 Repairs T Cell Activation in Chronic Lymphocytic Leukemia That Results in a Concomitant Increase in PD-1:PD-L1 and CTLA-4 Immune Checkpoint Expression at the Immunological Synapse
Authors: Apollonio Benedetta , Fanous Mariam , Benmebarek Mohamed-Reda , Devereux Stephen , Hagner Patrick , Pourdehnad Michael , Gandhi Anita K. , Patten Piers E. , Ramsay Alan G. 
Published: October 2019

Abstract
Immunomodulatory drugs (IMiDs®) such as lenalidomide and immune checkpoint blockade (ICB) antibodies can enhance autologous anti-tumor T cell immunity and have the potential to elicit durable control of disease in B cell malignancies. These immunotherapies are likely to be most effective when employed in treatment combinations. Thus, the goal of pre-clinical research should be to reveal mechanisms of action (MOA) in the tumor microenvironment (TME) and identify biomarkers to guide development of combination therapy for patients. CC-122 is a novel first-in-class pleiotropic pathway modifier (PPM®) that has potent anti-proliferative, anti-angiogenic and immunomodulatory activities and is currently in Phase I clinical trials for lymphoma and chronic lymphocytic leukemia (CLL). Here, we have utilized the immunological synapse bioassay to examine T cell interactions with CLL tumor cells (modeling anti-tumor T cell responses in the TME) following CC-122 treatment and measure the expression of co-signaling complexes at the synapse.
Conjugation assays and confocal imaging were used to visualize intercellular conjugate interactions and F-actin polymerization at the immune synapse between CD4+ and CD8+ T cells and autologous CLL tumor cells pulsed with superantigen (acting as antigen-presenting cells, APCs). Peripheral blood was obtained from treatment naive CLL patients (n=40) representative of disease heterogeneity. Treatment of both purified CLL cells and CD4+ or CD8+ T cells with CC-122 (0.01 - 1 μM for 24h) dramatically enhanced the number of T cells recognizing tumor cells (% conjugation) and increased the formation of F-actin immune synapses (area, μm2) compared to vehicle treated cells (P<.01). Notably, CC-122 treatment induced T cells to engage in multiple tumor cell synapse interactions that were more pronounced in restored CD8+ T cell lytic synapses. This immunomodulatory activity was detected across all CLL patient samples and drug concentrations tested. In addition, synapse strength as measured by total fluorescence intensity of F-actin per T cell:APC conjugate increased significantly with CC-122 (P<.01). A critical MOA of lenalidomide is activation of T cell immune synapse signaling. Here, our comparative studies revealed that CC-122 (0.1 - 1 μM) significantly enhanced autologous T cell synapse activity in CLL by 4 - 5 fold versus vehicle (P<.01), whereas lenalidomide (1 μM) enhanced activity by 3 fold vs vehicle. Moreover, CC-122 treatment resulted in increased expression and polarization of tyrosine-phosphorylated proteins at T cell synapses compared to lenalidomide and vehicle treatment (P<.01). This data provides evidence that CC-122 induces functional T cell synapses that control the assembly of signaling complexes between the T cell receptor (TCR) and the F-actin cytoskeletal layer. Following T cell recognition of APCs, co-signaling receptors co-localize at the immune synapse where they synergize with TCR signaling to promote (co-stimulatory receptors) or inhibit (co-inhibitory/'immune checkpoint' receptors) T cell activation. Quantitative image analysis studies revealed that restoration of T cell synapse activity with CC-122 was accompanied by an increased recruitment of inducible co-stimulator (ICOS) to the synapse that was dose-dependent (P<.01). CC-122 treatment also increased polarized expression of CTLA-4 and PD-1 immune checkpoint proteins at the synapse with PD-L1+ tumor cells. The observed up-regulation of co-inhibitory receptors led to combining CC-122 with anti-PD-L1, anti-PD-1 or anti-CTLA-4 blocking antibodies. Results show that these treatment combinations increased T cell synapse activity compared to using these immunotherapies alone (P<.01).
In conclusion, our results demonstrate for the first time that CC-122 can activate T cell immune synapse signaling against autologous CLL tumor cells and this immunomodulatory capability is more potent than lenalidomide. We further show that CC-122 activation of T cells is associated with enhanced expression of the co-stimulatory receptor ICOS and co-inhibitory checkpoints CTLA-4 and PD-1 at the synapse site. Importantly, our pre-clinical data demonstrates that this regulatory feedback inhibition can be exploited by the addition of anti-PD-L1, anti-PD-1 or anti-CTLA-4 ICB to CC-122 to more optimally stimulate T cell activity against immunosuppressive tumor cells.

Disclosures
Hagner: Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment. Gandhi:Celgene: Employment, Equity Ownership. Ramsay:MedImmune: Research Funding; Celgene: Research Funding.