Inherent intermediate-to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNg compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577-90. Ó2017 AACR.

Activation of co-stimulatory pathways in cytotoxic T lymphocytes expressing chimeric antigen receptors (CARs) have proven to boost effector activity, tumor rejection and long-term T cell persistence. When using antigen-specific T cell receptors (TCR) instead of CARs, the lack of co-stimulatory signals hampers robust antitumoral response, hence limiting clinical efficacy. In solid tumors, tumor stroma poses an additional hurdle through hindrance of infiltration and active inhibition. Our project aimed at generating chimeric co-stimulatory switch proteins (CSP) consisting of intracellular co-stimulatory domains (ICD) fused to extracellular protein domains (ECD) for which ligands are expressed in solid tumors. The ECD of CD40L was selected for combination with the ICD from the CD28 protein. With this approach, it was expected to not only provide co-stimulation and strengthen the TCR signaling, but also, through the CD40L ECD, facilitate the activation of tumor-resident antigen-presenting cells (APCs), modulate activation of tumor endothelium and induce TCR-MHC independent apoptotic effect on tumor cells. Since CD28 and CD40L belong to different classes of transmembrane proteins (type I and type II, respectively), creating a chimeric protein presented a structural and functional challenge. We present solutions to this challenge describing different CSP formats that were successfully expressed in human T cells along with an antigen-specific TCR. The level of surface expression of the CSPs depended on their distinct design and the state of T cell activation. In particular, CSPs were upregulated by TCR stimulation and downregulated following interaction with CD40 on target cells. Ligation of the CSP in the context of TCR-stimulation modulated intracellular signaling cascades and led to improved TCR-induced cytokine secretion and cytotoxicity. Moreover, the CD40L ECD exhibited activity as evidenced by effective maturation and activation of B cells and DCs. CD40L:CD28 CSPs are a new type of switch proteins designed to exert dual beneficial antitumor effect by acting directly on the gene-modified T cells and simultaneously on tumor cells and tumor-supporting cells of the TME. The observed effects suggest that they constitute a promising tool to be included in the engineering process of T cells to endow them with complementary features for improved performance in the tumor milieu.

<p>constructs, murine HCC model, spheroid and imaging, RCC tissue pathology</p>

<div>Abstract<p>Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. <i>Cancer Res; 77(13); 3577–90. ©2017 AACR</i>.</p></div>

<p>constructs, murine HCC model, spheroid and imaging, RCC tissue pathology</p>

Introduction: The use of cellular immunotherapies has led to impressive complete and durable clinical responses in patients with certain types of hematological cancers. However, positive clinical results in solid tumor indications are still rare and many patients are in urgent need of alternative treatment options for several different indications. It has become clear that expression of inhibitory immune checkpoint molecules as well as harsh metabolic conditions in the tumor microenvironment (TME) are responsible for lack of activity of T cell immunotherapies in several settings, especially solid tumors. Here additional strategies are necessary to efficiently employ cellular immunotherapies. With the aim to further enhance the clinical efficacy of TCR-based immunotherapies under immunosuppressive conditions found in tumors, we analyzed the ability of PD1-41BB, a chimeric co-stimulatory receptor, to reverse the natural inhibitory PD-1/PD-L1 interaction into a supporting co-stimulatory signal in TCR-modified T cells encountering tumor cells. Methods: We evaluated the ability of the chimeric co-stimulatory receptor PD1-41BB to improve activity of TCR-modified T cells using 2-dimensional or 3-dimensional in vitro assays that model different immunosuppressive conditions found in tumors. Results: We demonstrate that chronic stimulation as well as several immunosuppressive factors of the TME, such as tumor cell expression of inhibitory immune checkpoint molecules or glucose restriction, impede the ability of TCR-transduced T cells to produce inflammatory cytokines and to efficiently lyse tumor cells. By using a chimeric co-stimulatory receptor consisting of the extracellular part of PD-1 and the co-stimulatory domain of 4-1BB we reversed the naturally occurring inhibitory PD-1/PD-L1 interaction to provide a co-stimulatory signal for improved T cell activity under immunosuppressive conditions or chronic stimulation. Addition of the chimeric co-stimulatory receptor PD1-41BB to TCR-modified T cells led to enhanced release of Interferon-γ, increased tumor cell killing, T cell proliferation and persistence in these T cell-tumor cell models. Conclusions: These preclinical studies support our approach to enhance the clinical efficacy of TCR-T therapies in PD-L1-positive malignancies by reversing naturally occurring inhibitory signals enabling counteraction of checkpoint-mediated dysfunction and metabolic insufficiency. The chimeric co-stimulatory PD1-41BB receptor has the potential to further enhance the clinical efficacy of TCR-modified T cells in patients with PD-L1-positive malignancies. Further preclinical in vitro and in vivo studies are ongoing to investigate the safety and efficacy of PD1-41BB in combination with multiple TCR candidates to explore its feasibility for the treatment of various cancers. Citation Format: Nadja Sailer, Melanie Salvermoser, Maria Gerget, Sarah Thome, Angelika J. Fischbeck, Svenja Ruehland, Luis F. Olguín-Contreras, Maja Buerdek, Christian Ellinger, Elfriede Noessner, Dolores J. Schendel, Patrik Kehler. The chimeric co-stimulatory receptor PD1-41BB enhances the function of T cell receptor (TCR)-modified T cells targeting solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3231.