Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy.

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.

Tumor Lactic Acidosis: Protecting Tumor by Inhibiting Cytotoxic Activity Through Motility Arrest and Bioenergetic Silencing.

Adoptive T cell therapy (ACT) is highly effective in the treatment of hematologic malignancies, but shows limited success in solid tumors. Inactivation of T cells in the tumor milieu is a major hurdle to a wider application of ACT. Cytotoxicity is the most relevant activity for tumor eradication. Here, we document that cytotoxic T cells (CTL) in lactic acidosis exhibited strongly reduced tumor cell killing, which could be compensated partly by increasing the CTL to tumor cell ratio. Lactic acid intervened at multiple steps of the killing process. Lactic acid repressed the number of CTL that performed lytic granule exocytosis (degranulation) in tumor cell co-culture, and, additionally impaired the quality of the response, as judged by the reduced intensity of degranulation and lower secretion of cytotoxins (perforin, granzyme B, granzyme A). CTL in lactic acid switched to a low bioenergetic profile with an inability to metabolize glucose efficiently. They responded to anti-CD3 stimulation poorly with less extracellular acidification rate (ECAR). This might explain their repressed granule exocytosis activity. Using live cell imaging, we show that CTL in lactic acid have reduced motility, resulting in lower field coverage. Many CTL in lactic acidosis did not make contact with tumor cells; however, those which made contact, adhered to the tumor cell much longer than a CTL in normal medium. Reduced motility together with prolonged contact duration hinders serial killing, a defining feature of killing potency, but also locally confines cytotoxic activity, which helps to reduce the risk of collateral organ damage. These activities define lactic acid as a major signaling molecule able to orchestrate the spatial distribution of CTL inside inflamed tissue, such as cancer, as well as moderating their functional response. Lactic acid intervention and strategies to improve T cell metabolic fitness hold promise to improve the clinical efficacy of T cell-based cancer immunotherapy.

Progressive natural killer cell dysfunction associated with alterations in subset proportions and receptor expression in soft-tissue sarcoma patients.

Immunotherapy is currently investigated as treatment option in many types of cancer. So far, results from clinical trials have demonstrated that significant benefit from immunomodulatory therapies is restricted to patients with select histologies. To broaden the potential use of these therapies, a deeper understanding for mechanisms of immunosuppression in patients with cancer is needed. Soft-tissue sarcoma (STS) presents a medical challenge with significant mortality even after multimodal treatment. We investigated function and immunophenotype of peripheral natural killer (NK) cells from chemotherapy-naive STS patients (1st line) and STS patients with progression or relapse after previous chemotherapeutic treatment (2nd line). We found NK cells from peripheral blood of both STS patient cohorts to be dysfunctional, being unable to lyse K562 target cells while NK cells from renal cell cancer (RCC) patients did not display attenuated lytic activity. Ex vivo stimulation of NK cells from STS patients with interleukin-2 plus TKD restored cytotoxic function. Furthermore, altered NK cell subset composition with reduced proportions of CD56(dim) cells could be demonstrated, increasing from 1st- to 2nd-line patients. 2nd-line patients additionally displayed significantly reduced expression of receptors (NKG2D), mediators (CD3ζ), and effectors (perforin) of NK cell activation. In these patients, we also detected fewer NK cells with CD57 expression, a marker for terminally differentiated cytotoxic NK cells. Our results elucidate mechanisms of NK cell dysfunction in STS patients with advanced disease. Markers like NKG2D, CD3ζ, and perforin are candidates to characterize NK cells with effective antitumor function for immunotherapeutic interventions.

NK-cell dysfunction in human renal carcinoma reveals diacylglycerol kinase as key regulator and target for therapeutic intervention.

The relevance of NK cells in tumor control is well established in mouse models and human hematologic malignancies; however, their contribution to the control of human solid tumors remains disputed due to problems with in situ detection and reports of functional inactivity in the tumor milieu. In this study, we established a reliable in situ detection method for NK cells. Moreover, we performed analysis to elucidate mechanisms that impair NK-cell function in the tumor milieu and thereby identify therapeutic targets that allow recovery of NK-cell functionality. It was observed that NK cells from clear cell renal cell carcinoma (ccRCC), compared to NK cells from nontumor kidney and peripheral blood lymphocytes (PBLs), displayed conjoint phenotypic alterations and dysfunction induced by the tumor milieu, which were associated mechanistically with high levels of signaling attenuator diacylglycerol kinase (DGK)-α and blunted mitogen-activated protein kinase pathway activation (ERK1/2, Jun kinase). Reinstating NK-cell functionality was possible by DGK inhibition or brief IL-2 culture, interventions that de-repressed the ERK pathway. The extent of alteration and magnitude of recovery could be linked to NK-cell frequency within ccRCC-infiltrating lymphocytes, possibly explaining the observed survival benefit of patients with NK(high) tumors. In conclusion, DGK-mediated dampening of the ERK pathway ensuing in NK-cell dysfunction was identified as an important escape mechanism in ccRCC. DGK and the ERK pathway thus emerge as promising therapeutic targets to restore suppressed NK-cell activity for the improvement of antitumor immunity.

A novel CXCL10-based GPI-anchored fusion protein as adjuvant in NK-based tumor therapy.

BACKGROUND: Cellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor. METHODOLOGY/PRINCIPAL FINDINGS: A fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells. CONCLUSIONS: The results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings.

Intratumoral alterations of dendritic-cell differentiation and CD8(+) T-cell anergy are immune escape mechanisms of clear cell renal cell carcinoma.

Cytotoxic lymphocytes and dendritic cells infiltrating human renal cell carcinoma (RCC) are not sufficient to prevent tumor progression. Our studies identified alterations of the immune cell infiltrate as well as some of the underlying mechanisms. This knowledge should facilitate the development of anti-RCC therapies that achieve better tumor control.

High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention.

CD8(+) tumor-infiltrating T cells (CD8-TILs) are found in many types of tumors including human renal cell carcinoma. However, tumor rejection rarely occurs, suggesting limited functional activity in the tumor microenvironment. In this study, we document that CD8-TILs are unresponsive to CD3 stimulation, showing neither lytic activity, nor lytic granule exocytosis, nor IFN-γ production. Mechanistically, no deficits in TCR proximal signaling molecules (lymphocyte-specific protein tyrosine kinase, phospholipase Cγ) were identified. In contrast, distal TCR signaling was suppressed, as T cells of TILs showed strongly reduced steady-state phosphorylation of the MAPK ERK and were unable to increase phosphorylation of ERK and JNK as well as AKT and AKT client proteins (IκB, GSK3) after stimulation. These deficits were tumor-specific as they were not observed in CD8(+) T cells infiltrating non-tumor kidney areas (CD8(+) non-tumor kidney-infiltrating lymphocytes; CD8-NILs). Diacylglycerol kinase-α (DGK-α) was more highly expressed in CD8-TILs compared with that in CD8-NILs, and its inhibition improved ERK phosphorylation and lytic granule exocytosis. Cultivation of TILs in low-dose IL-2 reduced DGK-α protein levels, increased steady-state phosphorylation of ERK, improved stimulation-induced phosphorylation of ERK and AKT, and allowed more CD8-TILs to degranulate and to produce IFN-γ. Additionally, the protein level of the AKT client molecule p27kip, an inhibitory cell cycle protein, was reduced, whereas cyclin E, which promotes G1-S phase transition, was increased. These results indicate that the tumor-inflicted deficits of TILs are reversible. DGK-α inhibition and provision of IL-2 signals could be strategies to recruit the natural CD8(+) T cells to the anti-tumor response and may help prevent inactivation of adoptively transferred T cells thereby improving therapeutic efficacy.

Tumor lactic acidosis suppresses CTL function by inhibition of p38 and JNK/c-Jun activation.

Lactic acidosis is common to most solid tumors and has been found to affect infiltrating immune cells. Here we document effector phase inhibition of cytotoxic T cells (CTLs) involving complete blockage of cytokine production and partial impairment of lytic granule exocytosis. Lactic acidosis impaired TCR-triggered phosphorylation of JNK, c-Jun and p38, while not affecting MEK1 and ERK. The select targeting of signaling proteins involved in IFNγ production (JNK/c-Jun, p38) without affecting those jointly used in cytokine regulation and granule exocytosis (MEK1/ERK) explains the observed split effect of lactic acidosis on the CTL responses. CTL inhibition by lactic acidosis showed fast dynamics with immediate onset and reversion. Functional recovery by neutralizing the extracellular pH despite continuous presence of lactate holds promise that CTL activity can be improved in the milieu of solid tumors with appropriate anti-acidosis treatment, thereby increasing the efficacy of adoptive T cell therapy.