Metformin Sensitizes Leukemic Cells to Cytotoxic Lymphocytes by Increasing Expression of Intercellular Adhesion Molecule-1 (ICAM-1)

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The antidiabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention, although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1), a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of antiapoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels, which favors cytotoxic lymphocytes binding to tumor cells. Finally, metformin decreases the growth of human hematological tumor cells in xenograft models, mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy.

Enhancing Adoptive Cell Transfer with Combination BRAF-MEK and CDK4/6 Inhibitors in Melanoma

Despite the success of immune checkpoint inhibitors that target cytotoxic lymphocyte antigen-4 (CTLA-4) and programmed-cell-death-1 (PD-1) in the treatment of metastatic melanoma, there is still great need to develop robust options for patients who are refractory to first line immunotherapy. As such there has been a resurgence in interest of adoptive cell transfer (ACT) particularly derived from tumor infiltrating lymphocytes. Moreover, the addition of cyclin dependent kinase 4/6 inhibitors (CDK4/6i) have been shown to greatly extend duration of response in combination with BRAF-MEK inhibitors (BRAF-MEKi) in pre-clinical models of melanoma. We therefore investigated whether combinations of BRAF-MEK-CDK4/6i and ACT were efficacious in murine models of melanoma. Triplet targeted therapy of BRAF-MEK-CDK4/6i with OT-1 ACT led to sustained and robust anti-tumor responses in BRAFi sensitive YOVAL1.1. We also show that BRAF-MEKi but not CDK4/6i enhanced MHC Class I expression in melanoma cell lines in vitro. Paradoxically CDK4/6i in low concentrations of IFN-γ reduced expression of MHC Class I and PD-L1 in YOVAL1.1. Overall, this work provides additional pre-clinical evidence to pursue combination of BRAF-MEK-CDK4/6i and to combine this combination with ACT in the clinic.

Activation of anti-SARS-CoV-2 human CTLs by extracellular vesicles engineered with the N viral protein

We investigated an innovative anti-SARS-CoV-2 immune strategy finalized to oral administration of extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. Analysis of in vitro produced EVs proven the uploading of N protein also when fused with truncated Nefmut. Mice injected with DNA vectors expressing each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models.

Phase I study of single agent NIZ985, a recombinant heterodimeric IL-15 agonist, in adult patients with metastatic or unresectable solid tumors

BackgroundNIZ985 is a recombinant heterodimer of physiologically active interleukin (IL-)15 and IL-15 receptor alpha. In preclinical models, NIZ985 promotes cytotoxic lymphocyte proliferation, killing function, and organ/tumor infiltration, with resultant anticancer effects. In this first-in-human study, we assessed the safety, pharmacokinetics, and immune effects of NIZ985 in patients with metastatic or unresectable solid tumors.MethodsSingle agent NIZ985 dose escalation data are reported from a phase I dose escalation/expansion study of NIZ985 as monotherapy. Adult patients (N=14) received 0.25, 0.5, 1, 2 or 4 µg/kg subcutaneous NIZ985 three times weekly (TIW) for the first 2 weeks of each 28-day cycle, in an accelerated 3+3 dose escalation trial design. IL-15 and endogenous cytokines were monitored by ELISA and multiplexed electrochemiluminescent assays. Multiparameter flow cytometry assessed the frequency, phenotype and proliferation of peripheral blood mononuclear cells. Preliminary antitumor activity was assessed by overall response rate (Response Evaluation Criteria in Solid Tumors V.1.1).ResultsAs of March 2, 2020, median treatment duration was 7.5 weeks (range 1.1–77.1). Thirteen patients had discontinued and one (uveal melanoma) remains on treatment with stable disease. Best clinical response was stable disease (3 of 14 patients; 21%). The most frequent adverse events (AEs) were circular erythematous injection site reactions (100%), chills (71%), fatigue (57%), and fever (50%). Treatment-related grade 3/4 AEs occurred in six participants (43%); treatment-related serious AEs (SAEs) in three (21%). The per-protocol maximum tolerated dose was not reached. Pharmacokinetic accumulation of serum IL-15 in the first week was followed by significantly lower levels in week 2, likely due to more rapid cytokine consumption by an expanding lymphocyte pool. NIZ985 treatment was associated with increases in several cytokines, including interferon (IFN)-γ, IL-18, C-X-C motif chemokine ligand 10, and tumor necrosis factor-β, plus significant induction of cytotoxic lymphocyte proliferation (including natural killer and CD8+ T cells), increased CD16+ monocytes, and increased CD163+ macrophages at injection sites.ConclusionsSubcutaneous NIZ985 TIW was generally well tolerated in patients with advanced cancer and produced immune activation paralleling preclinical observations, with induction of IFN-γ and proliferation of cytotoxic lymphocytes. Due to delayed SAEs at the two highest dose levels, administration is being changed to once-weekly in a revised protocol, as monotherapy and combined with checkpoint inhibitor spartalizumab. These alterations are expected to maximize the potential of NIZ985 as a novel immunotherapy.Trial registration numberNCT02452268.

Breast Cancer Consensus Subtypes: A system for subtyping breast cancer tumors based on gene expression

Breast cancer is heterogeneous in prognoses and drug responses. To organize breast cancers by gene expression independent of statistical methodology, we identified the Breast Cancer Consensus Subtypes (BCCS) as the consensus groupings of six different subtyping methods. Our classification software identified seven BCCS subtypes in a study cohort of publicly available data (n = 5950) including METABRIC, TCGA-BRCA, and data assayed by Affymetrix arrays. All samples were fresh-frozen from primary tumors. The estrogen receptor-positive (ER+) BCCS subtypes were: PCS1 (18%) good prognosis, stromal infiltration; PCS2 (15%) poor prognosis, highly proliferative; PCS3 (13%) poor prognosis, highly proliferative, activated IFN-gamma signaling, cytotoxic lymphocyte infiltration, high tumor mutation burden; PCS4 (18%) good prognosis, hormone response genes highly expressed. The ER− BCCS subtypes were: NCS1 (11%) basal; NCS2 (10%) elevated androgen response; NCS3 (5%) cytotoxic lymphocyte infiltration; unclassified tumors (9%). HER2+ tumors were heterogeneous with respect to BCCS.

Mechanically active integrins direct cytotoxic secretion at the immune synapse

The secretory output of cell-cell interfaces must be tightly controlled in space and time to ensure functional efficacy. This is particularly true for the cytotoxic immune synapse (IS), the stereotyped junction formed between a cytotoxic lymphocyte and the infected or transformed target cell it aims to destroy. Cytotoxic lymphocytes kill their targets by channeling a mixture of granzyme proteases and the pore forming protein perforin directly into the IS. The synaptic secretion of these toxic molecules constrains their deleterious effects to the target cell alone, thereby protecting innocent bystander cells in the surrounding tissue from collateral damage. Despite the importance of this process for immune specificity, the molecular and cellular mechanisms that establish secretory sites within the IS remain poorly understood. Here, we identified an essential role for integrin mechanotransduction in cytotoxic secretion using a combination of single cell biophysical measurements, ligand micropatterning, and functional assays. Upon ligand-binding, the αLβ2 integrin LFA-1 functioned as a spatial cue, attracting lytic granules containing perforin and granzyme and inducing their fusion at closely adjacent sites within the synaptic membrane. LFA-1 molecules were subjected to pulling forces within these secretory domains, and genetic or pharmacological suppression of these forces abrogated cytotoxicity. We conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance both the potency and the security of their cytotoxic output.

Metformin Sensitizes Leukemic Cells to Cytotoxic Lymphocytes by Increasing Expression of Intercellular Adhesion Molecule-1 (ICAM-1)

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The antidiabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention, although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1), a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of antiapoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels, which favors cytotoxic lymphocytes binding to tumor cells. Finally, metformin decreases the growth of human hematological tumor cells in xenograft models, mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy.

Comparison of Wild Type DNA Sequence of Spike Protein from SARS-CoV-2 with Optimized Sequence on The Induction of Protective Responses Against SARS-Cov-2 Challenge in Mouse Model

COVID-19 caused by SARS-CoV-2 has been spreading worldwide. To date, several vaccine candidates moved into EUA or CA applications. Although DNA vaccine is on phase III clinical trial, it is a promised technology platform with many advantages. Here, we showed that the pGX9501 DNA vaccine encoded the spike full-length protein-induced strong humoral and cellular immune responses in mice with higher neutralizing antibodies, blocking the hACE2-RBD binding against live virus infection in vitro. Importantly, higher levels of IFN-γ expression in CD8+ and CD4+ T cell and specific cytotoxic lymphocyte (CTL) killings effect were also observed in the pGX9501-immunized group. It provided subsequent protection against virus challenges in the hACE2 transgenic mouse model. Overall, pGX9501 was a promising DNA vaccine candidate against COVID-19, inducing strong humoral immunity and cellular immunity that contributed to the vaccine's protective effects.

Metformin Sensitizes Leukemic Cells to Cytotoxic Lymphocytes by Increasing Expression of Intercellular Adhesion Molecule-1 (ICAM-1)

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The antidiabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention, although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1), a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of antiapoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels, which favors cytotoxic lymphocytes binding to tumor cells. Finally, metformin decreases the growth of human hematological tumor cells in xenograft models, mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy.

Enhancing the anti-leukemia immunity of leukemia-derived exosome-based vaccine by downregulation of PD-L1 expression

Cell-released nanovesicles can induce anti-leukemia immunity. Leukemia cell-derived exosomes (LEXs) are promising anti-tumor vaccine components for cancer immunotherapy. Nonetheless, LEX-based vaccines show modest potency in vivo, likely due to the presence of immunosuppressive PD-L1 proteins in the exosomes. We hypothesized that targeting exosomal PD-L1 could optimize LEX-based vaccines. To test this hypothesis, we compared the capacity of exosomes derived from PD-L1-silenced leukemia cells (LEXPD-L1si) and non-modified exosomes to induce anti-leukemia immunity.Lentivirus-mediated PD-L1 shRNA was used to downregulate PD-L1 expression in parental leukemia cells and LEXs. LEXPD-L1si were characterized by electron microscopy, western blotting, and flow cytometry, and their anti-leukemia immune effects were tested on immune cells and in animal models.In the present study, lentivirus-mediated PD-L1 shRNA successfully downregulated PD-L1 expression in parental leukemia cells and in LEXs. LEXPD-L1si induced better DC maturation and subsequently enhanced T-cell activation, as compared with non-modified LEXs. Consistently, immunization with LEXPD-L1si induced greater T-cell proliferation and Th1 cytokine release. LEXPD-L1si was a more potent inducer of antigen-specific cytotoxic lymphocyte (CTL) response. Finally, we vaccinated DBA/2 mice with exosome formulations to test their ability to induce both protective and therapeutic anti-tumor CTL responses in vivo. Vaccination with LEXPD-L1si strongly inhibited tumor growth and prolonged survival. Downregulation of exosomal PD-L1 expression in LEXs effectively induce more potent anti-leukemia immunity. Therefore our strategy for optimizing LEX-based vaccine has a potential application in leukemia immunotherapy.