Activation Of Immune Effector Cells Research Articles

Elevated Tumor Cell-Intrinsic STING Expression in Advanced Laryngeal Cancer

Laryngeal cancer is the second most common malignancy of the head and neck, worldwide. Immunotherapy targeting checkpoint inhibitors has been approved for the treatment of patients with recurrent or metastatic laryngeal cancer but has a relatively low response rate and outcomes that leave many patients underserved. Targeting the cGAS–STING signaling pathway can potentially improve the activation of immune effector cells, although its role in the development and progression of laryngeal cancer has not yet been investigated in depth. Fifty-nine tumor samples from patients with pathologically confirmed squamous cell carcinoma of the larynx, stage I–IV non-metastatic disease, who were treated at the University Hospital of Split, were immunohistochemically stained for the expression of STING, cGAS, CD8, CD68, and CD163. Elevated tumor cell-intrinsic STING expression was positively associated with stage IV (p = 0.0031), pT3, and pT4 laryngeal cancers (p = 0.0336) as well as with higher histological grades (G2 and G3) (p = 0.0204) and lymph node-positive tumors (p = 0.0371). After adjusting for age, sex, location, and cGAS expression, elevated STING expression was significantly associated with stage IV cancer in a multiple logistic regression model (β = 1.849, SE = ±0.8643, p = 0.0324). Elevated STING expression represents a potentially favorable predictive biomarker for new therapeutic approaches involving STING agonists combined with immunotherapy and DNA-damaging agents (radiotherapy, cisplatin, and PARP inhibitors) in laryngeal cancer.

Novel emerging nano-assisted anti-cancer strategies based on the STING pathway

Activation of simulator of interferon genes (STING), which induces the production of proinflammatory factors and immune effector cell activation, is considered a promising strategy for enhanced anti-cancer intervention. However, several obstacles prevent STING signaling in solid tumors, such as delivered molecules’ rapid degradation, restriction to tumor sites, insufficient intracellular concentrations, and low responsivity. Well-designed, multifunctional nano-formulations have emerged as optimized platforms for STING activation. Recently, a variety of nano-formulations have been developed and used in STING activation, thus facilitating immunotherapy in preclinical and clinical stages. Herein, we summarize recent advances in nanotechnology-based delivery, activation, and application strategies, which have advanced various aspects of immunotherapy. Novel STING agonists and their mechanisms in STING-activation-mediated tumor interventions are highlighted herein, to provide a comprehensive overview and discuss future directions for boosting immunotherapy via STING regulation.

Induction of T-helper-17-cell-mediated anti-tumour immunity by pathogen-mimicking polymer nanoparticles.

The effectivity of cancer immunotherapies is hindered by immunosuppressive tumour microenvironments that are poorly infiltrated by effector T cells and natural killer cells. In infection and autoimmune disease, the recruitment and activation of effector immune cells is coordinated by pro-inflammatory T helper 17 (TH17) cells. Here we show that pathogen-mimicking hollow nanoparticles displaying mannan (a polysaccharide that activates TH17 cells in microbial cell walls) limit the fraction of regulatory T cells and induce TH17-cell-mediated anti-tumour responses. The nanoparticles activate the pattern-recognition receptor Dectin-2 and Toll-like receptor 4 in dendritic cells, and promote the differentiation of CD4+ T cells into the TH17 phenotype. In mice, intra-tumoural administration of the nanoparticles decreased the fraction of regulatory T cells in the tumour while markedly increasing the fractions of TH17 cells (and the levels of TH17-cell-associated cytokines), CD8+ T cells, natural killer cells and M1-like macrophages. The anti-tumoural activity of the effector cells was amplified by an agonistic antibody against the co-stimulatory receptor OX40 in multiple mouse models. Nanomaterials that induce TH17-cell-mediated immune responses may have therapeutic potential.

Hemato-biochemical, endocrine and in vitro immune competence of lactating Ghungroo sows during different days of lactation cycle

Blood samples were collected from six Ghungroo sows on the day of farrowing, day 7, day 15, day 30, day 45 and day 60 post farrowing to investigate the alterations in the physio-biochemical, endocrine and in vitro activity of immune effector cells in Ghungroo sow during different days of lactation cycle. The hematological parameters were evaluated by standard hematological procedure. Blood biochemical and endocrine profiles were evaluated by commercially available kits. In vitro phagocytic activity and lymphocyte proliferation response were evaluated by colorimetric NBT and MTT assay respectively. Haemoglobin, packed cell volume (PCV) and total erythrocyte counts (TEC) increased significantly during the early lactation periods from the day of farrowing. The lymphocyte numbers decreased significantly from the day of farrowing to the 1st week of lactation cycle. Blood glucose and cholesterol level decreased significantly from the day of farrowing till first week of lactation cycle. The phagocytic activity of neutrophils was significantly higher on the day of farrowing and decreased till 30th day of lactation. The T4 level was higher on the day of farrowing and decreased significantly till 15th day of lactation. The data obtained in our investigation will help to formulate the managemental and therapeutic interventions during the period of transition from gestation to lactation in sows.

Cryoablation reshapes the immune microenvironment in the distal tumor and enhances the anti-tumor immunity.

As one of the local treatments, cryoablation plays an increasingly important role in the comprehensive treatment of malignant tumors with its advantages of less trauma, high reproducibility, and minimally invasive. Activation of anti-tumor immunity, another characteristic of cryoablation, has attracted more and more attention with the extensive application of immunotherapy. Unfortunately, the mechanism by which cryoablation enhances anti-tumor immunity is still unclear. In this study, we applied a multi-omics approach to investigate the effects of local cryoablation in the distal tumor microenvironment. The results revealed that large amounts of tumor antigens were released post-cryoablation, leading to a sterile inflammatory response in distant tumors. During this period, activated lysosome-related pathways result in over-expression of SNAP23 (Synaptosome associated protein 23) and STXBP2 (Syntaxin binding protein 2), activation of immune effector cells, suppression of the release of immunosuppressive factors, and finally enhancement of anti-tumor immunity, which shows a broad prospect in combined immunotherapy.

Hemagglutinin stalk-binding antibodies enhance effectiveness of neuraminidase inhibitors against influenza via Fc-dependent effector functions

SummaryThe conserved hemagglutinin stalk domain is an attractive target for broadly effective antibody-based therapeutics and next-generation universal influenza vaccines. Protection provided by hemagglutinin stalk-binding antibodies is principally mediated through activation of immune effector cells. Titers of stalk-binding antibodies are highly variable on an individual level and tend to increase with age as a result of increasing exposures to influenza virus. In our study, we show that stalk-binding antibodies cooperate with neuraminidase inhibitors to protect against influenza virus infection in an Fc-dependent manner. These data suggest that the effectiveness of neuraminidase inhibitors is likely influenced by an individual’s titers of stalk-binding antibodies and that neuraminidase inhibitors may enhance the effectiveness of future stalk-binding monoclonal antibody-based treatments.

Plasticity of monocytes and macrophages in cirrhosis of the liver.

Cirrhosis of the liver is a systemic condition with raising prevalence worldwide. Patients with cirrhosis are highly susceptible to develop bacterial infections leading to acute decompensation and acute-on-chronic liver failure both associated with a high morbidity and mortality and sparse therapeutic options other than transplantation. Mononuclear phagocytes play a central role in innate immune responses and represent a first line of defence against pathogens. Their function includes phagocytosis, killing of bacteria, antigen presentation, cytokine production as well as recruitment and activation of immune effector cells. Liver injury and development of cirrhosis induces activation of liver resident Kupffer cells and recruitment of monocytes to the liver. Damage- and pathogen-associated molecular patterns promote systemic inflammation which involves multiple compartments besides the liver, such as the circulation, gut, peritoneal cavity and others. The function of circulating monocytes and tissue macrophages is severely impaired and worsens along with cirrhosis progression. The underlying mechanisms are complex and incompletely understood. Recent 'omics' technologies help to transform our understanding of cellular diversity and function in health and disease. In this review we point out the current state of knowledge on phenotypical and functional changes of monocytes and macrophages during cirrhosis evolution in different compartments and their role in disease progression. We also discuss the value of potential prognostic markers for cirrhosis-associated immuneparesis, and future immunotherapeutic strategies that may reduce the need for transplantation and death.

Immunotherapy in colorectal cancer: an unmet need deserving of change.

The efficacy of immunotherapy in colorectal cancer is currently restricted to patients with metastatic colorectal cancer presenting deficient mismatch repair (dMMR) or microsatellite instability (MSI). So far, neither immunotherapy agents as monotherapy nor immunotherapy-based combinations have shown benefit for metastatic colorectal cancer with proficient mismatch repair (pMMR) or microsatellite stability (MSS), which are mostly immunologically cold. 1 Ganesh K Stadler ZK Cercek A et al. Immunotherapy in colorectal cancer: rationale, challenges and potential. Nat Rev Gastroenterol Hepatol. 2019; 16: 361-375 Crossref PubMed Scopus (480) Google Scholar However, the immune landscape of colorectal cancer is complex and heterogenous, and the development of these treatment strategies must consider, not only the histological context, but also its potential distinctive features. 2 Guinney J Dienstmann R Wang X et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015; 21: 1350-1356 Crossref PubMed Scopus (2403) Google Scholar In The Lancet Oncology, Carlotta Antoniotti and colleagues 3 Antoniotti C Rossini D Pietrantonio F et al. Upfront FOLFOXIRI plus bevacizumab with or without atezolizumab in the treatment of patients with metastatic colorectal cancer (AtezoTRIBE): a multicentre, open-label, randomised, controlled, phase 2 trial. Lancet Oncol. 2022; (published online May 27.)https://doi.org/10.1016/S1470-2045(22)00274-1 Summary Full Text Full Text PDF PubMed Google Scholar report the results of the addition of atezolizumab to front-line FOLFOXIRI (fluorouracil, leucovorin, oxaliplatin, and irinotecan) plus bevacizumab (the atezolizumab group) compared with FOLFOXIRI plus bevacizumab (the control group) in patients with metastatic colorectal cancer from the phase 2 AtezoTRIBE trial, irrespective of tumour microsatellite status. The superiority of FOLFOXIRI plus bevacizumab over FOLFIRI (leucovorin, fluorouracil, and irinotecan) plus bevacizumab as front-line treatment of metastatic colorectal cancer was previously demonstrated in the phase 3 TRIBE trial. Drawing from this rationale, in this multicentre, academic study, the investigators raised the question of whether the restoration of the anticancer immunity through inhibition of the PD-1–PD-L1 axis enhances efficacy of an upfront intensified chemotherapy regimen. The underlying hypothesis was that chemotherapy can induce tumour cells to release more neoantigens, leading to immunogenic cell death and activation of CD8 T lymphocytes, while bevacizumab-mediated vasculature normalisation upon VEGF or VEGFR blockade promotes increased tumour-infiltrating lymphocytes and activation of effector immune cells. 4 Kanterman J Sade-Feldman M Biton M et al. Adverse immunoregulatory effects of 5FU and CPT11 chemotherapy on myeloid-derived suppressor cells and colorectal cancer outcomes. Cancer Res. 2014; 74: 6022-6035 Crossref PubMed Scopus (113) Google Scholar , 5 Terme M Pernot S Marcheteau E et al. VEGFA–VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer. Cancer Res. 2013; 73: 539-549 Crossref PubMed Scopus (413) Google Scholar Given that no significant benefit has been derived from the combination of immunotherapy with non-intensified regimens, 6 Grothey A Tabernero J Arnold D et al. Fluoropyrimidine (FP) + bevacizumab (BEV) + atezolizumab vs FP/BEV in BRAFwt metastatic colorectal cancer (mCRC): findings from Cohort 2 of MODUL—a multicentre, randomized trial of biomarker-driven maintenance treatment following first-line induction therapy. Ann Oncol. 2018; 29: viii714-viii715 Google Scholar , 7 Lenz HJ Parikh AR Spigel DR et al. Nivolumab (NIVO) + 5-fluorouracil/leucovorin/oxaliplatin (mFOLFOX6)/bevacizumab (BEV) versus mFOLFOX6/BEV for first-line (1L) treatment of metastatic colorectal cancer (mCRC): phase 2 results from CheckMate 9X8. Proc Am Soc Clin Oncol. 2022; 40 (abstr).: 8 Crossref PubMed Google Scholar the use of FOLFOXIRI is a rational partner in an untreated RAS-mutated enriched metastatic colorectal cancer population. Upfront FOLFOXIRI plus bevacizumab with or without atezolizumab in the treatment of patients with metastatic colorectal cancer (AtezoTRIBE): a multicentre, open-label, randomised, controlled, phase 2 trialThe addition of atezolizumab to first-line FOLFOXIRI plus bevacizumab is safe and improved progression-free survival in patients with previously untreated metastatic colorectal cancer. Full-Text PDF

Immune cell-antibody interactions in health and disease.

SummaryThe human immune system safeguards against pathogens through a multitude of cellular and molecular signals, involving different components of the innate and adaptive response. Contrastingly, autoimmune diseases, allergic conditions, and cancer evoke different aspects of these otherwise protective processes. Understanding the immunological hallmarks for each pathological setting is essential for improving prevention, diagnosis, prognosis, and treatment. The activatory states of immune effector cells, especially in relation to their direct or indirect interactions with antibodies, are important determinants of an efficient, protective response that results in target clearance and improved clinical outcomes. Dysregulation of effector cells and their functions alongside alternatively activated humoral immune responses may contribute to several chronic diseases including allergic inflammation, autoimmune disorders and cancer. This Review Series brings to the forefront several key activation and regulatory features of immune effector cells in different diseases including cancer, infection allergy, and autoimmunity. Specific attention is drawn on how antibodies can impact effector cell states, and their pro-inflammatory and immune protective functions. Articles in this Series discuss different effector cells and antibody isotypes in infection, inflammation, tolerance and cancer immune surveillance, covering basic and translational mechanisms, clinical and epidemiological insights into these immune responses. Understanding the critical attributes of immune cells, especially those needed to effectively engage antibodies, will undoubtedly help better exploit their potential for disease management and therapy.

Catalytical nano-immunocomplexes for remote-controlled sono-metabolic checkpoint trimodal cancer therapy

Checkpoint immunotherapies have been combined with other therapeutic modalities to increase patient response rate and improve therapeutic outcome, which however exacerbates immune-related adverse events and requires to be carefully implemented in a narrowed therapeutic window. Strategies for precisely controlled combinational cancer immunotherapy can tackle this issue but remain lacking. We herein report a catalytical nano-immunocomplex for precise and persistent sono-metabolic checkpoint trimodal cancer therapy, whose full activities are only triggered by sono-irradiation in tumor microenvironment (TME). This nano-immunocomplex comprises three FDA-approved components, wherein checkpoint blockade inhibitor (anti-programmed death-ligand 1 antibody), immunometabolic reprogramming enzyme (adenosine deaminase, ADA), and sonosensitizer (hematoporphyrin) are covalently immobilized into one entity via acid-cleavable and singlet oxygen-activatable linkers. Thus, the activities of the nano-immunocomplex are initially silenced, and only under sono-irradiation in the acidic TME, the sonodynamic, checkpoint blockade, and immunometabolic reprogramming activities are remotely awakened. Due to the enzymatic conversion of adenosine to inosine by ADA, the nano-immunocomplex can reduce levels of intratumoral adenosine and inhibit A2A/A2B adenosine receptors-adenosinergic signaling, leading to efficient activation of immune effector cells and inhibition of immune suppressor cells in vivo. Thus, this study presents a generic and translatable nanoplatform towards precision combinational cancer immunotherapy.

Abstract 548: EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC

Abstract The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 548.

CD47xCD19 bispecific antibody triggers recruitment and activation of innate immune effector cells in a B-cell lymphoma xenograft model

BackgroundCD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model.MethodsThe contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments.ResultsWe showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells.ConclusionsThe study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).

Immunostimulatory Properties of Chemotherapy in Breast Cancer: From Immunogenic Modulation Mechanisms to Clinical Practice.

Breast cancer (BC) is the most common malignancy among females. Chemotherapy drugs remain the cornerstone of treatment of BC and undergo significant shifts over the past 100 years. The advent of immunotherapy presents promising opportunities and constitutes a significant complementary to existing therapeutic strategies for BC. Chemotherapy as a cytotoxic treatment that targets proliferation malignant cells has recently been shown as an effective immune-stimulus in multiple ways. Chemotherapeutic drugs can cause the release of damage-associated molecular patterns (DAMPs) from dying tumor cells, which result in long-lasting antitumor immunity by the key process of immunogenic cell death (ICD). Furthermore, Off-target effects of chemotherapy on immune cell subsets mainly involve activation of immune effector cells including natural killer (NK) cells, dendritic cells (DCs), and cytotoxic T cells, and depletion of immunosuppressive cells including Treg cells, M2 macrophages and myeloid-derived suppressor cells (MDSCs). Current mini-review summarized recent large clinical trials regarding the combination of chemotherapy and immunotherapy in BC and addressed the molecular mechanisms of immunostimulatory properties of chemotherapy in BC. The purpose of our work was to explore the immune-stimulating effects of chemotherapy at the molecular level based on the evidence from clinical trials, which might be a rationale for combinations of chemotherapy and immunotherapy in BC.

Abstract P198: MDNA11 is a long-acting ‘beta-only’ IL-2 agonist that demonstrates a safe and durable anti-tumor immune response

Abstract Background: MDNA11 is an engineered ‘beta-only’ IL-2 superkine with (1) enhanced affinity for IL-2Rb (CD122) to preferentially activate anti-cancer effector immune cells and (2) extended half-life by fusion to human albumin, avoiding the need for frequent administration. Methodology: MDNA11 was characterized in in vitro and in vivo studies including assessment of efficacy in syngeneic tumor models as single agent and in combination with immune checkpoint inhibitors (CPIs). A GLP study in non-human primate was conducted to evaluate the safety, pharmacodynamic and pharmacokinetic profiles of MDNA11. Results: MDNA11 does not bind IL-2Ra (CD25) but demonstrates a 30-fold higher affinity for IL-2Rb (CD122), resulting in enhanced in vitro STAT5 signaling in human NK and CD8+ T-cells with diminished activation in Tregs when compared to rhIL-2. In CT26 and MC38 syngeneic tumor models, MDNA11 demonstrated potent and durable anti-tumor activity as monotherapy (Q1W x 2) and synergized with anti-PD1 and anti-CTLA4 to achieve complete response and tumor clearance. These mice were protected against relapse and subsequent tumor re-challenges without any further treatment by inducing long-term antigen-specific CD8+ T-cells. MDNA11 binds to human and cynomolgus monkey IL-2 receptors with near-identical affinity. A GLP toxicology study involving intravenous administration on a Q2W x 3 schedule showed that MDNA11 was well tolerated up to the highest planned dose of 600 mcg/kg. The most common clinical observations were dehydration and diarrhea, which generally occurred following the first but not subsequent dose administration. There was a dose-proportional increase in Cmax and AUC. MDNA11 induced dose-dependent proliferation and expansion of peripheral CD4+ and CD8+ T-cells lasting beyond 7-days following each administration. Similar stimulatory effects on NK cells were observed but effects on Tregs were limited. Despite the robust immune response, there was no sign of cytokine release syndrome nor evidence of ADA. At the highest dose of MDNA11 (600 mcg/kg), an increase in eosinophil counts was observed following the second dose, but histopathological evaluation showed no evidence of pulmonary edema nor vascular leak syndrome in all organs examined. These data constitute a strong framework for the conduct of a phase 1/2 study in patients with advanced solid tumors that encompasses dose escalation, dose expansion, and combination with CPIs. Conclusions: MDNA11 is a long-acting ‘beta-only’ IL-2 superkine that exhibits robust anti-tumor activity in mouse tumor models as a single agent and in combination with anti-PD1 and anti-CTLA4. In a non-human primate GLP toxicology study, MDNA11 demonstrated durable and selective activation of immune effector cells (NK, CD4+ and CD8+ T-cells), and a favorable safety profile. Citation Format: Minh D. To, Fahar Merchant, Ma'an Muhsin, Carole Galligan, L. Bruce Pearce, Peter Lloyd, Rosemina Merchant. MDNA11 is a long-acting ‘beta-only’ IL-2 agonist that demonstrates a safe and durable anti-tumor immune response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P198.

P57.10 Clinicopathological Analysis of Anti-Tumor Immunology-Related Factors After Chemoradiotherapy for Lung Cancer

Recently, involvement of immune cells such as T cells, activation of immune effector cells, and reduction of immunosuppressive cells have been shown to be a part of mechanisms of antitumor effect of chemotherapy. Some chemotherapy and radiotherapy induce immunogenic cell death (ICD), leading to immune response against cancer cells. However, the expression of ICD related factors in clinical specimens are still unknown. We investigated the expression of ICD related factors before and after chemoradiotherapy in non-small cell lung cancer (NSCLC) and analyzed its relationships with other clinicopathological characteristics of lung cancer.

Down-regulation of IGHG1 enhances Protoporphyrin IX accumulation and inhibits hemin biosynthesis in colorectal cancer by suppressing the MEK-FECH axis.

Immunoglobulin γ-1 heavy chain constant region (IGHG1) is a functional isoform of immunoglobulins and plays an important role in the cytolytic activity of immune effector cells. Dysregulated IGHG1 was implicated in the occurrence and development of various tumors. Protoporphyrin IX (PpIX) is an endogenous fluorophore and is used in photodynamic therapy, which induces the generation of reactive oxygen species to initiate the death of tumor cells. However, the roles of IGHG1 in the colorectal cancer cell proliferation and PpIX accumulation have not been reported yet. Data from qRT-PCR and western blot analysis showed that IGHG1 was up-regulated in the colorectal cancer cells. Colorectal cancer cells were then transfected with shRNA targeting IGHG1 to down-regulate IGHG1 and conducted with Cell Counting Kit 8 (CCK8) and colony formation assays. Results demonstrated that shRNA-mediated down-regulation of IGHG1 decreased cell viability of colorectal cancer and suppressed cell proliferation. Moreover, PpIX accumulation was promoted and the hemin content was decreased by the silence of IGHG1. Interference of IGHG1 reduced the phosphorylated extracellular signal-regulated kinase (ERK) and ferrochelatase (FECH) expression, resulting in retarded cell proliferation in an MEK-FECH axis-dependent pathway.

Activating Natural Killer Cell Receptors, Selectins, and Inhibitory Siglecs Recognize Ebolavirus Glycoprotein

Expression of the extensively glycosylated Ebolavirus glycoprotein (EBOV-GP) induces physical alterations of surface molecules and plays a crucial role in viral pathogenicity. Here we investigate the interactions of EBOV-GP with host surface molecules using purified EBOV-GP, EBOV-GP-transfected cell lines, and EBOV-GP-pseudotyped lentiviral particles. Subsequently, we wanted to examine which receptors are involved in this recognition by binding studies to cells transfected with the EBOV-GP as well as to recombinant soluble EBOV-GP. As the viral components can also bind to inhibitory receptors of immune cells (e.g., Siglecs, TIM-1), they can even suppress the activity of immune effector cells. Our data show that natural killer (NK) cell receptors NKp44 and NKp46, selectins (CD62E/P/L), the host factors DC-SIGNR/DC-SIGN, and inhibitory Siglecs function as receptors for EBOV-GP. Our results show also moderate to strong avidity of homing receptors (P-, L-, and E-selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, to cells transfected with EBOV-GP, as well as to the envelope of a pseudotyped lentiviral vector carrying the EBOV-GP. The concomitant activation and inhibition of the immune system exemplifies the evolutionary antagonism between the immune system and pathogens. Altogether these interactions with activating and inhibitory receptors result in a reduced NK cell-mediated lysis of EBOV-GP-expressing cells. Modulation of these interactions may provide new strategies for treating infections caused by this virus.

Virulence of a virus: How it depends on growth rate, effectors, memory cells, and immune escape

A viral strain may infect a host, proliferate rapidly, become controlled by immune reactions, and eventually be eliminated from the body. The virulence, or the magnitude of harm to the host due to infection, depends on the abundance and duration of the viral strain in the body, and the importance of the damaged tissue of the host. In this study, we investigated how the cumulative viral load (time-integral of the number of infected cells) depends on various factors, such as the viral growth rate, the effectiveness of immune cells to kill infected cells, speed of immune activation, formation of memory cells, and longevity of immune cells. In addition, viruses may produce a mutant with different antigen types, escape the immune reaction targeting the original type, and inflate virulence. We succeeded in deriving simple and explicit formulas of the virulence in four different parameter regions. We found that, in some parameter region, enhancing memory cells is very effective in suppressing virulence, but direct activation of immune effector cells is not effective; while the opposite is the case in other regions. The result could be important in designing drug suppressing virus effectively. Additionally, we discussed the reported correlation between virulence and molecular evolution rate.

Abstract 1533: Vδ1γδ T-cells: A unique tissue-derived, allogeneic cell therapy platform for the treatment of cancer

Abstract The treatment of cancer with adoptive cell therapy is largely limited to platforms based on circulating, patient-derived, engineered autologous αβ T cells. Although successful in some hematological malignancies, this approach comes with challenges including associated toxicities, high production costs and a requirement to gene edit cells to avoid graft vs host disease if used in an allogeneic setting. While engineered αβ T cells have shown therapeutic activity in haematological malignancies, clinical activity in solid tumors has been challenging. In contrast to αβ T cells, Vδ1 γδ T cells are a subset of innate T cells defined by expression of T cell receptors composed of a γ chain paired to a Vδ1 chain. In mice, Vδ1 γδ T cells are predominantly tissue resident where they are highly protective against a broad spectrum of carcinomas by mediating anti-tumor responses via pattern and natural cytotoxicity receptor recognition. Similarly, in humans, Vδ1 γδ T-cell predominantly reside within epithelial tissues, mediate target cell recognition that is not MHC restricted and are not allo-HLA reactive. HLA matching of patients is therefore not required for γδ T-cell adoptive cell therapies. The innate Vδ1 γδ T-cell biology which enables antigen independent tumour recognition, lack of necessity for HLA matching, and inherent migration to and residence in human tissues makes Vδ1 γδ T-cells an attractive platform for cellular therapy. We have developed a good manufacturing practice (GMP) compliant, scalable processes to isolate tissue-resident Vδ1 γδ T-cells from healthy human skin tissue. Using organotypic culture methods, the process generates a mixture of tissue resident lymphocytes that is easily cryopreservable. This frozen intermediate (termed ‘DermaPack') serves as qualified starting material for expansion and highly efficient transduction of γδ T cells to generate allogeneic ‘off-the-shelf' CAR γδ T-cell therapies for the treatment of solid tumors. CAR Vδ1 γδ T-cells show excellent recoverability and viability post cryopreservation, an activated innate phenotype and produce high levels of IFNγ and chemokines which can attract and stimulate activation of other immune effector cells like APCs and αβ T cells. Vδ1 CAR-T cells show potent cytotoxic activity against solid tumor cell lines in vitro. These data demonstrate a novel method for a unique, off the shelf CAR-T cell platform for targeting solid tumor that combines the specificity of a CAR with the innate features of tissue resident Vδ1 γδ T cells. Citation Format: Andrew Hutton, Shristi Bhandari, Mahdieh Hassanjani, Daniel Adams, Madeline Dalziel, Peter Mitchell, Ines Sa Pereira, Sean O'Farrell, Rebecca Alade, Istvan Kovacs, Michael Koslowski, Alice C. Brown, Oliver Nussbaumer. Vδ1γδ T-cells: A unique tissue-derived, allogeneic cell therapy platform for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1533.

Engineering Human Induced Pluripotent Stem Cells with Novel Chimeric Antigen Receptors to Generate Natural Killer (NK) Cell Cancer Immunotherapies with Targeted Anti-Tumor Activity

Chimeric antigen receptors (CARs) provide a powerful strategy to direct and enhance anti-tumor activity of immune effector cells. While most studies have evaluated CAR-expression in T cells, here we evaluate and optimize CAR constructs that are specifically designed with natural killer (NK) cell transmembrane and signaling domains. Since NK cell-mediated cytotoxicity does not require self-HLA expression, derivation of NK cells from human induced pluripotent stem cells (iPSCs), combined with the use of NK cell-specific CARs, enables production of a standardized, targeted allogeneic effector cell population. After screening 10 combinations of CARs using an anti-mesothelin (meso) scFv to target ovarian cancer cells, we identified a CAR construct containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain that mediates a strong increase in intracellular NK cell signaling. iPSC-derived NK cells that express this novel CAR (NKCAR-iPSC-NK cells) have a surface phenotype similar to NK cells isolated from peripheral blood (PB-NK) and unmodified iPSC-derived NK cells. Interestingly, the NKCAR-iPSC-NK cells mediate improved killing of target-expressing targets more than iPSC-derived NK cells expressing a 3rd generation CAR with CD28, CD137, and ζ signaling elements optimized for T cell-mediated activity (TCAR-iPSC-NK cells). We examined intracellular signaling pathways stimulated by our NKG2D-2B4-ζ CAR construct. In NKCAR-iPSC-NK cells, antigen (meso)-expressing targets promoted phosphorylation of Syk and Erk. Site-directed mutagenesis studies demonstrate the requirement for the NKG2D and 2B4 domains to mediate release of cytotoxic granules (CD107a), cytokine (IFN-γ) release and robust tumor cell lysis. Using an ovarian cancer xenograft model, a single dose of NKCAR-iPSC-NK cells markedly inhibited tumor growth, and mediated significantly enhanced survival (84 days) compared to NK cells isolated from peripheral blood (PB-NK cells) (70 day survival), non-CAR-expressing iPSC-NK cells (57 days), and TCAR-iPSC-NK cells (63 days) (p < 0.002 for NKCAR-iPSC-NK cells compared to each of the other 3 NK cell populations). As an additional strategy, we have engineered iPSCs both with this novel CAR using an anti-CD19 scFv and with a high-affinity non-cleavable CD16 receptor (hnCD16 with F158V (high-affinity) and S197P (non-cleavable) substitutions). To prevent antigen loss variants that promote resistance of B cell malignancies to anti-CD19-CAR-T cell-based therapies, this approach now allows us to combine the use of anti-CD19-CAR with anti-CD20 antibody to target B cell malignancies using engineered iPSC-NK cells. This combinatorial targeting of Raji cells (CD19+CD20+ B cell lymphoma) demonstrates enhanced upregulation of CD107a and TNFα with improved elimination of CD19-negative target cells. Together, these strategies provide a comprehensive approach to utilize NKCAR-iPSC-NK cells as a novel strategy to produce “off-the-shelf”, targeted, allogeneic lymphocytes suitable to treat refractory solid tumor and hematologic malignancies. DisclosuresHermanson:Poseida Therapeutics: Employment. Bjordahl:Fate Therapeutics: Employment, Equity Ownership. Mahmood:Fate Therapeutics: Employment. Valamehr:Fate Therapeutics: Employment, Equity Ownership. Kaufman:Fate Therapeutics: Consultancy, Research Funding.