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Conventional immune checkpoint inhibitors (ICI) targeting CTLA-4 elicit durable survival, but primarily in patients with immune-inflamed tumors. Although the mechanisms underlying response to anti-CTLA-4 remain poorly understood, Fc-gamma receptor (FcγR) IIIA co-engagement appears critical for activity, potentially explaining the modest clinical benefits of approved anti-CTLA-4 antibodies. We demonstrate that anti-CTLA-4 engineered for enhanced FcγR affinity leverages FcγR-dependent mechanisms to potentiate T cell responsiveness, reduce intratumoral Tregs, and enhance antigen presenting cell activation. Fc-enhanced anti-CTLA-4 promoted superior efficacy in mouse models and remodeled innate and adaptive immunity versus conventional anti-CTLA-4. These findings extend to patients treated with botensilimab, an Fc-enhanced anti-CTLA-4 antibody, with clinical activity across multiple poorly immunogenic and ICI treatment-refractory cancers. Efficacy was independent of tumor neoantigen burden or FcγRIIIA genotype. However, FcγRIIA and FcγRIIIA expression emerged as potential response biomarkers. These data highlight the therapeutic potential of Fc-enhanced anti-CTLA-4 antibodies in cancers unresponsive to conventional ICI therapy.

TIGIT is an inhibitory receptor expressed on lymphocytes and can inhibit T cells by preventing CD226 co-stimulation through interactions in cis or through competition of shared ligands. Whether TIGIT directly delivers cell-intrinsic inhibitory signals in T cells remains unclear. Here we show, by analysing lymphocytes from matched human tumour and peripheral blood samples, that TIGIT and CD226 co-expression is rare on tumour-infiltrating lymphocytes. Using super-resolution microscopy and other techniques, we demonstrate that ligation with CD155 causes TIGIT to reorganise into dense nanoclusters, which coalesce with T cell receptor (TCR)-rich clusters at immune synapses. Functionally, this reduces cytokine secretion in a manner dependent on TIGIT's intracellular ITT-like signalling motif. Thus, we provide evidence that TIGIT directly inhibits lymphocyte activation, acting independently of CD226, requiring intracellular signalling that is proximal to the TCR. Within the subset of tumours where TIGIT-expressing cells do not commonly co-express CD226, this will likely be the dominant mechanism of action., (© 2023. Springer Nature Limited.)

In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies., Significance: Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Purpose: The presence and functional competence of intratumoral CD8 + T cells is often a barometer for successful immunotherapeutic responses in cancer. Despite this understanding and the extensive number of clinical-stage immunotherapies focused on potentiation (co-stimulation) or rescue (checkpoint blockade) of CD8 + T cell antitumor activity, dynamic biomarker strategies are often lacking. To help fill this gap, immuno-PET nuclear imaging has emerged as a powerful tool for in vivo molecular imaging of antibody targeting. Here, we took advantage of immuno-PET imaging using 89 Zr-IAB42M1-14, anti-mouse CD8 minibody, to characterize CD8 + T-cell tumor infiltration dynamics following ICOS (inducible T-cell co-stimulator) agonist antibody treatment alone and in combination with PD-1 blocking antibody in a model of mammary carcinoma., Procedures: Female BALB/c mice with established EMT6 tumors received 10 µg, IP of either IgG control antibodies, ICOS agonist monotherapy, or ICOS/PD-1 combination therapy on days 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 and 48 h post IV dose of 89 Zr IAB42M1-14. In addition to 89 Zr-IAB42M1-14 uptake in tumor and tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images to identify treatment effects. Imaging mass cytometry (IMC) and immunohistochemistry (IHC) was performed at end of study., Results: 89 Zr-IAB42M1-14 uptake in the tumor was observed by day 11 and was preceded by an increase in the TDLN as early as day 4. The spatial distribution of 89 Zr-IAB42M1-14 was more uniform in the drug treated vs. control tumors, which had spatially distinct tracer uptake in the periphery relative to the core of the tumor. IMC analysis showed an increased percentage of cytotoxic T cells in the ICOS monotherapy and ICOS/PD-1 combination group compared to IgG controls. Additionally, temporal radiomics analysis demonstrated early predictiveness of imaging features., Conclusion: To our knowledge, this is the first detailed description of the use of a novel immune-PET imaging technique to assess the kinetics of CD8 + T-cell infiltration into tumor and lymphoid tissues following ICOS agonist and PD-1 blocking antibody therapy. By demonstrating the capacity for increased spatial and temporal resolution of CD8 + T-cell infiltration across tumors and lymphoid tissues, these observations underscore the widespread potential clinical utility of non-invasive PET imaging for T-cell-based immunotherapy in cancer., (© 2022. The Author(s).)

NK cells have unique attributes to react towards cells undergoing malignant transformation or viral infection. This reactivity is regulated by activating or inhibitory germline encoded receptors. An impaired NK cell function may result from an aberrant expression of such receptors, a condition often seen in patients with hematological cancers. Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer worldwide and NK cells have emerged as crucial targets for developing immunotherapies. However, there are important gaps concerning the phenotype and behavior of NK cells during emergence of ALL. In this study we analyze the phenotype and function of NK cells from peripheral blood in pediatric patients with ALL at diagnosis. Our results showed that NK cells exhibited an altered phenotype highlighted by a significant reduction in the overall expression and percent representation of activating receptors compared to age-matched controls. No significant differences were found for the expression of inhibitory receptors. Moreover, NK cells with a concurrent reduced expression in various activating receptors, was the dominant phenotype among patients. An alteration in the relative frequencies of NK cells expressing NKG2A and CD57 within the mature NK cell pool was also observed. In addition, NK cells from patients displayed a significant reduction in the ability to sustain antibody-dependent cellular cytotoxicity (ADCC). Finally, an aberrant expression of activating receptors is associated with the phenomenon of leukemia during childhood., Competing Interests: Authors JDW, KWH, and KPM are employed by GlaxoSmithKline. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Valenzuela-Vázquez, Nuñez-Enriquez, Sánchez-Herrera, Medina-Sanson, Pérez-Saldivar, Jiménez-Hernández, Martiín-Trejo, Del Campo-Martínez, Flores-Lujano, Amador-Sánchez, Mora-Ríos, Peñaloza-González, Duarte-Rodríguez, Torres-Nava, Espinosa-Elizondo, Cortés-Herrera, Flores-Villegas, Merino-Pasaye, Almeida-Hernández, Ramírez-Colorado, Solís-Labastida, Medrano-López, Pérez-Gómez, Velázquez-Aviña, Martínez-Ríos, Aguilar-De los Santos, Santillán-Juárez, Gurrola-Silva, García-Velázquez, Mata-Rocha, Hernández-Echáurregui, Sepúlveda-Robles, Rosas-Vargas, Mancilla-Herrera, Jimenez-Morales, Hidalgo-Miranda, Martinez-Duncker, Waight, Hance, Madauss, Mejía-Aranguré and Cruz-Munoz.)

In recent years, a set of immune receptors that interact with members of the nectin/nectin-like (necl) family has garnered significant attention as possible points of manipulation in cancer. Central to this axis, CD226, TIGIT, and CD96 represent ligand (CD155)-competitive co-stimulatory/inhibitory receptors, analogous to the CTLA-4/B7/CD28 tripartite. The identification of PVRIG (CD112R) and CD112 has introduced complexity and enabled additional nodes of therapeutic intervention. By virtue of the clinical progression of TIGIT antagonists and emergence of novel CD96- and PVRIG-based approaches, our overall understanding of the 'CD226 axis' in cancer immunotherapy is starting to take shape. However, several questions remain regarding the unique characteristics of, and mechanistic interplay between, each receptor-ligand pair. This review provides an overview of the CD226 axis in the context of cancer, with a focus on the status of immunotherapeutic strategies (TIGIT, CD96, and PVRIG) and their underlying biology (i.e., cis / trans interactions). We also integrate our emerging knowledge of the immune populations involved, key considerations for Fc gamma (γ) receptor biology in therapeutic activity, and a snapshot of the rapidly evolving clinical landscape., Competing Interests: All authors are employees of GlaxoSmithKline., (Copyright © 2022 Conner, Hance, Yadavilli, Smothers and Waight.)

Decision making in immuno-oncology is pivotal to adapt therapy to the tumor microenvironment (TME) of the patient among the numerous options of monoclonal antibodies or small molecules. Predicting the best combinatorial regimen remains an unmet medical need. Here, we report a multiplex functional and dynamic immuno-assay based on the capacity of the TME to respond to ex vivo stimulation with twelve immunomodulators including immune checkpoint inhibitors (ICI) in 43 human primary tumors. This "in sitro" (in situ/in vitro) assay has the potential to predict unresponsiveness to anti-PD-1 mAbs, and to detect the most appropriate and personalized combinatorial regimen. Prospective clinical trials are awaited to validate this in sitro assay., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The co-engagement of fragment crystallizable (Fc) gamma receptors (FcγRs) with the Fc region of recombinant immunoglobulin monoclonal antibodies (mAbs) and its contribution to therapeutic activity has been extensively studied. For example, Fc-FcγR interactions have been shown to be important for mAb-directed effector cell activities, as well as mAb-dependent forward signaling into target cells via receptor clustering. Here we identify a function of mAbs targeting T cell-expressed antigens that involves FcγR co-engagement on antigen-presenting cells (APCs). In the case of mAbs targeting CTLA-4 and TIGIT, the interaction with FcγR on APCs enhanced antigen-specific T cell responses and tumoricidal activity. This mechanism extended to an anti-CD45RB mAb, which led to FcγR-dependent regulatory T cell expansion in mice., (Copyright © 2018 Elsevier Inc. All rights reserved.)

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

Co-stimulatory tumor necrosis factor receptors (TNFRs) can sculpt the responsiveness of T cells recognizing tumor-associated antigens. For this reason, agonist antibodies targeting CD137, CD357, CD134 and CD27 have received considerable attention for their therapeutic utility in enhancing anti-tumor immune responses, particularly in combination with other immuno-modulatory antibodies targeting co-inhibitory pathways in T cells. The design of therapeutic antibodies that optimally engage and activate co-stimulatory TNFRs presents an important challenge of how to promote effective anti-tumor immunity while avoiding serious immune-related adverse events. Here we review our current understanding of the expression, signaling and structural features of CD137, CD357, CD134 and CD27, and how this may inform the design of pharmacologically active immuno-modulatory antibodies targeting these receptors. This includes the integration of our emerging knowledge of the role of Fcγ receptors (FcγRs) in facilitating antibody-mediated receptor clustering and forward signaling, as well as promoting immune effector cell-mediated activities. Finally, we bring our current preclinical and clinical knowledge of co-stimulatory TNFR antibodies into the context of opportunities for next generation molecules with improved pharmacologic properties.

CD4(+) regulatory T cells (Tregs) are critical for maintaining self-tolerance and function to prevent autoimmune disease. High densities of intratumoral Tregs are generally associated with poor patient prognosis, a correlation attributed to their broad immune-suppressive features. Two major populations of Tregs have been defined, thymically derived natural Tregs (nTregs) and peripherally induced Tregs (iTregs). However, the relative contribution of nTregs versus iTregs to the intratumoral Treg compartment remains controversial. Demarcating the proportion of nTregs versus iTregs has important implications in the design of therapeutic strategies to overcome their antagonistic effects on antitumor immune responses. We used epigenetic, phenotypic, and functional parameters to evaluate the composition of nTregs versus iTregs isolated from mouse tumor models and primary human tumors. Our findings failed to find evidence for extensive intratumoral iTreg induction. Rather, we identified a population of Foxp3-stable nTregs in tumors from mice and humans., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Tyrosine kinase inhibitors such as imatinib can effectively target the BCR-ABL oncoprotein in a majority of patients with chronic myeloid leukemia (CML). Unfortunately, some patients are resistant primarily to imatinib and others develop drug resistance, prompting interest in the discovery of new drug targets. Although much of this resistance can be explained by the presence of mutations within the tyrosine kinase domain of BCR-ABL, such mutations are not universally identified. Interferon regulatory factor-8 (IRF-8) is a transcription factor that is essential for myelopoiesis. Depressed IRF-8 levels are observed in a majority of CML patients and Irf-8(-/-) mice exhibit a CML-like disease. The underlying mechanisms of IRF-8 loss in CML are unknown. We hypothesized that BCR-ABL suppresses transcription of IRF-8 through STAT5, a proximal BCR-ABL target. Treatment of primary cells from newly diagnosed CML patients in chronic phase as well as BCR-ABL(+) cell lines with imatinib increased IRF-8 transcription. Furthermore, IRF-8 expression in cell line models was necessary for imatinib-induced antitumor responses. We have demonstrated that IRF-8 is a direct target of STAT5 and that silencing of STAT5 induced IRF-8 expression. Conversely, activating STAT5 suppressed IRF-8 transcription. Finally, we showed that STAT5 blockade using a recently discovered antagonist increased IRF-8 expression in patient samples. These data reveal a previously unrecognized BCR-ABL-STAT5-IRF-8 network, which widens the repertoire of potentially new anti-CML targets., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20-26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30-31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8(+) T lymphocytes and CD8(+) T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.

Myeloid-derived suppressor cells (MDSCs) comprise immature myeloid populations produced in diverse pathologies, including neoplasia. Because MDSCs can impair antitumor immunity, these cells have emerged as a significant barrier to cancer therapy. Although much research has focused on how MDSCs promote tumor progression, it remains unclear how MDSCs develop and why the MDSC response is heavily granulocytic. Given that MDSCs are a manifestation of aberrant myelopoiesis, we hypothesized that MDSCs arise from perturbations in the regulation of interferon regulatory factor-8 (IRF-8), an integral transcriptional component of myeloid differentiation and lineage commitment. Overall, we demonstrated that (a) Irf8-deficient mice generated myeloid populations highly homologous to tumor-induced MDSCs with respect to phenotype, function, and gene expression profiles; (b) IRF-8 overexpression in mice attenuated MDSC accumulation and enhanced immunotherapeutic efficacy; (c) the MDSC-inducing factors G-CSF and GM-CSF facilitated IRF-8 downregulation via STAT3- and STAT5-dependent pathways; and (d) IRF-8 levels in MDSCs of breast cancer patients declined with increasing MDSC frequency, implicating IRF-8 as a negative regulator in human MDSC biology. Together, our results reveal a previously unrecognized role for IRF-8 expression in MDSC subset development, which may provide new avenues to target MDSCs in neoplasia.

Myeloid-derived suppressor cells (MDSC) induced during neoplasia display potent pro-tumorigenic activities. Tumor-derived factors influence MDSC development, yielding monocytic and granulocytic subsets. In contrast to monocytic MDSC, little is known about how granulocytic MDSC develop. We demonstrated that tumor-derived G-CSF drives granulocytic MDSC formation, thus providing new insights into myeloid-tumor biology.

Myeloid-derived suppressor cells (MDSC) are induced under diverse pathologic conditions, including neoplasia, and suppress innate and adaptive immunity. While the mechanisms by which MDSC mediate immunosuppression are well-characterized, details on how they develop remain less understood. This is complicated further by the fact that MDSC comprise multiple myeloid cell types, namely monocytes and granulocytes, reflecting diverse stages of differentiation and the proportion of these subpopulations vary among different neoplastic models. Thus, it is thought that the type and quantities of inflammatory mediators generated during neoplasia dictate the composition of the resultant MDSC response. Although much interest has been devoted to monocytic MDSC biology, a fundamental gap remains in our understanding of the derivation of granulocytic MDSC. In settings of heightened granulocytic MDSC responses, we hypothesized that inappropriate production of G-CSF is a key initiator of granulocytic MDSC accumulation. We observed abundant amounts of G-CSF in vivo, which correlated with robust granulocytic MDSC responses in multiple tumor models. Using G-CSF loss- and gain-of-function approaches, we demonstrated for the first time that: 1) abrogating G-CSF production significantly diminished granulocytic MDSC accumulation and tumor growth; 2) ectopically over-expressing G-CSF in G-CSF-negative tumors significantly augmented granulocytic MDSC accumulation and tumor growth; and 3) treatment of naïve healthy mice with recombinant G-CSF protein elicited granulocytic-like MDSC remarkably similar to those induced under tumor-bearing conditions. Collectively, we demonstrated that tumor-derived G-CSF enhances tumor growth through granulocytic MDSC-dependent mechanisms. These findings provide us with novel insights into MDSC subset development and potentially new biomarkers or targets for cancer therapy.

Bacterial growth from a single flake of tobacco was documented for cigarettes that had been purchased recently from local vendors and from cigarettes that had been stored for more than six years in a warehouse. In a novel tobacco flake assay, a pack of cigarettes was opened within the sterile environment of a laminar flow hood. A single flake of tobacco was collected randomly and aseptically from the middle of the cigarette column and placed onto the surface of a blood agar plate. The test cigarettes included eight different popular US brands, and these were from three different tobacco companies. After 24 hours of incubation at 37 degrees C, the plates showed bacterial growth for tobacco from all brands of cigarettes. Further, more than 90% of the individual tobacco flakes of a given brand grew bacteria. Likewise, bacteria grew from microparticulate tobacco that had been sieved from cigarettes. Tobacco flakes were observed lying loosely on the cut surface of the filter of cigarettes in newly opened packs, and bacteria grew from cigarette filters that had been touched to the surface of a blood agar plate. In conclusion, the results of these studies predict that diverse microbes and microbial toxins are carried by tobacco microparticulates that are released from the cigarette during smoking, and carried into mainstream smoke that is sucked deep into the lung.