Activating Inducible T-cell Costimulator Yields Antitumor Activity Alone and in Combination with Anti-PD-1 Checkpoint Blockade.

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.

Immuno-PET Monitoring of CD8+ T Cell Infiltration Post ICOS Agonist Antibody Treatment Alone and in Combination with PD-1 Blocking Antibody Using a 89Zr Anti-CD8+ Mouse Minibody in EMT6 Syngeneic Tumor Mouse.

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 89Zr-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 89Zr IAB42M1-14. In addition to 89Zr-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: 89Zr-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 89Zr-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.

Author Correction: Design of amidobenzimidazole STING receptor agonists with systemic activity.

Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

Tumor-immune profiling of murine syngeneic tumor models as a framework to guide mechanistic studies and predict therapy response in distinct tumor microenvironments.

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.

Preclinical activity of eltrombopag (SB-497115), an oral, nonpeptide thrombopoietin receptor agonist.

Eltrombopag is a first-in-class, orally bioavailable, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR, which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34(+) bone marrow cells into CD41(+) megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. The in vivo activity of eltrombopag was demonstrated by an increase of up to 100% in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion, eltrombopag interacts selectively with the TpoR without competing with Tpo, leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production.

New benzimidazoles as thrombopoietin receptor agonists.

A novel benzimidazole series of small-molecule thrombopoietin receptor agonists has been discovered. Herein, we discuss the preliminary exploration of structure-activity relationships within this chemotype.

Discovery and characterization of a selective, nonpeptidyl thrombopoietin receptor agonist.

OBJECTIVE: Peptide and other small molecule agonists have been described for several cytokines and growth factors. Hydrazone compounds described here as thrombopoietin receptor agonists were identified as activating STAT proteins in a Tpo responsive cell line. METHODS: STAT activation and analysis of signal transduction pathways in cell lines and normal human platelets was elucidated by Western blot and electrophoretic mobility shift assays. Proliferation assays in cell types responsive to other cytokines determined specificity for Tpo receptor. Flow cytometry quantified differentiation of CD34(+) cells into CD41(+) megakaryocytes and platelet production in vitro. RESULTS: Activation of STAT5, mitogen-activated protein kinase, p38, and early response genes by SB 394725 was similar to that induced by Tpo. SB 394725 induced a reporter gene response under a STAT activation promoter as well as the megakaryocyte-specific gpIIb promoter. The compound induced proliferation of Tpo responsive lines but demonstrated no activity in cell lines responding to other cytokines, i.e., erythropoietin, granulocyte-colony stimulating factor, interleukin-3, interferon-gamma. The response of normal human Tpo receptors was elucidated by measuring growth and differentiation of human bone marrow in vitro. Activation of endogenous Tpo receptors by SB 394725 was demonstrated in human and chimp platelets, but not in platelets of other species including mouse, dog, rabbit, or cynomolgus monkey. CONCLUSIONS: SB 394725, a small molecule with a molecular weight of 452 Da, is capable of activating Tpo-specific signal transduction, proliferation, and differentiation responses similar to the responses and functions of the protein growth factor, Tpo.

Characterization of Siglec-5 (CD170) expression and functional activity of anti-Siglec-5 antibodies on human phagocytes.

OBJECTIVE: The Siglec family of proteins consists of at least 10 members with immunoglobulin and lectin domains and with similar sialic acid-binding properties. Many Siglec family members are expressed on hematopoietic cells and are involved in cell/cell interactions. Some family members are suspected of regulating cellular processes through specific signaling pathways. Monoclonal antibodies were generated against specific epitopes of Siglec-5 (CD170) and were used to determine expression of Siglec-5 on normal blood and marrow cells and cell lines. The antibodies also were used to elucidate functional activity for Siglec-5 on blood neutrophils. METHODS: Flow cytometry and ELISA were used to determine the specificity of the monoclonal antibodies for Siglec-5 and to determine expression patterns. Chemiluminescence assays were employed to measure the oxidative burst activity of whole blood or purified neutrophils following treatment with the anti-Siglec-5 antibodies. RESULTS: Cell surface expression analysis demonstrated that the protein was expressed on gated human neutrophil and monocyte populations, both in the blood and bone marrow. Expression on neutrophils was enhanced by one-hour treatment with fMLP or TNF-alpha. Epitope-specific anti-Siglec-5 monoclonal antibodies did not directly activate human neutrophils; however, antibody binding augmented neutrophil oxidative burst activity as determined by fMLP-induced luminol-dependent chemiluminescence. CONCLUSION: Data demonstrating expression of Siglec-5 on cells of the myelomonocytic lineage and alteration of its expression by inflammatory stimuli suggest a role for this protein in cell/cell interactions following microbial exposure.