Activation Of Costimulatory Molecules Research Articles

Abstract 4524: Non-genetic generation of IgG-like immune cell-redirecting antibodies lacking effector function

Abstract Since the early approval of catumaxomab and blinatumomab, T cell-redirecting antibodies have emerged as a promising biologics in cancer treatment. New technologies are overcoming the limitations of the 1st generation T-cell redirecting bispecifics, especially extending half-life to allow intermittent dosing, reducing immunogenicity and improving the safety profile. Today, T cell-engagers are generated as a wide variety of molecular architectures, typically IgG-type. However, nearly without exception, these bispecifics are generated by genetic fusion of a cancer-binding and an anti-CD3 antibody, which inevitably requires genetic reengineering of an existing targeting antibody and/or extensive optimization of antibody format. We have shown earlier that the native glycan of a monoclonal antibody provides a privileged site for controlled attachment of small-molecule cytotoxic drugs.1 The resulting antibody-drug conjugates (ADCs) were found to display significantly improved therapeutic index versus conventional technologies, while improving manufacturability. This proprietary technology, known as GlycoConnect™, has been adapted by various ADC developers, and entered clinical phase early 2019. Here we present the adaption of GlycoConnect™ technology for the generation of immune cell-redirecting antibodies, without requiring prior protein reengineering. Instead, a two-stage chemoenzymatic process is applied that allows the conversion of any IgG isotype into a T cell-engager by selective attachment of anti-CD3 scFv to the antibody glycan. Studies are presented to precisely control conjugation stoichiometry, providing molecular architectures with defined 2:1 or 2:2 ratio (target-binding:CD3-binding), as well as functional studies to corroborate the biological activity of these GlycoConnect™ bispecifics. Finally, early results will be presented regarding extension of the concept to bispecifics designed to redirect natural killer (NK) cells, or to engage T-cells by activation of costimulatory molecules, such as CD137 (4-1BB). 1 van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved NGlycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. Citation Format: Floris L. van Delft, Remon van Geel. Non-genetic generation of IgG-like immune cell-redirecting antibodies lacking effector function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4524.

Advanced Foot-And-Mouth Disease Vaccine Platform for Stimulation of Simultaneous Cellular and Humoral Immune Responses.

Currently available commercial foot-and-mouth disease (FMD) vaccines have various limitations, such as the slow induction and short-term maintenance of antibody titers. Therefore, a novel FMD vaccine that can rapidly induce high neutralizing antibody titers to protect the host in early stages of an FMD virus infection, maintain high antibody titers for long periods after one vaccination dose, and confer full protection against clinical symptoms by simultaneously stimulating cellular and humoral immunity is needed. Here, we developed immunopotent FMD vaccine strains A-3A and A-HSP70, which elicit strong initial cellular immune response and induce humoral immune response, including long-lasting memory response. We purified the antigen (inactivated virus) derived from these immunopotent vaccine strains, and evaluated the immunogenicity and efficacy of the vaccines containing these antigens in mice and pigs. The immunopotent vaccine strains A-3A and A-HSP70 demonstrated superior immunogenicity compared with the A strain (backbone strain) in mice. The oil emulsion-free vaccine containing A-3A and A-HSP70 antigens effectively induced early, mid-term, and long-term immunity in mice and pigs by eliciting robust cellular and humoral immune responses through the activation of co-stimulatory molecules and the secretion of proinflammatory cytokines. We successfully derived an innovative FMD vaccine formulation to create more effective FMD vaccines.

An Integrative Network Modeling Approach to T CD4 Cell Activation.

The adaptive immune response is initiated by the interaction of the T cell antigen receptor/CD3 complex (TCR) with a cognate peptide bound to a MHC molecule. This interaction, along with the activity of co-stimulatory molecules and cytokines in the microenvironment, enables cells to proliferate and produce soluble factors that stimulate other branches of the immune response for inactivation of infectious agents. The intracellular activation signals are reinforced, amplified and diversified by a complex network of biochemical interactions, and includes the activity of molecules that modulate the activation process and stimulate the metabolic changes necessary for fulfilling the cell energy demands. We present an approach to the analysis of the main early signaling events of T cell activation by proposing a concise 46-node hybrid Boolean model of the main steps of TCR and CD28 downstream signaling, encompassing the activity of the anergy factor Ndrg1, modulation of activation by CTLA-4, and the activity of the nutrient sensor AMPK as intrinsic players of the activation process. The model generates stable states that reflect the overcoming of activation signals and induction of anergy by the expression of Ndrg1 in the absence of co-stimulation. The model also includes the induction of CTLA-4 upon activation and its competition with CD28 for binding to the co-stimulatory CD80/86 molecules, leading to stable states that reflect the activation arrest. Furthermore, the model integrates the activity of AMPK to the general pathways driving differentiation to functional cell subsets (Th1, Th2, Th17, and Treg). Thus, the network topology incorporates basic mechanism associated to activation, regulation and induction of effector cell phenotypes. The model puts forth a conceptual framework for the integration of functionally relevant processes in the analysis of the T CD4 cell function.

CD5, an Undercover Regulator of TCR Signaling.

T cells are critical components of adaptive immunity. As such, their activation is regulated by the T cell receptor (TCR) that constantly scan peptides associated with major histocompatibility complexes (MHC). TCR engagement initiates a series of molecular events leading to cytokine secretion, proliferation, and differentiation of T cells. As a second coincident event, activation of co-stimulatory molecules, such as CD28, synergize with the TCR in order to prolong and/or amplify intracellular signals. With the recent advances in immunotherapies targeting T cells, co-inhibitory receptors are of growing interest for immunologists due to their potential modulatory properties on T cell functions. However, special attention should be dedicated to avoid unwanted clinical outcomes (1). In particular, Manichean categorization of receptors based on incomplete functional knowledge can lead to an over-simplistic view of complex cellular regulations. Thus, analysis of the functions that characterize these receptors in diverse physiological contexts remains essential for their rational use in therapeutic protocols. Here we focus on CD5, a transmembrane receptor that regulates T cell functions and development but remains poorly characterized at the molecular level. We will review its roles in physiological conditions and suggest potential molecular effectors that could account for CD5-dependent regulation of TCR signaling.

Abstract 2746: Synergistic immunotherapeutic effects of TLR7/8 agonist on low-dose cyclophosphamide-treated C26 model

Abstract In recent years, low dose cyclophosphamide has been used clinically either as a single agent or as combination therapy to treat lymphomas, and breast and ovarian cancers. The efficacy of low dose cyclophosphamide is primarily due to its ability to immune-modulate in addition to its direct anti-tumor effects. This ability highlights the potential for synergism between conventional chemotherapy and novel immunotherapy. Toll-like receptors (TLRs) are a crucial part of the innate immunity and present the first line of defense against pathogens. Resiquimod is the ligand for TLR7 and 8 and directly activate innate immune cells, including myeloid dendritic cells, plasmacytoid dendritic cells, and monocytes/macrophages. This activation may result in activation of co-stimulatory molecules, production of antiviral cytokines, and stimulation of cell-mediated NK and T cell immune responses. The aim of this study was to analyze whether immunopotentiating TLR7/8 agonist might synergize with low doses of cyclophosphamide in the treatment of colorectal carcinoma. The effect of combining a weekly dosing of cyclophosphamide intraperitoneally with systemic administration of Resiquimod was evaluated in an in vivo murine C26 synergistic model. Systemic administration of Resiquimod in mice induced significant systemic immune responses as evidenced by upregulating IFNα inducible gene clusters. The combined therapy with Resiquimod and cyclophosphamide was superior to each single treatment and increased efficacy of tumor inhibition and prolonged their survival significantly. Importantly, the combined treatment generated a significant numbers of tumor-specific T cell infiltration in tumor microenvironment. Our data suggest that systemic administration of TLR7/8 agonist enhances anti-tumor effect of low dose of cyclophosphamide. The data supports the use of a combination of TLR7/8 agonist and cyclophosphamide as a novel therapeutic strategy against colorectal carcinoma. Citation Format: Jing Yang, Patrick Z. Li, Xiangyun Yin, Xiaohua Nie, Jingshu Wang, Walter Lau, Liguo Zhang, Lixin Li. Synergistic immunotherapeutic effects of TLR7/8 agonist on low-dose cyclophosphamide-treated C26 model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2746.

Abstract B183: Treg inhibition by anti-GITR therapy evokes therapeutic antitumor responses selectively in the context of a pro-inflammatory tumor microenvironment

Abstract The recent clinical successes of CTLA4 and PD-1 blockade have clearly demonstrated the therapeutic efficacy to enhance anticancer immune responses. Novel targets of immunomodulation are now under investigation to increase the number of immunotherapeutic options available for relapsing/refractory patients. Activation of co-stimulatory molecules with agonist monoclonal antibody (mAbs) represents the most rationale alternative option. Based on the impressive preclinical activity of stimulating the glucocorticoid-induced TNF receptor (GITR) with the mAb DTA-1, an agonist anti-human GITR mAb has very recently entered the clinical evaluation. Although DTA-1, as a monotherapy, proved effective in curing established immunogenic tumors, it failed against advanced poorly immunogenic models, such as B16 melanoma. As anti-GITR therapy is translated into clinic, defining the basis of its activity and failure is key to identify prognostic markers and more effective combination therapies. We thus studied how GITR stimulation with the agonist antibody DTA-1 affects T cells in B16 melanoma-bearing mice treated in curative (early) or refractory (advanced) condition, and verified their functional changes in vitro. In vivo, the comparison of advanced versus early B16 microenvironment before treatment revealed a significant increase in Treg frequency, which caused the Teff/Treg ratio to decrease, and a dysfunctional Teff phenotype. Moreover, B16 cells, in the setting of established tumors, expressed lower levels of MHC-I and MHC-II molecules. DTA-1 consistently reduced intratumor Treg frequency and expression of suppression markers in both curative and refractory conditions. However, Teff activation and tumor-associated MHCs were induced only when DTA-1 was administered in curative conditions. In vitro, pre-incubation of Tregs with DTA-1 completely abolished their ability to inhibit proliferation of target Teff, without affecting Treg expression of the lineage markers Foxp3 and Helios. In addition, treatment with DTA-1, in comparison with the matched IgG isotype control, boosted the release of IFN-gamma, TNF-alpha, IL-17 and IL-6 cytokines in cultures of Tregs, pointing to the promotion of a Th1/Th17 differentiation profile following GITR engagement. Interestingly, when B16 cells were added in culture with Tregs and incubated with DTA-1, they up-regulated MHC-I and MHC-II molecules in a Treg and/or DTA-1 dependent manner. The presence of tumor cells during exposure of Tregs to anti-GITR stimulation did not further alter their function and phenotype, indicating a major direct effect of DTA-1 on Tregs. To confirm the functional modulation of GITR stimulation in human Tregs, we tested their activity in vitro upon pre-incubation with a recombinant human GITR ligand (GITRL). We consistently found that proliferation of activated Teff was no longer suppressed if Tregs were previously treated with GITRL. These results validate GITR as a target to revert immune tolerance in the human setting. Our data indicate that anti-GITR therapy counteracts Treg-mediated immunosuppression independently from the stage of the disease, whereas T-cell activation and tumor immune recognition are favored selectively in the presence of low tumor burden. These findings suggest that, in the setting of advanced and poorly immunogenic tumors, anti-GITR therapy should be exploited in combination with treatments promoting Teff functions and/or tumor killing. Citation Format: Roberta Zappasodi, Sadna Budhu, Cailian Liu, Kyle Draleau, David Schaer, Hong Zhong, Xia Yang, Taha Merghoub, Jedd D. Wolchok. Treg inhibition by anti-GITR therapy evokes therapeutic antitumor responses selectively in the context of a pro-inflammatory tumor microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B183.

Census of cytosolic aminopeptidase activity reveals two novel cytosolic aminopeptidases

Activation of CD8(+) cytotoxic T cells is crucial for the adaptive immune response against viral infections and the control of malignant transformed cells. Together with activation of costimulatory molecules like CD3 and CD28, CD8(+) T cells need activation of their unique T cell receptor via recognition of foreign peptide epitopes in combination with major histocompatibility complexes class I on the cell surface of professional antigen-presenting cells. Presentation of pathogen-associated proteins is the result of a complex proteolytic process. It starts with the breakdown of proteins by a cytosolic endopeptidase, the proteasome, and is continued by subsequent N-terminal trimming events in the cytosol and/or the endoplasmic reticulum. Analysis of the proteolytic aminopeptidase activity in the former cellular compartment showed that the cytosol harbors a multitude of aminopeptidases that have singular specificities, but on the other hand also show redundancy in the trimming of N-terminal residues. The observed pattern of the overall trimming in the cytosol is reflected by the activity of the four identified aminopeptidases, and the administration of protease inhibitors made it possible to assign specificity of cleaving of proteinogenic amino acids to one or more identified aminopeptidase. The only exception was the cleavage of aspartic acid, which is performed by one yet unidentified enzyme.

Role of human neutrophil peptides in the initial interaction between lung epithelial cells and CD4+ lymphocytes

Human neutrophil peptides (HNP) exert immune-modulating effects. We hypothesized that HNP link innate and adaptive immunity through activation of costimulatory molecules. Human lung epithelial cells and CD4+ lymphocytes were treated with HNP separately or in coculture. Stimulation with HNP induced an increase in cell surface expression of CD54 (ICAM-1), CD80, and CD86 on lung epithelial cells and the corresponding major ligands, CD11a (LFA-1), CD152 (CTLA-4), and CD28 on CD4+ lymphocytes. There was an increased nuclear expression of the transcription factor p53 in human alveolar A549 cells and an elevated NF-kappaB (p50) and a degradation of I-kappaB protein in CD4+ lymphocytes following HNP stimulation. HNP enhanced the interaction between A549 cells and CD4+ lymphocytes by increasing cell adhesion and release of IFN-gamma, IL-2, and IL-8. This was attenuated by using an alpha1-proteinase inhibitor to neutralize HNP. We conclude that HNP play an important role in linking innate to acquired immunity by activation of costimulatory molecules in lung epithelial cells and CD4+ lymphocytes.

Costimulation: critical pathways in the immunologic regulation of asthma

T cells play an important role in the pathogenesis of allergy and asthma. T-cell receptor engagement by antigenic peptides presented to major histocompatibility complex (MHC) class II molecules and activation of costimulatory molecules are crucial in the regulation of T-cell immune responses. Costimulatory molecules are responsible for second signals that induce T-cell activation and proliferation. The best characterized costimulatory pathways include CD80/CD86 interacting with CD28, and a number of additional costimulatory molecules have recently been identified, including members of the tumor necrosis family. The positive signals induced by these molecules are counterbalanced by other members of the costimulatory family, including cytotoxic T lymphocyte-associated antigen (CTLA)-4, programmed death (PD)-1, and B and T lymphocyte attenuator (BTLA), which dampen immune responses. In this review, we describe the fundamental properties of costimulatory molecules and address the influence of costimulatory signals on allergic responses.

Recognition of single-stranded RNA viruses by Toll-like receptor 7.

Viral infection of mammalian host results in the activation of innate immune responses. Toll-like receptors (TLRs) have been shown to mediate the recognition of many types of pathogens, including viruses. The genomes of viruses possess unique characteristics that are not found in mammalian genomes, such as high CpG content and double-stranded RNA. These genomic nucleic acids serve as molecular signatures associated with viral infections. Here we show that TLR7 recognizes the single-stranded RNA viruses, vesicular stomatitis virus and influenza virus. The recognition of these viruses by plasmacytoid dendritic cells and B cells through TLR7 results in their activation of costimulatory molecules and production of cytokines. Moreover, this recognition required intact endocytic pathways. Mice deficient in either the TLR7 or the TLR adaptor protein MyD88 demonstrated reduced responses to in vivo infection with vesicular stomatitis virus. These results demonstrate microbial ligand recognition by TLR7 and provide insights into the pathways used by the innate immune cells in the recognition of viral pathogens.

The molecular and cellular pathology of inflammatory muscle diseases.

The inflammatory muscle diseases dermatomyositis, polymyositis and inclusion body myositis are of unknown cause, but immune mechanisms are strongly implicated. Progress in the past two years has led to an improved understanding of the main molecular events involved in the immunological synapse between muscle and autoinvasive T cells. In particular, we now have a better understanding of TCR gene rearrangement in endomysial T cells, regulation of MHC expression, activity of co-stimulatory molecules, and the signalling cascades activated by cytokines, chemokines and metalloproteinases. Recent reports of an upregulation of strong anti-apoptotic molecules on the surface of muscle fibers identifies the end result of these disease processes, loss of muscle cells, as through necrosis, and not apoptosis. Such progress in molecular immunopathology has generated the interest to apply semispecific immunotherapies with the hope of halting disease progression or improving the strength of patients unresponsive to currently available non-specific immunotherapeutic interventions.

Expression of CD28 and CD86 by human eosinophils and role in the secretion of type 1 cytokines (interleukin 2 and interferon gamma): inhibition by immunoglobulin a complexes.

Eosinophils are the source of various immunoregulatory cytokines, but the membrane molecules involved in their secretion have not been clearly identified. Here we show that peripheral blood eosinophils from hypereosinophilic patients could express membrane CD86 but not CD80. The T cell costimulatory molecule CD28 is also detected on the eosinophil surface. CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected. In contrast to T cells requiring two signals for effective stimulation, CD28 ligation alone was sufficient for optimal eosinophil activation. Eosinophil-derived IL-2 and IFN-gamma were biologically active, as supernatants from anti-CD28-treated cells were able to induce CTLL-2 proliferation and major histocompatibility complex class II expression on the colon carcinoma cell line Colo 205, respectively. Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release. These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals. They confirm that through activation of costimulatory molecules, eosinophils could function as an immunoregulatory cell involved in the release of both type 1 and type 2 cytokines.

T Cell and cytokine basis of host variability in response to intestinal nematode infections

Infection by a variety of species of intestinal nematode infection gives rise to a wide variation in parasite load within a host population. There has been much investigation into the basis of this variation and is thought to involve several factors. Studies of infections of gut dwelling nematodes in laboratory rodents has clearly demonstrated that this variation may be due to the production of cytokines produced as part of the host immune response to infection. More specifically, activation of distinct T helper cell subsets leads to the generation of effective or ineffective responses resulting in clearance of the parasite load or maintenance of chronic infection. The induction of differential responses remains to be determined but is likely to be influenced at a number of levels including involvement of accessory cells and activation of co-stimulatory molecules on antigen presenting cells. Moreover, it appears that these parasites may actively interfere with the host cytokine response to promote their own survival. This review concentrates on recent findings of cytokine mediated control of intestinal nematodes highlighting a central role for the immune system in regulating both acute and chronic infection by these parasites.