Antigenic Modulation of CD6 By Itolizumab Is a New Mechanism for Effector T Cell Inhibition

Introduction Acute Graft-Versus-Host Disease (aGVHD) is a serious complication of hematopoietic stem cell transplantation (HSCT) that is primarily driven by alloreactive T cells. CD6 is a costimulatory receptor primarily expressed on T cells that promotes synapse formation, T cell activation, and migration into tissues by engaging with its ligand, activated leukocyte cell adhesion molecule (ALCAM). CD6 is expressed on reconstituting T cells soon after HSCT (Rambaldi et al., 2019) and early studies have shown that ex-vivo depletion of CD6 + donor cells prior to HSCT decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998). The reduced levels of aGVHD were attributed to an increased prevalence of CD6 - T cells that were less alloreactive (Rasmussen et al., 1994). Therefore, modulating activity of the CD6-ALCAM pathway may ameliorate aGVHD. Itolizumab is a humanized anti-CD6 monoclonal antibody that was previously described to block the engagement of ALCAM, thereby inhibiting T cell activity and trafficking. Here, we elucidate a second and highly novel mechanism in which antibody-mediated loss of CD6 from the surface of T cells results in CD6 low T cells that are hyporesponsive to T cell stimulation. Methods To assess the molecular mechanisms for itolizumab-induced loss of cell surface CD6, peripheral blood mononuclear cells (PBMCs) were exposed to different conditions including treatment with selected protease inhibitors, a membrane inhibitor, or Fc receptor blocking antibodies in the presence of itolizumab. Furthermore, B cells, NK cells, and monocytes were isolated from PBMCs and mixed with T cells in combination with itolizumab to evaluate cell contact requirements for loss. Following treatment, cell surface levels of CD6 protein were assessed by flow cytometry using a non-competing anti-CD6 detection antibody while analysis of full length CD6 was detected by western blot from total protein lysate. The soluble form of CD6 (sCD6) was analyzed by electrochemiluminescence and immunoprecipitation from supernatant of treated cells. Results Following treatment with itolizumab, the levels of cell surface CD6 was reduced as assessed by flow cytometry and western blot. Concurrent with a loss of cell surface CD6, levels of sCD6 in the cell supernatant increased, suggesting CD6 is primarily cleaved from the cell surface rather than internalized. Characterization of the cleaved sCD6 product by western blot revealed a 30KDa product. Itolizumab-induced changes in both levels of cell surface CD6 and sCD6 was abrogated by both 4-benzenesulfonyl fluoride hydrochloride (AEBSF), a serine protease inhibitor and cytochalasin D, an inhibitor of actin polymerization that prevents movement within the cell membrane and blocks endocytic trafficking. This suggests that a membrane-bound serine protease is responsible for cleavage. Itolizumab-induced CD6 cleavage did not occur with T cells alone and required the presence of other immune cells including monocytes and NK cells, but not B cells (Figure 1). In addition, when monocytes and T cells were separated by a membrane, cleavage of CD6 was not observed in the presence of itolizumab, indicating that cell-to-cell contact is required. Furthermore, blocking antibodies indicated that functional Fc receptors, especially FcγRIA, were required, suggesting that binding of itolizumab to both an Fc receptor on monocytes and CD6 on T cells predicated the cleavage event. The resulting CD6 low T cells were hyporesponsive to T cell stimulation, even in the absence of itolizumab, as indicated by reduced expression of PD-1, CD71 and CD25 as well as inflammatory cytokines (Figure 2). Inhibition was observed for both naïve and effector/memory T cell subsets. Conclusions Our results reveal a novel mechanism of antigenic modulation by itolizumab in which CD6 is cleaved from the T cell surface and released in a soluble form. Cleavage of CD6 occurs via a membrane-bound serine protease and appears dependent upon the engagement of itolizumab with Fc receptor(s) present on monocytes and NK cells. The loss of cell surface CD6 results in T cells with reduced responses to TCR-mediated stimulation. As such, treatment of aGVHD patients with itolizumab may reduce the alloreactivity of donor T cells, ameliorating disease symptoms and improving the clinical outcomes in these patients. Figure 1 Figure 1. Disclosures Chu: Equillium: Current Employment. Marrocco: Equillium, Inc.: Current Employment. Tiet: Equillium, Inc.: Current Employment. Ampudia: Equillium, Inc.: Current Employment. Connelly: Equillium: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Ng: Equillium: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months.

IgG surface mobility promotes antibody-dependent cellular phagocytosis by Syk and Arp2/3 mediated reorganization of Fcγ receptors in macrophages.

By visualizing the movements of Rituximab during Antibody dependent cellular phagocytosis (ADCP) of B lymphoma cells by macrophages, we found that Fcγ receptors (FcγR) on the macrophage surface microcluster, recruit Syk and undergro large-scale reorganization at the phagocytic synapse prior to and during engulfment of the target cell. Given these dramatic rearrangements, we analyzed how the surface mobility of Rituximab contributes to FcγR signal amplification and ADCP efficiency. Depolymerization of the target cell actin cytoskeleton resulted in free diffusion of Rituximab docked to CD20, enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of Rituximab by chemical fixation impaired microcluster formation and diminished Syk recruitment and ADCP. In macrophages lacking Syk, Rituximab accumulated at the base of the phagosome and were trogocytosed, consistent with Syk kinase activity being necessary to trigger the redistribution of Rituximab-FcγR during engulfment and to prevent antigenic modulation of the target. Total internal reflection fluorescence analysis of FcγR-IgG on fluid supported lipid bilayers revealed a membrane topography displaying inward reaching leading edges and protruding contact sites reminiscent of podosomes. This topography was distinct from the closely apposed macrophage/target membranes observed during engagement of IgG displayed on immobile supported lipid bilayers. The organization of this contact, pseudopod extension and the rearrangement of microclusters depended critically on Arp 2/3. Thus, Syk and Arp2/3 coordinate actin rearrangements and FcγR-IgG complexes that were of previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens. ADCP is an important effector mechanism for the removal of malignant, immunologically aberrant, and infected cells during treatment with therapeutic antibodies or adaptive immune responses. Most transmembrane protein antigens are mobile with transient confinement from the actin of the target cell. This work demonstrates that macrophage forces overcome these confinements to rearrange FcγR-IgG-antigen complexes before and during ADCP. Thus, new paradigms are needed as ADCP has largely been studied using model target particles that display immobile antigens. Moreover, we found that the mobility of the therapeutic antibody, Rituximab, on the surface of B lymphoma cells foretells ADCP efficacy, with lower densities of IgG mediating ADCP on increasingly mobile antigens.

Checkpoint Inhibitors and Hepatotoxicity

Uncontrolled immune response to a pathogen or any protein can lead to tissue damage and autoimmune diseases, that represent aberrant immune responses of the individual to its own cells and/or proteins. The immune checkpoint system is the regulatory mechanism that controls immune responses. Tumor cells escape the immune surveillance mechanism, avoiding immune detection and elimination by activating these checkpoints and suppressing the anti-tumor response, thus allowing formation of tumors. Antigenic modulation facilitates masking and contributes to the escape of tumor cells. In addition, there are growing cell promoters, like transforming growth factor β (TGF-β), contributing to escape mechanisms. Targeting the immunological escape of malignant cells is the basis of immune oncology. Checkpoint inhibitors, cytokines and their antibodies may enhance the immune system’s response to tumors. Currently, immunomodulatory agents have been designed, evaluated in clinical trials and have been approved by both European and United States Drug Agencies. The present review is a reflection of the increasingly important role of the checkpoint inhibitors. Our aim is to review the side effects with the emphasis on hepatic adverse reactions of these novel biological drug interventions.

Antibody-induced crosslinking and cholesterol-sensitive, anomalous diffusion of nicotinic acetylcholine receptors

Synaptic strength depends on the number of cell-surface neurotransmitter receptors in dynamic equilibrium with intracellular pools. Dysregulation of this homeostatic balance occurs e.g. in myasthenia gravis, an autoimmune disease characterized by a decrease in the number of postsynaptic nicotinic acetylcholine receptors (nAChRs). Monoclonal antibody mAb35 mimics this effect. Here we use STORM nanoscopy to characterize the individual and ensemble dynamics of mAb35-crosslinked receptors in the clonal cell line CHO-K1/A5, which robustly expresses adult muscle-type nAChRs. Antibody labeling of live cells results in 80% receptor immobilization. The remaining mobile fraction exhibits a heterogeneous combination of Brownian and anomalous diffusion. Single-molecule trajectories exhibit a two-state switching behavior between free Brownian walks and anticorrelated walks within confinement areas. The latter act as permeable fences (∼34 nm radius, ∼400 ms lifetime). Dynamic clustering, trapping and immobilization also occur in larger nanocluster zones (120-180 nm radius) with longer lifetimes (11 ± 1 s), in a strongly cholesterol-sensitive manner. Cholesterol depletion increases the size and average duration of the clustering phenomenon; cholesterol enrichment has the opposite effect. The disclosed high proportion of mAb35-crosslinked immobile receptors, together with their anomalous, cholesterol-sensitive diffusion and clustering, provides new insights into the antibody-enhanced antigenic modulation that leads to physiopathological internalization and degradation of receptors in myasthenia.A preliminary version of this work has appeared in the biorXiv repository: https://www.biorxiv.org/content/10.1101/744664v1. The study was not pre-registered.

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies

Key Points Antigenic modulation significantly impacts natural killer cell and macrophage ability to mediate Fc γ receptor-dependent killing. hIgG1 mAbs are unable to elicit natural killer–mediated ADCC in the mouse, supporting ADCP as the dominant effector mechanism.

Fcγ-receptor–mediated trogocytosis impacts mAb-based therapies: historical precedence and recent developments

A specialized form of trogocytosis occurs when Fcγ receptors on acceptor cells take up and internalize donor cell-associated immune complexes composed of specific monoclonal antibodies (mAbs) bound to target antigens on donor cells. This trogocytosis reaction, an example of antigenic modulation, has been described in recent clinical correlative studies and in vitro investigations for several mAbs used in cancer immunotherapy, including rituximab and ofatumumab. We discuss the impact of Fcγ-receptor–mediated trogocytosis on the efficacy of cancer immunotherapy and other mAb-based therapies.