Despite pre-clinical murine data supporting T regulatory (Treg) cell depletion as a major mechanism by which anti-CTLA-4 antibodies function in vivo, the two main antibodies tested in patients (ipilimumab and tremelimumab) have failed to demonstrate similar effects. We report analogous findings in an immunocompetent murine model humanized for CTLA-4 and Fcy receptors (hCTLA-4/hFcyR mice), where both ipilimumab and tremelimumab fail to show appreciable Treg depletion. Immune profiling of the tumor microenvironment (TME) in both mice and human samples revealed upregulation of the inhibitory Fcy receptor, FcyRIIB, which limits the ability of the antibody Fc fragment of human anti-CTLA-4 antibodies to induce effective antibody dependent cellular cytotoxicty/phagocytosis (ADCC/ADCP). Blocking FcyRIIB in humanized mice rescues Treg depleting capacity and anti-tumor activity of ipilimumab. For another target, CC motif chemokine receptor 8 (CCR8), which is selectively expressed on tumor infiltrating Tregs, we show that Fc engineering to enhance binding to activating Fc receptors, while limiting binding to the inhibitory Fc receptor, leads to consistent Treg depletion and single-agent activity across multiple tumor models, including B16, MC38 and MB49. These data reveal the importance of reducing engagement to the inhibitory Fc receptor to optimize Treg depletion by TME targeting antibodies. Our results define the inhibitory FcyRIIB receptor as a novel immune checkpoint limiting antibody-mediated Treg depletion in tumors, and demonstrate Fc variant engineering as a means to overcome this limitation and augment efficacy for a repertoire of antibodies currently in use or under clinical evaluation in oncology.
FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies
Knorr D., Leidner R., Jensen S., Meng R., Jones A., Ballesteros-Merino C., Bell R. B., Baez M., Sprott D., Bifulco C., Piening B., Dahan R., Fox B. A. & Ravetch J. (2023) bioRxiv : the preprint server for biology. Abstract
Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy
Cohen Saban N., Yalin A., Landsberger T., Salomon R., Alva A., Feferman T., Amit I. & Dahan R. (2023) Science immunology. Abstract
FDA-approved anti-PD-L1 monoclonal antibodies (mAbs) bear the IgG1 isotype, whose scaffolds are either wild-type (e.g., avelumab) or Fc-mutated and lacking Fcγ receptor (FcγR) engagement (e.g., atezolizumab). It is unknown whether variation in the ability of the IgG1 Fc region to engage FcγRs renders mAbs with superior therapeutic activity. In this study, we used humanized FcγR mice to study the contribution of FcγR signaling to the antitumor activity of human anti-PD-L1 mAbs and to identify an optimal human IgG scaffold for PD-L1 mAbs. We observed similar antitumor efficacy and comparable tumor immune responses in mice treated with anti-PD-L1 mAbs with wild-type and Fc-mutated IgG scaffolds. However, in vivo antitumor activity of the wild-type anti-PD-L1 mAb avelumab was enhanced by combination treatment with an FcγRIIB-blocking antibody, which was co-administered to overcome the suppressor function of FcγRIIB in the tumor microenvironment (TME). We performed Fc glycoengineering to remove the fucose subunit from the Fc-attached glycan of avelumab to enhance its binding to the activating FcγRIIIA. Treatment with the Fc-afucosylated version of avelumab also enhanced antitumor activity and induced stronger antitumor immune responses compared with the parental IgG. The enhanced effect by afucosylated PD-L1 antibody was dependent on neutrophils and associated with decreased frequencies of PD-L1 myeloid cells and increased infiltration of T cells in the TME. Our data reveal that the current design of FDA-approved anti-PD-L1 mAbs does not optimally harness FcγR pathways and suggest two strategies to enhance FcγR engagement to optimize anti-PD-L1 immunotherapy.
Bispecific antibodies increase the therapeutic window of CD40 agonists through selective dendritic cell targeting
Salomon R., Rotem H., Katzenelenbogen Y., Weiner A., Cohen Saban N., Feferman T., Amit I. & Dahan R. (2022) Nature Cancer. Abstract
Therapeutic use of agonistic anti-CD40 antibodies is a potentially powerful approach for activation of the immune response to eradicate tumors. However, the translation of this approach to clinical practice has been substantially restricted due to the severe dose-limiting toxicities observed in multiple clinical trials. Here, we demonstrate that conventional type 1 dendritic cells are essential for triggering antitumor immunity but not the toxicity of CD40 agonists, while macrophages, platelets and monocytes lead to toxic events. Therefore, we designed bispecific antibodies that target CD40 activation preferentially to dendritic cells, by coupling the CD40 agonist arm with CD11c-, DEC-205- or CLEC9A-targeting arms. These bispecific reagents demonstrate a superior safety profile compared to their parental CD40 monospecific antibody while triggering potent antitumor activity. We suggest such cell-selective bispecific agonistic antibodies as a drug platform to bypass the dose-limiting toxicities of anti-CD40, and of additional types of agonistic antibodies used for cancer immunotherapy.
Next Generation CD40 Agonistic Antibodies for Cancer Immunotherapy
Salomon R. & Dahan R. (2022) Frontiers in Immunology. Abstract
The clinical use of anti-CD40 agonist monoclonal antibodies (mAbs) is aimed at recruiting the immune system to fight the tumor cells. This approach has been demonstrated to be effective in various preclinical models. However, human CD40 Abs displayed only modest antitumor activity in cancer patients, characterized by low efficacy and dose-limiting toxicity. While recent studies highlight the importance of engineering the Fc region of human CD40 mAbs to optimize their agonistic potency, toxicity remains the main limiting factor, restricting clinical application to suboptimal doses. Here, we discuss the current challenges in realizing the full potential of CD40 mAbs in clinical practice, and describe novel approaches designed to circumvent the systemic toxicity associated with CD40 agonism.
Artificial Antigen Presenting Cells for Detection and Desensitization of Autoreactive T cells Associated with Type 1 Diabetes
Artzy-Schnirman A., Abu-Shah E., Chandrawati R., Altman E., Yusuf N., Wang S., Ramos J., Hansel C. S., Haus-Cohen M., Dahan R., Arif S., Dustin M. L., Peakman M., Reiter Y. & Stevens M. M. (2022) Nano Letters. Abstract
Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen-presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC, we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients, and using mouse models, we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.
Therapeutic antibody activation of the glucocorticoid-induced TNF receptor by a clustering mechanism
He C., Maniyar R. R., Avraham Y., Zappasodi R., Rusinova R., Newman W., Heath H., Wolchok J. D., Dahan R., Merghoub T. & Meyerson J. (2022) Science advances. Abstract
GITR is a TNF receptor, and its activation promotes immune responses and drives antitumor activity. The receptor is activated by the GITR ligand (GITRL), which is believed to cluster receptors into a high-order array. Immunotherapeutic agonist antibodies also activate the receptor, but their mechanisms are not well characterized. We solved the structure of full-length mouse GITR bound to Fabs from the antibody DTA-1. The receptor is a dimer, and each subunit binds one Fab in an orientation suggesting that the antibody clusters receptors. Binding experiments with purified proteins show that DTA-1 IgG and GITRL both drive extensive clustering of GITR. Functional data reveal that DTA-1 and the anti-human GITR antibody TRX518 activate GITR in their IgG forms but not as Fabs. Thus, the divalent character of the IgG agonists confers an ability to mimic GITRL and cluster and activate GITR. These findings will inform the clinical development of this class of antibodies for immuno-oncology.
Human antibodies targeting a Mycobacterium transporter protein mediate protection against tuberculosis
Watson A., Li H., Ma B., Weiss R., Bendayan D., Abramovitz L., Ben-Shalom N., Mor M., Pinko E., Bar Oz M., Wang Z., Du F., Lu Y., Rybniker J., Dahan R., Huang H., Barkan D., Xiang Y., Javid B. & Freund N. T. (2021) Nature Communications. Abstract
Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.
Anti-SARS-CoV-2 antibodies elicited by COVID-19mRNA vaccine exhibit a unique glycosylation pattern
Farkash I., Feferman T., Cohen-Saban N., Avraham Y., Morgenstern D., Mayuni G., Barth N., Lustig Y., Miller L., Shouval D. S., Biber A., Kirgner I., Levin Y. & Dahan R. (2021) Cell reports (Cambridge). Abstract
Messenger RNA-based vaccines against COVID-19 induce a robust anti-SARS-CoV-2 antibody response with potent viral neutralization activity. Antibody effector functions are determined by their constant region subclasses and by their glycosylation patterns, but their role in vaccine efficacy is unclear. Moreover, whether vaccination induces antibodies similar to those in patients with COVID-19 remains unknown. We analyze BNT162b2 vaccine-induced IgG subclass distribution and Fc glycosylation patterns and their potential to drive effector function via Fcy receptors and complement pathways. We identify unique and dynamic pro -inflammatory Fc compositions that are distinct from those in patients with COVID-19 and convalescents. Vaccine-induced anti-Spike IgG is characterized by distinct Fab-and Fc-mediated functions between different age groups and in comparison to antibodies generated during natural viral infection. These data highlight the heterogeneity of Fc responses to SARS-CoV-2 infection and vaccination and suggest that they support long-lasting protection differently.
Unified platform for genetic and serological detection of COVID-19 with single-molecule technology
Furth N., Shilo S., Cohen N., Erez N., Fedyuk V., Schrager A. M., Weinberger A., Dror A. A., Zigron A., Shehadeh M., Sela E., Srouji S., Amit S., Levy I., Segal E., Dahan R., Jones D., Douek D. C. & Shema E. (2021) PLoS ONE. Abstract
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
Testing IgG antibodies against the RBD of SARS-CoV-2 is sufficient and necessary for COVID-19 diagnosis
Indenbaum V., Koren R., Katz-Likvornik S., Yitzchaki M., Halpern O., Regev-Yochay G., Cohen C., Biber A., Feferman T., Saban N. C., Dahan R., Levin T., Gozlan Y., Weil M., Mor O., Mandelboim M., Sofer D., Mendelson E. & Lustig Y. (2020) PLoS ONE. Abstract
The COVID-19 pandemic and the fast global spread of the disease resulted in unprecedented decline in world trade and travel. A critical priority is, therefore, to quickly develop serological diagnostic capacity and identify individuals with past exposure to SARS-CoV-2. In this study serum samples obtained from 309 persons infected by SARS-CoV-2 and 324 of healthy, uninfected individuals as well as serum from 7 COVID-19 patients with 4-7 samples each ranging between 1-92 days post first positive PCR were tested by an "in house"ELISA which detects IgM, IgA and IgG antibodies against the receptor binding domain (RBD) of SARS-CoV-2. Sensitivity of 47%, 80% and 88% and specificity of 100%, 98% and 98% in detection of IgM, IgA and IgG antibodies, respectively, were observed. IgG antibody levels against the RBD were demonstrated to be up regulated between 1-7 days after COVID-19 detection, earlier than both IgM and IgA antibodies. Study of the antibody kinetics of seven COVID 19 patients revealed that while IgG levels are high and maintained for at least 3 months, IgM and IgA levels decline after a 35-50 days following infection. Altogether, these results highlight the usefulness of the RBD based ELISA, which is both easy and cheap to prepare, to identify COVID-19 patients even at the acute phase. Most importantly our results demonstrate that measuring IgG levels alone is both sufficient and necessary to diagnose past exposure to SARS-CoV-2.
Single-cell genomic approaches for developing the next generation of immunotherapies
Yofe I., Dahan R. & Amit I. (2020) Nature Medicine. Abstract
Recent progress in single-cell genomics urges its application in drug development, particularly of cancer immunotherapies. Current immunotherapy pipelines are focused on functional outcome and simple cellular and molecular readouts. A thorough mechanistic understanding of the cells and pathways targeted by immunotherapy agents is lacking, which limits the success rate of clinical trials. A large leap forward can be made if the immunotherapy target cells and pathways are characterized at high resolution before and after treatment, in clinical cohorts and model systems. This will enable rapid development of effective immunotherapies and data-driven design of synergistic drug combinations. In this Perspective, we discuss how emerging single-cell genomic technologies can serve as an engine for target identification and drug development.Amit and colleagues discuss where single-cell genomic technologies can be applied both in trial design and in the clinical trial stage to improve the development of immunotherapies.
BCR affinity differentially regulates colonization of the subepithelial dome and infiltration into germinal centers within Peyer's patches
Biram A., Stromberg A., Winter E., Stoler-Barak L., Salomon R., Addadi Y., Dahan R., Yaari G., Bemark M. & Shulman Z. (2019) Nature Immunology. Abstract
Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer's patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.
Toxicity of an Fc-engineered anti-CD40 antibody is abrogated by intratumoral injection and results in durable antitumor immunity
Knorr D. A., Dahan R. & Ravetch J. (2018) Proceedings Of The National Academy Of Sciences Of The United States Of America-Physical Sciences. Abstract
Immune stimulation has emerged as a promising approach to the treatment of neoplastic diseases. Currently approved therapeutics, such as anti-CTLA4 and anti-PD1, are primarily aimed at blocking inhibitory signaling by immune cells. An alternative and potentially synergistic approach would involve activation of immune pathways by agonism of stimulatory receptors, such as CD40. Agonistic antibodies, while promising in principle, have encountered significant barriers in clinical trials limited by the systemic toxicity of such approaches. Using a mouse model humanized for both Fc receptors and CD40, we previously demonstrated enhanced antitumor activity with an Fc-modified antibody. We now demonstrate that this model recapitulates the platelet and hepatic toxicities seen with anti-CD40 antibodies in patients, providing a predictive measure of the dose-limiting activity of this approach. We further show that such toxicity can be circumvented and durable systemic antitumor immunity achieved by intratumoral delivery of an Fc-engineered anti-CD40 agonistic antibody.
Signaling by Antibodies: Recent Progress
Bournazos S., Wang T. T., Dahan R., Maamary J. & Ravetch J. V. (2017) ANNUAL REVIEW OF IMMUNOLOGY.
Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors
Vargas F. A., Furness A. J. S., Solomon I., Joshi K., Mekkaoui L., Lesko M. H., Rota E. M., Dahan R., Georgiou A., Sledzinska A., Ben Aissa A. A., Franz D., Sunderland M. W., Wong Y. N. S., Henry J. Y., O'Brien T., Nicol D., Challacombe B., Beers S. A., Turajlic S., Gore M., Larkin J., Swanton C., Chester K. A., Pule M., Ravetch J. V., Marafioti T., Peggs K. S. & Quezada S. A. (2017) Immunity.
Co-targeting of Adenosine Signaling Pathways for Immunotherapy: Potentiation by Fc Receptor Engagement
Dahan R. & Ravetch J. V. (2016) Cancer Cell.
Reprogramming Tumor-Associated Macrophages by Antibody Targeting Inhibits Cancer Progression and Metastasis
Georgoudaki A., Prokopec K. E., Boura V. F., Hellqvist E., Sohn S., Ostling J., Dahan R., Harris R. A., Rantalainen M., Klevebring D., Sund M., Brage S. E., Fuxe J., Rolny C., Li F., Ravetch J. V. & Karlsson M. C. I. (2016) Cell Reports.
Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective Fc gamma R Engagement
Dahan R., Barnhart B. C., Li F., Yamniuk A. P., Korman A. J. & Ravetch J. V. (2016) Cancer Cell.
Fc gamma Rs Modulate the Anti-tumor Activity of Antibodies Targeting the PD-1/PD-L1 Axis
Dahan R., Sega E., Engelhardt J., Selby M., Korman A. J. & Ravetch J. V. (2015) Cancer Cell.
HLA-DR alpha 1 Constructs Block CD74 Expression and MIF Effects in Experimental Autoimmune Encephalomyelitis
Meza-Romero R., Benedek G., Yu X., Mooney J. L., Dahan R., Duvshani N., Bucala R., Offner H., Reiter Y., Burrows G. G. & Vandenbark A. A. (2014) Journal of Immunology.
Type I and type II Fc receptors regulate innate and adaptive immunity
Pincetic A., Bournazos S., DiLillo D. J., Maamary J., Wang T. T., Dahan R., Fiebiger B. & Ravetch J. V. (2014) Nature Immunology.
A novel regulatory pathway for autoimmune disease: Binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T-cell tolerance
Vandenbark A. A., Meza-Romero R., Benedek G., Andrew S., Huan J., Chou Y. K., Buenafe A. C., Dahan R., Reiter Y., Mooney J. L., Offner H. & Burrows G. G. (2013) Journal of Autoimmunity.
Antigen-specific immunomodulation for type 1 diabetes by novel recombinant antibodies directed against diabetes-associates auto-reactive T cell epitope
Dahan R., Gebe J. A., Preisinger A., James E. A., Tendler M., Nepom G. T. & Reiter Y. (2013) Journal of Autoimmunity.
T-cell-receptor-like antibodies - generation, function and applications
Dahan R. & Reiter Y. (2012) Expert Reviews in Molecular Medicine.
TCR-like antibodies distinguish conformational and functional differences in two- versus four-domain auto reactive MHC class II-peptide complexes
Dahan R., Tabul M., Chou Y. K., Meza-Romero R., Andrew S., Ferro A. J., Burrows G. G., Offner H., Vandenbark A. A. & Reiter Y. (2011) European Journal of Immunology.
Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: Predominance of strand transfer
Izhar L., Goldsmith M., Dahan R., Geacintov N., Lloyd R. G. & Livneh Z. (2008) Journal of Molecular Biology. Abstract
Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichia coli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism. (C) 2008 Elsevier Ltd. All rights reserved.
A promising therapeutic approach for multiple sclerosis: recombinant T-cell receptor ligands modulate experimental autoimmune encephalomyelitis by reducing interleukin-17 production and inhibiting migration of encephalitogenic cells into the CNS
Sinha S., Subramanian S., Proctor T. M., Kaler L. J., Grafe M., Dahan R., Huan J., Vandenbark A. A., Burrows G. G. & Offner H. (2007) Journal of Neuroscience.
HLA-DRB1*1501 risk association in multiple sclerosis may not be related to presentation of myelin epitopes
Finn T., Jones R., Rich C., Dahan R., Link J., David C., Chou Y., Offner H. & Vandenbark A. (2004) Journal of Neuroscience Research.