Fc-FcγR Interactions Research Articles

Fc-FcγR interactions during infections: From neutralizing antibodies to antibody-dependent enhancement.

Advances in antibody technologies have resulted in the development of potent antibody-based therapeutics with proven clinical efficacy against infectious diseases. Several monoclonal antibodies (mAbs), mainly against viruses such as SARS-CoV-2, HIV-1, Ebola virus, influenza virus, and hepatitis B virus, are currently undergoing clinical testing or are already in use. Although these mAbs exhibit potent neutralizing activity that effectively blocks host cell infection, their antiviral activity results not only from Fab-mediated virus neutralization, but also from the protective effector functions mediated through the interaction of their Fc domains with Fcγ receptors (FcγRs) on effector leukocytes. Fc-FcγR interactions confer pleiotropic protective activities, including the clearance of opsonized virions and infected cells, as well as the induction of antiviral T-cell responses. However, excessive or inappropriate activation of specific FcγR pathways can lead to disease enhancement and exacerbated pathology, as seen in the context of dengue virus infections. A comprehensive understanding of the diversity of Fc effector functions during infection has guided the development of engineered antiviral antibodies optimized for maximal effector activity, as well as the design of targeted therapeutic approaches to prevent antibody-dependent enhancement of disease.

Antibody-mediated control mechanisms of viral infections.

Antibodies generated after vaccination or natural pathogen exposure are essential mediators of protection against many infections. Most studies with viruses have focused on antibody neutralization, in which protection is conferred by the fragment antigen binding region (Fab) through targeting of different steps in the viral lifecycle including attachment, internalization, fusion, and egress. Beyond neutralization, the fragment crystallizable (Fc) region of antibodies can integrate innate and adaptive immune responses by engaging complement components and distinct Fc gamma receptors (FcγR) on different host immune cells. In this review, we discuss recent advances in our understanding of antibody neutralization and Fc effector functions, and the assays used to measure them. Additionally, we describe the contexts in which these mechanisms are associated with protection against viruses and highlight how Fc-FcγR interactions can improve the potency of antibody-based therapies.

Functional Fc receptors are crucial in antibody-mediated protection against cytomegalovirus.

Human cytomegalovirus is a medically important pathogen. Previously, using murine CMV (MCMV), we provided evidence that both neutralizing and nonneutralizing antibodies can confer protection from viral infection in vivo. In this study, we report that serum derived from infected animals had a greater protective capacity in MCMV-infected RAG-/- mice than serum from animals immunized with purified virus. The protective activity of immune serum was strictly dependent on functional Fcγ receptors (FcγR). Deletion of individual FcγRs or combined deletion of FcγRI and FcγRIV had little impact on the protection afforded by serum. Adoptive transfer of CD115-positive cells from noninfected donors demonstrated that monocytes represent important cellular mediators of the protective activity provided by immune serum. Our studies suggest that Fc-FcγR interactions and monocytic cells are critical for antibody-mediated protection against MCMV infection in vivo. These findings may provide new avenues for the development of novel strategies for more effective CMV vaccines or antiviral immunotherapies.

Broadly Reactive Nanobody Targeting the H3 Hemagglutinin of the Influenza A Virus.

Monoclonal antibodies and recombinant antibody fragments are a very promising therapeutic tool to combat infectious diseases. Due to their unique paratope structure, nanobodies (VHHs) hold several advantages over conventional monoclonal antibodies, especially in relation to viral infections. Influenza A viruses (IAVs) remain a major threat to public health. The hemagglutinin (HA) protein is the main protective and immunodominant antigen of IAVs. In this study, three broadly reactive nanobodies (D9.2, E12.2, and D4.2) to H3N2 influenza strains were isolated and Fc-fusion proteins (VHH-Fcs) were obtained and characterized in vitro. This modification improved the nanobodies' binding activity and allowed for their interaction with a wider range of strains. The D9.2-Fc antibody showed a 100% protection rate against mortality in vivo in a mouse lethal model. Furthermore, we demonstrated that the observed protection has to do with Fc-FcγR interactions. These results indicate that D9.2-Fc can serve as an effective antiviral agent against the H3N2 influenza infection.

Abstract CT008: A phase 2, two-stage study of mirvetuximab soravtansine (IMGN853) in combination with pembrolizumab in patients with microsatellite stable (MSS) recurrent or persistent endometrial cancer

Abstract Background & Objectives: Folate receptor-alpha (FRα) expression is associated with poor prognosis in endometrial cancer (EC). Mirvetuximab soravtansine (MIRV), an antibody drug conjugate (ADC) comprising a FRα-binding antibody, cleavable linker, and the tubulin-disrupting maytansinoid DM4, showed tolerability and single agent activity in a Phase 1 dose expansion study in FRα-positive advanced/recurrent EC (NCT01609556). In addition to target-mediated cytotoxicity, MIRV activates monocytes and promotes phagocytosis of tumor cells through Fc-FcγR interactions. We therefore hypothesized that MIRV would improve the low response to immune checkpoint inhibitors (ICI) in MSS/pMMR EC and conducted this two-stage, phase 2, single cohort study evaluating the activity of MIRV in combination with pembrolizumab in patients with recurrent/persistent EC (NCT03835819). Methods: Patients with advanced or recurrent MSS/pMMR, FRα positive (defined as ≥50% of tumor cells with ≥2+ staining on IHC by Ventana, Inc.) serous EC received MIRV 6 mg/kg AIBW and pembrolizumab 200 mg every 21 days until disease progression or unacceptable toxicity. Patients could have received 1-3 prior lines of therapy; prior ICI therapy was allowed. The co-primary endpoints were objective response rate (ORR) as measured by RECIST 1.1 and frequency of patients surviving progression-free for 6 months (PFS6). In the first stage of the study, 16 patients were enrolled; if there were ≥2 objective responses or ≥2 PFS6 responses, an additional 19 patients would be enrolled in Stage 2. If a total of 4 objective responses or 8 PFS6 events were achieved, the combination would be considered worthy of further study. Data: Among 130 patient tumors prescreened for FRα expression, the median FRα (% of tumor cells with ≥2+ staining) was 30% (IQR: 5-55) and 43 (33.1%) were FRα positive (PS 2+ ≥50%). As of data-cutoff in November 2023, a total of 16 patients had received study treatment with median follow-up of 4.7 months (IQR 2.9, 17.3). Of 16 treated patients, 6 patients (37.5%, 95% CI: 15.2-64.6%) achieved an objective response, including 1 patient achieving a confirmed CR and 5 patients achieving a PR [3 confirmed PR (18.8%) and 2 unconfirmed PR (12.5%)] (Fig. 1). An additional 31.3% (5/16) had stable disease and two patients were alive and progression free at 6 months. The most common TRAEs included AST elevation (50%), blurred vision (44%), fatigue (44%) and diarrhea (43%); grade 3 TRAEs occurred in 2 patients (12.5%). Ocular toxicities of any grade were reported in 56.3% of patients. Updated efficacy and safety data as well as ongoing correlative studies will be reported at the time of the meeting. Conclusions: In this investigator-initiated study, the combination of MIRV and pembrolizumab met the co-primary endpoint to be considered worthy of further study in FRα-positive recurrent MSS/pMMR serous EC. Investigations into subpopulations most likely to derive clinical benefit from this combination are ongoing. Citation Format: Rebecca L. Porter, Niya Xiong, Nabihah Tayob, Madeline Polak, Hannah Sawyer, Martin Hayes, Jeanette Gardner, Susana Campos, Neil Horowitz, Carolyn Krasner, Elizabeth K. Lee, Joyce F. Liu, Elizabeth H. Stover, Jennifer Veneris, Alexi A. Wright, Ursula A. Matulonis, Panagiotis Konstantinopoulos. A phase 2, two-stage study of mirvetuximab soravtansine (IMGN853) in combination with pembrolizumab in patients with microsatellite stable (MSS) recurrent or persistent endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT008.

The presence of CpGs in AAV gene therapy vectors induces a plasmacytoid dendritic cell-like population very early after administration

AAV-mediated gene transfer is a promising platform still plagued by potential host-derived, antagonistic immune responses to therapeutic components. CpG-mediated TLR9 stimulation activates innate immune cells and leads to cognate T cell activation and suppression of transgene expression. Here, we demonstrate that CpG depletion increased expression of an antibody transgene product by 2–3-fold as early as 24 h post-vector administration in mice. No significant differences were noted in anti-transgene product/ anti-AAV capsid antibody production or cytotoxic gene induction. Instead, CpG depletion significantly reduced the presence of a pDC-like myeloid cell population, which was able to directly bind the antibody transgene product via Fc-FcγR interactions. Thus, we extend the mechanisms of TLR9-mediated antagonism of transgene expression in AAV gene therapy to include the actions of a previously unreported pDC-like cell population.

Fcγ receptors and immunomodulatory antibodies in cancer.

The discovery of both cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) as negative regulators of antitumour immunity led to the development of numerous immunomodulatory antibodies as cancer treatments. Preclinical studies have demonstrated that the efficacy of immunoglobulin G (IgG)-based therapies depends not only on their ability to block or engage their targets but also on the antibody's constant region (Fc) and its interactions with Fcγ receptors (FcγRs). Fc-FcγR interactions are essential for the activity of tumour-targeting antibodies, such as rituximab, trastuzumab and cetuximab, where the killing of tumour cells occurs at least in part due to these mechanisms. However, our understanding of these interactions in the context of immunomodulatory antibodies designed to boost antitumour immunity remains less explored. In this Review, we discuss our current understanding of the contribution of FcγRs to the in vivo activity of immunomodulatory antibodies and the challenges of translating results from preclinical models into the clinic. In addition, we review the impact of genetic variability of human FcγRs on the activity of therapeutic antibodies and how antibody engineering is being utilized to develop the next generation of cancer immunotherapies.

The antitumor activities of anti-CD47 antibodies require Fc-FcγR interactions

While anti-CD47 antibodies hold promise for cancer immunotherapy, early-phase clinical trials have shown limited clinical benefit, suggesting that CD47 blockade alone might be insufficient for effective tumor control. Here, we investigate the contributions of the Fc domain of anti-CD47 antibodies required for optimal invivo antitumor activity across multiple species-matched models, providing insights into the mechanisms behind the efficacy of this emerging class of therapeutic antibodies. Using a mouse model humanized for CD47, SIRPα, and FcγRs, we demonstrate that local administration of Fc-engineered anti-CD47 antibodies with enhanced binding to activating FcγRs promotes tumor infiltration of macrophages and antigen-specific Tcells, while depleting regulatory Tcells. These effects result in improved long-term systemic antitumor immunity and minimal on-target off-tumor toxicity. Our results highlight the importance of Fc optimization in the development of effective anti-CD47 therapies and provide an attractive strategy to enhance the activity of this promising immunotherapy.

Fc gamma receptors promote antibody-induced LILRB4 internalization and immune regulation of monocytic AML.

The immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4) is found specifically on the cell surface of acute monocytic leukemia (monocytic AML), an aggressive and common subtype of AML. We have developed a humanized monoclonal IgG1 LILRB4-blocking antibody (h128-3), which improved immune regulation but reduced cell surface expression of LILRB4 in monocytic AML models by 40-60%. Interestingly, most of this effect was neutralized by mutation of the Fc region of the antibody (h128-3/N297A), which prevents interaction with Fc gamma receptors (FcγRs). This suggested that there is FcγR-dependent antigenic modulation underlying h128-3's effects, a mechanism known to alter the function of antibodies targeting B-cell malignancies. We disrupted the Fc-FcγR interaction pharmacologically and with stable CRISPR-Cas9-mediated genetic knockout of FcγRs in monocytic AML cell lines to investigate the role of FcγR-dependent antigenic modulation in the regulation of LILRB4 by h128-3. When FcγRI is inhibited or removed from the surface of monocytic AML cells, h128-3 cannot optimally perform its blocking function, resulting in activation of the LILRB4 inhibitory receptor and leading to a 15-25% decrease in T-cell-mediated cytotoxicity in vitro. In the absence of FcγRI, scaffolding by FcγRIIa allows h128-3 to maintain LILRB4-blocking function. Here we define a FcγR-dependent antigenic modulation mechanism underlying the function of an immunoreceptor blocking antibody for the first time in myeloid malignancy. This research will facilitate the development of safe, precision-targeted antibody therapeutics in myeloid malignancies with greater potency and efficacy.

Antibodies elicited in humans upon chimeric hemagglutinin-based influenza virus vaccination confer FcγR-dependent protection in vivo.

Antibody responses against highly conserved epitopes on the stalk domain of influenza virus hemagglutinin (HA) confer broad protection; however, such responses are limited. To effectively induce stalk-specific immunity against conserved HA epitopes, sequential immunization strategies have been developed based on chimeric HA (cHA) constructs featuring different head domains but the same stalk regions. Immunogenicity studies in small animal models, as well as in humans, revealed that cHA immunogens elicit stalk-specific IgG responses with broad specificity against heterologous influenza virus strains. However, the mechanisms by which these antibodies confer in vivo protection and the contribution of their Fc effector function remain unclear. To characterize the role of Fc-FcγR (Fcγ receptor) interactions to the in vivo protective activity of IgG antibodies elicited in participants in a phase I trial of a cHA vaccine candidate, we performed passive transfer studies of vaccine-elicited IgG antibodies in mice humanized for all classes of FcγRs, as well as in mice deficient for FcγRs. IgG antibodies elicited upon cHA vaccination completely protected FcγR humanized mice against lethal influenza virus challenge, while no protection was evident in FcγR-deficient mice, suggesting a major role for FcγR pathways in the protective function of vaccine-elicited IgG antibodies. These findings have important implications for influenza vaccine development, guiding the design of vaccination approaches with the capacity to elicit IgG responses with optimal Fc effector function.

Distinct impact of IgG subclass on autoantibody pathogenicity in different IgG4-mediated diseases.

IgG4 is the least potent human IgG subclass for the FcγR-mediated antibody effector function. Paradoxically, IgG4 is also the dominant IgG subclass of pathogenic autoantibodies in IgG4-mediated diseases. Here, we show that the IgG subclass and Fc-FcγR interaction have a distinct impact on the pathogenic function of autoantibodies in different IgG4-mediated diseases in mouse models. While IgG4 and its weak Fc-FcγR interaction have an ameliorative role in the pathogenicity of anti-ADAMTS13 autoantibodies isolated from thrombotic thrombocytopenic purpura (TTP) patients, they have an unexpected exacerbating effect on anti-Dsg1 autoantibody pathogenicity in pemphigus foliaceus (PF) models. Strikingly, a non-pathogenic anti-Dsg1 antibody variant optimized for FcγR-mediated effector function can attenuate the skin lesions induced by pathogenic anti-Dsg1 antibodies by promoting the clearance of dead keratinocytes. These studies suggest that IgG effector function contributes to the clearance of autoantibody-Ag complexes, which is harmful in TTP, but beneficial in PF and may provide new therapeutic opportunity.

Fc-Dependent Immunomodulation Induced by Antiviral Therapeutic Antibodies: New Perspectives for Eliciting Protective Immune Responses.

The multiple mechanisms of action of antiviral monoclonal antibodies (mAbs) have made these molecules a potential therapeutic alternative for treating severe viral infections. In addition to their direct effect on viral propagation, several studies have shown that mAbs are able to enhance the host’s adaptive immune response and generate long-lasting protective immunity. Such immunomodulatory effects occur in an Fc-dependent manner and rely on Fc-FcγR interactions. It is noteworthy that several FcγR-expressing cells have been shown to play a key role in enhancing humoral and cellular immune responses (so-called “vaccinal effects”) in different experimental settings. This review recalls recent findings concerning the vaccinal effects induced by antiviral mAbs, both in several preclinical animal models and in patients treated with mAbs. It summarizes the main cellular and molecular mechanisms involved in these immunomodulatory properties of antiviral mAbs identified in different pathological contexts. It also describes potential therapeutic interventions to enhance host immune responses that could guide the design of improved mAb-based immunotherapies.

Fc gamma receptor is not required for in vivo processing of radio- and drug-conjugates of the dead tumor cell-targeting monoclonal antibody, APOMAB®

The Fc region of a monoclonal antibody (mAb) can play a crucial role in its biodistribution and therapeutic activity. The chimeric mAb, chDAB4 (APOMAB®), which binds to dead tumor cells after DNA-damaging anticancer treatment, has been studied pre-clinically in both diagnostic and therapeutic applications in cancer. Given that macrophages contribute to the tumor accumulation of chDAB4 and its potency as an antibody drug conjugate in vivo, we next wanted to determine whether the Fc region of the chDAB4 mAb also contributed. We found that, regardless of prior labeling with chDAB4, dead EL4 lymphoma or Lewis Lung (LL2) tumor cells were phagocytosed equally by wild-type or Fcγ knock-down macrophage cell lines. A similar result was seen with bone marrow-derived macrophages from wild-type, Fcγ knock-out (KO) and NOTAM mice that express Fcγ but lack immunoreceptor tyrosine-based activation motif (ITAM) signaling. Among EL4 tumor-bearing wild-type, Fcγ KO or NOTAM mice, no differences were observed in post-chemotherapy uptake of 89Zr-labeled chDAB4. Similarly, no differences were observed between LL2 tumor-bearing wild-type and Fcγ KO mice in post-chemotherapy uptake of 89Zr-chDAB4. Also, the post-chemotherapy activity of a chDAB4-antibody drug conjugate (ADC) directed against LL2 tumors did not differ among tumor-bearing wild-type, Fcγ KO and NOTAM mice, nor did the proportions and characteristics of the LL2 tumor immune cell infiltrates differ significantly among these mice. In conclusion, Fc-FcγR interactions are not essential for the diagnostic or therapeutic applications of chDAB4 conjugates because the tumor-associated macrophages, which engulf the chDAB4-labelled dead cells, respond to endogenous ‘eat me’ signals rather than depend on functional FcγR expression for phagocytosis.

Monoclonal antibody Daratumumab promotes macrophage-mediated anti-myeloma phagocytic activity via engaging FC gamma receptor and activation of macrophages.

Daratumumab (DAR) is novel human anti-CD38 IgG1, high-affinity human monoclonal antibody having broad-spectrum killing activity. The antibody is recommended to treat multiple myeloma. Recently Antibody-dependent cellular phagocytosis (ADCP) have been identified as the potential mechanism of DAR in addition to complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In the present study we evaluated the effect of Daratumumab on other effector cells of multiple myeloma. Luciferase+ MM.1R GFP cells were selected for the study. For immune-compromised multiple myeloma tumour xenograft mouse model we used severe combined immunodeficient beige (SCID-beige), NOD SCID gamma (NSG) and C57Bl/6j mice. Bioluminescence imaging was carried by injecting luciferin, and in vivo confocal microscopy was done for tracing bone marrow niches. Spleen and tumours were submitted to immunophenotypic analysis. MTT assay was done for cell proliferation studies. We established tumour xenograft mouse model. It was found that DAR showed significant anti-tumour effect in tumour xenograft multiple myeloma mice. We found that DAR showed anti-tumour activity via Fc-FcγR interaction with macrophages. DAR induced phenotypic activation of macrophages in mice and resulted in ADCP of cancerous cells via interacting Fc-FcγR in vitro. The study suggested that DAR exerted anti-tumour activity in multiple myeloma by interacting with Fc-FcγR.

Requirement of Fc-Fc Gamma Receptor Interaction for Antibody-Based Protection against Emerging Virus Infections.

Identification of therapeutics against emerging and re-emerging viruses remains a continued priority that is only reinforced by the recent SARS-CoV-2 pandemic. Advances in monoclonal antibody (mAb) isolation, characterization, and production make it a viable option for rapid treatment development. While mAbs are traditionally screened and selected based on potency of neutralization in vitro, it is clear that additional factors contribute to the in vivo efficacy of a mAb beyond viral neutralization. These factors include interactions with Fc receptors (FcRs) and complement that can enhance neutralization, clearance of infected cells, opsonization of virions, and modulation of the innate and adaptive immune response. In this review, we discuss recent studies, primarily using mouse models, that identified a role for Fc-FcγR interactions for optimal antibody-based protection against emerging and re-emerging virus infections.

A phase 2, two-stage study of mirvetuximab soravtansine (IMGN853) in combination with pembrolizumab in patients with microsatellite stable (MSS) endometrial cancer (EC).

TPS5611 Background: Folate receptor-alpha (FRα) is expressed on endometrial cancer (EC) cells and is associated with poor prognosis. Mirvetuximab soravtansine (ImmunoGen), an antibody drug conjugate (ADC) comprising a FRα-binding antibody, cleavable linker, and the tubulin-disrupting maytansinoid DM4, showed tolerability and single agent activity in a Phase 1 study with dose expansion in FRα+ advanced/recurrent EC (NCT01609556) and also when combined with chemotherapy, bevacizumab as well as pembrolizumab (NCT02606305). In addition to having direct target-mediated cytotoxicity, ADCs also stimulate the local tumor immune microenvironment. Mirvetuximab soravtansine has been shown to activate monocytes and promote phagocytosis of mirvetuximab-treated FRα-positive tumor cells through a mechanism of Fc-FcγR interaction. Further, the combination of ADCs with immune checkpoint inhibitors (ICI) can overcome primary resistance to immunotherapy in murine models. Given the low response of MSS endometrial cancers to PD-1 blockade, we hypothesized that addition of mirvetuximab may enhance response of these tumors to immunotherapy. Methods: This is a Phase 2, single cohort study of mirvetuximab soravtansine with pembrolizumab in recurrent or persistent EC. Patients must have advanced or recurrent MSS serous endometrial cancer with at least 1 and up to 3 prior lines of therapy. Confirmation of FRα expression (with PS2+ staining intensity in ≥ 50% of cells, performed centrally at Ventana Medical Systems, Inc) is required. Prior receipt of ICI is excluded. Patients will receive the combination of mirvetuximab soravtansine 6 mg/kg AIBW IV and pembrolizumab 200 mg IV administered every 21 days. The co-primary endpoint is progression-free survival at 6 months (PFS6) and objective response rate (ORR) by RECIST 1.1. Translational objectives include assessment of tumor infiltrating lymphocytes (TILs), expression of immune checkpoint markers, and whole exome sequencing (WES) for DNA repair pathway mutations, neoantigens, and polymorphisms in immunologically relevant genes. Statistical considerations are for a Simon two-stage optimal design with 16 patients in Stage 1 and 19 patients in Stage 2, to a total of 35. Prespecified activity for the first stage of accrual was met, and second stage accrual began November 2020. Clinical trial information: NCT03835819.

Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

AbstractAntibodies that bind CD47 on tumor cells and prevent interaction with SIRPα on phagocytes are active against multiple cancer types including T-cell lymphoma (TCL). Here we demonstrate that surface CD47 is heterogeneously expressed across primary TCLs, whereas major histocompatibility complex (MHC) class I, which can also suppress phagocytosis, is ubiquitous. Multiple monoclonal antibodies (mAbs) that block CD47-SIRPα interaction promoted phagocytosis of TCL cells, which was enhanced by cotreatment with antibodies targeting MHC class I. Expression levels of surface CD47 and genes that modulate CD47 pyroglutamation did not correlate with the extent of phagocytosis induced by CD47 blockade in TCL lines. In vivo treatment of multiple human TCL patient-derived xenografts or an immunocompetent murine TCL model with a short course of anti-CD47 mAb markedly reduced lymphoma burden and extended survival. Depletion of macrophages reduced efficacy in vivo, whereas depletion of neutrophils had no effect. F(ab′)2-only fragments of anti-CD47 antibodies failed to induce phagocytosis by human macrophages, indicating a requirement for Fc-Fcγ receptor interactions. In contrast, F(ab′)2-only fragments increased phagocytosis by murine macrophages independent of SLAMF7-Mac-1 interaction. Full-length anti-CD47 mAbs also induced phagocytosis by Fcγ receptor-deficient murine macrophages. An immunoglobulin G1 anti-CD47 mAb induced phagocytosis and natural killer cell-mediated cytotoxicity of TCL cells that was augmented by cotreatment with mogamulizumab, an anti-CCR4 mAb, or a mAb blocking MHC class I. These studies help explain the disparate activity of monotherapy with agents that block CD47 in murine models compared with patients. They also have direct translational implications for the deployment of anti-CD47 mAbs alone or in combination.

Differential requirements for FcγR engagement by protective antibodies against Ebola virus

Ebola virus (EBOV) continues to pose significant threats to global public health, requiring ongoing development of multiple strategies for disease control. To date, numerous monoclonal antibodies (mAbs) that target the EBOV glycoprotein (GP) have demonstrated potent protective activity in animal disease models and are thus promising candidates for the control of EBOV. However, recent work in a variety of virus diseases has highlighted the importance of coupling Fab neutralization with Fc effector activity for effective antibody-mediated protection. To determine the contribution of Fc effector activity to the protective function of mAbs to EBOV GP, we selected anti-GP mAbs targeting representative, protective epitopes and characterized their Fc receptor (FcγR) dependence in vivo in FcγR humanized mouse challenge models of EBOV disease. In contrast to previous studies, we find that anti-GP mAbs exhibited differential requirements for FcγR engagement in mediating their protective activity independent of their distance from the viral membrane. Anti-GP mAbs targeting membrane proximal epitopes or the GP mucin domain do not rely on Fc-FcγR interactions to confer activity, whereas antibodies against the GP chalice bowl and the fusion loop require FcγR engagement for optimal in vivo antiviral activity. This complexity of antibody-mediated protection from EBOV disease highlights the structural constraints of FcγR binding for specific viral epitopes and has important implications for the development of mAb-based immunotherapeutics with optimal potency and efficacy.

Antibodies targeting sialyl Lewis A mediate tumor clearance through distinct effector pathways.

Sialyl Lewis A (sLeA, also known as CA19-9), a tetrasaccharide selectively and highly expressed on advanced adenocarcinomas including colon, stomach, and pancreatic cancers, has long been considered as an attractive target for active and passive vaccination. While progress in antibodies targeting tumor-associated protein antigens resulted in an impressive array of therapeutics for cancer treatment, similar progress in exploiting tumor-associated carbohydrate antigens, such as sLeA, has been hampered by the lack of a detailed understanding of the singular characteristics of these antigens. We have addressed this issue by analyzing antibodies derived from patients immunized with an sLeA/KLH vaccine. These antibodies were engineered to mediate tumor clearance in vivo in preclinical models through Fc-FcγR interactions. However, in contrast to protein antigens in which hFcγRIIIA engagement was both necessary and sufficient to mediate tumor clearance in both preclinical and clinical settings, a similar selective dependence was not seen for anti-sLeA antibodies. Thus, re-engineering the Fc portion of sLeA-targeting antibodies to broadly enhance their affinity for activating FcγRs led to an enhanced therapeutic effect. These findings will facilitate the development of more efficient anticancer therapies and further advance this promising class of therapeutic antibodies into clinical use.

Abstract 2390: FcgR co-engagement by anti-TIGIT monoclonal antibodies enhances T cell functionality and antitumor immune responses

Abstract T-cell immunoreceptor with Ig and ITIM domains (TIGIT) has emerged as an important regulator of the cancer-immunity cycle. Preclinical studies have demonstrated that TIGIT antibodies can enhance T and NK cell anti-tumor immunity, and therapeutic antibodies targeting TIGIT are advancing in the clinic. Nonetheless, questions on the optimal format for TIGIT therapeutics remain unresolved. Preclinical studies have demonstrated that anti-TIGIT antibodies enhance anti-tumor immunity by (1) blocking inhibitory signaling downstream of TIGIT/PVR and TIGIT/PVRL2 receptor-ligand interactions and (2) redirecting PVR/PVRL2 ligand binding to the co-stimulatory receptor CD226. Here we describe a novel and unanticipated mechanism of action of anti-TIGIT antibodies in which interactions between the antibody Fc domain and select FcγRs, particularly FcγRIIIA, dramatically improve T cell activation and effector function. In mouse tumor models, TIGIT antibodies that promote FcγR interactions enhance anti-tumor responses, whereas Fc-inert TIGIT antibodies show no such benefit. Moreover, TIGIT antibodies that enhance binding to human FcγRIIIA or mouse FcγRIV promote superior single agent activity and combination activity with other checkpoint modulators in antigen-stimulation assays and mouse tumor models respectively. Notably, this mechanism of action is independent of regulatory T cell depletion. Our findings support a dependence on Fc-FcγR interaction for promoting T cell responsiveness and effector function upon TIGIT antagonism. We further demonstrate that this novel mechanism also extends to anti-CTLA-4, but not anti-PD-1 or anti-LAG-3 antibodies. Altogether, our data describe a novel FcγR-dependent mechanism of action that may enhance the therapeutic activity of anti-TIGIT antibodies, deepen our understanding of this class of therapies, and inform on the optimal design for a new class of Fc-engineered antibodies that could be leveraged to potentially enhance antitumor immune responses. Citation Format: Dhan Chand, Jeremy D. Waight, Elena Paltrinieri, Sylvia Dietrich, Mark Bushell, Mathew Costa, Randi Gombos, Nicholas S. Wilson, Jennifer S. Buell, Robert B. Stein, Alexander Duncan, David A. Savitsky. FcgR co-engagement by anti-TIGIT monoclonal antibodies enhances T cell functionality and antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2390.