Fc-dependent Manner Research Articles

A broadly reactive antibody targeting the N-terminal domain of SARS-CoV-2 spike confers Fc-mediated protection

Most neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) target the receptor binding domain (RBD) of the spike (S)protein. Here, we characterize a panel of mAbs targeting the N-terminal domain (NTD) or other non-RBD epitopes of S. A subset of NTD mAbs inhibits SARS-CoV-2 entry at a post-attachment step and avidly binds the surface of infected cells. One neutralizing NTD mAb, SARS2-57, protects K18-hACE2 mice against SARS-CoV-2 infection in an Fc-dependent manner. Structural analysis demonstrates that SARS2-57 engages an antigenic supersite that is remodeled by deletions common to emerging variants. In neutralization escape studies with SARS2-57, this NTD site accumulates mutations, including a similar deletion, but the addition of an anti-RBD mAb prevents such escape. Thus, our study highlights a common strategy of immune evasion by SARS-CoV-2 variants and how targeting spatially distinct epitopes, including those in the NTD, may limit such escape.

Fc-competent multispecific PDL-1/TIGIT/LAG-3 antibodies potentiate superior anti-tumor T cell response

The landscape of current cancer immunotherapy is dominated by antibodies targeting PD-1/PD-L1 and CTLA-4 that have transformed cancer therapy, yet their efficacy is limited by primary and acquired resistance. The blockade of additional immune checkpoints, especially TIGIT and LAG-3, has been extensively explored, but so far only a LAG-3 antibody has been approved for combination with nivolumab to treat unresectable or metastatic melanoma. Here we report the development of a PDL1 × TIGIT bi-specific antibody (bsAb) GB265, a PDL1 × LAG3 bsAb GB266, and a PDL1 × TIGIT × LAG3 tri-specific antibody (tsAb) GB266T, all with intact Fc function. In in vitro cell-based assays, these antibodies promote greater T cell expansion and tumor cell killing than benchmark antibodies and antibody combinations in an Fc-dependent manner, likely by facilitating T cell interactions (bridging) with cancer cells and monocytes, in addition to blocking immune checkpoints. In animal models, GB265 and GB266T antibodies outperformed benchmarks in tumor suppression. This study demonstrates the potential of a new generation of multispecific checkpoint inhibitors to overcome resistance to current monospecific checkpoint antibodies or their combinations for the treatment of human cancers.

Abstract 4423: XMT-2056, a HER2-targeted STING agonist antibody-drug conjugate, exhibits ADCC function that synergizes with STING pathway activation and contributes to anti-tumor responses

Abstract XMT-2056 is a systemically administered Immunosynthen STING agonist antibody-drug conjugate (ADC) that targets a novel HER2 epitope and induces complete tumor regressions with a single dose in multiple tumor models. We have previously shown that XMT-2056 delivers its STING agonist payload into tumor cells and FcγRI (CD64)-expressing myeloid cells, activating STING signaling in both cell types, leading to type I interferon (IFN) and anti-tumor innate immune responses. Here, we demonstrate that XMT-2056 exhibits ADCC (antibody-dependent cell-mediated cytotoxicity) function, which synergizes with STING pathway activation and induces potent cancer cell-killing activity in co-cultures of HER2-expressing cancer cells and FcγRIII+ (CD16+) immune cells. We show that both XMT-2056 and HT-19 (the unconjugated parental antibody) retain significant cancer cell-killing activity in an Fc-dependent manner in PBMC co-cultures depleted of FcγRI-expressing myeloid cells. This activity is abrogated by co-depletion of FcγRIII+ immune cells, illustrating the ADCC function of XMT-2056. In this setting, XMT-2056 cancer cell-killing activity was significantly increased compared to HT-19, suggesting that the STING payload contributes to the differential activity observed with XMT-2056 treatment. Indeed, co-treatment of cancer cell and immune cell co-cultures with HT-19 and free STING agonist payload enhanced the anti-tumor responses, although to a lesser extent than XMT-2056, suggesting a synergy between the ADCC function and STING pathway activation. Consistently, XMT-2056 treatment of HER2-expressing cancer cells co-cultured with unstimulated CD56+/FcγRIII+ NK cells induced granzyme b and IFN-γ cytokine production, expression of NK cell activation markers, and cancer cell-killing activity. The ADCC activity of HT-19 was comparable to that of trastuzumab in NK cell co-cultures. Finally, we found that depletion of FcγRI+ cells inhibited the cancer cell-killing activity of XMT-2056 in cancer cell and PBMC co-cultures more substantially compared to depletion of FcγRIII+ cells or CD56+ NK cells, indicating a greater contribution of myeloid cells to the XMT-2056 mechanism of action in this setting. Notably, XMT-2056 was capable of engaging both FcγRI+ myeloid cells and FcγRIII+ NK cells, activating both STING-mediated innate immune responses and ADCC function in triple cultures with HER2-expressing cancer cells. Collectively, our data reveal a synergy between ADCC function and STING pathway induction both mediated by XMT-2056, which enhances the cancer cell-killing activity of FcγRIII+ cells. This additional mechanism of action of XMT-2056 can potentially impact the overall anti-tumor immune responses in tumors infiltrated by FcγRIII+ cells. Citation Format: Jahna Soomer-James, Marc Damelin, Naniye Malli. XMT-2056, a HER2-targeted STING agonist antibody-drug conjugate, exhibits ADCC function that synergizes with STING pathway activation and contributes to anti-tumor responses. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4423.

Hemagglutinin stalk-binding antibodies enhance effectiveness of neuraminidase inhibitors against influenza via Fc-dependent effector functions

SummaryThe conserved hemagglutinin stalk domain is an attractive target for broadly effective antibody-based therapeutics and next-generation universal influenza vaccines. Protection provided by hemagglutinin stalk-binding antibodies is principally mediated through activation of immune effector cells. Titers of stalk-binding antibodies are highly variable on an individual level and tend to increase with age as a result of increasing exposures to influenza virus. In our study, we show that stalk-binding antibodies cooperate with neuraminidase inhibitors to protect against influenza virus infection in an Fc-dependent manner. These data suggest that the effectiveness of neuraminidase inhibitors is likely influenced by an individual’s titers of stalk-binding antibodies and that neuraminidase inhibitors may enhance the effectiveness of future stalk-binding monoclonal antibody-based treatments.

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.

An Fc-Competent Anti-Human TIGIT Blocking Antibody Ociperlimab (BGB-A1217) Elicits Strong Immune Responses and Potent Anti-Tumor Efficacy in Pre-Clinical Models.

TIGIT (T-cell immunoglobulin and ITIM domain) has emerged as a promising target in cancer immunotherapy. It is an immune “checkpoint” inhibitor primarily expressed on activated T cells, NK cells and Tregs. Engagement of TIGIT to its ligands PVR and PVR-L2 leads to inhibitory signaling in T cells, promoting functional exhaustion of tumor-infiltrating T lymphocytes. Here, we described the pre-clinical characterization of Ociperlimab (BGB-A1217), a novel humanized IgG1 anti-TIGIT antibody (mAb), and systemically evaluated the contribution of Fc functions in the TIGIT mAb-mediated anti-tumor activities. BGB-A1217 binds to the extracellular domain of human TIGIT with high affinity (KD = 0.135 nM) and specificity, and efficiently blocks the interaction between TIGIT and its ligands PVR or PVR-L2. Cell-based assays show that BGB-A1217 significantly enhances T-cell functions. In addition, BGB-A1217 induces antibody dependent cellular cytotoxicity (ADCC) against Treg cells, activates NK cells and monocytes, and removes TIGIT from T cell surfaces in an Fc-dependent manner, In vivo, BGB-A1217, either alone or in combination with an anti-PD-1 mAb elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models. Moreover, the Fc effector function is critical for the anti-tumor activity of BGB-A1217 in a syngeneic human TIGIT-knock-in mouse model. The observed anti-tumor efficacy is associated with a pharmacodynamic change of TIGIT down-regulation and Treg reduction. These data support the selection of BGB-A1217 with an effector function competent Fc region for clinical development for the treatment of human cancers.

Stereotyped B-cell response that counteracts antigenic variation of influenza viruses.

Influenza A subtypes are categorized into group 1 and group 2 based on the hemagglutinin (HA) sequence. Owing to the phylogenetic distance of HAs in different groups, antibodies that bind multiple HA subtypes across different groups are extremely rare. In this study, we demonstrated that an immunization with acid-treated HA antigen elicits germinal center (GC) B cells that bind multiple HA subtypes in both group 1 and group 2. The cross-group GC B cells utilized mostly one VH gene (1S56) and exhibited a sign of clonal evolution within GCs. The 1S56-lineage IgGs derived from GC B cells were able to bind to HA protein on the infected cell surface but not to the native form of HA protein, suggesting the cryptic nature of the 1S56 epitope and its exposure in infected cells. Finally, the 1S56-lineage IgGs provided protection against lethal infection in an Fc-dependent manner, independent of the virus-neutralizing activity. Thus, we identified 1S56-lineage antibodies as a unique stereotype for achieving cross-group influenza specificity. The antigens exposing the 1S56 epitope may be good candidates for broadly protective immunogens.

MICA immune complex formed with alpha 3 domain-specific antibody activates human NK cells in a Fc-dependent manner

BackgroundOne of the mechanisms by which tumors evade immune surveillance is through shedding of the major histocompatibility complex (MHC) class I chain-related protein A and B (MICA/B) from their cell surface. MICA/B are ligands for the activating receptor NKG2D on NK and CD8 T cells. This shedding reduces cell surface levels of MICA/B and impairs NKG2D recognition. Shed MICA/B can also mask NKG2D receptor and is thought to induce NKG2D internalization, further compromising immune surveillance by NK cells.MethodsWe isolated human primary NK cells from normal donors and tested the suppressive activity of soluble recombinant MICA in vitro. Utilizing a panel of novel anti-MICA antibodies, we further examined the stimulatory activities of anti-MICA antibodies that reversed the suppressive effects of soluble MICA.ResultsWe show that suppressive effects of soluble MICA (sMICA) on NK cell cytolytic activity was not due to the down-regulation of cell surface NKG2D. In the presence of an α3 domain-specific MICA antibody, which did not obstruct NKG2D binding, sMICA-mediated NK cell suppression was completely reversed. Reversal of NK cell inhibition by sMICA was mediated by immune complex formation that agonized NKG2D signaling. Furthermore, this restorative activity was dependent on antibody Fc effector function as the introduction of Fc mutations to abrogate Fc receptor binding failed to reverse sMICA-mediated NK cell suppression. Furthermore, MICA immune complexes preformed with an α3 domain-specific antibody (containing a wild-type Fc) induced IFN-γ and TNF-α secretion by NK cells in the absence of cancer cells, whereas MICA immune complexes preformed with the Fc effectorless antibody failed to induce IFN-γ and TNF-α secretion. Finally, we demonstrated that MICA immune complexes formed with the α3 domain-specific antibody activates NKG2D on NK cells leading to the release of IFN-γ.ConclusionsOur results demonstrate that an α3 domain-specific MICA antibody can circumvent sMICA-mediated suppression of NK cell cytolytic activity. Moreover, our data suggest that MICA immune complexes formed with α3-specific antibodies can activate NKG2D receptor and restore NK cell function in a Fc-dependent manner. The clinical utility of α3 domain-specific MICA/B antibodies may hold great promise as a new strategy for cancer immunotherapy.

Donor-Derived Factors Influence NK Cell Effector Activity in a Model of Antibody-Mediated Chronic Rejection

Purpose Natural killer (NK) cells are key components of the innate immune system that contribute to acute and chronic allograft rejection. Previous studies have shown that donor-specific antibodies (DSA) in concert with NK cells can be sufficient to inflict chronic allograft vasculopathy (CAV) in an Fc dependent manner. However, it has been observed clinically that detectable DSA does not always correlate with demonstrable injury. The modern understanding of NK cell triggering suggests that the transition from quiescence to activation is mediated by a network of activating and inhibitory receptors; it is the integration of the sum of these signals that determines the final NK cell response. Our study aimed to investigate if donor-derived factors can alter the balance of the NK cell effector response. Methods We utilized a murine model of cardiac transplant with C3H/HeJ (C3H, H-2k), BALB/cByJ (BALB/c, H-2d), or C3HxBALB/c F1 (F1) generation donors engrafted into C57Bl/6.rag-/- (B6.rag) recipients. Recipients had no functional T or B cells, but an intact innate immune system. Experimental animals received monoclonal IgG2a Class I DSA (anti-H2k or anti-H2d). Allografts were subsequently recovered and examined after 60 days for the presence of CAV (defined by calculation of the neointimal index). Results Initial studies verified that NK cells alone could not cause significant vascular injury. When C3H mice were used as donors (n=7), administration of Class I DSA resulted in marked neointimal changes consistent with CAV. However, when BALB/c mice were used as donors (n=6), administration of Class I DSA did not result in development of CAV, despite the fact that this donor strain also represented a fully allogeneic mismatch from the recipient. When F1 mice were utilized as donors (allografts expressed both H-2k and H-2d MHC molecules), neither administration of anti-H2k (n=7) nor anti-H2d (n=6) Class I DSA resulted in development of CAV. Conclusion Donor strain determines if the activating signal provided to NK cells by binding to Class I DSA, coupled with the lack of inhibition from “missing self,” is sufficient to result in NK cell triggering with initial results suggesting that certain donor / recipient MHC combinations demonstrate a permissive phenotype towards NK cell quiescence.

Non-neutralizing Antibodies Alter the Course of HIV-1 Infection In Vivo

Non-neutralizing antibodies (nnAbs) to HIV-1 show little measurable activity in prevention or therapy in animal models yet were the only correlate of protection in the RV144 vaccine trial. To investigate the role of nnAbs on HIV-1 infection invivo, we devised areplication-competent HIV-1 reporter virus that expresses a heterologous HA-tag on the surface ofinfected cells and virions. Anti-HA antibodies bindto, but do not neutralize, the reporter virus invitro. However, anti-HA protects against infection in humanized mice and strongly selects for nnAb-resistant viruses in an entirely Fc-dependent manner. Similar results were also obtained with tier 2 HIV-1 viruses using a human anti-gp41 nnAb, 246D. While nnAbs are demonstrably less effective than broadly neutralizing antibodies (bNAbs) against HIV-1 in vitro and invivo, the data show that nnAbs can protect against and alter the course of HIV-1 infection invivo. PAPERCLIP.

Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner.

ABSTRACTBroadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

An internalizing antibody induces the production of pro-inflammatory cytokines via Fc-dependent pathway in THP-1 cell.

Abstract Conventionally, the extracellular effector functions of antibodies are mediated by the heavy chain constant regions of Ig molecules, but recently, intracellular effector functions of antibodies have been reported. After antibodies binds to the virus and the antibody-virus complex enters the cytosol, the complex can be detected by TRIM21 that is an intracellular Fc receptor, leading to the degradation of the virus and activation of transcription factors related to immune response. In this process, the minimum requirement is the presence of antibody in the cytosol for interaction with TRIM21. We previously have shown that 3D8 IgG originated from an autoimmune-prone MRL-lpr/lpr mouse is an internalizing antibody that can spontaneously enter the cytosol. Here, we investigated whether 3D8 IgG regulates the immune response in diverse cells without viral infection. Treatment of 3D8 IgG wild type induces pro-inflammatory cytokines (IL-8 and TNF-α) in human monocyte cell line THP-1. In contrast, treatment of 3D8 IgG mutant which does not bind to TRIM21 up-regulates production of the cytokines, compared to 3D8 IgG wild type. Moreover, 3D8 scFv (single chain fragment variable) which is devoid of Fc region did not induce the production of cytokines. Our results demonstrate that the production pathway(s) of cytokines are TRIM21-independent and Fc-dependent manner. We suppose that Fc-interacting unknown molecules would induce the secretion of pro-inflammatory cytokines. Our study would provide a new insight to the pathogenesis of autoimmune disease.

Myelin-reactive antibodies initiate T cell-mediated CNS autoimmune disease by opsonization of endogenous antigen.

In the pathogenesis of central nervous system (CNS) demyelinating disorders, antigen-specific B cells are implicated to act as potent antigen-presenting cells (APC), eliciting waves of inflammatory CNS infiltration. Here, we provide the first evidence that CNS-reactive antibodies (Ab) are similarly capable of initiating an encephalitogenic immune response by targeting endogenous CNS antigen to otherwise inert myeloid APC. In a transgenic mouse model, constitutive production of Ab against myelin oligodendrocyte glycoprotein (MOG) was sufficient to promote spontaneous experimental autoimmune encephalomyelitis (EAE) in the absence of B cells, when mice endogenously contained MOG-recognizing T cells. Adoptive transfer studies corroborated that anti-MOG Ab triggered activation and expansion of peripheral MOG-specific T cells in an Fc-dependent manner, subsequently causing EAE. To evaluate the underlying mechanism, anti-MOG Ab were added to a co-culture of myeloid APC and MOG-specific T cells. At otherwise undetected concentrations, anti-MOG Ab enabled Fc-mediated APC recognition of intact MOG; internalized, processed and presented MOG activated naïve T cells to differentiate in an encephalitogenic manner. In a series of translational experiments, anti-MOG Ab from two patients with an acute flare of CNS inflammation likewise facilitated detection of human MOG. Jointly, these observations highlight Ab-mediated opsonization of endogenous CNS auto-antigen as a novel disease- and/or relapse-triggering mechanism in CNS demyelinating disorders.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1559-8) contains supplementary material, which is available to authorized users.

Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an in vitro bioassay, depending on the experimental conditions, while, when injected in mice together with human GM-CSF, they lead to the accumulation of a large pool of circulating GM-CSF that remains partially bioavailable. In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies.

Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody.

Microglia have been shown to contribute to the clearance of brain amyloid β peptides (Aβ), the major component of amyloid plaques, in Alzheimer’s disease (AD). However, it is not known whether microglia play a similar role in the clearance of tau, the major component of neurofibrillary tangles (NFTs). We now report that murine microglia rapidly internalize and degrade hyperphosphorylated pathological tau isolated from AD brain tissue in a time-dependent manner in vitro. We further demonstrate that microglia readily degrade human tau species released from AD brain sections and eliminate NFTs from brain sections of P301S tauopathy mice. The anti-tau monoclonal antibody MC1 enhances microglia-mediated tau degradation in an Fc-dependent manner. Our data identify a potential role for microglia in the degradation and clearance of pathological tau species in brain and provide a mechanism explaining the potential therapeutic actions of passively administered anti-tau monoclonal antibodies.

Glycoprotein Ibα Clustering Induces Macrophage-Mediated Platelet Clearance in the Liver

Objective: Immune thrombocytopenia (ITP) is an autoimmune bleeding disease in which autoantibodies are directed against the patient’s own platelets, leading to platelet destruction and bleeding. Currently, platelet destruction in ITP is thought to be mediated by monocytic and phagocytic cells, and the antibody-coated platelets are removed from circulation by the spleen in an Fc-dependent manner. Conventional treatments for ITP therefore aim at reducing platelet destruction, either by immunosuppression or splenectomy. However, many ITP patients, particularly patients with anti-glycoprotein (GP) Ib-IX autoantibodies, do not respond to conventional treatments such as intravenous immunoglobulin G (IVIG). However, the underlying mechanism remains unclear.Methods: Anti-GPIb-IX antibodies-induced platelet aggregation was detected by an optical aggregometer. GPIbα clustering was measured by Fluorescence Resonance Energy Transfer (FRET) by flow cytometry. Antibodies-induced platelet depletion and rescue experiments were performed in guinea pigs and cynomolgus macaques. Platelet distribution after antibody-mediated depletion was detected by in vivo Imaging System (IVIS), immunohistochemistry and immunofluorescence microscopy.Results: We found that anti-GPIbα N-terminus antibody AN51, but not other anti-GPIbα antibodies (AK2, HIP1, VM16d, or WM23), induced integrin αⅡbβ3-dependent platelet aggregation. FRET result showed that AN51 induced obvious GPIbα clustering but not VM16d. After intravenous injection, AN51 dose-dependently induced thrombocytopenia in guinea pigs, and the platelets were mainly removed by macrophages in the liver. N-acetyl-D-glucosamine (GlcNAc), previously shown to inhibited αMβ2-mediated phagocytosis of refrigerated platelets, showed dose-dependent inhibition of AN51-induced platelet destruction. Furthermore, AN51 but not VM16d, induced rapid platelet clearance in the liver of cynomolgus macaques. Five of twenty-two chronic ITP patients had anti-GPIbα autoantibodies, and the autoantibodies from four of the five patients competed with AN51 for binding to platelets.Conclusion: GPIbα clustering induced by anti-GPIbα N-terminus antibody causes integrin αⅡbβ3-dependent platelet aggregation, phagocytosis, and rapid platelet clearance in the liver. Our findings reveal a novel Fc-independent mechanism underlying the pathogenesis of ITP, and suggest new therapeutic strategies for ITP patients with anti-GPIbα autoantibodies. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5035: Blockade of VEGF with ziv-aflibercept (VEGF Trap) enhances anti-tumor efficacy of CTLA-4 blocking antibody in an Fc dependent manner

Abstract VEGF-dependent angiogenesis is required for tumor progression. Ziv-aflibercept (VEGF Trap) is a VEGF inhibitor, which exhibits potent antitumor activity in preclinical models and was approved by the FDA in combination with FOLFIRI for patients with metastatic colorectal cancer (mCRC) who did not respond or progressed following an oxaliplatin-containing regimen. Recent data suggest that VEGF blockade may also modulate tumor immunity by promoting necrosis and inflammation, however the precise mechanism is not fully understood. CTLA-4 blockade promotes tumor immunity by, in part, reversing suppressive effects of CTLA-4 receptor on T cell activation, and exhibits synergistic effects with other immunomodulatory agents in both clinical and experimental settings. In this study, we examined the combination of ziv-aflibercept (at 10 mg/kg) and CTLA-4 blocking antibody (at 200 ug/mouse) administered five times within two weeks in two mouse colon carcinoma models (Colon26 and MC38). Prophylactic therapy with either single agent inhibited Colon26 tumor growth. Responses to ziv-aflibercept were associated with reduction of tumor vascularity, while those to α-CTLA-4 murine IgG2b antibody were associated with enrichment of tumor CD8, but not CD4, T cells as measured by flow cytometry. Combination therapy resulted in complete tumor regression and significantly improved survival (0%, 30% and 70% survival in ziv-aflibercept, α-CTLA-4 and combination groups respectively). Pathological evaluation of ziv-aflibercept treated tumors by histostaining revealed increased tumor necrosis, which became more severe in the combination group. α-CTLA-4 antibody did not promote definitive tumor necrosis, but resulted in increased immune cell infiltrate composed mainly of lymphocytes and macrophages. To address the role of the immunoglobulin constant region of α-CTLA-4 antibody, we compared the effect of murine IgG2a and IgG2b antibody versions with high and low effector function respectively. α-CTLA-4 IgG2a antibody was significantly more potent than IgG2b, resulting in eradication of established Colon26 tumors and partial growth inhibition of established MC38 tumors, whereas these established tumors were resistant to α-CTLA4 IgG2b therapy. Co-administration of ziv-aflibercept significantly improved α-CTLA4 IgG2a antibody efficacy against established MC38 tumors. These results suggest that combining immunotherapy and ziv-aflibercept may be beneficial for the treatment of established tumors in a clinical setting. Citation Format: Elena Burova, Ella Ioffe, Chandrika Taduriyasas, Omaira Allbritton, Gaurav Tyagi, Lekan Oyejide, John Rudge, Israel Lowy, Gavin Thurston. Blockade of VEGF with ziv-aflibercept (VEGF Trap) enhances anti-tumor efficacy of CTLA-4 blocking antibody in an Fc dependent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5035. doi:10.1158/1538-7445.AM2014-5035

Rituximab mediates loss of CD19 on B cells in the absence of cell death

To evaluate loss of the B cell-specific marker CD19 after the addition of rituximab (RTX) to healthy donor blood and to determine the role of complement-mediated cytotoxicity in these cells. Whole blood and peripheral blood mononuclear cells (PBMCs) from healthy donors were evaluated for the loss of CD19 in the presence of RTX using flow cytometry. The effect of complement on CD19 loss was examined using serum-free media, C3- and C5-deficient sera, and a C5-blocking antibody. Evidence of B cell death was evaluated by measuring messenger RNA (mRNA) levels as well as by flow cytometry. Transfer of CD19 antigen to monocytes and neutrophils was evaluated by flow cytometry and confocal microscopy. RTX induced a rapid decrease in CD19 count (mean 51%; n = 37) in PBMCs. This reduction occurred in the absence of complement. Despite the decrease in CD19 expression, B cell death did not occur, as evidenced by a lack of change in CD19 or CD20 mRNA levels and a lack of change in CD19 levels determined by intracellular staining and through the use of viability dyes. The CD19 antigen was shown to be transferred to monocytes and neutrophils in an Fc-dependent manner. Our findings indicate that the addition of RTX to healthy donor PBMCs in vitro results in complement-independent loss of CD19 without causing B cell death. CD19 is transferred from B cells to monocytes and neutrophils during shaving of the RTX-CD20 complex in an Fc-dependent manner. These data suggest that monitoring the effect of RTX by measuring the CD19+ cell count may be compromised by this activity.

A Novel Pathway of Chronic Allograft Rejection Mediated by NK Cells and Alloantibody

Chronic allograft vasculopathy (CAV) in murine heart allografts can be elicited by adoptive transfer of donor specific antibody (DSA) to class I MHC antigens and is independent of complement. Here we address the mechanism by which DSA causes CAV. B6.RAG1(-/-) or B6.RAG1(-/-)C3(-/-) (H-2(b)) mice received B10.BR (H-2(k)) heart allografts and repeated doses of IgG2a, IgG1 or F(ab')(2) fragments of IgG2a DSA (anti-H-2(k)). Intact DSA regularly elicited markedly stenotic CAV in recipients over 28 days. In contrast, depletion of NK cells with anti-NK1.1 reduced significantly DSA-induced CAV, as judged morphometrically. Recipients genetically deficient in mature NK cells (γ-chain knock out) also showed decreased severity of DSA-induced CAV. Direct NK reactivity to the graft was not necessary. F(ab')(2) DSA fragments, even at doses twofold higher than intact DSA, were inactive. Graft microvascular endothelial cells responded to DSA in vivo by increased expression of phospho-extracellular signal-regulated kinase (pERK), a response not elicited by F(ab')(2) DSA. We conclude that antibody mediates CAV through NK cells, by an Fc dependent manner. This new pathway adds to the possible mechanisms of chronic rejection and may relate to the recently described C4d-negative chronic antibody-mediated rejection in humans.

Abstract A135: T-DM1-induced thrombocytopenia results from impaired platelet production in a HER2-independent manner.

Abstract Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising trastuzumab, a stable thioether linker, and the maytansine derivative DM1, a microtubule inhibitor. T-DM1 has been evaluated for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer in several phase 2 trials. Unexpectedly, the dose-limiting toxicity in patients treated with T-DM1 was dose- and duration-dependent thrombocytopenia, even though HER2 is not detected on platelets. To understand the mechanism of T-DM1-induced thrombocytopenia, we evaluated the effect of T-DM1 on platelet (PLT) function and formation from hematopoietic stem cells (HSCs). Using PLT-rich plasma (PRP) or washed PLT (WP) from healthy human donors, PLT function was assessed by aggregometry and flow cytometry to evaluate activation markers (PAC1; CD62P) in the presence of T-DM1, trastuzumab, control ADC (anti-CD22 conjugated to DM1), free DM1, or vehicle. Human HSCs (CD133+/CD34+) were purified from healthy donor bone marrow, expanded in hematopoietic expansion media, and transferred to megakaryocyte (Mk) differentiation media for 14 days to obtain mature Mks, in the presence of T-DM1, trastuzumab, ADC control (5B6, anti-gD conjugated to DM1), and vehicle. T-DM1 uptake and catabolism in Mks were evaluated by incubating the cells with T-[3H]DM1. Incubation with T-DM1, free DM1, or any controls at clinically relevant concentrations had no observable effect on platelet aggregation in PRP or WP and no direct impact on PLT activation (PAC1 binding or CD62P expression). Neither T-DM1 nor free DM1 inhibited PLT aggregation and activation induced by the PLT agonists collagen and TRAP6. Treatment of mature Mks with T-DM1 and control ADC resulted in a decrease in Mk number and a significant decrease (∼3-fold) in Mk production from HSC precursors, as measured by a reduction in CD41+/CD61+ cells. Incubation of Alexa488-conjugated trastuzumab and T-DM1 with Mks resulted in surface binding and internalization as assessed by immunofluorescence and flow cytometry. Preincubation with anti-CD32 decreased Alexa488-conjugated T-DM1 antibody binding and uptake (∼2-fold), suggesting that FcR IIb contributes to this process. To determine the fate of the internalized antibody, we quantified the uptake and catabolism of T-DM1 in Mks using T-[3H]DM1. Total cellular T-[3H]DM1 increased over the 3-day incubation with peak concentrations occurring during the first 8 hours of incubation, and a peak total uptake in the Mks of 0.4% of the total dose. The metabolized intracellular⌝-free [3H]DM1 levels were 0.1% of the total dose. By quantification of the HSCs and various ploidy populations, T-DM1 was found to primarily inhibit Mk production from HSCs rather than directly impact mature Mks. Furthermore, the similarity of effects between T-DM1 and 5B6-DM1 suggests that the DM1 moiety is responsible for the observed platelet reduction. Our experiments show that T-DM1 did not have a direct effect on PLT function but did impair Mk and PLT production. DM1-conjugated antibodies were internalized in a target-independent, partially Fc-dependent manner into Mks, and intracellular release of DM1 resulted in a reduction of the stem cell population. These data support the hypothesis that the thrombocytopenia observed in clinical trials may be a consequence of impaired PLT production from Mks in the bone marrow. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A135.