Role Of Antibody-dependent Cellular Cytotoxicity Research Articles

Roles of microglia/macrophage and antibody in cell sheet transplantation in the central nervous system

BackgroundWe previously established a human mesenchymal stem cell (MSC) line that was modified to express trophic factors. Transplanting a cell sheet produced from this line in an amyotrophic lateral sclerosis mouse model showed a beneficial trend for mouse life spans. However, the sheet survived for less than 14 days, and numerous microglia and macrophages were observed within and adjacent to the sheet. Here, we examined the roles of microglia and macrophages as well as acquired antibodies in cell sheet transplantation.MethodsWe observed the effects of several MSC lines on macrophages in vitro, that is, phenotype polarization (M1 or M2) and migration. We then investigated how phenotypic polarization affected MSC survival using antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). We also confirmed the role of complement on cytotoxicity. Lastly, we selectively eliminated microglia and macrophages in vivo to determine whether these cells were cytoprotective to the donor sheet.ResultsIn vitro co-culture with MSCs induced M2 polarization in macrophages and facilitated their migration toward MSCs in vitro. There was no difference between M1 and M2 phenotypes on ADCC and ADCP. Cytotoxicity was observed even in the absence of complement. Eliminating microglia/macrophage populations in vivo resulted in increased survival of donor cells after transplantation.ConclusionsAcquired antibodies played a role in ADCC and ADCP. MSCs induced M2 polarization in macrophages and facilitated their migration toward MSCs in vitro. Despite these favorable characteristics of microglia and macrophages, deletion of these cells was advantageous for the survival of donor cells in vivo.

Abstract 553: Combined use of human iPSC-derived natural killer cells with macrophages and anti-CD47 blockade to improve killing of acute myeloid leukemia

Abstract Despite many recent advances, treatment of acute myeloid leukemia (AML) remains challenging. To generate an improved cell-based therapy against AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from combination with other therapies. Based on studies showing that targeting the CD47 pathway on macrophages (Mϕ) improves anti-tumor activity in AML, we investigated the combination of iPSC-NK cells with iPSC-Mϕ with and without CD47 blockade for the treatment of AML. To determine if the addition of iPSC-Mϕ can improve the cytotoxicity of NK cells against AML blasts, we co-cultured iPSC-NK cells and MOLM13 or MV-4-11 AML cells with iPSC-Mϕ in standard cytotoxicity assays. While Mϕ alone did not kill AML blasts, the addition of iPSC-Mϕ to iPSC-NK cells significantly improved killing of AML blasts by 50% (p<0.01). Addition of an anti-CD47 mAb (B6H12) further increased killing of AML blasts by an additional 23% (p<0.01). Addition of the CD47 mAb to iPSC-Mϕ without NK cells did not result in increased killing. We also demonstrated that blockade of SIRPα (receptor for CD47) significantly increased NK cell killing of AML blasts by 16% (p<0.05). Furthermore, the combination of iPSC-NK cells + iPSC-Mϕ + SIRPα mAb led to a 37% increase in cytotoxicity compared to iPSC-NK cells + iPSC-Mϕ alone (p<0.01). To confirm that addition of anti-CD47 or anti-SIRPα antibodies increased killing via loss of the inhibitory CD47-SIRPα interaction on NK cells and not via another mechanism, we tested the effect of adding anti-CD47 mAb and SIRPα antibodies simultaneously. Compared to addition of either mAb alone, the combination induced no additional increase in anti-AML activity by the NK cells. We also found the addition of the SIRPα mAb to iPSC-Mϕ alone did not increase cytotoxicity. We next evaluated if antibody dependent cellular cytotoxicity (ADCC) mediated by anti-CD47 mAb binding AML cells could account for the increase in cytotoxicity and found that blockade of Fc-receptors on NK cells does not diminish the increase in cytotoxicity, indicating no role for ADCC. Finally, we showed that direct contact between NK cells and Mϕ is required to increase killing of AML in a transwell assay. Separation of iPSC-Mϕ from iPSC-NK cells by a transwell insert abrogated the increase in anti-AML cytotoxicity seen with addition of iPSC-Mϕ to NK cells without the insert. In vivo studies testing the combination of iPSC-NK cells, iPSC-Mϕ and CD47 mAb against human AML in mouse xenograft-models are ongoing. Together our results indicate that CD47 blockade combined with iPSC-Mϕ leads to increased NK cell-mediated anti-tumor activity. iPSC-NK cells and iPSC-Mϕ provide an important, standardized, allogeneic cell therapy approach. Citation Format: Benjamin Goldenson, Manuel Fierro, Somayeh Pouyanfard, Dan S. Kaufman. Combined use of human iPSC-derived natural killer cells with macrophages and anti-CD47 blockade to improve killing of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 553.

IMMU-29. B-CELL-BASED VACCINE PRODUCES GLIOBLASTOMA-REACTIVE ANTIBODIES THAT CONTRIBUTE TO TUMOR CLEARANCE

Abstract Glioblastomas (GBM) are characterized by a strong immunosuppressive environment, contributing to their poor prognosis and limited therapeutic response to immunotherapies. B-cells represent a unique opportunity to promote immunotherapy due to their potential to kill tumors by both cellular and humoral immunity. To generate our B-cell-based vaccine (BVax) platform, we activated 41BBL+ B cells from tumor bearing mice or GBM patient blood with BAFF, CD40, and IFNg. We have previously demonstrated that BVax potentiates radiation therapy, temozolomide and checkpoint blockade in murine models of GBM via enhancement of CD8+ T-cell based immunity. The aim of this current study is to evaluate the humoral effector functions of BVax. We examined the antibody (Ab) repertoire in vivo from serum of tumor-bearing B-cell knockout mice treated with BVax or by ex vivo stimulation of patient-derived BVax. Upon systemic administration, BVax infiltrates the tumor where it differentiates into plasmablasts. Murine BVax- and BNaive-derived serum immunoglobulin generated in vivo showed that the majority of murine BVax-derived Ab were IgG isotype, while BNaive mainly produced IgM isotype. Transfer of IgG from BVax treated mice directly into tumors of recipient animals significantly prolonged their survival, demonstrating anti-tumor cytotoxicity directly through humoral immunity. Patient-derived BVax activated ex vivo showed a plasmablast phenotype and the Ab repertoire supports the previous findings seen in our murine model. Our work suggests BVax-derived IgGs role in antibody-dependent cellular cytotoxicity and improved survival in murine models. This function, in addition to its role in cellular immunity against GBM, renders BVax a potentially effective alternative immunotherapeutic option for GBM patients.

Considerations of Antibody Geometric Constraints on NK Cell Antibody Dependent Cellular Cytotoxicity.

It has been well-established that antibody isotype, glycosylation, and epitope all play roles in the process of antibody dependent cellular cytotoxicity (ADCC). For natural killer (NK) cells, these phenotypes are linked to cellular activation through interaction with the IgG receptor FcγRIIIa, a single pass transmembrane receptor that participates in cytoplasmic signaling complexes. Therefore, it has been hypothesized that there may be underlying spatial and geometric principles that guide proper assembly of an activation complex within the NK cell immune synapse. Further, synergy of antibody phenotypic properties as well as allosteric changes upon antigen binding may also play an as-of-yet unknown role in ADCC. Understanding these facets, however, remains hampered by difficulties associated with studying immune synapse dynamics using classical approaches. In this review, I will discuss relevant NK cell biology related to ADCC, including the structural biology of Fc gamma receptors, and how the dynamics of the NK cell immune synapse are being studied using innovative microscopy techniques. I will provide examples from the literature demonstrating the effects of spatial and geometric constraints on the T cell receptor complex and how this relates to intracellular signaling and the molecular nature of lymphocyte activation complexes, including those of NK cells. Finally, I will examine how the integration of high-throughput and “omics” technologies will influence basic NK cell biology research moving forward. Overall, the goal of this review is to lay a basis for understanding the development of drugs and therapeutic antibodies aimed at augmenting appropriate NK cell ADCC activity in patients being treated for a wide range of illnesses.

Infiltration of CD4, CD8, CD56, and Fox-P3-positive lymphocytes in breast carcinoma tissue after neoadjuvant chemotherapy with or without trastuzumab.

Trastuzumab (Tz) is assumed to prime antibody-dependent cellular cytotoxicity (ADCC); however, it remains unclear whether Tz therapy can clinically induce adaptive cellular immunity. Adaptive Cellular Immune Effect of Tz Therapy. This study included 29 surgical invasive breast carcinomas administered neoadjuvant chemotherapy with Tz (15 cases) or without Tz (14 cases). The numbers of immunoreactive cells (CD4, CD8, CD56, and Fox-P3) in three different compartments (intratumoral, adjacent stromal, and distant stromal) were determined. The average number of adjacent stromal CD4-positive, CD8-positive, and Fox-P3-positive cells in the Tz+ group was significantly greater than that in the Tz- group (p =0.036, 0.0049, and 0.043, respectively). However, the number of Fox-P3-positive cells was much less than that of CD4-positive cells. Moreover, distant stromal CD4-positive and CD8-positive cells in the Tz+ group was also significantly greater than that of the Tz- group (p =0.029 and 0.032, respectively). Only a small number of CD56-positive natural killer cells, playing a main role in ADCC, accumulated at the tumor site after Tz therapy. The results suggest that Tz therapy induces adaptive cellular immunity, including infiltration of both CD4-positive helper T cells and CD8-positive cytotoxic T cells into the breast carcinoma lesion.

Highlights of This Issue

AKT kinase, as a component of the PI3K/mTOR/AKT cascade, mediates chemotherapy resistance in ovarian cancer. Following preclinical evidence that AKT inhibition restores chemotherapy efficacy, Blagden and colleagues conducted a Phase IB combination study of the AKT inhibitor afuresertib given with carboplatin and paclitaxel chemotherapy in 29 patients with platinum-resistant ovarian cancer. A 32% response rate was observed, and the combination showed acceptable tolerability. These findings support a future randomized evaluation of this combination in treatment-resistant ovarian cancer.CNS involvement worsens the prognosis of patients with T-cell acute lymphoblastic leukemia (T-ALL), because most anticancer drugs cannot pass the blood brain barrier. The histone demethylase LSD1 is considered to be a therapeutic target of T-ALL, in which LSD1 is overexpressed and acts as a founder abnormality. T-ALL cells, however, are resistant to tranylcypromine-based conventional LSD1 inhibitors. Saito and colleagues developed a novel LSD1 inhibitor, S2157, via N-alkylation of tranylcypromine and demonstrated its ability to eradicate CNS leukemia in a murine T-ALL model. The brain-permeable LSD1 inhibitor S2157 would be highly beneficial for T-ALL patients with CNS involvement.JX-594 (Pexa-vec) is one of the most promising oncolytic virus platforms in clinical development as one of the few oncolytic viruses in phase III clinical trials. Chon and colleagues show that a murine version of JX-594 (JX) remodels the tumor microenvironment by facilitating the accumulation of T cells. As a result, poorly immunogenic tumors become sensitive to cancer immunotherapy. Of note, the triple-combination therapy of JX, αPD-1, and αCTLA-4 maximizes anticancer immunity and induces durable regression with improved overall survival. These findings demonstrate the potential of JX in combination with immune checkpoint inhibitors for improving anticancer immune responses.Tumor-infiltrating NK cell (TI-NK) numbers were an independent predictor of pathological complete response, a surrogate marker of patient outcome, to anti-HER2 antibody-based treatment in primary HER2 positive breast cancer. The findings of Muntasell and colleagues add strong support for a role of antibody-dependent cellular cytotoxicity for the antitumor effects of anti-HER2 antibodies and, hence, for future research strategies to overcome resistance directed at the recruitment of NK cells in NK “cold” tumors. Furthermore, combination of HLA class I expression and TI-NK cell numbers improved the ability to predict long-term clinical outcomes, suggesting new ways to tailor anti-HER2 therapeutic strategies.

Abstract CT147: Safety and efficacy of high purity and activity NK cells therapy in combination with IgG1 antibody in patients with gastric or colorectal cancer: A phase I clinical trial

Abstract Background; Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells, and produce numerous cytokines resulting in the activation of the adoptive immune systems. Thus, NK cell is considered ideal cell for adoptive cancer immunotherapy. But the difficulty of obtaining large numbers of NK cells that are safe to administer deters its clinical use. We successfully generated large numbers of activated NK cells from small quantities of blood and also determined that the expanded cells were safe to administer in a monotherapy (J Transl Med (2015) 13:277). Since NK cells play a key role in antibody-dependent cellular cytotoxicity (ADCC), administration of NK cells would be expected to enhance the efficacy of IgG1 monoclonal antibodies by augmenting ADCC. This phase I clinical trial evaluated the safety, toxicity, and immunological response of the adoptive NK therapy in combination with IgG1 antibodies. Patients and Methods; Patients with unresectable advanced gastric or colorectal cancer who have administered or have planned to administer IgG1 antibody (i.e. trastuzumab for HER2 positive gastric cancer or cetuximab for RAS-wild colorectal cancer). NK cells were expanded from PBMCs with the same method in previous clinical trial, briefly ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296-induced T cells. Expanded NK cells were intravenously injected 3 days after IgG1 antibody administration in a dose-escalating manner (dose 0.5 × 109, 1.0 × 109, 2.0 × 109 cells/injection, three patients/one cohort), every 3 weeks for 3 cycles. We evaluated the safety and efficacy of the combination therapy. To assess the immunological response, immunomonitoring was performed. Results; Nine eligible patients were enrolled. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D(97.6%) and CD16(69.6%). This combination therapy was well tolerated with no severe adverse events. Among six evaluable patients, 4 presented SD, 2 presented PD. Of the four SD patients, 3 showed an overall decrease in tumor size after combination therapy. In immunomonitoring analysis, whole blood IFN-gamma production level was increased and the proportion of Treg (Treg/CD4) in peripheral blood was decreased after the combination therapy. Conclusions; This is the first clinical trial of the combination of adoptive NK cell therapy and IgG1 monoclonal antibodies. Our data provide evidence of good tolerability and preliminary anti-tumor activity for this combination therapy. Moreover, immunological monitoring revealed that the combination therapy induced Th1-type immune response and reduced peripheral Tregs. However, given the limited activity observed in this clinical trial, additional studies are necessary to develop these combination approaches. Citation Format: Takeshi Ishikawa, Tetsuya Okayama, Naoyuki Sakamoto, Mitsuko Ideno, Kaname Oka, Tatsuji Enoki, Junichi Mineno, Hideyuki Konishi, Satoshi Kokura, Yuji Naito, Yoshito Itoh. Safety and efficacy of high purity and activity NK cells therapy in combination with IgG1 antibody in patients with gastric or colorectal cancer: A phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT147.

NK cells inhibit Plasmodium falciparum growth in red blood cells via antibody-dependent cellular cytotoxicity.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite-host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

Characterization of Therapeutic Monoclonal Antibodies by Using FcγR-expressing Reporter Cell Lines.

Fcγ receptors (FcγRs), which bind to the Fc regions of antibodies, play an important role in antibody effector functions. In humans, there are four types of activating FcγRs: FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb. These are expressed on various effector cells such as natural killer (NK) cells, neutrophils and macrophages. FcγRIIIa expressed on NK cells is known to play a pivotal role in antibody-dependent cellular cytotoxicity (ADCC) by therapeutic monoclonal antibodies (mAbs). To assess the ADCC activity of mAbs, the killing of target cells is often measured using human peripheral mononuclear blood cells (hPBMCs) or isolated primary NK cells as effector cells. These assays can directly assess the cytotoxicity induced by mAbs, but require fresh blood from donors, and are insufficiently reproducible due to differences in effector cell activity among donors. We developed a cell-based assay using reporter cell lines expressing human FcγR and a nuclear factor of activated T cells (NFAT)-driven luciferase reporter gene (Jurkat/FcγR/NFAT-Luc), which can estimate the activation of various FcγRs by antigen-bound mAbs in vitro, with high reproducibility. The usefulness of this assay was confirmed by comparing mAbs activity with different abilities to activate FcγRs, including Fc-engineered anti-CD20 mAbs and anti-EGFR mAbs with different IgG subclasses. We also confirmed the application of this assay for the characterization of mAbs product-related substances. Our FcγR reporter assay is a promising new tool for the characterization of therapeutic mAbs in various stages of mAbs development.

Paring Down HIV Env: Design and Crystal Structure of a Stabilized Inner Domain of HIV-1 gp120 Displaying a Major ADCC Target of the A32 Region

SummaryEvidence supports a role of antibody-dependent cellular cytotoxicity (ADCC) toward transitional epitopes in the first and second constant (C1-C2) regions of gp120 (A32-like epitopes) in preventing HIV-1 infection and in vaccine-induced protection. Here, we describe the first successful attempt at isolating the inner domain (ID) of gp120 as an independent molecule that encapsulates the A32-like region within a minimal structural unit of the HIV-1 Env. Through structure-based design, we developed ID2, which consists of the ID expressed independently of the outer domain and stabilized in the CD4-bound conformation by an inter-layer disulfide bond. ID2 expresses C1-C2 epitopes in the context of CD4-triggered full-length gp120 but without any known neutralizing epitope present. Thus, ID2 represents a novel probe for the analysis and/or selective induction of antibody responses to the A32 epitope region. We also present the crystal structure of ID2 complexed with mAb A32, which defines its epitope.

Natural killer cell education does not affect the magnitude of granzyme B delivery to target cells by antibody-dependent cellular cytotoxicity.

Interest in the role of antibody-dependent cellular cytotoxicity (ADCC) in protection from HIV infection has grown since analyses of the RV144 HIV vaccine trial results found ADCC correlated with protection. Natural killer (NK) cells are among the effector cells that mediate ADCC. The level of antibody-induced NK cell activation depends on NK cell education through inhibitory NK cell receptor human leukocyte antigen (HLA) ligand interactions. Here, we investigated the impact of NK cell education on the delivery of Granzyme B (GzB) to target cells. Lymphocytes from 50 HIV-uninfected [30 Bw4 (Bw4) and 20 Bw4 (Bw6)] KIR3DL1 homozygote persons were used as effectors and cocultured with gp120-coated target cells in the presence of a single source of anti-HIV gp120 antibody to ascertain whether NK cell education status influenced the level of GzB delivered to target cells. The GTL assay assessed the frequency of GzB-positive (%GzB) CEM.NKr.CCR5 target cells generated by effectors from each individual. The frequency of CD107a, interferon (IFN)-γ and CCL4 NK cells was assessed as a measure of antibody-induced NK cell activation. KIR3DL1 NK cells from the Bw4 group were more functional than KIR3DL1 NK cells. Despite this, the %GzB target cells generated in the GTL assay did not differ according to the KIR3DL1-HLA-B genotype of the effector cells. The %GzB cells positively correlated with the frequency of CD16KIR3DL1 NK cells in the effector population. ADCC potency does not depend on NK cell education.

Development of a cell-based assay measuring the activation of FcγRIIa for the characterization of therapeutic monoclonal antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.

High Antibody-Dependent Cellular Cytotoxicity Responses Are Correlated with Strong CD8 T Cell Viral Suppressive Activity but Not with B57 Status in HIV-1 Elite Controllers

The role of Antibody-dependent cellular cytotoxicity (ADCC) responses in HIV-1 controllers is still unclear due to the heterogeneity of these patients. We analyzed 67 HIV-1 controllers and found significantly higher levels of ADCC antibodies in controllers versus viremic subjects (p = 0.017). Moreover, multivariate analysis revealed significantly higher ADCC titers in HLA B57- controllers compared to HLA-B57+ ones (p = 0.0086). These data suggest a role for ADCC in immune control of HIV, especially in HLA B57 negative controllers.

Abstract 2847: Molecular determinants of sensitivity and resistance to tumor-directed antibody-dependent cellular cytotoxicity (ADCC).

Abstract Monoclonal antibodies can induce antibody-dependent cellular cytotoxicity (ADCC) through immune effector cells, such as natural killer (NK) cells. Although clinical evidence suggests a role for ADCC in effective antibody therapy of cancer, molecular determinants of ADCC responsiveness—in oncogenic signaling networks, for example—have not been well characterized. Using an in vitro model system including an NK-like cell line (NK92-CD16V), cetuximab, and A431 cells, we screened 60 genes from an EGFR gene network by RNAi to assess for synthetic enhancement of ADCC. Primary and validation screens, and correlative characterization studies, identified four genes whose knockdown enhanced ADCC: GRB7, PRKCE, RET, and ABL1. Inhibition of Abl kinase activity enhanced ADCC, phenocopying the effects of ABL1 knockdown and providing pre-clinical evidence for combining Abl inhibitors and cetuximab. Next, we derived an ADCC-resistant cell population (A431/ADCC-R) by treating parental A431 cells with over 30 successive ADCC treatments. We describe the development, screening, and characterization of A431/ADCC-R. Our screening strategy has revealed molecular determinants of both tumor cell sensitivity and resistance to ADCC in vitro that may translate into increased monoclonal antibody efficacy in the clinic. Citation Format: Joseph C. Murray, Rochelle E. Nasto, Sandra A. Jablonski, Yong Tang, Louis M. Weiner. Molecular determinants of sensitivity and resistance to tumor-directed antibody-dependent cellular cytotoxicity (ADCC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2847. doi:10.1158/1538-7445.AM2013-2847

Specific antibody‐dependent cellular cytotoxicity responses associated with slow progression of HIV infection

Antibody-dependent cellular cytotoxicity (ADCC) is potentially an effective adaptive immune response to HIV infection. However, little is understood about the role of ADCC in controlling chronic infection in the small number of long-term slow-progressors (LTSP) who maintain a relatively normal immunological state for prolonged periods of time. We analysed HIV-specific ADCC responses in sera from 139 HIV(+) subjects not on antiretroviral therapy. Sixty-five subjects were LTSP, who maintained a CD4 T-cell count > 500/μl for over 8 years after infection without antiretroviral therapy and 74 were non-LTSP individuals. The ADCC responses were measured using an natural killer cell activation assay to overlapping HIV peptides that allowed us to map ADCC epitopes. We found that although the magnitude of ADCC responses in the LTSP cohort were not higher and did not correlate with CD4 T-cell depletion rates, the LTSP cohort had significantly broader ADCC responses compared with the non-LTSP cohort. Specifically, regulatory/accessory HIV-1 proteins were targeted more frequently by LTSP. Indeed, three particular ADCC epitopes within the Vpu protein of HIV were recognized only by LTSP individuals. Our study provides evidence that broader ADCC responses may play a role in long-term control of HIV progression and suggests novel vaccine targets.

Critical role for monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity

Background Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. Considerable evidence supports a role for ADCC activity in the control of HIV-1 infection and in the context of vaccination. One method widely used to assess the role of ADCC is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. In the RFADCC assay specific killing of target cells by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26 +), which is assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We have revisited this assay to assess the role of effector cells in mediating ADCC.

Genetic polymorphisms of FCGR2A encoding Fcγ receptor IIa in a Japanese population and functional analysis of the L273P variant

Fcγ receptor IIa (FcγRIIa) plays an important role in antibody-dependent cellular cytotoxicity and inflammation. Changes in FcγRIIa expression levels or activity caused by genetic polymorphisms in FCGR2A, the gene encoding FcγRIIa, may lead to differences in disease progression as well as efficacy of antibody therapeutics between individuals. In this study, we sequenced the 5'-flanking region along with all exons and their flanking regions of FCGR2A from 111 Japanese subjects. Forty-eight genetic variations were found including 12 novel ones. Beside the well-known functional 497A > G (H166R) polymorphism, we detected 818T > C (L273P) at 0.005 frequency. Since the functional significance of this polymorphism has not been revealed, we next assessed the functions of the L273P substitution by expressing wild-type and the variant proteins in human Jurkat cells. The L273P variant protein showed similar cell surface expression and IgG-binding properties as the wild-type, but it exhibited a stronger signal transduction activity based on the approximately 2-fold enhancement of tyrosine phosphorylation of FcγRIIa itself. The current results suggest that L273P could have functional significance in the antibody-dependent clinical responses through FcγRIIa.

Abstract LB-128: Targeted RNAi screening of an EGFR network reveals molecular determinants of tumor cell sensitivity to antibody-dependent cellular cytotoxicity (ADCC)

Abstract Monoclonal antibodies can modulate signal transduction and survival of tumor cells, and can induce antibody-dependent cellular cytotoxicity (ADCC) through immune effectors, such as natural killer (NK) cells. Although clinical evidence suggests a role for ADCC in effective antibody therapy, molecular determinants of ADCC responsiveness—in oncogenic signaling networks, for example—have not been identified. Using a novel high-throughput screening platform, siRNAs targeting 60 genes of an EGFR network were reverse transfected into A431 cells, and cetuximab and an NK-like cell line (NK92-CD16V) were used to induce ADCC. Primary and validation screens identified four genes whose knockdown enhanced ADCC: GRB7, PRKCE, RET and ABL1. Pharmacological inhibition of Abl kinase activity enhanced ADCC, recapitulating the effects of ABL1 gene knockdown. Additional studies revealed distinct mechanisms associated with enhanced ADCC, including increased EGFR surface expression and enhanced susceptibility to apoptosis. This screening strategy has revealed putative molecular determinants of tumor cell sensitivity to ADCC that may translate to increased monoclonal antibody efficacy in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-128. doi:1538-7445.AM2012-LB-128

Immune escape from HIV-specific antibody-dependent cellular cytotoxicity (ADCC) pressure

Effective immunity to HIV is poorly understood. In particular, a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV is controversial. We hypothesized that significant pressure from HIV-specific ADCC would result in immune-escape variants. A series of ADCC epitopes in HIV-infected subjects to specific consensus strain HIV peptides were mapped using a flow cytometric assay for natural killer cell activation. We then compared the ADCC responses to the same peptide epitope derived from the concurrent HIV sequence(s) expressed in circulating virus. In 9 of 13 epitopes studied, ADCC antibodies were unable to recognize the concurrent HIV sequence. Our studies suggest ADCC responses apply significant immune pressure on the virus. This result has implications for the induction of ADCC responses by HIV vaccines.

The broadly neutralizing HIV-1 IgG 2F5 elicits gp41-specific antibody-dependent cell cytotoxicity in a FcγRI-dependent manner

A role for antibody-dependent cellular cytotoxicity (ADCC) in controlling initial development of HIV-1 infection is supported by a growing number of studies. 2F5, a broadly HIV-1-neutralizing IgG specific for HIV-1 envelope gp41, has been extensively studied in vitro and in vivo for its neutralizing and transcytosis-blocking activities. In the present paper, we have studied the in vitro ADCC potential of 2F5. We have developed an ADCC model based on either monocytic cell line THP1 or monocytes, both FcγRI(+) FcγRIII(-) as effector cells, and natural killer resistant-CEM (NKr-CEM) either coated with HIV envelope subunit, or stably expressing an X4 tropic HIV-1 envelope as target cells. Finally, in order to better simulate the in vivo situation, we used R5-tropic JR-CSF HIV-1-infected NKr-CEM as targets. ADCC was monitored using a fluorescently based, nonradioactive, easy to use assay. 2F5 triggered ADCC of HIV-1 envelope subunit coated cells. Remarkably, 2F5 at ng/ml concentration elicited ADCC of both X4-tropic HIV-1 envelope-expressing cells, and R5-HIV-infected cells. ADCC relied on binding to the FcγRI on effector cell and was abolished by preincubation of 2F5 with its cognate epitope ELDKWA. The capacity of the broadly neutralizing 2F5 to elicit ADCC, and thereby linking adaptive and innate immunity, expands its prophylactic potential. Raising antibodies to the membrane proximal region of HIV-1 envelope with similar ADCC properties, in addition to neutralization, should be taken into account in HIV-1 vaccine design.