Increase In Antibody-dependent Cellular Cytotoxicity Research Articles

Assessment of Broadly Reactive Responses in Patients With MERS-CoV Infection and SARS-CoV-2 Vaccination

In the ongoing COVID-19 pandemic, there remain unanswered questions regarding the nature and importance of the humoral immune response against other coronaviruses. Although coinfection of the Middle East respiratory syndrome coronavirus (MERS-CoV) with the SARS-CoV-2 has not been documented yet, several patients previously infected with MERS-CoV received the COVID-19 vaccine; data describing how preexisting MERS-CoV immunity may shape the response to SARS-CoV-2 following infection or vaccination are lacking. To characterize the cross-reactive and protective humoral responses in patients exposed to both MERS-CoV infection and SARS-CoV-2 vaccination. This cohort study involved a total of 18 sera samples collected from 14 patients with MERS-CoV infection before (n = 12) and after (n = 6) vaccination with 2 doses of COVID-19 mRNA vaccine (BNT162b2 or mRNA-1273). Of those patients, 4 had prevaccination and postvaccination samples. Antibody responses to SARS-CoV-2 and MERS-CoV were assessed as well as cross-reactive responses to other human coronaviruses. The main outcomes measured were binding antibody responses, neutralizing antibodies, and antibody-dependent cellular cytotoxicity (ADCC) activity. Binding antibodies targeting SARS-CoV-2 main antigens (spike [S], nucleocapsid, and receptor-binding domain) were detected using automated immunoassays. Cross-reactive antibodies with the S1 protein of SARS-CoV, MERS-CoV, and common human coronaviruses were analyzed using a bead-based assay. Neutralizing antibodies (NAbs) against MERS-CoV and SARS-CoV-2 as well as ADCC activity against SARS-CoV-2 were assessed. A total of 18 samples were collected from 14 male patients with MERS-CoV infection (mean [SD] age, 43.8 [14.6] years). Median (IQR) duration between primary COVID-19 vaccination and sample collection was 146 (47-189) days. Prevaccination samples had high levels of anti-MERS S1 immunoglobin M (IgM) and IgG (reactivity index ranging from 0.80 to 54.7 for IgM and from 0.85 to 176.3 for IgG). Cross-reactive antibodies with SARS-CoV and SARS-CoV-2 were also detected in these samples. However, cross-reactivity against other coronaviruses was not detected by the microarray assay. Postvaccination samples showed significantly higher levels of total antibodies, IgG, and IgA targeting SARS-CoV-2 S protein compared with prevaccination samples (eg, mean total antibodies: 8955.0 AU/mL; 95% CI, -5025.0 to 22936.0 arbitrary units/mL; P = .002). In addition, significantly higher anti-SARS S1 IgG levels were detected following vaccination (mean reactivity index, 55.4; 95% CI, -9.1 to 120.0; P = .001), suggesting potential cross-reactivity with these coronaviruses. Also, anti-S NAbs were significantly boosted against SARS-CoV-2 (50.5% neutralization; 95% CI, 17.6% to 83.2% neutralization; P < .001) after vaccination. Furthermore, there was no significant increase in antibody-dependent cellular cytotoxicity against SARS-CoV-2 S protein postvaccination. This cohort study found a significant boost in cross-reactive NAbs in some patients exposed to MERS-CoV and SARS-CoV-2 antigens. These findings suggest that isolation of broadly reactive antibodies from these patients may help guide the development of a pancoronavirus vaccine by targeting cross-reactive epitopes between distinct strains of human coronaviruses.

KLRG1 Depletion Is a Novel Therapeutic Strategy for Patients with Mature T and NK/T-Cell Lymphomas

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Killer cell lectin-like receptor G1 (KLRG1) is a co-inhibitory immune checkpoint receptor expressed on late differentiated CD8+ T effector memory (TEM) and TEMRA cells. Monotherapy with KLRG1 neutralizing antibodiesextended survival in solid tumor models. Here, we report the therapeutic potential of a novel and distinct strategy characterized by depletion of KLRG1+ malignant cells in hematologic malignancies and specifically in TCLs. We defined the molecular epidemiology of KLRG1 by determining tumor cell-specific RNA and protein expression on T cell subsets from 8 healthy donors, 26 TCL lines, 15 patient-derived xenografts (PDXs), and 165 tumor and blood specimens representing various subtypes of T and NK/TCLs. Microarray andRNA-seqdata demonstrated highertranscript levels of KLRG1 in patients with T-LGLL and PDXs of T-PLL and HSTCL relative to other subtypes, which were corroborated by protein expression. We quantified tumor cell-specific protein expression of KLRG1 by IHC by generating H scores (% of KLRG1+ tumor cells × measure of staining intensity) ranging from 0-300 across 141 primary TCL biopsies. PTCL-NOS and AITL had heterogeneous KLRG1 expression, whereas ALCL, CTCL, and EATL demonstrated none. The majority of patients with T-PLL (median 180), δ/γ TCLs (median 140), and NK/TCLs (median 100) exhibited higher H scores relative to other subtypes. Up to 99% of the CD3+/CD8+/CD57+ and up to 97% of the CD3-/CD56+/CD57+/CD16+/CD94+ leukemic cells in the blood of patients with T-LGLL (n=12) and CLPD-NK (n=12) respectively expressed KLRG1 protein (Fig.1). None of the TCL cell lines expressed surface KLRG1. Therefore, SMZ1 cells (TP63 rearranged PTCL-NOS line) were stably transduced to express KLRG1. We utilized two human KLRG1 binding antibodies, 13F12F2 (a commercial mIgG2a Ab) and mAb208 (a humanized afucosylated mIgG2a Ab) for in vitro and in vivo studies. Antibody binding capacity assays confirmed a higher number of KLRG1 receptors per cell on SMZ1 KLRG1+ (175,800), HSTCL (23,586), and CD8+ T cells (16,326) compared with B-cell lymphoma cells (242). Competition binding assays demonstrated that concentrations of 0.1 ug/ml of mAb208 saturated receptors on SMZ1 KLRG1+ cells. mAb208 induced a 2-5-fold dose-dependent increase in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), but not apoptosis of SMZ1 KLRG1+ cells relative to SMZ1 KLRG1- WT and isotype control (CTL). This was exhibited across a variety of human and murine effectors such as human PBMCs, human and murine macrophages, and human and murine complements. Reporter-based murine FcγRIV ADCC bioassay demonstrated a dose-dependent 2-6 fold induction of bioluminescence by mAb208 in SMZ1 KLRG1+ cells relative to WT (p=<0.0001). In 4/4 tested T-LGLL patient samples, mAb208 induced ADCC of CD8+/CD57+ LGLs with autologous PBMCs (p=0.03) relative to CTL. Finally, mAb208 induced selective ADCC of KLRG1+ CD8+ TEM and TEMRA cells while sparing KLRG1- naive and central memory T cells in 3 healthy donors (p=0.06). Duvelisib (Duv), a dual PI3K-δ/γ inhibitor (PI3Ki), has demonstrated a high ORR of 50% in phase II trials in TCLs and its ability to reprogram macrophages to an M1 phenotype in TCL PDXs. Thus, we hypothesized that the combination of PI3Ki with anti-KLRG1 mAbs would enhance the clearance of apoptotic and opsonized KLRG1+ TCL cells by proinflammatory macrophages in several PTCL subtypes. We tested this combination in vivo using SMZ1 KLRG1+ subcutaneous xenografts in NSG mice. After 17 days of treatment, combination (mAb208 + Duv)- treated mice had a marked reduction in their mean tumor volumes (159 mm3) relative to mAb208 (1003 mm3 ; p=0.03), Duv (465.6 mm3 ; p=0.03) and CTL (2104 mm3 ; p=0.03) mice resulting in prolonged survival (Fig. 2). Studies in PDX models of T-PLL and HSTCL with varying levels of KLRG1 expression (30% and 80% respectively) have been initiated and will be reported at the meeting. Selective depletion of tumor cells with an anti-KLRG1 depleting mAb in specific subtypes of TCLs, T-LGLL, and CLPD-NK is effective in vitro and in vivo, and its combination with Duv has marked activity in an aggressive PTCL-NOS xenograft model. A multicenter phase I/II trial of the KLRG1 depleting antibody ABC008 (Abcuro) in patients with R/R T-LGLL has been initiated. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Bicistronic Vector Expressing CD16 and a Membrane Bound IL-15 Construct in iPSC Derived NK Cells Increased Cytotoxicity and Persistence

Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability.Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence.CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity.The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time.In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. DisclosuresAllen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Antibody–receptor interactions mediate antibody-dependent cellular cytotoxicity

Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel mAb biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated monoclonal antibodies demonstrate increased affinity toward the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity. Crystal structure analysis has led to the hypothesis that afucosylation in the Fc region results in reduced steric hindrance between antibody–receptor intermolecular glycan interactions, enhancing receptor affinity; however, solution-phase data have yet to corroborate this hypothesis. In addition, recent work has shown that the fragment antigen-binding (Fab) region may directly interact with Fc receptors; however, the biological consequences of these interactions remain unclear. By probing differences in solvent accessibility between native and afucosylated immunoglobulin G1 (IgG1) using hydroxyl radical footprinting–MS, we provide the first solution-phase evidence that an IgG1 bearing an afucosylated Fc region appears to require fewer conformational changes for FcγRIIIa binding. In addition, we performed extensive molecular dynamics (MD) simulations to understand the molecular mechanism behind the effects of afucosylation. The combination of these techniques provides molecular insight into the steric hindrance from the core Fc fucose in IgG1 and corroborates previously proposed Fab–receptor interactions. Furthermore, MD-guided rational mutagenesis enabled us to demonstrate that Fab–receptor interactions directly contribute to the modulation of antibody-dependent cellular cytotoxicity activity. This work demonstrates that in addition to Fc–polypeptide and glycan-mediated interactions, the Fab provides a third component that influences IgG–Fc receptor biology.

A general Fc engineering platform for the next generation of antibody therapeutics.

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform.Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8+ T cell response model in FcγR/CD40-humanized mice.Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models.Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.

Abstract B082: Reovirus treated NK cells exhibit enhanced cetuximab mediated antibody- dependent cellular cytotoxicity against colorectal cancer cell lines

Abstract The naturally occurring oncolytic virus, reovirus, exhibits cytotoxic effects on cancer cells with an activated RAS-signaling pathway. In addition to their direct cytotoxic effects they also activate the innate and adaptive immune responses to facilitate tumor clearance. Reovirus is safe, and currently is in clinical testing for the treatment of multiple clinical cancer histologies. NK cells are innate immune effectors that mediate antibody dependent cellular cytotoxicity (ADCC) against tumor cells. Here we investigated the direct effect of reovirus on NK cell mediated ADCC against two EGFR (Epidermal Growth Factor) positive colorectal cancer cell lines: DLD-1 (KRAS mut) and Caco-2 (KRAS WT). NK cells isolated from human PBMCs were cultured with 1pfu of reovirus for 12 hrs. These reovirus treated NK cells were co-cultured with DLD-1 cells or Caco2 cells (E:T-1:1) coated with increasing concentrations anti-EGFR antibody cetuximab. ADCC was measured after 4hrs using a lactate dehydrogenase (LDH) based cytotoxicity assay. We observed that the reovirus treated NK cells (Reo-NK cells) exhibited a ~16-fold increase in cytotoxicity against DLD-1 (16.3% ±1.5, n=3) compared to untreated NK cells (NK cells), even in the absence of any cetuximab antibody. In the presence of cetuximab antibody, NK cells showed a dose dependent increase in ADCC, with maximum ADCC, observed at 0.1 µg/ml of cetuximab (DLD-NK: 33.4%± 7.1, Caco2-NK: 5.7%± 0.071, n=3). Interestingly, Reo-NK cells showed maximum ADCC even at 0.01 µg /ml of cetuximab (DLD1-Reo-NK: 39.1±7.4, DLD1-NK: 26.7±2.4%; Caco2-Reo-NK: 9.2±0.4, Caco2-NK: 4.0±0.8, n=3). To further investigate the factors contributing to the increased cytotoxic potential of Reo-NK cells we performed flow cytometry analysis to determine the expression of activation and degranulation markers on NK cells. We observed that in presence of tumor cells Reo-NK cells exhibited a 2-fold increased expression of activation marker CD69 when compared to untreated NK cells (Reo-NK:70.4%, NK:35.2%). A similar increase was also observed when Reo-NK cells were co-cultured with target cells coated with 0.01 µg /ml of cetuximab (Reo-NK: 85.1%; NK: 72.7%). Expression of the degranulation marker CD107a is increased on NK cells during ADCC. There was a ~3-fold increase in expression of CD107a on Reo-NK cells when compared to NK cells (Reo-NK: 14.6%; NK: 4.45%). To further investigate if perforin-based cytotoxicity was responsible for the increased ADCC by Reo-NK cells, we treated Reo-NK and NK cells with concanamycin A (CMA), an inhibitor of perforin. We found that CMA treatment reduced Reo-NK cell mediated ADCC by 1.5 fold (CMA treated-28.2%, CMA untreated 18.9%) indicating a perforin-mediated cytotoxicity contributes to the increased cytotoxicity of Reo-NK cells. Thus, in this study our results demonstrated that human NK cells when treated with reovirus show increases in activation, degranulation and cytotoxicity when compared to untreated NK cells. Importantly, reovirus treated NK cells lowered the threshold of cetuximab required to achieve maximum ADCC. We propose that reovirus activated NK cells are a potential candidate for cell based immunotherapy in combination with FDA approved tumor targeting antibodies. Further studies are ongoing to investigate the underlying mechanisms that contribute to the increase in cytotoxicity by NK cells treated with reovirus. Citation Format: Xing Zhao, Narendiran Rajasekaran, Cariad Chester, Atsushi Yonezawa, Suparna Dutt, Matt Coffey, Holbrook Kohrt. Reovirus treated NK cells exhibit enhanced cetuximab mediated antibody- dependent cellular cytotoxicity against colorectal cancer cell lines. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B082.

Natural Killer Cells Activated By Oncolytic Reovirus Enhance Cetuximab Mediated Antibody Dependent Cellular Cytotoxicity in an in Vitro and In Vivo Model of Colorectal Cancer

The naturally occurring oncolytic virus, reovirus, exhibits cytotoxic effects on cancer cells. Reovirus is currently being tested in multiple clinical trials for the treatment of different cancers. In addition, they also activate the innate and adaptive immune responses targeting immune cells like dendritic cells and macrophages. In this study we investigated the direct effect of reovirus on Natural Killer cells (NK cells) and its effect on NK cell mediated antibody dependent cellular cytotoxicity (ADCC) against the EGFR (Epidermal Growth Factor) positive colorectal cancer cell line: DLD-1 (KRAS mutant). NK cells isolated from human PBMCs were cultured with 1pfu of reovirus for 12 hrs and subsequently co-cultured with DLD-1 cells coated with increasing concentrations anti-EGFR antibody cetuximab. ADCC was measured after 4 hrs using a lactate dehydrogenase (LDH) based cytotoxicity assay. We observed that the reovirus treated NK cells (Reo-NK cells) exhibited a ~16-fold increase in cytotoxicity against DLD-1 (16.3% ±1.5, n=3) compared to untreated NK cells (NK), even in the absence of any cetuximab. In the presence of cetuximab, NK cells showed a dose dependent increase in ADCC, with maximum ADCC, observed at 0.1 µg/ml of cetuximab (DLD-1+NK: 33.4%± 7.1, n=3). Interestingly, Reo-NK cells showed maximum ADCC even at 0.01 µg /ml of cetuximab (DLD-1+Reo-NK: 39.1±7.4, DLD-1+NK: 26.7±2.4%, n=3). Reo-NK cells also exhibited an increased expression of activation marker CD69 (Reo-NK: 70.4%, NK: 35.2%) and degranulation maker CD107a (Reo-NK: 14.6%; NK: 4.45%) compared to the untreated NK cells. We further characterized the Reo-NK cells by using the HIMChip microarray platform; a custom Agilent SurePrint HD 8x15k format array containing over 7,000 unique probes for over 4,274 human immune-related genes. In ingenuity pathway analysis, we observed that the Interferon pathway (2.13E-20) and pathway controlling activation of IRF by cytosolic pattern recognition receptors (1.27E-11) were the predominant pathways observed in the Reo-NK cells. These results suggest an interferon-mediated response could be contributing to the increased cytoxicity of the NK cells. In an in vivo study, DLD-1 cells were grown subcutaneously in athymic nude mice and injected intravenously with reovirus (5x 108 pfu), followed by intraperitoneal injection of Cetuximab (200 ug/mice) every week. We observed a significant regression of tumors in the Reovirus+Cetuximab combination group compared to the Reovirus treated (Reovirus+Cet: 349.9 mm3, Reovirus: 623.8 mm3; n=9; P=0.0028) or Cetuximab treated (Reovirus+Cet: 349.9 mm3, Cet: 730.5 mm3;n=9; P= 0.030) groups on day 28 post treatment. Thus, in this study our results demonstrated that human NK cells when treated with reovirus show increases in activation, degranulation and cytotoxicity when compared to untreated NK cells. Further, in the in vivo model we observed increased tumor regression in mice treated with reovirus in combination with cetuximab. We propose that reovirus activated NK cells are a potential candidate for cell based immunotherapy in combination with FDA approved tumor targeting antibodies to treat malignancies, including lymphomas. Further studies are ongoing to investigate the underlying mechanisms that contribute to the increase in cytotoxicity by NK cells treated with reovirus. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1332: Interleukin-15 enhances rituximab-dependent cytotoxicity ex vivo and in vivo against a mouse lymphoma expressing human CD20

Abstract Rituximab (RTX), an anti-CD20 antibody, revolutionized treatment for B-cell malignancies, but it is not without its own shortcomings, most notably tumor relapse. Recent research has provided evidence supporting the increased efficacy of RTX when combined with interleukin-15 (IL-15). IL-15 enhances antibody-dependent cellular cytotoxicity (ADCC), an important mechanism of RTX, by increasing the proliferation and activation of natural killer (NK) cells, as well as monocytes and macrophages. However, the majority of this evidence has been obtained through in vitro experiments and in vivo models using xenografts in immuno-deficient mice. Given the complexity of the immune system, we used an immuno-competent, syngeneic mouse model of human B-cell lymphoma to further investigate the effect of combining IL-15 with RTX to enhance ADCC. Wild-type (WT) C57BL/6 mice (n = 40) were distributed into treatment groups of 10 mice each, and inoculated intravenously with EL4-CD20 cells, a mouse lymphoma line transfected with human CD20. IL-15 was given five times per week for 4 weeks (5μg/mouse), starting on day 3 after tumor inoculation, and RTX was given once per week for 4 weeks (100 μg/mouse), starting on day 5. While IL-15 and RTX individually prolonged survival of the mice when compared with the control (p&amp;lt;0.05), the combination of IL-15 and RTX showed an additive effect, prolonging mouse survival significantly when compared with the monotherapies alone (p&amp;lt;0.01). At day 75 after tumor inoculation, 90% of the mice in the combination group were still alive. In comparison, only 30% of the mice in each of the IL-15 and RTX groups and none of the mice in the control group were alive. In contrast, treatment of EL4-CD20-bearing FcRγ-/- mice with RTX, under the same dose and dosing schedule as those used in WT mice, did not show a therapeutic effect when compared with the control. We further confirmed the role of ADCC by performing an ex vivo experiment using NK cells purified from splenocytes of either WT or FcRγ-/- mice treated with IL-15 five days before extraction. NK cells were cultured in triplicate with 51Cr-labeled EL4-CD20 cells with or without RTX and lysis was evaluated using a chromium-51 release assay. NK cells from IL-15-treated WT mice combined with RTX resulted in a three-fold increase in ADCC when compared with the monotherapies alone (16% combination vs. 6% IL-15 and 5% RTX, at a 10:1 E:T ratio). In contrast, NK cells from FcRγ-/- mice showed no difference in ADCC with or without RTX treatment. In agreement with previously published research, our study gives further evidence that IL-15 increases the efficacy of RTX, primarily through enhancing ADCC. Confirming this synergistic effect in an immuno-competent mouse model better supports a future study of RTX with IL-15 in human clinical trials for indolent B-cell lymphomas. Citation Format: Bernard Wen, Meili Zhang, David Dilillo, Jeffrey V. Ravetch, Thomas A. Waldmann. Interleukin-15 enhances rituximab-dependent cytotoxicity ex vivo and in vivo against a mouse lymphoma expressing human CD20. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1332. doi:10.1158/1538-7445.AM2015-1332

HIV-specific ADCC improves after antiretroviral therapy and correlates with normalization of the NK cell phenotype.

Natural killer (NK) cell phenotype and function have recently gained much attention as playing crucial roles in antibody-dependent cellular cytotoxicity (ADCC). We investigated NK cell function, as measured by ADCC, in HIV-1-positive individuals before and 6 months after highly active antiretroviral therapy (HAART) initiation. The ability of antibodies and NK cells to mediate ADCC was investigated separately and in combination in an autologous model. The NK cell subset distribution and NK cell phenotype (ie, expression of maturation and activation markers within NK cell subsets) were analyzed. The ability of NK cells to mediate ADCC was significantly increased after only 6 months of HAART and was not explained by a normalization of NK cell subsets (CD56 CD16 and CD56 CD16 NK cells) but rather by normalization in the frequency of NK cells expressing CCR7 and CD27. For individuals with no increase in ADCC after 6 months of HAART, the frequency of NK cells expressing NKp46 was downregulated. The ability of antibodies to mediate ADCC alone and in combination in an autologous model was not improved. HAART improves the ability of NK cells to mediate ADCC after 6 months. This improvement does not correlate with general immune restoration, as measured by CD4 T-cell counts, but rather to a decrease in the frequency of NK cells expressing CCR7 and CD27.

Effect of trastuzumab on antibody-dependent cellular cytotoxicity (ADCC) in HER2 nonamplified (non-amp) breast cancer (BC) cells.

659 Background: Trastuzumab (T) is currently indicated for the treatment of Her-2 positive breast cancers. The NSABP B-31 adjuvant trial suggested a possible clinical benefit for the addition of T to chemotherapy in patients whose tumors were Her-2 negative (low Her-2 expressing, non-amplified by FISH). ADCC mediated by CD56+ natural killer (NK) cells is known to play a role in response to T in Her-2 positive tumors. To investigate the possibility that T may induce ADCC against Her-2 negative tumors, we examined NK cell-mediated ADCC in T-treated, non-amp, low Her-2 expressing BC cell lines. Methods: A membrane/cytosol separation kit and immunoblotting were used to assess surface Her-2 levels in the non-amp BC cell lines CAMA-1, T47D, EFM19, MCF-7 and CAL-51. The positive control for Her-2 expression and amplification was HCC1954 and MDA-MB-468 was the negative control. PBMCs were isolated from whole blood of 8 healthy volunteers using a Ficoll-Plaque method. CD56+ NK cells were isolated from the PBMC fraction using magnetic bead based positive selection. Direct NK activity against the K562 cell line and the ability of the NK cells to induce T-related ADCC were measured using flow cytometry. Results: Membrane expression of Her-2 was detected in all cell lines examined except the MDA-MB-468 cell line. The direct cytotoxicity of NK cells ranged from 42% to 73% (ratio 10:1, NK: K562). T induced a significant ADCC response in the positive control HCC1954 (p value 0.002) and in each of the five non-amp, low Her-2 expressing cell lines, but not in the negative control MDA-MB-468 (Table). Conclusions: These results suggest that Her-2 non-amp BC cells, with low but detectable levels of Her-2 protein, can bind T and initiate ADCC. Future research will include determination of T-induced ADCC response by NK cells from patients with Her-2 non-amp BC. % increase in ADCC in response to 200 nM T (Ratio 1:1, NK: Target cell) HCC1954 CAMA-1 T47D EFM19 MCF-7 CAL-51 MDA-MB-468 Total Her-2 +++ + + + + + - % Increase ADCC ± std. dev. 29 ± 3 43 ± 13 21 ± 3 25 ± 7 31 ± 15 33 ± 8 0.6 ± 1 p value* 0.002 0.019 0.037 0.015 0.026 0.005 0.627 * p value – Student's t test, % ADCC for each cell line ± T in triplicate p<0.05 significant. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Roche Roche

Enhancement of trastuzumab-mediated cellular cytotoxicity by interleukin-21

2567 Background: Interleukin-21, a cytokine produced by activated T cells, has potent effects on growth, survival and activation status of T, B and natural killer (NK) cells. In vitro studies show increased NK cytolytic function following combined human recombinant (rIL-21) stimulation and Fc receptor engagement. We studied the effect of rIL-21 on antibody-dependent cellular cytotoxicity (ADCC) against trastuzumab-coated breast cancer targets. Methods: ADCC assays were performed on peripheral blood mononuclear cell preparations (MNCs) from cynomolgus monkeys and on enriched human NK cells, following in vitro treatment with rIL-21. Cynomolgus monkeys were treated with rIL-21 as part of an in vivo model of ADCC (Hughes, et al., ASCO submitted), and the ex vivo ADCC activity of MNCs from these animals was tested. Whole blood and MNCs were characterized by flow cytometry. Results: Human NK cells incubated with rIL-21 had 2 to 4-fold increased ADCC against breast cancer targets including Her2/neu-low lines MCF7 and HCC38 (p<.02). Cynomolgus MNCs cultured with rIL-21 showed variable ADCC activation. In animals treated with rIL-21, an immediate decline in ADCC activity correlated with the extravasation of NK cells from the peripheral blood. This was followed by a substantial increase in ADCC against BT-474 cells at later timepoints. Normalization for NK cell numbers showed increased lytic efficiency of NK cells, especially after two cycles of treatment. The percentage of perforin-positive MNCs increased following treatment, as did other biomarkers of immune activation including FcγRI and FcγRIII expression. Conclusion: Treatment of cynomolgus monkeys with rIL-21 increased the activity of MNCs in trastuzumab-mediated ex vivo ADCC assays. This lytic activation is consistent with the enhanced effector function observed after in vitro incubation of NK cells or MNCs with rIL-21. Thus, adjuvant treatment with rIL-21 may enhance monoclonal antibody therapy by improving ADCC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ZymoGenetics ZymoGenetics

Complement Activation by HIV-1-Infected Target Cells Enhances IL-2-Stimulated but Not Unstimulated ADCC Activity Mediated by peripheral Blood Mononuclear Cells

The goal of this study was to determine whether deposition of complement C3 breakdown products on the surface of HIV-infected target cells could augment the levels of antibody-dependent cellular cytotoxicity (ADCC) mediated by peripheral blood mononuclear cells (PBMC). Although C3 was deposited on the surface of infected cells in the presence of anti-HIV antiboby from infected persons, no increase in the levels f ADCC mediated by freshly isolated PBMC was seen with either infected H9 or CEM-NKrtraget cells. However, a significant increase in ADCC was observed due to deposition of C3 on target cells with Il-2-stimulated effector cells. these results show that C3 deposition on target cells can increase ADCC cytotoxicity under certain conditions. Complement maythus contribute to destruction of HIV-infected cells through this mechanismin vivo, although these experiments suggest that specific antibody is the major targeting molecule for ADCC.

IL-8 enhances antibody-dependent cellular cytotoxicity in human neutrophils.

Human neutrophils are able to kill in vitro colorectal carcinoma cell line SW11-16 coated with mAb 17-1A, but they are not cytotoxic towards a non-immunized tumour target. Neutrophil exposure to the inflammatory cytokine, IL-8, produces a significant increase in antibody-dependent cellular cytotoxicity, which is related to IL-8 concentration. Oxyradical production is one of the lytic mechanisms used by phagocytes, and IL-8 is shown to activate this function, which does not occur if neutrophils are pretreated with the protein kinase C inhibitor, staurosporine, but is increased by R59022, a dyacylglycerol kinase inhibitor. The IL-8 effect is mediated by protein kinase C, which is potentiated by the calcium flux induced by the interaction between antibody coating tumour target and Fc gamma RIII on effector cells, as previously demonstrated. Data suggest a possible new role for IL-8 in tumour surveillance.

Immunologic Effects of Levamisole in Mice and Humans

Selected immunomodulatory effects of levamisole were studied in patients with asymptomatic metastatic colon cancer and in a preclinical model (CF1 female mice treated with methyl-azoxymethanol acetate) for colon tumors. In the patient population studied, there was no augmentation of cellular cytotoxicity or alteration in lymphocyte subpopulations that participate in these functions. An increase in Fc receptor binding on circulating monocytes was apparent at the 4-week timepoint; however, a corresponding increase in antibody-dependent cellular cytotoxicity was observed in only one of the six patients studied. In most patients, cellular cytotoxicity diminished with time. No significant effects on cellular immunity or carcinogenesis were observed in our murine studies. However, treatment with levamisole did increase circulating immunoglobulin levels and IgM response in mice immunized with the T-dependent antigen keyhole limpet hemocyanin. This parameter was not tested in the human trial. Failure to demonstrate antitumor effects on cellular immunity by levamisole in both human and murine studies suggests that these effects, if they do exist, may involve immunological parameters that were not tested using our methods or that may not be apparent in patients with more advanced malignancy.

Characterization of a human macrophage-like cell line stimulated in vitro: a model of macrophage functions.

Abstract When cells of human macrophage-like cell line U937 are cultured in the presence of medium conditioned by mixed lymphocyte culture (MLC), PHA- or Con A-stimulated lymphocytes they demonstrate morphologic and functional characteristics of stimulation. The cells become larger, their surface more villous, and the cytoplasm has increased numbers of lysosomes and phagosomes. There is a marked (at least 10-fold) increase in antibody-dependent cellular cytotoxicity (ADCC) against both erythroid and tumor targets accompanied by increased expression of Fc receptors (FcR). There is also an augmentation of antibody-dependent phagocytosis. Medium conditioned by unstimulated lymphocytes, a B lymphoblastoid cell line or a fibroblast cell line produce very little stimulation of U937 cells. U937 cells gradually lose their increased ADCC capacity if they are washed free of conditioned medium (CM) but can be maintained in a stimulated state for long periods of time by culturing them in medium supplemented with PHA-CM at a concentration of 2%. Stimulation of macrophage-like cell line U937 may provide a useful model for further study of mechanisms of macrophage cytotoxicity and its modulation by products of activated lymphocytes.

Comparison of the effector cells in human spontaneous cellular cytotoxicity and antibody-dependent cellular cytotoxicity: differential sensitivity of effector cells to in vivo and in vitro corticosteroids.

The effector cells mediating antibody dependent cellular cytotoxicity (ADCC) and spontaneous cellular cytotoxicity (SCC) in humans have been reported to possess similar characteristics. Multiple cell separation techniques were employed in an attempt to physically separate and distinguish the effector cells in these two types of cellular cytotoxicity. Subpopulations of mononuclear cells obtained by a variety of fractionation procedures which either enriched or depleted monocytes, lymphocytes bearing a receptor for sheep erythrocytes (SRBC), a receptor for complement (CRL) or an Fc receptor for IgG always had similar effects on both ADCC and SCC. Aggregated gamma globulin blockade of Fc receptors produced similar dose-dependent depressions of ADCC and SCC. Despite our inability to physically separate the effector cells of ADCC and SCC, administration of in vivo dexamethasone caused a relative increase in ADCC but a profound decrease in SCC. Furthermore, in vitro dexamethasone in pharmacologic and suprapharmacologic concentrations caused no change in ADCC but significantly decreased SCC. This study demonstrates that although the effector cells cannot be physically separated, ADCC and SCC are differentially sensitive to corticosteroids and are hence functionally distinct either on the basis of different subsets of effector cells with similar surface markers or different mechanisms of cytotoxicity by the same effector cell.