Killer Antibody-dependent Cellular Cytotoxicity Research Articles

Abstract 6582: Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing tumors

Abstract Background: Nectin-4 is a cell membrane adhesion protein overexpressed in several solid tumors, including urothelial, breast, lung, ovarian, and pancreatic cancers, with limited expression in normal tissues, making it an ideal target for antibody-drug conjugates (ADC). Enfortumab Vedotin (EV, PADCEV®) is an ADC targeting Nectin-4 with a monomethyl auristatin E (MMAE) payload approved for the treatment of patients with urothelial carcinoma (UC), but with limited clinical activity reported beyond UC. In addition, EV causes peripheral neuropathy and skin toxicity, leading to treatment discontinuation. To overcome these limitations and the mechanism of resistance to EV, IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 with an improved therapeutic index, was developed. Methods: In vitro studies were performed to investigate internalization, tumor cell killing and bystander effect, antibody-dependent cellular cytotoxicity (ADCC) and complement activation. In vivo efficacy studies were performed on cell line-derived xenografts (CDX) of triple negative breast cancer, patient-derived xenografts (PDX) of urothelial carcinoma and syngeneic tumor models expressing human Nectin-4. IPH45 efficacy was compared to EV. Toxicology studies to determine the dose range were conducted in rat and non-human primates (NHP). Results: IPH45 consists of a humanized IgG1 targeting a unique epitope in human Nectin-4, non-overlapping with EV, a cleavable linker, and a topoisomerase I inhibitor with a high drug-to-antibody ratio (DAR). In vitro, IPH45 is internalized upon binding to its target and induces direct killing of Nectin-4 expressing tumor cells as well as bystander killing of Nectin-4 non-expressing tumor cells in mixed culture. IPH45 mediates ADCC and complement-mediated killing. IPH45 demonstrates activity in CDX and PDX models with similar effective dose as EV. Moreover, IPH45 shows anti-tumor efficacy in vitro and in vivo in primary and secondary EV resistant models. In dose-range finding toxicology studies in rats and NHP, IPH45, unlike EV, does not elicit skin toxicity. IPH45 remains well tolerated up to the highest dose tested, suggesting a potentially larger therapeutic index than MMAE-based ADCs. Pharmacokinetic analyses in mice, rats, and NHP show minimal release of free toxin in the serum and half-life compatible with Q2W or less frequent dosing schedules in clinic. Conclusion: IPH45 is a topoisomerase I ADC targeting Nectin-4 with the potential for larger therapeutic index than EV, improved safety and dosing regimen, and ability to overcome primary and secondary resistance to EV or other Nectin-4 ADC in development. IPH45 is progressing to the clinic where the translatability of this unique profile into improved patient outcomes will be assessed across indications. Citation Format: Romain Remark, Cécile Bonnafous, Laura Chiossone, Caroline Soulas, Cyril Perrier, Guillaume Habif, Sivan Bokobza, Aurélie Maguer, Rachel Courtois, Julie Lopez, Grégory Fenaux, Olivier Benac, Barbara Carrette, Robin Letay-Drouet, Raja Bonifay, Séverine Augier, Léa Simon, Benjamin Rossi, Ariane Morel, Agnès Represa, Nicola Beltraminelli, Yannis Morel, Carine Paturel, Eric Vivier. Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6582.

Engineering Superaffinity Antibody Dependent Cellular Cytotoxcity Receptors into iPSC-Derived NK Cells As Next-Generation Immunotherapies for Cancer

Engineered immune cells have provided an important new approach to treating cancer. Most of the progress so far has focused on autologous CAR-T cell therapy, although this approach has been met with several limitations including high-cost and complexity of manufacturing, donor-to-donor variability, and severe toxicities in some patients. Allogenic natural killer (NK) cell therapies have also been shown to mount potent responses against hematologic malignancies but are less likely to cause high-grade toxicities and can be mass produced for off-the-shelf usage. We have developed an induced pluripotent stem cell (iPSC) derived NK (iNK) platform amenable to engineering multiple features in immune cells to improve efficacy against both solid and liquid tumors. Our previous studies showed that knocking out the CISH gene, which encodes a key regulator of activation, in iNK cells significantly improves their anti-cancer activity, in vivo persistence, metabolic fitness, polyfunctional cytokine production, and resistance to cell exhaustion. In these CISH Knockout (KO) iNK cells, we have screened a library of CAR constructs containing domains from diverse immune cell signaling receptors including NK cell activating receptors, cytokine receptors, and integrins. Our screen identified SLNK12, a CAR signaling domains that performed better than both T cell CARs and previously reported NK cell CARs in the context of CISH KO iNK cells. In this study, we describe the development of iNK cells with enhanced antibody dependent cellular cytotoxicity (ADCC) activity. NK cells normally bind antibodies via their CD16A receptor, which has medium-to-low affinity for the antibody Fc domain and is also typically expressed at a low level in unmodified iNKs. We have constructed a recombinant Fc-receptor that combines a high-affinity Fc-binding domain (to improve antibody binding) with our NK cell optimized SLNK12 CAR signaling domain to enhance ADCC-mediated killing. When co-cultured with multiple NC cell resistant tumor lines such as BT-474 (breast cancer) or FaDu (squamous cell carcinoma), these SuperAffinity ADCC receptor CISH KO iNK cells possess potent anti-cancer activity in combination with several different therapeutic monoclonal antibodies, including anti-EGFR (Cetuximab) and anti-HER2 (Trastuzumab) amongst others (Figure 1). We have assessed efficacy in a variety of different 2D and 3D spheroid cytotoxicity assays, as well as cytokine release and CAR activation assays. In addition, we demonstrate that our SuperAffinity receptor mediates tumor cell killing at lower antibody concentrations than do CD16A variants. Furthermore, introduction of serum IgG inhibits, but does not stop ADCC killing for both our SuperAffinity ADCC receptor and the CD16A receptor. Taken together, our results show that engineering CISH KO iNK cells with NK-optimized CARs for enhanced ADCC improves anti-tumor activity against a variety of solid tumor target lines. Future work will focus on advancing the new cellular therapies into clinical development.

CD47-SIRPα Controls ADCC Killing of Primary T Cells by PMN Through a Combination of Trogocytosis and NADPH Oxidase Activation

Immunotherapies targeting the “don’t eat me” myeloid checkpoint constituted by CD47 SIRPα interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti–CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies and the strong ADCC (antibody-dependent cellular cytotoxicity) effector function of polymorphonuclear cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb–dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRPα checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRPα blockade, and ROS induction. Although previous studies had concluded that disruption of SIRPα signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRPα also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRPα immune checkpoint and to design agents able to enhance myeloid cell function while limiting adverse effects on healthy cells able to participate in the anti-tumor immune response.

Optimizing Ex-Vivo Expanded NK Cell- Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Combined with NKTR-255 in Chronic Lymphocytic Leukemia (CLL), Follicular Lymphoma (FL), and Burkitt Lymphoma (BL)

Background: The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Rituximab, a monoclonal chimeric anti-CD20 antibody, has been widely used as a chemoimmunotherapeutic regimen in the frontline therapy for patients with CD20+ BL and diffuse large B-cell lymphoma. The addition of rituximab to the CHOP backbone or to standard FAB/LMB therapy has greatly improved outcomes without significantly increasing toxicity in patients with B-NHL (Goldman/Cairo, Leukemia, 2013, Coiffier et al, NEJM, 2002). However, patients who relapse have a poor clinical response to rituximab retreatment. Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully expanded functional and active peripheral blood NK cells PBNKwith irradiated feeder cells to target B-NHL (Chu/Cairo, et al, Can Imm Res 2015). We previously demonstrated that obinutuzumab has significantly enhanced expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and expand natural killer (NK) cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, and enhances long-term immunological memory which may lead to sustained anti-tumor immune response. Objective: To investigate the effects of NKTR-255 on the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Dean Lee, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFNg, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, ASH, 2018). MEC-1 (CLL), PGA-1 (CLL), DOHH2 (FL) and Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Results: NKTR-255 significantly enhanced the in vitro cytotoxicity of expanded NK cells when combined with rituximab against MEC-1 (E:T=3:1, p<0.001), PGA-1 (E:T=3:1, p<0.001), and DOHH2 (E:T=3:1, p<0.001) as compared to the control groups (Fig.1A). NKTR-255 also significantly enhanced granzyme and perforin release from expanded NK cells when combined with rituximab against MEC-1 (granzyme: p<0.05; perforin: p<0.001), PGA-1(granzyme: p<0.05; perforin: p<0.05), DOHH2 (granzyme: p<0.05; perforin: p<0.001) as compared to controls. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab agains rituximab-resistant BL cells like Raji-2R (E:T=3:1, p <0.01), and Raji-4RH (E:T=3:1, p<0.01) as compared to the control groups (Fig.1B). NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.01 and perforin: p<0.01) as compared to controls. Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells in vitro with enhanced IFN-g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells using humanized NSG models are under investigation. Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Madakamutil:Nektar Therapeutics: Current Employment. Marcondes:Nektar Therapeutics: Current Employment. Klein:Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Cairo:Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding.

Evaluation of the Anti-Tumor Activity of the Humanized Monoclonal Antibody NEO-201 in Preclinical Models of Ovarian Cancer

Purpose: Despite high initial response rates with cytoreductive surgery, conventional chemotherapy and the incorporation of biologic agents, ovarian cancer patients often relapse and die from their disease. New approaches are needed to improve patient outcomes. This study was designed to evaluate the antitumor activity of NEO-201 monoclonal antibody (mAb) in preclinical models of ovarian cancer where the NEO-201 target is highly expressed.Experimental Design: Functional analysis of NEO-201 against tumor cell lines was performed by antibody-dependent cellular cytotoxicity (ADCC) assays. Binding of NEO-201 to tumor tissues and cell lines were determined by immunohistochemistry (IHC) and flow cytometry, respectively. Further characterization of the antigen recognized by NEO-201 was performed by mass spectrometry. Ovarian cancer models were used to evaluate the anti-tumor activity of NEO-201 in vivo. NEO-201 at a concentration of 250 g/mouse was injected intraperitoneally (IP) on days 1, 4, and 8. Human PBMCs were injected IP simultaneously as effector cells.Results: Both IHC and flow cytometry revealed that NEO-201 binds prominently to the colon, pancreatic, and mucinous ovarian cancer tissues and cell lines. Immunoprecipitation of the antigen recognized by NEO-201 was performed in human ovarian, colon, and pancreatic cancer cell lines. From these screening, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 were identified as the most likely targets of NEO-201. Our results confirmed that NEO-201 binds different types of cancers; the binding is highly selective for the tumor cells without cross reactivity with the surrounding healthy tissue. Functional analysis revealed that NEO-201 mediates ADCC killing against human ovarian and colorectal carcinoma cell lines in vitro. In addition, NEO-201 inhibited tumor growth in the presence of activated human PBMCs in orthotopic mouse models of both primary and metastatic ovarian cancer. Importantly, NEO-201 prolonged survival of tumor-bearing mice.Conclusions: These data suggested that NEO-201 has an antitumor activity against tumor cells expressing its antigen. Targeting an antigen expressed in tumors, but not in normal tissues, allows patient selection for optimal treatment. These findings strongly indicate that NEO-201 warrants clinical testing as both a novel therapeutic and diagnostic agent for treatment of ovarian carcinomas. A first in human clinical trial evaluating NEO-201 in adults with chemo-resistant solid tumors is ongoing at the NIH clinical Center.

Efficient ADCC killing of meningioma by avelumab and a high-affinity natural killer cell line, haNK.

Meningiomas are the most common adult primary tumor of the central nervous system, but there are no known effective medical therapies for recurrent meningioma, particularly for World Health Organization grade II and III tumors. Meningiomas arise from the meninges, located outside the blood-brain barrier, and therefore may be directly targeted by antibody-mediated immunotherapy. We found that programmed cell death ligand 1 (PD-L1) was highly expressed in multiple human malignant meningioma cell lines and patient tumor samples. PD-L1 was targeted with the anti-PD-L1 antibody avelumab and directed natural killer cells to mediate antibody-dependent cellular cytotoxicity (ADCC) of PD-L1-expressing meningioma tumors both in vitro and in vivo. ADCC of meningioma cells was significantly increased in target cells that upregulated PD-L1 expression and, conversely, abrogated in tumor cells that were depleted of PD-L1. Additionally, the high-affinity natural killer cell line, haNK, outperformed healthy donor NK cells in meningioma ADCC. Together, these data support a clinical trial designed to target PD-L1 with avelumab and haNK cells, potentially offering a novel immunotherapeutic approach for patients with malignant meningioma.

Abstract 2370: Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC)

Abstract Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that targets folate receptor alpha (FRA), a cell surface protein highly overexpressed on a large number of cancers. Clinical evidence suggests that FAR enhances the anti-tumor effects of carboplatin and taxane in ovarian cancer patients whose tumors express low CA125 levels. In light of the recent evidence that CA125 has a direct effect on suppressing the humoral immune mechanisms [antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)] of FAR (1, 2) and the findings that chemotherapeutic agents can enhance the ADCC activity of certain mAbs, we tested the potential synergistic effects that carboplatin may have on FAR immune effector function. Here we show the immunosuppressive effects that CA125 has on FAR-mediated tumor cell killing in vivo and demonstrate that carboplatin is able to suppress cell surface CA125 expression leading to enhanced FAR ADCC on FRA-expressing ovarian tumor cells. CA125 has been previously found to suppress FAR-mediated tumor cell killing in vitro. To determine its effect in vivo, we tested single agent FAR against isogenic tumor cells in mouse xenografts. OVCAR-3 is a FRA positive ovarian tumor cell line that expresses high levels of CA125 on its cell surface. A CA125 shRNA knockdown line (OV-KD) has been previously generated and shown to be more susceptible to FAR ADCC than its parental counterpart. Xenograft studies using these lines found that FAR had significant single agent tumor killing in the OV-KD xenografts in contrast to OVCAR-3 xenografts. We next tested FAR in vivo killing when used in combination with carboplatin. These data showed that carboplatin had a synergistic effect when combined with FAR treatment as compared to single agent treatment alone. To determine if this mechanism involved enhanced ADCC, we conducted similar studies in vitro whereby OVCAR-3 cells were treated with a sublethal concentration of carboplatin for 72 hrs, washed and then tested for FAR ADCC killing. These data found that FAR had enhanced ADCC activity on carboplatin treated cells as compared to untreated cells. Finally, to explore the potential mechanism by which this effect may occur, we examined the steady-state levels of both the mannose-6-phosphate receptor (M6PR), reported to be upregulated by chemotherapeutic drugs leading to enhanced ADCC via higher granzyme B uptake and the CA125 immunosuppressive factor. Our data found that both M6PR and CA125 expression were significantly suppressed following carboplatin exposure. These data support a mechanism by which carboplatin may enhance the humoral immune response for FAR and potentially other anti-tumor mAbs via downregulation of the immunosuppressive CA125 protein. 1. OncoTarget 8:66747-57, 20172. 2. EJI 201847707, 2018 Citation Format: J. Bradford Kline, Keiji Furuuchi, James Fulmer, Jennifer McDonough, Luigi Grasso, Nicholas C. Nicolaides. Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2370.

Evaluation of Different Parameters of Humoral and Cellular Immune Responses in HIV Serodiscordant Heterosexual Couples: Humoral Response Potentially Implicated in Modulating Transmission Rates

As the HIV/AIDS pandemic still progresses, understanding the mechanisms governing viral transmission as well as protection from HIV acquisition is fundamental. In this context, cohorts of HIV serodiscordant heterosexual couples (SDC) represent a unique tool. The present study was aimed to evaluate specific parameters of innate, cellular and humoral immune responses in SDC. Specifically, plasma levels of cytokines and chemokines, HIV-specific T-cell responses, gp120-specific IgG and IgA antibodies, and HIV-specific antibody-dependent cellular cytotoxicity (ADCC) activity were assessed in nine HIV-exposed seronegative individuals (ESN) and their corresponding HIV seropositive partners (HIV+-P), in eighteen chronically infected HIV subjects (C), nine chronically infected subjects known to be HIV transmitters (CT) and ten healthy HIV− donors (HD). Very low magnitude HIV-specific cellular responses were found in two out of six ESN. Interestingly, HIV+-P had the highest ADCC magnitude, the lowest IgA levels and the highest IgG/IgA ratio, all compared to CT. Positive correlations between CD4+ T-cell counts and both IgG/IgA ratios and %ADCC killing uniquely distinguished HIV+-P. Additionally, evidence of IgA interference with ADCC responses from HIV+-P and CT is provided. These data suggest for the first time a potential role of ADCC and/or gp120-specific IgG/IgA balance in modulating heterosexual transmission. In sum, this study provides key information to understand the host factors that influence viral transmission, which should be considered in both the development of prophylactic vaccines and novel immunotherapies for HIV-1 infection.

Comparing high-throughput methods to measure NK cell-mediated antibody dependent cellular cytotoxicity during HIV-infection

HIV-specific binding antibody responses, including those mediating antibody-dependent cellular cytotoxicity (ADCC), provided the best functional correlate of lower risk of infection in the RV144 HIV-1 vaccine clinical trial. The aim of this study was to compare two high-throughput flow cytometry based methods to measure HIV-specific ADCC responses, the GranToxilux and PanToxilux assays. Plasma from nine HIV-1 seropositive individuals was screened for binding antibody titres against HIV-1 subtype C gp120 by ELISA and western blot. Plasma from six HIV-negative individuals was included as controls. Both ADCC assays used subtype C gp120-coated CEM.NKRCCR5 cells as targets. The PanToxilux assay (which measured both granzyme B and caspase activity) measured higher levels of direct natural killer (NK) cell killing of K562 tumour cells than the GranToxilux assay (granzyme B alone; p<0.05). In ADCC assays in which NK cell killing was directed against gp120-coated CEM.NKRCCR5 cells in an antibody-dependent manner, plasma from HIV-positive individuals yielded significantly higher levels of ADCC activity than the HIV-negative controls. In contrast to direct killing, the GranToxilux assay measured similar levels of ADCC killing as the PanToxilux assay but had significantly lower background cytotoxicity against target cells coated with HIV negative serum. In conclusion, the PanToxilux assay was more sensitive for detecting direct NK cell killing of K562 cells than the GranToxilux assay, although the GranToxilux assay performed better at detecting HIV-specific ADCC activity, because of lower background cytotoxicity from HIV-negative serum. This is the first study to compare GranToxilux and PanToxilux ability to detect ADCC during HIV infection.

The adaptor protein shc is involved in the negative regulation of NK cell-mediated cytotoxicity.

The activation of protein tyrosine kinase(s) (PTK) is a critical event required for the development of NK cell-mediated cytotoxicity. Here we demonstrate that the adaptor protein shc undergoes tyrosine phosphorylation during the generation of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we report that, upon direct or antibody-dependent target cell interaction, shc coprecipitates with the Src homology 2 (SH2)-containing inositol phosphatase, SHIP. To gain information on the functional role of shc in NK cytotoxicity, we overexpressed wild-type or dominant negative shc constructs in the human NKL cell line. Our findings show a consistent shc-mediated down-regulation of ADCC and natural killing. Such functional effect correlates with a perturbation of the phosphoinositide (PI) metabolism by means of a shc-mediated negative regulation of inositol 1,4,5 triphosphate (IP3) generation and intracellular calcium flux upon CD16 ligation. Furthermore, our data show that dominant-negative shc-mediated perturbation of shc/SHIP interaction leads to inhibition of ligand-dependent SHIP recruitment to CD16 zeta chain. We suggest that shc plays a role of negative adaptor by modulating SHIP recruitment to activation receptors involved in the generation of NK cytotoxic function.

The Yin and Yang of ADCC-Mediating Antibodies

Antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) have been of increasing interest since their identification as a protective immune correlate in the RV144 clinical vaccine efficacy trial (Haynes et al., 2012). Earlier studies associated these non-neutralizing antibodies with protection against HIV infection and disease progression in both humans and non-human primates (for review see Vargas-Inchaustegui and Robert-Guroff, 2013). Therefore it is somewhat surprising to find them now linked to HIV pathogenesis. HIV disease progression is marked by a gradual loss of CD4+ T cells during which a small proportion of HIV-infected CD4+ T cells and a much larger proportion of uninfected “bystander” CD4+ T cells are killed. While numerous mechanisms have been described for infected cell killing, the mechanism of bystander killing has remained controversial. In this issue, Richard et al. (2016) attribute bystander killing largely to antibodies mediating ADCC. They further suggest that a small CD4 mimetic developed by the Sodroski group might be used to alter this pathogenic mechanism for therapeutic benefit. How can the conflicting findings regarding ADCC antibodies be explained? And what caveats should be considered in using CD4 mimetics? As reported by Richard et al. in this issue of EBioMedicine, the external envelope of HIV is labile and continually sheds the gp120 component which then binds the primary viral receptor, CD4, on bystander uninfected T cells. A subsequent conformational change opens the co-receptor binding site exposing an epitope recognized by cluster A antibodies, including the prototypic ADCC-mediating monoclonal antibody, A32. Killing of the bystander CD4+ T cells follows. Concurrently, the virus subverts killing of infected cells by down-regulating CD4, inhibiting tetherin, and internalizing Env (as cited in von Bredow et al., 2015). Consequently, less Env is expressed on the infected-cell surface, less CD4 is available to complex with gp120, and less expression of Env in the open conformation occurs. Thus ADCC killing of the infected cell is diminished in favor of bystander killing. CD4 mimetics were previously shown to interact with the viral envelope, inducing conformational changes, exposing the co-receptor binding site, and rendering the virus susceptible to neutralization by CD4-induced antibodies (Madani et al., 2008, Madani et al., 2014). The Finzi laboratory also previously reported that gp120/CD4 binding within the same infected cell led to exposure of ADCC epitopes (Veillette et al., 2014) and that the CD4 mimetic sensitized infected cells to ADCC killing (Richard et al., 2015). In the current paper they show that the mimetic decreases binding of shed gp120 to bystander CD4 cells, inferring that less ADCC-mediated killing of these uninfected cells will result. They also suggest that binding of the mimetic to envelope remaining on the infected cell surface should “redirect” ADCC-mediating antibodies to the infected cell away from bystanders, as stated above. Recent support of this hypothesis showed ADCC killing of primary HIV-infected T cells was enhanced in vitro by co-culturing with the CD4 mimetic (Lee et al., 2015). The current study was conducted primarily with the A32 antibody, together with a small number of sera from HIV-infected individuals. ADCC antibodies are among the first elicited following infection, as the gp120–CD4 interaction leads to exposure of cluster A epitopes. Not all such CD4-induced epitopes elicit equivalent ADCC activities, due to subtle differences in antibody-epitope recognition and/or Fc differences (Gohain et al., 2015). Moreover, antibodies targeting other Env sites, including some with neutralizing activity such as 2G12 and b12, also mediate ADCC. The relative abundance of all these antibodies in the human sera tested was not determined. Further, the degree of gp120 shedding and levels of CD4 and Env present on the cells tested in vitro compared to uncultured, primary HIV-infected cells was not reported. It will be critical to confirm the observations made with in vivo studies using the SHIV model. Similarly, the in vivo effects of CD4 mimetic administration must be determined. It is an obvious therapeutic candidate, however, the possibility that it might facilitate greater CD4-independent infection must be considered. Vaccine-elicited ADCC antibodies recognizing cluster A epitopes might well be protective, as they target an early epitope uncovered during virus transmission. Use of the CD4 mimetic in vaccine design might also stimulate development of such ADCC-mediating antibodies. However, if incomplete protection and break-through infection occurred following administration of such a vaccine, accelerated CD4 T cell loss and disease progression might occur by the mechanism reported by Richard et al.. To circumvent this possibility, vaccine design should include several immune targets to achieve balanced protection against HIV infection.

Glycan Elongation Beyond the Mucin Associated Tn Antigen Protects Tumor Cells from Immune-Mediated Killing

Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr), STn (NeuAcα2-6GalNAc-Ser/Thr), T (Galβ1–3GalNAc-Ser/Thr), and ST (NeuAcα2-6Galβ1–3GalNAc-Ser/Thr) antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn). Glyco-engineering was performed by zinc finger nuclease (ZFN) knockout (KO) of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC) and cytotoxic T lymphocyte (CTL)-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.

Final Results of a Phase I Study of the Fc Engineered CD19 Antibody XmAb®5574 (MOR00208) in Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL) or Small Lymphocytic Lymphoma (SLL).

AbstractAbstract 2894Antibody (Ab) therapies such as the CD20 monoclonal abs rituximab and ofatumumab are commonly used in CLL alone and in combination with chemotherapy, however, CD20 density is low on CLL cells, suggesting this may not be the ideal target. CD19, which is ubiquitously expressed on CLL cells and those of other B cell malignancies is a reasonable candidate for ab targeting. XmAb5574 is a novel humanized IgG1 CD19 monoclonal ab with an engineered Fc region to enhance Fc gamma receptor binding affinity. In vitro, this ab demonstrates direct cytotoxicity and antibody dependent cellular phagocytosis similar to rituximab, however, shows enhanced natural killer antibody dependent cellular cytotoxicity compared to other therapeutic abs used in CLL (Awan, FT Blood 2009). We have performed a first in human trial of this ab as a single agent in relapsed or refractory (R/R) CLL, and present the results in this report.This study is a multi-institutional phase I trial of XmAb5574 in patients (pts) with R/R CLL. Eligible pts were those with CLL who had at least 1 prior therapy and required treatment by International Working Group on CLL (IWCLL) 2008 Guidelines (Hallek, M Blood 2008), had Eastern Cooperative Oncology Group Performance Status <3, had platelets ≥50,000/mm3, and had adequate organ function. Primary endpoints were to determine maximal tolerated dose (MTD), describe toxicity, and characterize pharmacokinetics (PK). A secondary endpoint was to explore efficacy. An accelerated titration design was used in which 1 pt was accrued to the first two dose levels provided there were no dose limiting toxicities (DLT) or ≥ grade 2 adverse events (AE), and then a standard 3×3 design was employed from dose level 3 forward. Dose levels included 0.3, 1, 3, 6, 9, and 12 mg/kg with an expansion to a total of 16 pts at the MTD. XmAb5574 was administered as an intravenous infusion on days 1, 4, 8, 15, and 22 of cycle (C) 1, and on days 1, 8, 15, and 22 of C2. Toxicity was assessed using the National Cancer Institute’s Common Criteria for Adverse Events v4.0 for non-hematologic toxicity, and IWCLL 2008 guidelines for hematologic toxicity. Disease response assessment by physical exam was performed on C1D28, C2D28, and 4, 8, and 12 weeks after the end of C2. Radiographic assessment was performed C2D28.27 pts were enrolled to this phase I trial. The median age of all pts was 66 years (range 40–84). The pts were generally high risk: 14 (52%) had high-risk disease by Rai stage, 8 (30%) had del(11q22.3) and 10 (37%) had del(17p13.1) by FISH, and 24 (89%) had IgVH unmutated disease. The median number of prior therapies was 4 (range 1–14). Toxicity with this agent was modest. Dose escalation continued without dose limiting toxicity (DLT) until the highest dose level, in which one patient experienced grade 4 neutropenia associated with febrile neutropenia which required dose discontinuation. 100% of patients experienced any AE, with the majority of AE being grade 1–2. The most common AEs were infusion reactions in 18 patients (67%), all of which were grade 1 or 2. Treatment-related Grade 3 or 4 AEs occurred in 5 pts (19%), and included neutropenia (n=3), thrombocytopenia (n=2), increased aspartate aminotransferase (AST) (n=1), febrile neutropenia (n=1), and tumor lysis syndrome (n=1). All were on the 12 mg/kg dose level except one pt receiving 1mg/kg who experienced neutropenia.Overall response rate by IWCLL 2008 criteria is 11%, all of which have been partial responses (PR). Using IWCLL 1996 response criteria which does not include CT scan assessment of disease resulted in a PR in 13 pts (42%). Only 2 pts had PD at the 8 week evaluation point. Responses occurred at the 6, 9, and 12 mg/kg dose levels. All objective responses were in pts categorized as CLL as opposed to SLL, and no patients with lymph nodes >5cm responded.PK was best modeled by a two-compartment model. Half-life was 14 days, with clearance 5mL/day/kg that was not dose-dependent. Across the dose range, area under the curve increased in a dose-proportional manner, while maximum concentration increased in a less than proportional manner. A steady-state was reached at or before infusion 9.XmAb5574 shows acceptable toxicity and signs of preliminary efficacy in patients with high-risk, heavily pretreated CLL. These results justify movement into phase II study in CLL as well as other B cell malignancies. Modest toxicity, in particular infectious toxicity, will potentially allow combinations with other active agents in CLL. Disclosures:Enoch:Xencor, Inc.: Employment. Foster:Xencor, Inc: Consultancy.

CD37 Is a Potential Therapeutic Target for B-Cell Non-Hodgkin Lymphoma

AbstractAbstract 3098 Background:The Non-Hodgkin Lymphomas (NHLs) are a heterogeneous group of malignancies with approximately 85% of NHL belonging to the B-cell lineage. The use of monoclonal antibodies (mAb), including the anti-CD20 therapy rituximab, has become a standard of care for the treatment of many forms of NHL. However, only a subset of patients respond to rituximab and the majority of those eventually relapse after treatment. Additional therapeutic options are necessary for these patients. Here we report the expression of CD37, a lineage-specific B-cell antigen, in different subtypes of B-cell NHL tissues and evaluate the functional activity of an anti-CD37 scFv-Fc fusion protein, known as a SMIP ™ protein (CD37-SMIP) against NHL cells. Methods:Fresh NHL tissues or peripheral blood from leukemia-stage lymphoma patients were used for flow cytometric assay of CD37 expression. Monotypic kappa or lambda IgG light chain positive malignant B-cells were gated for this assay. Immunohistochemical (IHC) staining of formalin fixed paraffin-embedded (FFPE) NHL tissues was performed for CD37 expression. Fresh primary NHL cells were isolated and used as targets in assays to measure antibody dependent cellular cytotoxicity (ADCC) activity as well as direct cell death mediated by CD37-SMIP. The effects of CD37-SMIP on multiple B-cell NHL lines was also determined by measuring cell proliferation, Annexin V staining, caspase3/7 release and changes in mitochondria membrane potentials using JC-1 staining. Results:Eighteen cases of NHL (follicular lymphoma (2), chronic lymphocytic leukemia/small lymphocytic lymphoma (3), mantle cell lymphoma (5), persistent marginal zone lymphoma (1), diffuse large B-cell lymphoma (1) and other types (6)) were analyzed by flow cytometry. Six of the eighteen cases had a history of rituximab treatment. Flow data showed malignant B-cells from these NHL specimens were strongly positive for CD37, while other cell populations had minimal to low levels of CD37 expression. IHC results on FFPE samples (n=170) indicated 89% (151 of 170 cases) of stained lymphoma tissues were positive for CD37, including follicular lymphoma (80/88), mantle cell lymphoma (39/44), DLBCL (32/38), and marginal zone lymphoma (5/5). Twenty seven B-cell lymphoma cell lines representing MCL, DLBCL, FL, and Burkitt's lymphoma subtypes were evaluated for CD37 expression and functional response to CD37-SMIP. CD37-SMIP mediated cell death in a wide range of NHL cell lines. The response to CD37-SMIP correlated with the levels of CD37 surface expression in NHL cell lines (Pearson analysis, r=0.78) The CD37-SMIP mediated response was rapid, with Annexin V staining and mitochondria depolarization observed in 3 hours; however, no caspase activity was observed at this time point. Two of ten NHL cell lines had modest caspase 3/7 activity at 24 h post treatment. However, caspase inhibitors did not suppress the CD37-SMIP mediated response. ADCC and direct killing activities elicited by SMIP-CD37 on freshly isolated primary NHL cells are under evaluation. Conclusion:CD37 is highly expressed on majority of B-cell NHL tissues, including those from patients treated with rituximab. CD37-SMIP mediated rapid cell death in NHL cell lines with mitochondria dysfunction but appears to be independent of caspase activity. The expression pattern of CD37 and functional effects of CD37-SMIP supports the targeting of CD37 in B-cell NHL. TRU-016, a humanized SMIP protein specific for CD37, is currently being evaluated in relapsed CLL and NHL patients in a phase 1 trial. Disclosures:No relevant conflicts of interest to declare.

Multiple Vaccine-Elicited Nonneutralizing Antienvelope Antibody Activities Contribute to Protective Efficacy by Reducing both Acute and Chronic Viremia following Simian/Human Immunodeficiency Virus SHIV89.6PChallenge in Rhesus Macaques

We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)(89.6P) challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.

Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing.

An understanding of model systems of trastuzumab (Herceptin) resistance is of great importance since the humanized monoclonal antibody is now used as first line therapy with paclitaxel in patients with metastatic Her2 overexpressing breast cancer, and the majority of their tumors has innate resistance or develops acquired resistance to the treatment. Previously, we selected trastuzumab-resistant clonal cell lines in vitro from trastuzumab-sensitive parental BT-474 cells and showed that cloned trastuzumab-resistant cell lines maintain similar levels of the extracellular Her2 receptor, bind trastuzumab as efficiently as the parental cells, but continue to grow in the presence of trastuzumab and display cell cycle profiles and growth rates comparable to parental cells grown in the absence of trastuzumab (Kute et al. in Cytometry A 57:86-93, 2004). We now show that trastuzumab-resistant and trastuzumab-sensitive cells both surprisingly display trastuzumab-mediated growth inhibition in athymic nude mice. This demonstrates that resistance developed in vitro is not predictive of resistance in vivo. The observation that in vitro resistant cells are sensitive to trastuzumab in vivo could be explained by antibody dependent cellular cytotoxicity (ADCC). Therefore, both parental and trastuzumab-resistant cells were assayed for ADCC in real time on electroplates with and without trastuzumab in the presence of a natural killer cell line (NK-92), and granulocyte or mononuclear cellular fractions isolated from human peripheral blood. Mononuclear cells and NK-92 cells were more effective in killing both parental and trastuzumab-resistant cells in the presence of trastuzumab. Both trastuzumab-resistant cells and trastuzumab-sensitive cells showed similar susceptibility to ADCC despite displaying divergent growth responses to trastuzumab. The granulocyte fraction was able to kill these cells with equal efficacy in the presence or absence of trastuzumab. These results support a model of trastuzumab tumor cell killing in vivo mediated primarily by ADCC from the mononuclear fraction of innate immune cells and suggest that in the clinical setting not only should changes in signaling transduction pathways be studied in acquired tumor resistance to trastuzumab, but also mechanisms by which tumors impede immune function should be evaluated.

A Fully Human Anti-CD40 Antagonistic Antibody, CHIR-12.12, Inhibit the Proliferation of Human B Cell Non-Hodgkin's Lymphoma.

Immunotherapy using anti-tumor antibodies has become a feasible alternative for treating patients with lymphoma. These anti-tumor antibodies may target a specific receptor to disrupt proliferative signaling or mediate their anti-tumor effect by antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated killing. The CD40 antigen is a good target for such anti-tumor antibodies for several reasons: CD40 is expressed on the vast majority of the non-Hodgkin's B cell lymphomas and it has been proposed that the CD40/CD40L interaction provides a critical survival or proliferative signal for B cell lymphoma, especially the low-grade follicular lymphoma. In addition, B lymphoma cell lines become less sensitive to chemotherapy-induced apoptosis after CD40 cross-linking in an in vitro study. Therefore, an anti-CD40 antagonist that disrupts the CD40/CD40L interaction and mediates effector mechanism could have a therapeutic advantage. In this report, we describe a fully human anti-CD40 antagonistic IgG1 monoclonal antibody, CHIR-12.12 that was generated from mice with a human immunoglobulin gene loci (XenoMouse®mice, Abgenix Inc.). We first compared the antigen expression level of CD40 to the level of CD20, the target for rituximab, on primary lymphoma cells. While the expression level of CD40 was similar between different samples of primary follicular lymphoma cells, it was 10 fold less than the level of CD20. The expression of CD40 and CD20 on chronic lymphocytic leukemia/small lymphocytic lymphoma cells (CLL/SLL) was more variable. However, the level of CD20 was still significantly higher than the level of CD40 in all samples tested (2.4 to 13 fold). While CHIR-12.12 binds to primary lymphoma cells similarly to several other anti-CD40 antibodies, CHIR-12.12 did not induce proliferation of these primary tumore cells. By contrast, an agonist anti-CD40 antibody induced proliferation of these lymphoma cells up to 6-fold over baseline. To study the ability of CHIR-12.12 to interrupt the CD40-CD40L interaction, we cultured lymphoma cells with CD40L-transfected feeder cells in the presence of control IgG1, CHIR-12.12 or rituximab. In this system, the lymphoma cells proliferate in response to CD40-CD40L interaction. The addition of rituximab did not influence the proliferation. However, CHIR-12.12 inhibited the proliferation of follicular lymphoma and of CLL/SLL cells in a dose-dependent manner. The inhibition was observed with antibody concentration at 1 μg/ml and reached maximum of 90% inhibition at 10 μg/ml. We then evaluated the ability of CHIR-12.12 to elicit complement-mediated killing or ADCC. In vitro, rituximab induced complement-mediated cytotoxicity, while CHIR-12.12 did not. However, both CHIR-12.12 and rituximab induced effective ADCC of primary follicular lymphoma cells using purified NK cells from a healthy donor. Even though the level of CD40 is 10-fold less than the level of CD20 on the cell surface of these tumor cells, CHIR-12.12 induced the same degree of ADCC killing as did rituximab. Thus, this novel antagonist CHIR-12.12 antibody both blocks tumor-stimulatory CD40/CD40L interaction and mediates ADCC in the presence of a low number of target antigen. Our results support further development of this antibody to treat patients with B cell lymphoma.

The adaptor protein shc is involved in the negative regulation of NK cell-mediated cytotoxicity

The activation of protein tyrosine kinase(s) (PTK) is a critical event required for the development of NK cell-mediated cytotoxicity. Here we demonstrate that the adaptor protein shc undergoes tyrosine phosphorylation during the generation of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we report that, upon direct or antibody-dependent target cell interaction, shc coprecipitates with the Src homology 2 (SH2)-containing inositol phosphatase, SHIP. To gain information on the functional role of shc in NK cytotoxicity, we overexpressed wild-type or dominant negative shc constructs in the human NKL cell line. Our findings show a consistent shc-mediated down-regulation of ADCC and natural killing. Such functional effect correlates with a perturbation of the phosphoinositide (PI) metabolism by means of a shc-mediated negative regulation of inositol 1,4,5 triphosphate (IP3) generation and intracellular calcium flux upon CD16 ligation. Furthermore, our data show that dominant-negative shc-mediated perturbation of shc/SHIP interaction leads to inhibition of ligand-dependent SHIP recruitment to CD16 zeta chain. We suggest that shc plays a role of negative adaptor by modulating SHIP recruitment to activation receptors involved in the generation of NK cytotoxic function.

Improved Lymphocyte Cytotoxicity Against Murine Renal Cell Carcinoma

In this paper we describe the generation of antibody dependent cellular cytotoxicity against a murine renal cell carcinoma. Using human recombinant interleukin-2 and in vitro adherence to plastic, we generated lymphokine activated killer and adherent lymphokine activated killer cells. Adherent lymphokine activated killer cells had significant (p < 0.05) higher unrestricted cytotoxicity than LAK cells. Using a rabbit antibody against Renca developed in our laboratory, we induced significant (p < 0.01) antibody dependent cellular cytotoxicity using fresh spleen, lymphokine activated killer and adherent lymphokine activated killer cells. The strongest antibody dependent cellular cytotoxicity killing was mediated by adherent lymphokine activated killer cells and was restricted only to the renal cell carcinoma target. Using FACS cell surface analysis and antibody and complement depletion of selected effector cell subsets, we also demonstrate that the antibody dependent cellular cytotoxicity effector cell population consists of asialoGMU Lyt 2.1“ natural killer cells. This first description of antibody dependent cellular cytotoxicity against renal cell carcinoma by activated natural killer cells suggests a novel method for more efficient use of cytotoxic effector cells against this type of cancer.

Detection of antibodies which mediate human immunodeficiency virus-specific cellular cytotoxicity (ADCC) in vitro

A method to detect antibodies which mediate antibody-dependent cellular cytotoxicity (ADCC) against HIV-infected target cells was developed. Normal lymphocytes were used as effector cells and different HIV-infected cell lines as target cells. The level of ADCC varied depending on both the effector cell function and the target cell susceptibility. For evaluation of HIV-specific ADCC, the effector function was simultaneously tested against a standard antigen ( β 2 microglobulin), expressed both on infected and uninfected target cells. The HIV-infected monocytoid cell line U937, clone 2, was found to be the most useful target cell in the present system. The detection of antibodies which induced HIV-specific ADCC killing will permit clinical and experimental analysis of the possible importance of such antibodies in protection against HIV-associated disease symptoms.