Monoclonal anti-human tumor antibodies of six isotypes in cytotoxic reactions with human and murine effector cells

Eighty-seven murine monoclonal antibodies (MAb) produced against human tumors of various origins and representing six different immunoglobulin classes were tested for antitumor reactivity in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. Mouse splenocytes, thioglycolate-elicited mouse peritoneal macrophages, freshly obtained nonadherent human peripheral blood lymphocytes, and human monocytes were used as effector cells, and human or rabbit serum as the source of complement. Of all four effector cell types tested, mouse macrophages showed the highest cytotoxic activity, based on net cytotoxicity, minimum requirement for Mab concentration, and effector cell number. Different immunoglobulin classes were associated with characteristic patterns of reactivity with the various effector cells or complement, independent of the target cell type used. MAb able to mediate ADCC were found among all IgG subclasses, with IgG2a and IgG3 MAb inducing lysis with all effector cell types. IgM and IgA MAb were nonreactive in the various ADCC assays, but IgM MAb were highly cytotoxic with complement.     

Cytolytic activity of murine anti-dog lymphoma monoclonal antibodies with canine effector cells and complement

Peripheral blood leukocytes (PBL), nonadherent lymphocytes, and adherent monocytes separated from freshly isolated blood of 15 dogs were analyzed for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in combination with murine anti-tumor monoclonal antibodies (MAbs). Canine monocytes isolated from most donors by adherence to gelatin-fibronectin-coated plastic surface presented high ADCC activity against the canine lymphoma 17-71 tumor cell line in combination with antilymphoma MAbs 231 (IgG2a) and 234-2a (IgG2a). Canine lymphocytes generally showed lower ADCC activity than total PBL or monocytes. Canine PBL effector cells showed high ADCC activity against the human colorectal carcinoma SW948 cell line using the Y-6-specific MAb isotype switch variants 55-2 IgG3, 55-2 IgG1, 55-2 IgG2b, and 55-2 IgG2a. Analysis of the role of murine MAb isotypes on ADCC activity against tumors by canine cells using anti-human tumor class-switch variant MAbs and a panel of anti-canine lymphoma MAbs of different IgG subclass revealed the highest ADCC activity with MAbs of the IgG2a and IgG3 subclasses. IgG2a antilymphoma MAbs were also able to lyse tumor cells in complement-dependent cytotoxicity (CDC) assay. These results suggest the potential value of MAbs of IgG3 and IgG2a subclasses in immunotherapy against canine lymphoma.     

Lenalidomide enhances antibody-dependent cellular cytotoxicity of solid tumor cells in vitro: influence of host immune and tumor markers

We evaluated the effect of combining lenalidomide with therapeutic antibodies on antibody-dependant cell-mediated cytotoxicity (ADCC) of solid tumor cells, and the requirement for expression of natural killer (NK) cell-activating receptors and their solid tumor surface ligands. Twenty-three human tumor cell lines (colon, breast, lung, head and neck, ovary, and bone sarcoma) were analyzed. NK effector cells were isolated from healthy donors, pre-treated with and without lenalidomide, and incubated with antibody-coated tumor cells to determine ADCC. In blocking experiments, NK cells were pre-incubated with anti-DNAM-1 or anti-NKG2D antibodies, and target colorectal cells were pre-incubated with anti-CD155 (PVR), anti-MIC-A/B, or anti-ULBP 3 antibodies. Differences between groups were assessed using unpaired and paired Student’s t test and one-way ANOVA. Lenalidomide enhanced NK cell-mediated ADCC of trastuzumab- and cetuximab-coated tumor cells. Activity against colorectal cancer cells was dependent on target antigen expression, but independent of KRAS status and FcγRIIIa genotype. The extent of ADCC and its enhancement by lenalidomide correlated with NK cell expression of NKG2D and DNAM-1, and tumor cell expression of PVR and MIC-A. Blocking of NKG2D and, to a lesser extent, DNAM-1 inhibited ADCC. Anti-MIC-A/B monoclonal antibody blocked natural cytotoxicity, but not ADCC. Lenalidomide enhances the ability of IgG1-isotype antibodies to mediate ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D–NKG2D ligand interactions, but appears to be independent of MIC-A/B. This provides a rationale for exploratory clinical studies and an assessment of potential biomarkers predictive of clinical benefit.     

Resolution of adhesion- and activation-associated components of monoclonal antibody-dependent human NK cell-mediated cytotoxicity.

Flow cytometry was used to investigate two functional parameters of human natural-killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC): (i) the frequency of NK cells which formed conjugates (NKC) with autologous monoclonal antibody (mAb)-coated lymphocyte target cells, a measure of the avidity of CD16-dependent cell-cell adhesion, and (ii) the rise in the intracellular concentration of ionized calcium ([Ca2+]i) elicited in NKC by contact with target cells, a measure of CD16-dependent NK cell activation. For each of four rat IgG2b mAb directed against target cell antigens CDw52, CD5, CD45, and class I HLA, there existed quantitatively similar relationships between ADCC and rise in NKC[Ca2+]i but significant inter-mAb differences with respect to the ADCC vs the NKC frequency relationship. Cytolytic efficiencies of mAb appeared to be determined at the level of the NK cell, dependent upon CD16 and LFA-1, but restricted with respect to quantitative levels of NKC[Ca2+]i. In concert with this notion, targets coated with an IgG1 isotype-switch variant alpha CDw52 mAb promoted significant conjugate formation but failed to elicit a rise in NKC[Ca2+]i or ADCC. Thus, Fc regions of antibodies make contacts with NK cell CD16 which may strengthen cell-cell adhesion without eliciting an activation stimulus, a finding which supports a complexity of CD16 functional regulation of probable significance in the clinical consequences of antibody responses or therapeutic mAb manipulations.     

Therapeutic potential of rat monoclonal antibodies: isotype specificity of antibody-dependent cell-mediated cytotoxicity with human lymphocytes

The ability of rat monoclonal antibodies to promote antibody-dependent cell-mediated cytotoxicity with human effector cells was tested by using a variety of antibodies against different human and mouse leukocyte antigens. It was found that only IgG2b antibodies were effective. This isotype has already been shown to be efficient in fixing human complement, which suggests that among rat monoclonal antibodies, the IgG2b subclass might be a good choice for attempts at serotherapy. Further studies with other antibody-mediated effector mechanisms as well as suitable clinical trials are merited.     

Compromised NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Chronic SIV/SHIV Infection

Increasing evidence indicates that antibody-dependent cellular cytotoxicity (ADCC) contributes to the control of HIV/SIV infection. However, little is known about the ADCC function of natural killer (NK) cells in non-human primate model. Here we demonstrated that ADCC function of NK cells was significantly compromised in chronic SIV/SHIV infection, correlating closely with the expression of FcγRIIIa receptor (CD16) on NK cells. CD32, another class of IgG Fc receptors, was identified on NK cells with higher expression in the infected macaques and the blockade of CD32 impacted the ability of NK cells to respond to antibody-coated target cells. The inhibition of matrix metalloproteases (MMPs), a group of enzymes normally involved in tissue/receptor remodeling, could restore NK cell-mediated ADCC with increased CD16 expression on macaque NK cells. These data offer a clearer understanding of NK cell-mediated ADCC in rhesus macaques, which will allow us to evaluate the ADCC repertoire arising from preclinical vaccination studies in non-human primates and inform us in the future design of effective HIV vaccination strategies.     

Effects of interleukin-18 on natural killer cells: costimulation of activation through Fc receptors for immunoglobulin

The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.     

Cytotoxic activities of normal cultured human T cells.

Normal human T cells grown in continued cultures in medium containing conditioned medium (CM) from PHA-stimulated lymphocytes were studied for their ability to manifest three known forms of cell-mediated cytotoxicity: lectin-induced cellular cytotoxicity (LICC), natural killer cell (NK) activity, and antibody-dependent cellular cytotoxicity (ADCC). The cultured T cells (CTC) were very effective mediators of LICC, being cytotoxic even at very low attacker-target cell ratios in the presence of different lectins, and against different types of targets. When tested without the addition of lectin, the CTC demonstrated a low degree of spontaneous cytotoxicity. This spontaneous cytotoxicity might not be due to conventional NK cells however, since the CTC failed to show significant numbers of cells with Fc receptors (FcR) for IgG, and had no detectable ADCC activity. CTC could represent a population enriched in polyclonal activated T cells with low spontaneous cytotoxicity against a variety of allogeneic target cells, which is greatly enhanced by the addition of lectins dur ing the 51Cr release assay.     

A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK) Cells

The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.     

Enhanced antitumor activity of a combination treatment with a mouse/human chimeric anti-MK-1 antibody and lymphokine-activated killer cells in vitro and in a severe combined immunodeficient mouse xenograft model

Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarcinoma, recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas and seems to be valuable in immunodiagnosis and immunotherapy of various cancers. In a recent study, we constructed a mouse/human chimeric antibody, designated Ch FU-MK-1, by fusing the FU-MK-1 V_H and Vκ genes to the human Cγ1 and Cκ genes, respectively. In the present study, we tested combination immunotherapy of Ch FU-MK-1 with human lymphokine-activated killer (LAK) cells in vitro and in mice with severe combined immunodeficiency (SCID) bearing human MK-1-expressing tumors. In in vitro experiments, Ch FU-MK-1 effectively mediated antibody-dependent cell-mediated cytotoxicity (ADCC) against MK-1-expressing MKN-74 cells, which was completely blocked by an anti-FcR antibody. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by LAK cells and Ch FU-MK-1 against MKN-74 cells. The implication of the apoptosis during ADCC was demonstrated by means of both a terminal-deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay and a propidium iodide staining method. In vivo antitumor activity of combination treatment with LAK cells and Ch FU-MK-1 was estimated using SCID mice inoculated s.c. with MKN-74 cells. The i.v. administration of LAK cells and i.p. administration of Ch FU-MK-1 and interleukin-2 (IL-2) produced a marked growth inhibition of MKN-74 tumors in SCID mice. When the actual tumor weights were measured 16 days after initiation of treatment, more than 70% reduction was observed in the group receiving LAK cells plus Ch FU-MK-1 plus IL-2 as compared to the control untreated group. Together these results suggest that Ch FU-MK-1 may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors. Received: 27 November 1998 / Accepted: 23 February 1999     

An NK cell line (NK92-41BB) expressing high levels of granzyme is engineered to express the high affinity chimeric genes CD16/CAR

Natural killer (NK) cells are known to play a role in mediating innate immunity and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) based on the reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, devoid of CD16 and derived from a lymphoma patient, has been well characterized. The adoptive transfer of irradiated NK-92 cells demonstrated safety and showed preliminary evidence of clinical benefit for cancer patients. The molecules 41BB and CD3 are commonly used as stimulators in the CAR structure, and their expression in NK cells can promote the activation of NK cells, leading to the enhanced perforin- and granzyme-mediated lysis of tumor cells. This study showed that genetically modified NK-92 cells combined with antibody-mediated ADCC using rituximab and trastuzumab monoclonal antibodies lysed tumor cells more efficient than the NK-92 cell lines. It also showed that the anti-tumor activity of chimeric stimulator molecules of the CAR-modified CD16 receptor was stronger than that of CD16 (allotype V158). These studies provide a rationale for the use of genetically modified NK-92 cells in combination with IgG1 anti-tumor monoclonal antibodies. We also provide a rationale for the chimeric modified CD16 receptor that can improve the anti-tumor effect of NK92 cells via ADCC.

     

Human large granular lymphocytes express high affinity receptors for murine monoclonal antibodies of the IgG3 subclass

We have evaluated the ability of murine monoclonal antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by human lymphoid cells. Purified large granular lymphocytes (LGL) and interleukin 2-dependent cloned LGL lines having a CD2+/CD16+/CD3- phenotype were tested as effector cells in an ADCC assay by using a family of IgG isotype switch variant anti-Thy-1.1 antibodies against 51Cr-labeled Thy-1.1+ murine SL2 thymoma target cells, a system in which human cells have no spontaneous cytotoxicity. Cytotoxicity was greatest when using the IgG3, followed in rank order by the IgG2a and IgG2b. No cytotoxicity was observed with the IgG1 antibody. Because the antigen-binding regions of the antibodies are identical, the differences in cytotoxicity directly reflect the relative affinity of LGL Fc receptors for each antibody isotype.     

Augmentation of cellular immune response by Ipomoea obscura and Ipobscurine alkaloid attenuates tumor growth in mice

The immune status of the host plays a crucial role in controling the process of carcinogenesis. General or selective activation of various immunocompetent cells and their secretory function to maintain a healthy immune status may help in cancer prophylaxis, as well as therapy. The present study focused on the effect of Ipomoea obscura and Ipobscurine on cell-mediated immune response. In this study we evaluated the effect of I.?obscura and an indole alkaloid fraction from I. obscura on effector mechanisms of cell-mediated immune response by analyzing cytotoxic T lymphocyte (CTL) activity, natural killer (NK) cell activity, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent complement-mediated cytotoxicity (ACC). The effect of I. obscura and Ipobscurine on interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels was also analyzed. In the in vitro and in vivo systems, I. obscura and Ipobscurine treatment augmented cell-mediated immune response by enhancing the killing activity of CTL and NK cells from splenocytes in normal as well as tumor-bearing mice. ADCC and ACC were also enhanced significantly in both normal and tumor-bearing animals after drug administration, compared with untreated control. Administration of I. obscura and Ipobscurine significantly enhanced the production of IL-2 and IFN-γ in normal as well as tumor-bearing animals. This study reveals that both I. obscura and Ipobscurine have the potential to augment immune response through the enhanced secretion of IL-2 and IFN-γ by T cells and thereby inhibit tumor growth and as an alternative medicine for cancer treatment.     

Interaction of monocytes with tumor cells coated with complement with or without antibody

Raji, a human B lymphoblastoid cell line has the ability to activate the complement cascade by alternate pathway mechanisms with subsequent fixation of C3 to receptors on the Raji cell membrane. Using this property, we examined the role that complement plays in mediating a cytolytic event between human peripheral blood monocytes and Raji cells coated with C3b, antibody, or both. Presence of C3 was confirmed by immune adherence. IgG bound to the Raji membrane was quantitated using I¹²⁵ Staphylococcal protein A assay. The presence of alternate pathway-activated C3 on Raji cells failed to produce monocyte-mediated cytotoxicity. These same target cells subsequently coated with antibody concentration ranging from 200 to >600,000 SPA molecules per Raji cell produced neither enhancement nor inhibition of antibody-dependent, cell-mediated cytotoxicity (ADCC). ADCC was enhanced by complement when complement activation and binding of C3 to the cell surface occurred by classical pathway mechanisms. ADCC of 32% ± 3.2 occurred with undiluted antiserum (625,000 SPA molecules bound/Raji cell) with enhancement to 52% ± 1.1 in the presence of C3. IgG inhibition of ADCC was unaffected by the presence of membrane-bound C3.     

An Fc engineering approach that modulates antibody-dependent cytokine release without altering cell-killing functions

Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.     

Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis

KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model. Received: 10 July 1999 / Accepted: 21 January 2000     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Identification of five human lymphocyte subpopulations by their differential binding of various strains of bacteria.

The ability of human peripheral blood lymphocytes to kill antibody-coated Chang liver cells in antibody-dependent cell-mediated cytotoxicity (ADCC) can be blocked with aggregated IgG (agg-IgG) or by soluble immune complexes. Dissociation of aggregates of IgG or immune complexes from the cell surface, however, resulted in partial recovery of the ability both to bind agg-IgG and to kill in the ADCC assay. Our results indicate that “unblocking” of effector cells could occur in vivo when the concentration of circulating immune complexes is lowered.     

The human natural killer cytotoxic cell line NK-92, once armed with a murine CD16 receptor,represents a convenient cellular tool for the screening of mouse mAbs according to their ADCC potential

To take advantage of the large number of well-characterized mouse immunoglobulins (IgGs) for the study of antibody-dependent cell-mediated cytotoxicity (ADCC) in human cells, we armed human cytotoxic lymphocytes with a mouse receptor for the Fc portion of IgG antibodies. The human ΝΚ−92 natural killer cell line was transduced with a mouse receptor gene (mCD16), which was stably expressed on the cell surface (referred to as NK-92^mCD16). When tested against a B-lymphoblastoid cell line (BLCL) coated with mouse anti-CD20 IgG1, IgG2a or IgG2b monoclonal antibodies (mAbs), the newly expressed mouse Fc receptor enabled the NK-92^mCD16 cells to kill the BLCL by ADCC. Next, using the NK-92^mCD16 we compared mouse mAbs directed at B lineage specific CD antigens for their ability to induce ADCC against human Epstein-Barr virus- infected B lymphoblastoid (for anti-CD19, -CD20 and -CD21) or against myeloma (for anti-CD38 and –CD138) target cells. Our results demonstrated that the “NK-92^mCD16 assay” allows convenient and sensitive discrimination of mouse mAbs for their ability to mediate ADCC in a human cellular system. In addition, our results provide examples of dissociation between opsonization and target cell killing through ADCC. These “murinized” human effector cells thus represent a convenient cellular tool for the study of ADCC.     

Studies of natural killer cell activity and antibody-dependent cell-mediated cytotoxicity among patients with acute leukemia in complete remission

Summary Low natural killer (NK) cell activity against the K-562 leukemia cell line was observed in patients with acute leukemia in the early stages of remission, i.e., 2–4 months (11.3%±7.95% specific target cell lysis). This parameter was found to be normal among leukemia patients after a longer time in remission (19.53%±7.55%) when compared with healthy donors (18.46%±12.98%). A similar pattern of activity was observed in studies of antibody-dependent cell-mediated cytolysis (ADCC) to the CEM lymphoid tumor cell line in the same group (37.58%±12.4% vs. 51%±6.79% specific target cell lysis).ADCC to chicken red blood cells (CRBC) and to human red blood cells (HRBC) was not significantly different from that for healthy controls at either duration of remission.Nine patients relapsed over a follow-up period of 9 months. They were found to have slightly lower NK activity (14.4%±9.3%) and ADCC to CEM (41.4%±8.5%) than the patients who remained in remission (17.1%±6.8%; 48.7%±9.7%, respectively).These data indicate a lymphocyte deficit which may persist for some time after remission has been induced, and which may be due to the effect of leukemic cell burden or the effect of aggressive chemotherapy.