Ocaratuzumab,an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells.

In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells.

Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab.

The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.     

Ocaratuzumab,an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells. In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.     

Vaccination of Stage IV patients with allogeneic IL-4- or IL-2-gene-transduced melanoma cells generates functional antibodies against vaccinating and autologous melanoma cells

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.     

Neutrophils are cytotoxic and growth-inhibiting for neuroblastoma cells with an anti-GD2 antibody but, without cytotoxicity, can be growth-stimulating

Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells. To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside (GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence of target cells in 96-well plates after 6–18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive: SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by DIMSCAN, was correlated with neuroblastoma cell number in both assays (100–2000 cells/well). In the cytotoxicity test, both neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5–50:1) and mAb ch.14.18 dose (0.1–10 μg/ml). ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 μg/ml). Without antibody, neutrophils inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion, neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell growth if antibody is not present or if GD2 is not expressed. Received: 18 November 1998 / Accepted: 24 September 1999     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen-I (OFA-I)

Summary IgG anti-OFA-I found in melanoma patients was tested for its ability to lyse human tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC). Sera from 89 stage II melanoma patients which contained non-HLA-related IgG antibody to an OFA-I-positive melanoma cell line (M14) as tested by indirect membrane immunofluorescence (IMI) were originally chosen as possible sources of IgG anti-OFA-I. Of those tested for specific IgG activity to OFA-I by IMI, anti-OFA-I was found only in those patients immunized with OFA-I-positive tumor cells. When the same sera were tested in ADCC, no non-HLA-related activity could be demonstrated. This result was confirmed with purified IgG fractions that could, nevertheless, show anti-OFA-I reactivity in a complement-dependent cytotoxicity assay. The fact that naturally occurring IgG anti-OFA-I antibody was not readily detectable in patients' sera and that induced IgG anti-OFA-I did not participate in ADCC indicates that OFA-I-related tumor cell lysis via ADCC is an unlikely phenomenon in cancer patients.     

Soluble ICAM-1 in breast cancer: clinical significance and biological implications

OBJECTIVES: In previous experiments, we demonstrated a decreased expression of intercellular adhesion molecule I (ICAM-1) on both tumour cells and antigen-presenting cells derived from patients with breast cancer, resulting in an abrogation of antigen presentation and tumour cell lysis. Recently, increased levels of a soluble isoform of ICAM-1 (sICAM-1) have been detected in the sera of breast cancer patients. The present investigation was performed in order to investigate the biological relevance of serum concentrations and the effects of sICAM-1 in patients with breast cancer. PATIENTS AND METHODS: sICAM-1 was determined using a sandwich enzyme immunoassay on sera from 88 patients with various stages of breast cancer and correlated with clinical parameters. The effect of sICAM-1 present in the sera of patients with breast cancer upon unspecific and anti-Her-21/neu antibody-mediated cytotoxicity (ADCC), as well as upon antigen presentation, was determined using a 51Cr-release assay and [3H]thymidine-uptake of T cells after co-incubation with tetanus-toxoid-pulsed antigen-presenting cells. RESULTS: In patients with early breast cancer, serum levels of sICAM-1 were significantly lower compared to patients with metastatic disease, but did not correlate with usual clinical parameters. In patients with metastatic breast cancer, a significant correlation of sICAM-1 with tumour markers CEA and CA 15-3 was observed. No influence of sICAM-1 upon unspecific cytotoxicity, ADCC, or the ability to present antigen was observed. DISCUSSION: The origin of sICAM-1 in the sera of patients with breast cancer remains unknown. In contrast to its membrane-bound isoform, sICAM-1 was increased in the sera of patients with various stages of breast cancer, but its presence did not influence unspecific cytotoxicity, ADCC, or antigen-induced T cell proliferation.     

Cell-mediate cytotoxicity in vitro of human lymphocytes against a tissue culture melanoma cell line (igr3).

Purified peripheral blood lymphocytes from 13 healthy donors, 6 melanoma patients and 1 halo nevus patient were tested for cytotoxic activity against an allogeneic melanoma cell line (IGR3) in, at least, one of the following assays: cell-mediated cytotoxicity (ADCC) and microcytotoxicity assays (ma). The lymphocytes were isolated by Ficoll-Triosil gradient centrifugation (fraction F) followed by removal of iron-phagocytosing and adherent cells (fraction FFF) and by subsequent passage through anti-IgG columns (fraction FFF-C). Leukocytes of each fraction were identified by different methods including morphology, rosette-formation, phagocytic activity, and membrane fluorescence. CMC activity paralled ADCC activity at a log lower level of sensitivity. In both assays lymphocytes of fractions F and FFF had the highest activity, whereas in fraction FFF-C cytotoxicity was strongly reduced. In all three lymphocyte fractions CMC and ADCC activity could be blocked by preincubation of the effector cells in aggregated IgG. Furthermore, depletion of E rosette-forming lymphocytes slightly increased ADCC and CMC activity, whereas depletion of EA and EAC rosette-forming lymphocytes strongly decreased it. Our results therefore indicate that in both CMC and ADCC assays, non-adherent, non-phagocytic Fc receptor-bearing lymphocytes ("K" cells) were the active cytotoxic cells. In MA, on the other hand, mononuclear phagocytes seemed to be the most active cell population. So far no significant difference was observed in CMC, ADCC, and MA between control persons and melanoma patients     

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

Protein glycosylation often changes during cancer development, resulting in the expression of cancer-associated carbohydrate antigens. In particular mucins such as MUC1 are subject to these changes. We previously identified an immunodominant Tn-MUC1 (GalNAc-α-MUC1) cancer-specific epitope not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis demonstrated high affinity binding (K_D?=?1.7 nM) of mAb 5E5 to its target, Tn-MUC1. Immunolabelling with mAb 5E5 revealed surface expression of the Tn-MUC1 epitope in breast cancer tissue and cell lines, and mAb 5E5 induced ADCC in two human breast cancer cell lines, MCF7 and T47D. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation     

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. VII. The effect of immunodeficiency disease.

Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and antibody-dependent cellular cytotoxicity (ADCC) was assessed in 13 patients with immunodeficiency diseases—immunodeficiency-thymoma syndrome (1), Bruton type agammaglobulinemia (3), and common variable hypogammaglobulinemia (9). SLMC and ADCC function were intact (and possibly enhanced) in the patient with immunodeficiency thymoma. Both ADCC and SLMC were detectable in the three patients with X-linked agammaglobulinemia, one of whom had lower than expected SLMC. In all of the immunodeficient patients, the relative inability of B lymphocytes to produce immunoglobulin in vivo or in vitro did not consistently affect the ability of (presumably) other lymphocytes to mediate SLMC and ADCC, although in three of the CVH patients this was lower than normal. In every case, removal of Fc receptor-bearing cells from the patients' lymphocyte preparations severely depleted SLMC (and ADCC when tested), but cytotoxicity was either unchanged or enhanced by depletion of E rosette forming T cells. The effects of Fc receptor-positive cell depletion, T-cell depletion, culture serum variation, or the addition of antibody-coated erythrocytes to the assay were similar on both SLMC and ADCC effector cells (“NK” and “K” cells), and whether patients' or normal lymphocytes were tested. The possible significance of the results with respect to surveillance against cancer is discussed.     

Decreased monocyte antibody-dependent cell-mediated toxicity in stage I–II malignant melanoma

Summary Lymphocyte and monocyte antibody-dependent cell-mediated cytotoxicity (ADCC) against human red blood cells was examined in 28 stage-I-II malignant melanoma patients. Eighteen were studied at various time intervals after receiving SC Corynebacterium parvum (C. parvum); 10 were untreated. Fifteen normal age-matched controls were also studied. Monocyte ADCC was significantly decreased in untreated patients compared with controls (P<0.005) and was significantly increased above controls and untreated patients in individuals treated with C. parvum (P<0.008). No significant differences in lymphocyte ADCC were seen. Optimal enhancement of monocyte ADCC by C. parvum occurred from 2 weeks to 1 month after treatment. Significant decreases in ADCC to baseline levels occurred in patients studied from 3 to 6 months beyond treatment.     

Antibody-dependent cellular cytotoxicity against HIV-coated target cells by peripheral blood monocytes from HIV seropositive asymptomatic patients

Cytotoxic effector cells like cytotoxic T cells, NK cells, monocytes/macrophages, and neutrophils can lyse directly HIV-infected or HIV-coated cells in the absence or presence of anti-HIV antibodies. Therefore, these cytotoxic mechanisms can be invoked either in the control of HIV infection at early stages of the disease or in the generalized immunosuppression observed at later stages of the disease. The relationship between anti-HIV effector mechanisms and disease, however, remains elusive. The present study investigates in HIV+ seropositive asymptomatic patients peripheral blood monocytes (PBM)-mediated antibody dependent cellular cytotoxicity (ADCC) against HIV-coated target cells in the presence of heterologous or autologous anti-HIV serum. To test for specific ADCC against HIV Ag, a T4+ CEM.TR line resistant to TNF and macrophage-mediated cytotoxicity was selected in vitro. ADCC was performed in an 18-h 51Cr-release assay using CEM.TR cells coated with inactivated HIV. Unlike PBM from normal controls, significant ADCC was observed by PBM from HIV+ seropositive patients in the presence of pooled HIV+ antiserum. The ADCC activity was specific for HIV and was dependent on the E:T ratio and the antiserum dilution used. Upon activation of PBM with rIFN-gamma, both normal and HIV+ PBM-mediated ADCC against HIV-coated CEM.TR. Furthermore, ADCC activity by PBM from HIV+ seropositive patients in the presence of their autologous serum was examined. Significant ADCC activity was observed and was dependent on the E:T ratio and serum dilution used. The findings demonstrating anti-HIV ADCC activity by PBM from HIV+ seropositive individuals and their autologous sera support the notion that monocyte-mediated ADCC may be operative in vivo.     

Depressed lectin-dependent and enhanced antibody-dependent cell-mediated cytotoxicity in patients with stage I cancer of the larynx

Lectin-dependent cell-mediated cytotoxicity (LDCC) of peripheral blood mononuclear cells (PBMC) from patients with stage I cancer of the larynx (LC) was evaluated using human adherent 3H-TdR-prelabeled HEp-2 carcinoma cells as targets at 50:1 effector-target ratio with 25 micrograms/ml concanavalin A (Con A) in a 24-hour assay. Under these conditions, but without Con A, no considerable natural cell-mediated cytotoxicity (NCMC) was performed by PBMC either from control or from LC donors. Depressed levels of LDCC, but augmented ADCC to chicken red blood cells were detected in LC patients. Natural killer activity to K562 targets was not different from that of control subjects. In parallel studies, normal Con A-induced blastogenesis and B cell counts, low T, and active T cell counts, as well as high Leu-11a+ cell counts were detected in patients with LC. The relationship between depressed LDCC and low T, and active T cell counts, and enhanced ADCC and high Leu-11a+ cell counts is suggested in stage I LC patients.     

Antibody-dependent cell-mediated cytotoxicity using a murine monoclonal antibody against human colorectal cancer in cancer patients

Summary Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by a murine monoclonal antibody against human colerectal carcinoma, antibody 19–9, with human effector cells was tested in 33 patients with various carcinomas, 16 patients with benign lesions, and 13 normal controls, using a 12-h ⁵¹Cr release assay using human colorectal cancer cells as targets. Peripheral blood mononuclear cells (PBM) from these groups of patients and normal controls achieved moderate levels of target cell lysis in the presence of the monoclonal antibody at the high effector to target cell ratio of 200:1. The ADCC activity of PBM in cancer patients was significantly higher than that in either normal persons or patients with benign lesions. Since the ADCC was shown to be mainly mediated by adherent monocytes in the PBM, ADCC activity of monocytes from cancer patients was compared to those from control groups at an effector to target cell ratio of 30:1. The results also showed that the lytic capacity of monocytes was significantly higher in cancer patients than that in the control populations.     

Demonstration of the specificity of a monkey antiserum against human melanoma

Summary The reactivity spectrum of a monkey antiserum raised against in vitro-cultured human melanoma cells was compared by means of three different assays: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and mixed hemadsorption (MHA). After absorption with a pool of cells from T- and B-lymphoid cell lines the antiserum was specifically cytotoxic in CDC for cultured melanoma cells. The melanoma specificity of the antiserum was confirmed by quantitative absorption experiments with cultured melanoma and nonmelanoma cells. When the lymphoid cell-absorbed antiserum was assayed by MHA, however, a high reactivity against both melanoma and nonmelanoma cells was observed. Further absorption with nonmelanoma tumor cells removed the reactivity of the antiserum with different nonmelanoma cell lines, but did not abolish its reactivity with melanoma cells. After this second absorption, the antiserum remained cytolytic against melanoma cells in CDC. In ADCC experiments this antiserum was not able to induce any cytotoxicity even before absorption.Analysis of Sephadex G 200 fractions of the antiserum revealed that the melanoma-specific antibodies cytotoxic in CDC were localized exclusively in the IgM peak. This finding was confirmed in similar studies with four other nonhuman primate melanoma antisera. Abbreviations used in this paper: CDC, complement dependent cytotoxicity: ADCC, antibody dependent cell mediated cytotoxicity; MHA, mixed hemadsorption; FCS, fetal calf serum.     

The role of apoptosis in antibody-dependent cellular cytotoxicity

Apoptosis in three lymphoma cell lines has been studied following cytotoxicity induced in vitro by normal human blood lymphocytes utilizing either natural killer (NK) or antibody-dependent cellular cytotoxic (ADCC) mechanisms. Guinea-pig L₂C leukaemic lymphocytes, but not the human cell lines Daudi and Jurkat, revealed a degree of time- and temperature-dependent apoptotic death upon simple culture in vitro. NK cytotoxicity at low effector: target ratios (E: T) induced both release of⁵¹Cr and apoptosis. However NK cytotoxicity at higher E : T, and ADCC at all E : T, increased the level of⁵¹Cr release while reducing the level of apoptosis. The findings were consistent with the apoptotic process being cut short by intervention of necrotic death. The same characteristics accompanied ADCC whether the effectors were recruited by Fc regions of antibody coating the targets, or by bispecific antibodies attaching one arm to the targets and the other to Fc receptors type III on effectors. This finding, and the high level of cytotoxicity elicited by the bispecific method, confirm the belief that NK cells, in addition to exerting NK cytotoxicity, represent the principal effectors for ADCC among blood mononuclear cells. Our results suggest that NK cells have both apoptotic and necrotic mechanisms available for killing their targets, but use only the latter for ADCC.     

Immunological effects of cancer chemotherapy in malignant melanoma

Summary The effects on the immune system of highdose cyclical combination chemotherapy were studied in nine patients with advanced malignant melanoma. Chemotherapy consisted of monthly cycles of dimethyl triazeno imidazole carboxamide 150 mg/m² i.v. daily from days 1–5, cyclophosphamide 1000 mg/m² i.v. on day 5, and vincristine 1.4 mg/m² i.v. on day 5.Immunological testing was carried out prior to treatment and at weekly intervals during the first month.B, T and non-B, non-T cell numbers all tended to fall early in the cycle as did the phytohaemagglutinin(PHA)-induced transformation and PHA-induced cytotoxicity to chicken red cells. Although PHA-induced transformation and cytotoxicity usually returned to normal by day 29, B and T cell numbers often remained subnormal. In contrast, levels for antibody-dependent, cell-mediated cytotoxicity (ADCC) were relatively stable throughout the cycle. Two patients with subsequent tumour response to therapy had rebound supranormal PHA transformations between weeks 1 and 3 of the first cycle. No other changes correlated with prognosis in individual patients.Analysis of the temporal relationships between PHA transformation, PHA-induced cytotoxicity, and ADCC supported the concept that the three assays reflect the function of separate mononuclear cell subpopulations.The stability of ADCC is of particular interest in view of other work suggesting that this function may be important in immune responses to tumours, including melanoma.Work was supported by grants from the National Health and Medical Research Council and the Western Australian Arthritis and Rheumatism Foundation, and the Cancer Council of Western Australia     

Human polymorphonuclear leukocyte-mediated cytotoxicity against varicella-zoster virus-infected fibroblasts

Polymorphonuclear leukocytes (PMN) were studied for their ability to mediate cytotoxicity against varicella-zoster virus (VZV)-infected and uninfected human fibroblasts in 51Cr release assays. PMN were capable of mediating antibody-dependent cellular cytotoxicity (ADCC) against VZV-infected targets. Maximal ADCC was obtained with effector-to-target ratios of 100:1 and 18 h of incubation. Percent 51Cr release for 26 normal adults was 14.1 +/- 0.6 (mean +/- standard error) in the presence of pooled human seropositive sera (final dilution, 1:100) and 0.5 +/- 0.6 in the presence of pooled human seronegative sera. Addition of phorbol myristate acetate (PMA) enhanced PMN-mediated cytotoxicity against VZV-infected and uninfected targets. PMA-stimulated cytotoxicity was optimal with PMA concentrations of 200 ng/ml and effector-to-target ratios of 10:1, and antibody was not required; killing was detected as early as 3 h after incubation and was maximal after 18 h. Highly purified PMN were capable of mediating both ADCC and PMA-stimulated lysis. Catalase completely inhibited PMA-stimulated PMN cytotoxicity, but had no effect on PMN-mediated ADCC. PMN from patients with chronic granulomatous disease were capable of mediating ADCC, but not PMA-stimulated killing, against VZV-infected targets. Thus, PMN could kill VZV-infected targets by two different mechanisms: ADCC, which required antibody but not hydrogen peroxide (H2O2), and PMA-stimulated cytotoxicity, which required H2O2 but not antibody.     

Antibody-dependent cellular cytotoxicity to virus-infected target cells: role of nylon wool-adherent T cells as effectors

This study was designed to elucidate whether populations of human blood lymphocytes other than non-T, non-B, Fc receptor-positive K cells can mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells acutely infected with type 1 herpes simplex virus. With appropriate technical precautions, a subset of E rosette-positive, nylon wool-adherent T cells were found to be effective in killing antibody-coated target cells. Thus, there appears to be at least two relatively distinct populations of lymphocytes in human peripheral blood that function as effectors in ADCC: one that consists of E rosette-negative cells, the other of E rosette-positive cells. These findings suggest a possible relationship between “classical” E rosette-negative K cells and E rosette-positive T cells.     

Intramucosal bacteria in colon cancer and their elimination by probiotic strainEnterococcus faecium M-74 with organic selenium

Intraepithelial bacteria were isolated by the gentamicin protection assay (GPA) from biopsy samples obtained at colonoscopy (colon cancer, n = 10 patients; colonic adenoma, n = 20; control group, n = 20; cancer patients without gastrointestinal tract GIT malignancy, n = 10). After a three-month administration of E. faecium M-74 to patients with positive GPA biopsies, 172 biopsy specimens from 60 patients were examined with the GPA. The number of biopsies with intracellular bacteria was significantly higher in adenoma and carcinoma group than in control group (26 vs. 10%; p = 0.004); in cancer patients without GIT malignancy the difference was nonsignificant. E. faecium M-74 was also administered to 5 patients with colonic adenoma; according to a control colonoscopy the number of biopsies with intracellular bacteria was significantly lower after probiotic administration (48 vs. 16%; p = 0.03). A striking prevalence of intraepithelial bacteria was also showed in patients with large bowel adenoma and carcinoma. The administration of probiotic strain M-74 can thus be considered to be an effective and promising method for elimination of pathogenic bacteria in the case of inflammatory bowel disease and colon cancer.