Cytotoxic properties of a ricin A chain immunotoxin recognising the cluster-5A antigen associated with human small-cell lung cancer

Summary The cytotoxic properties of a ricin A chain immunotoxin made with the mouse monoclonal antibody SWA20, recognising a family of sialoglycoprotein antigens selectively expressed by human small-cell lung cancer (SCLC), were examined using a panel of tumour cell lines in tissue culture. SWA20—ricin-A-chain was selectively toxic to the SW2, NCI-H69 and GLC-8 SCLC cell lines, inhibiting the incorporation of [³H]leucine by 50% at a concentration of 0.2–2 nM, but had no selective activity against the NCI-H23 and NCI-H125 lung adenocarcinoma or the control CEM T-lymphoblastoid cell lines. The SWA20 immunotoxin intoxicated the SW2 cell line rapidly, inhibiting [³H]leucine incorporation by 50% within 2 h compared with 0.5 h for ricin. Analysis of the effects of SWA20—ricin-A-chain on the growth of SW2 cells using a limiting-dilution clonogenic assay revealed that the immunotoxin could eliminate 95% of clonogenic malignant cells. Although SWA20—ricin-A-chain was found to be rapidly active against the majority of tumour cells, its action was limited by the presence of insensitive cells expressing low levels of the target antigen.     

In vitro and in vivo properties of an anti-CD5—momordin immunotoxin on normal and neoplastic T lymphocytes

An anti-CD5 monoclonal antibody (mAb) was linked to the plant toxin momordin, a type-1 ribosome-inactivating protein purified fromMomordica charantia. The in vitro cytotoxicity of the immunotoxin was evaluated as the inhibition of protein and/or DNA synthesis on isolated peripheral blood mononuclear cells (PBMC) and on human T cell leukemia Jurkat. The potency of the immunotoxin on PBMC was very high (IC₅₀ = 1–10 pM) and was not affected by blood components. The conjugate was also very efficient in the inhibition of the proliferative response in a mixed lymphocyte reaction (IC₅₀ = 10 pM). Moreover, the in vitro performances of the immunotoxin compared favourably with those reported for other anti-CD5-based immunoconjugates containing ricin A chain. The in vivo activity of the immunotoxin was assessed in the model ofnu/nu mice bearing Jurkat leukemia. A significant inhibition of the tumour development (80%,P <0.01) in the animals treated with immunotoxin was observed. Taken together, the in vitro and in vivo results suggest that the anti-CD5-momordin conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5-positive leukemias and lymphomas.     

Blocked ricin-conjugated T cell immunotoxins: Effect of anti-CD6-blocked ricin on normal T cell function

Summary The biological properties of an immunotoxin composed of an anti-CD6 monoclonal antibody conjugated to whole ricin, which had been modified so that the galactose-binding sites of the B chain were blocked (blocked ricin), were examined. Treatment of peripheral blood lymphocytes with anti-CD6-blocked ricin for a 24-h period prevented T cell proliferation induced by phytohemagglutinin in a dose-dependent manner with concentrations causing 50% inhibition (IC₅₀) ranging from 5 pM to 30 pM. In contrast, treatment with either blocked ricin alone or with a control immunotoxin prepared with a B-cell-lineage-restricted monoclonal antibody gave IC₅₀ values of approximately 2 nM. Although shortening the duration of the anti-CD6-blocked ricin treatment to as little as 3 h had little significant effect on the observed inhibition, T cell viability experiments demonstrated that the magnitude of immunotoxin-induced killing after a given time period is significantly higher when the target cells become activated. Thus, from the initial concentration of cells treated with anti-CD6-blocked ricin placed in culture, 40%–45% viable cells remained after 2 days yet only 3%–9% remained if phorbol ester and Ca²⁺ ionophore were added; activation of T cells after mock treatment using blocked ricin plus nonconjugated anti-CD6 demonstrated that this effect was not the result of activation alone. The toxicity of anti-CD6-blocked ricin was also measured by inhibition of PHA-induced clonogenic growth of normal T cells. Continuous treatment of the cells using anti-CD6-blocked ricin at 0.1 nM resulted in a surviving fraction of about 3.5 × 10^–3; when immunotoxin treatment was for 24 h or less, the surviving fraction was only about 10^–1. As an indication of the unique specificity of anti-CD6-blocked ricin, immunotoxin pretreatment of potential responder cells prevented the generation of allogeneic cytolytic T lymphocytes in mixed lymphocyte cultures yet had little effect on the generation of interleukin-2-induced lymphokine-activated killer cell activity. We conclude that anti-CD6-blocked ricin demonstrates a cellular specificity and potency that make it a highly promising anti-T cell reagent.     

Anti-CD30 immunotoxins with native and recombinant dianthin 30

Immunotoxins were prepared with a Ber-H2 (anti-CD30) monoclonal antibody and native or recombinant dianthin 30, a ribosome-inactivating protein fromDianthus caryophyllus (carnation). Both immunotoxins selectively inhibited protein synthesis by CD30⁺ cell lines D430B (lymphoblastoid, infected with Epstein-Barr virus). L428 and L540 (both from Hodgkin's lymphoma). IC₅₀ values (concentrations, as dianthin, causing 50% inhibition) ranged from 324 pM to 479 pM (immunotoxin with native dianthin 30) or from 45 pM to 182 pM (immunotoxin with recombinant dianthin 30). The effect of either immunotoxin on protein synthesis by the CD30^– cell line K562 (from a chronic myeloid leukaemia) was not different from that of free dianthin (IC₅₀ higher than nM).     

Immunotoxin-mediated elimination of clonogenic tumor cells in the presence of human bone marrow

An immunotoxin was synthesized with pokeweed antiviral protein and an IgG1 monoclonal antibody directed against human B and pre-B cells. The B43 murine monoclonal antibody does not react with normal human bone marrow precursor cells. The immunotoxin bound to all Burkitt's lymphoma cell lines that were tested but not to human peripheral blood T cells. The ability of antibody-toxin conjugate to inhibit human lymphoblast cell lines was checked in a clonogenic assay system. The immunotoxin in the presence of chloroquine elicited 5.8 logs of killing of Burkitt's lymphoma cells (B-ALL). The efficient inhibition of clonogenic growth of B-ALL cells was not affected by the presence of normal bone marrow cells. The immunotoxin was not very toxic to pluripotent stem cells; less than 50% of the stem cells were lost under conditions where 5.6 logs of clonogenic lymphoma cells were eliminated from a 100-fold excess of normal marrow cells. Further, when assayed by long-term human bone marrow cultures, immunotoxin treatment did not result in a significant loss of pluripotent precursor cells.     

Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin

Summary An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [³H]leucine by CEM cells was inhibited by 50% at an M-T151-ricin-A-chain concentration (IC₅₀) of 4.6 nM compared with an IC₅₀ of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC₅₀ values of 20–30 pM. The addition of ricin B chain to CEM cells treated with M-T151—ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly.Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151—ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [³H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

Carbohydrate vaccines that induce antibodies against cancer. 1. Rationale

  Resistance to chemotherapy is a major cause for failure in the treatment of lung cancer. Compared to conventional cytotoxic drugs, immunotoxins act by different mechanisms and thus might be promising for the treatment of chemoresistant cancer. The monoclonal antibody MOC31 recognises the epithelial glycoprotein-2 (EGP-2), a cell-surface antigen associated with small-cell lung cancer (SCLC) and a major fraction of lung adenocarcinomas. An immunotoxin composed of MOC31 and a recombinant form of Pseudomonas exotoxin A lacking the cell-binding domain (ETA_{252 – 613}) was prepared, and its effect on lung cancer cell lines examined. MOC31-ETA_{252 – 613} was selectively cytotoxic to EGP-2-positive SCLC and adenocarcinoma cell lines inhibiting proliferation by 50% at concentrations ranging from 0.01 nM to 0.3 nM. Moreover, the immunotoxin reduced the numberof clonogenic tumour cells from cultures by factors of 10⁴ and 10⁵ during a 24-h and a 3-week exposure respectively. In athymic mice, the immunotoxin, which revealed a serum half-life of approximately 4 h, caused substantial regression of small (40 mm³) chemoresistant tumour xenografts and significantly delayed the growth of larger tumours (120 mm³). This finding indicates that MOC31-ETA_{252 – 613} may be useful for the treatment of lung cancer in the setting of chemoresistant minimal residual disease. Received: 31 October 1996 / Accepted: 5 December 1996     

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2

Summary Monoclonal antibody 14G2a (anti-GD₂) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD₂. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagentN-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor and interferon and chemotherapeutic agents such as cisplatinum andN,N-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.Research conducted, in part, by the Clayton Foundation for Research     

In vitro purging of clonogenic leukaemic cells from human bone marrow by interferon-γ-activated monocytes

With a view to the immunologically mediated purging of autologous bone marrow transplants in acute myeloid leukaemia, the efficacy of cytotoxic monocytes to cradicate leukaemic cells has been studied using clonogenic assays. U937 cells were found to be sensitive to highly purified and interferon--activated human monocytes whereas HL60 cells were rather resistant as measured in an MTT-based cytotoxicity assay under liquid conditions. A spectrophotometric clonogenic assay measured almost complete inhibition of clonogenic activity for U937 cells at low effector-to-target cell (E/T) ratios of at least 0.1. Limiting dilution analysis detected a 2–3 log₁₀ unit reduction in clonogenic activity. In an experimental mixture of U937 cells with a 20-fold excess of normal bone marrow nuclear cells a maximum 2-log₁₀-unit killing could be measured at E/T=10. Only at high E/T ratios could a reduction in granulocyte/macrophage-colony-forming units (cfu) be observed with only marginal effects on erythroid cfu and erythroid burst-forming. In conclusion, cytotoxic monocytes are highly potent anti-leukaemic effector cells, as measured in clonogenic assays, that do not compromise normal human progenitors.     

An immunotoxin for the treatment of T-acute lymphoblastic leukemic meningitis: studies in rhesus monkeys

Summary Monoclonal antibody WT1 (anti-CD7), conjugated to ricin A chain, was administered intrathecally to rhesus monkeys to test its suitability for use in the therapy of leukemic meningitis. The WT1-SMPT-dgRTA conjugate was cytotoxic to CEM (T-lymphoblastic leukemia) cells in vitro with an ID₅₀ of 53 pM. Immunoperoxidase testing showed no binding of WT1 to normal human tissues other than lymph nodes. Thirteen animals received one or more intrathecal 60-g doses of WT1-SMPT-dgRTA. All monkeys receiving repeated doses developed a cerebrospinal fluid (CSF) pleocytosis (primarily eosinophils), which was generally resolving by 3–4 weeks after therapy. Pharmacokinetic studies showed a half-life of 99 min, consistent with CSF clearance by bulk flow. Peak CSF immunotoxin concentrations exceeded the ID₅₀ for CEM cells by more than 2 log units and a concentration exceeding the ID₅₀ was maintained for as long as 24 h. All eight monkeys receiving repeated doses of immunotoxin developed serum antibodies against both WT1 and ricin A chain. In six of these monkeys antibodies were also present in the CSF. Both anti-WT1 and anti-(ricin A chain) antibodies were able to inhibit in vitro cytotoxicity of the immunotoxin for CEM cells; however, only anti-WT1 antibodies could block immunotoxin binding to the cell surface. No monkey developed anti-immunotoxin antibodies fewer than 7 days after the initiation of therapy, suggesting that repeated doses could be administered for up to 1 week without inhibition of clinical activity.     

CYTOTOXIC EFFECTS OF COLCEMID OR HIGH SPECIFIC ACTIVITY TRITIATED THYMIDINE ON CLONOGENIC CELL SURVIVAL IN B6CF1 MICE

High specific activity tritiated thymidine (HSA-[³H]TdR) and colcemid were given in cytotoxic doses and regimens to B6CF₁/Anl mice. The number of cells per intestinal crypt was reduced by the S-phase-specific HSA-[³H]TdR and the metaphase blocking and cytotoxic effect of multiple injections of colcemid. In 50-day old mice, the cytotoxic effect of multiple injections of colcemid reduced both the number of cells per crypt and the clonogenic cell survival. However, the number of surviving intestinal clonogenic or stem cells, assayed by the micro-colony technique, did not change in 110–130-day old mice. These data suggest that most of the cells at risk from these cytotoxic agents are not clonogenic in adult 110–130-day old mice but are the cells in amplification division. However, since the stem cells of young mice are more susceptible to colcemid, they are apparently in a more rapid cell cycle than those of older mice. The clonogenic cell survival measured in 110–130-day old mice after a single radiation dose of 14 Gy (1400 rad) responded in a non-linear way to increasing time of continuous colcemid cytotoxicity. These data suggest that the intestinal stem cells can respond to amplification compartment cell death by a shortening of their cell cycle and thus, over time, the number of stem cells at risk to colcemid cytotoxicity increases.     

Interferon β enhances the natural killer activity of patients with bladder carcinoma

We have investigated the effect of interferon (INFß) on the natural killer (NK) cytotoxic activity of peripheral blood mononuclear cells (PBMC) from patients with superficial and infiltrative transitional-cell carcinoma of the bladder (TCC) against both NK-sensitive and NK-resistant target cells. The normal NK activity found in PBMC from these patients can be significantly enhanced by short-term incubation (18 h) with INFß (P<0.05). The depressed NK cytotoxic activity found in PBMC from patients with infiltrative TCC can also be significantly enhanced, but not normalized, by short-term incubation with INFß (P<0.05). In kinetic studies we found that the maximal levels of the INFß-promoted cytotoxic activity against NK-sensitive and against NK-resistant target cells in PBMC from TCC patients were reached after 18 h of culture. Short-term-INFß-incubated PBMC from patients with TCC of the bladder also showed marked cytotoxic activity against NK-resistant target cells. The effector cells of the INFß-induced cytotoxic activity in PBMC from patients with TCC were CD16⁺ CD3^– NK cells. This cytotoxic inducer effect of INFß synergized with that of interleukin-2. In conclusion, INFß can enhance the NK activity of PMBC from patients with TCC of the bladder.     

Endocytosis of an antibody ricin A-chain conjugate (immuno-A-toxin) adsorbed on colloidal gold. Effects of ammonium chloride and monensin

An immunotoxin (IT) formed by a specific antibody coupled to the ricin A chain was adsorbed on colloidal gold particles (IT-Au). Binding and internalization of IT-Au in human lymphoblastic CEM cells were studied using electron microscopy. IT-Au showed specific cytotoxic activity toward the target cells. After 1 h at 4 degrees C, IT-Au were linked diffusely to the plasma membrane with 45% of the particles regrouped in clusters. Upon transfer to 37 degrees C, the particles carrying the ligand were regrouped more frequently and internalized into the cell by endocytosis through smooth microinvaginations or coated pits of the plasma membrane. After 15 min, IT-Au was observed in endocytic vacuoles, or receptosomes, in tubular structure near the Golgi apparatus and in lysosomes. Entry of IT-Au into lysosomes was rapid (around 50% of intracellular IT-Au particles after 30 min). NH4Cl or monensin, well-known potentiators of immunotoxin activity, when present in incubation medium, altered neither the processes nor the rate of IT-Au endocytosis. In the presence of either of these substances, IT-Au accumulated in the normal or often enlarged endocytic vacuoles, and entry into the lysosomes was slowed down (50% of particles after 2 h 15 min). We conclude that this intense slowing-down in the speed of IT-Au transportation into lysosomes and the functional modifications of these organelles help to explain the increased efficacy of immunotoxins in the presence of potentiators.     

Intracellular delivery of ceramide lipids via liposomes enhances apoptosis in vitro

Ceramide lipids have emerged as important intracellular signalling molecules that mediate diverse cellular effects, of which programmed cell death, or apoptosis, has attracted significant interest. Although the exact mechanism(s) by which ceramides trigger apoptosis is not fully understood, there is considerable evidence that they are key mediators of this response. Exogenously applied, cell-permeable ceramides have been shown to induce apoptosis when incubated with cells in culture. We examined here the cytotoxicity of ceramides with varying acyl chain lengths in order to determine whether acyl chain length affects pro-apoptotic activity within the concentration range of 0-100 μM. We found that for C₆-, C₈-, C₁₀-, C₁₄- and C₁₆-ceramide, the chain length was inversely proportional to cytotoxic activity, with C₆-ceramide being most active (IC₅₀ values in the 3-14 μM range) and C₁₆-ceramide being least active (IC₅₀ values in excess of 100 μM) in the MDA435/LCC6 human breast cancer and J774 mouse macrophage cell lines investigated. Using these two ceramide forms we were able to correlate the observed cytotoxicity with cellular uptake, and we observed that a lack of intracellular delivery may be responsible for the weak activity of C₁₆-ceramide. We therefore investigated the possibility of incorporating ceramide lipids into liposome bilayers to enhance this delivery. We demonstrate that stable, ceramide-containing liposomes can be formulated, and that they are cytotoxic when taken up by cells in vitro. These results provide an increased understanding of the differences in cytotoxic activity of exogenous short- and long-chain ceramide lipids, and their incorporation into biologically active liposomal formulations opens new avenues for apoptosis induction.     

Interleukin-4 receptor expression on AIDS-associated Kaposi's sarcoma cells and their targeting by a chimeric protein comprised of circularly permuted interleukin-4 and Pseudomonas exotoxin.

BACKGROUND: AIDS-associated Kaposi's sarcoma (AIDS-KS) represents one of the most common malignancies associated with human immunodeficiency virus infection. To target effective therapeutic agents to AIDS-KS, we have identified a new target in the form of interleukin-4 receptors (IL-4R). MATERIALS AND METHODS: The expression of IL-4R on AIDS-KS cells and their subunit structure was determined by radioligand receptor binding, cross-linking and Northern and RT-PCR analyses. The in vitro effect of IL-4 and recombinant fusion protein made up of circularly permuted IL-4 and a mutated form of Pseudomonas exotoxin, IL-4(38-37)-PE38KDEL, was examined by clonogenic and protein synthesis inhibition assays. RESULTS: Five AIDS-KS cell lines expressed high-affinity IL-4R with a Kd of 23.5-219 pM. IL-4 appeared to cross-link to one major protein corresponding to 140 kDa and a broad band corresponding to 60-70 kDa. Both cross-linked proteins were immunoprecipitated with an antibody to human IL-4R beta chain. AIDS-KS cells exhibited IL-4R beta-specific mRNA. IL-4 caused a modest inhibition (31-34%) of colony formation in two AIDS-KS cell lines tested. IL-4(38-37)-PE38KDEL was found to be highly effective in inhibiting the protein synthesis in all five AIDS-KS examined. The IC50 ranged from 32 to 1225 pM. The cytotoxic action of IL-4 toxin was blocked by an excess of IL-4, exhibiting the specificity of IL-4(38-37)-PE38KDEL. The cytotoxicity of IL-4 toxin observed by a clonogenic assay corroborated well with the IC50 obtained by protein synthesis inhibition assay. Normal human endothelial cells expressed a negligible number of IL-4R (< 50 sites/cell) and were less sensitive or not sensitive to IL-4(38-37)-PE38KDEL. CONCLUSION: The presence of a new plasma membrane protein in the form of IL-4R on AIDS-KS cells may be targeted by IL-4(38-37)-PE38KDEL for its potential implication in the treatment of AIDS-KS.     

Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing internalization rates

 Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin, 7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfR^neg) and two lines transfected with either wild-type TfR (Tfr^wt) or an internalization-deficient (TfR^{δ7 – 58del}) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfR^wt cells (ID₅₀<1 nM) while both TfR^neg cells and TfR^{δ7 – 58del} were only minimally affected (ID₅₀>100 nM). Butyrate up-regulation substantially increased receptor expression on the TfR^wt and TfR^{δ7 – 58del} cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by co-treatment of TfR^wt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfR^{δ7 – 58del} cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin efficacy than receptor number. Received: 15 March 1996 / Accepted: 28 May 1996     

Comparison between clonogenic and cytotoxic assays for measuring LAK cell activity

The antiproliferative effect of lymphokine-activated killer (LAK) cells was studied using a clonogenic assay in an attempt to find a model for predicting this effect in vivo or ex vivo (in the case of purging) in cancer treatment. The results were compared with the standard 51Cr-release cytotoxic assay. Cells from clonogenic neoplastic cell lines (K562 and HL-60) were plated in methylcellulose with LAK cells obtained from ten different donors in various effector-to-target (E:T) ratios. At E:T ratios of 16:1, elimination of greater than 90% of the clonogenic cells was seen in 20 of 21 experiments, whereas such lysis was incidentally found in the 51Cr-release assay. In almost all paired combinations, clonogenic cells tested in a colony assay were more sensitive to kill by LAK cells than the whole tumor cell suspensions measured in the 51Cr-release assay.     

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.     

Study of the cytotoxic activity of Styrax camporum extract and its chemical markers,egonol and homoegonol

The benzofuran lignans egonol and homoegonol are found in all species of the genus Styrax. Since natural products are important sources of new anticancer drugs, this study evaluated the cytotoxic activity of a hydroalcoholic extract of the stems of S. camporum (SCHE) and their chemical markers, egonol (EG) and homoegonol (HE), against different tumor cell lines (B16F10, MCF-7, HeLa, HepG2, and MO59J). A normal human cell line (GM07492A) was included. Cytotoxic activity was evaluated at different treatment times (24, 48 and 72 h) using the XTT assay. More effective results were observed after 72 h of treatment. The lowest IC₅₀ values were found for the HepG2 cell line, ranging from 11.2 to 55.0 µg/mL. The combination of EG and HE exerted higher cytotoxic activity than SCHE or treatment with either lignan alone, with the lowest IC₅₀ (13.31 µg/mL) being observed for the MCF-7 line. Furthermore, treatment with these lignans was significantly more cytotoxic for some tumor cell lines compared to the normal cell line, GM07492A, indicating selectivity. These results suggest that these lignans may be used to treat cancer without affecting normal cells.