5-Aminolevulinic acid-based photochemical internalization of the immunotoxin MOC31-gelonin generates synergistic cytotoxic effects in vitro.

Photochemical internalization (PCI) is a novel method for the endosomal or lysosomal release of membrane-impermeable molecules into the cytosol of target cells. This novel technology is based on the photodynamically induced rupture of endocytic vesicles preloaded with molecules of therapeutic interest. PCI of the ribosome-inactivating plant toxin gelonin and the immunotoxin monoclonal antibody 31 (MOC31) gelonin has been performed previously by the use of the endocytic vesicle-localizing photosensitizers TPPS2a and AIPcS2a and light, demonstrating synergistic toxicity against the more than 20 different cell lines tested, most of them of neoplastic origin. In this study we demonstrate that 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) is also capable of inducing PCI of MOC31-gelonin in the human colon adenocarcinoma cell line WiDr. The cells were incubated with 1 mM 5-ALA for up to 8 h in serum-free medium and from 24 to 96 h in serum-containing medium. Fluorescence microscopical studies indicate a partial plasma membrane localization of PpIX when 5-ALA was applied under serum-free conditions. This plasma membrane localization was not seen when 5-ALA was given in the presence of serum. There was a granular component of the PpIX localization in addition to a diffuse cytoplasmic localization. The granular component resembled the localization of the fluorescent dye conjugate Alexa-gelonin and the lysosomal localizing dye acridine orange. Our present results provide evidence for an endocytic vesicle-associated fraction of PpIX after 5-ALA incubation of the WiDr cells. We demonstrate that PCI, by combining 5-ALA, MOC31-gelonin and light, induces a synergistic cytotoxic effect against the WiDr cells.

In vivo documentation of photochemical internalization, a novel approach to site specific cancer therapy.

Photochemical internalization (PCI) is a unique procedure for site-specific delivery of several types of membrane-impermeable molecules to the cytosol of target cells. The technology is based on photochemical-induced release of endocytosed macromolecules from endosomes and lysosomes into the cytosol. The purpose of this study was to evaluate the therapeutic potential of PCI of the type I ribosomal-inactivating protein gelonin in an animal model. The photosensitizer aluminum phthalocyanine disulfonate (AlPcS(2a)) was injected intraperitoneally (10 mg/kg) into athymic female BALB/c (nu/nu) nude mice (8-9 mice per group) with subcutaneously growing human adenocarcinoma (WiDr) tumors 48 hr before exposure to 135 J/cm(2) of red light focused on the tumor. Six hours before light exposure a single dose of 50 microg gelonin was administrated intratumorally. Tumor growth was measured at least twice a week. After immunomagnetic separation of in vivo growing tumor cells the subcellular localization of the photosensitizer was evaluated by fluorescence microscopy. The photosensitizer localized in endocytic vesicles in in vivo growing WiDr cells. Furthermore, it was found that in vitro gelonin treatment of WiDr cells isolated from photosensitizer-treated mice potentiated a light-induced decrease of clonal survival. Complete remission in 6 of 9 (67%) of the treated mice were induced. Our findings indicate that photochemical treatment with the photosensitizer AlPcS(2a) activates the cytotoxic potential of gelonin in vivo. These results demonstrate that the synergistic effect of combining photoactivation of photosensitizer located in endocytic vesicles and gelonin is indeed a result of PCI of gelonin.

Protection against Helicobacter pylori and other bacterial infections by garlic.

Louis Pasteur was the first to describe the antibacterial effect of onion and garlic juices. Historically, garlic has been used worldwide to fight bacterial infections. Allium vegetables, particularly garlic (Allium sativum L.) exhibit a broad antibiotic spectrum against both gram-positive and gram-negative bacteria. Noteworthy results published include the following: 1) raw juice of garlic was found to be effective against many common pathogenic bacteria-intestinal bacteria, which are responsible for diarrhea in humans and animals; 2) garlic is effective even against those strains that have become resistant to antibiotics; 3) the combination of garlic with antibiotics leads to partial or total synergism; 4) complete lack of resistance has been observed repeatedly; 5) even toxin production by microorganisms is prevented by garlic. Helicobacter pylori (H. pylori) is a bacterium implicated in the etiology of stomach cancer and ulcers. The incidence of stomach cancer is lower in populations with a high intake of allium vegetables. We have demonstrated in vitro that H. pylori is susceptible to garlic extract at a fairly moderate concentration. Even some antibiotic-resistant H. pylori strains are susceptible to garlic. Clinical trials are necessary to explore the possibility of using garlic as a low-cost remedy for eradicating H. pylori.

Systemic immunotoxin treatment inhibits formation of human breast cancer metastasis and tumor growth in nude rats.

Adjuvant chemotherapy in breast cancer patients has had limited success, which is possibly because of lack of effect on non-proliferating cells accompanied by the emergence of drug-resistant cell clones. Since immunotoxins (ITs) are known to exert proliferation-independent cytotoxicity, we investigated the efficacy of systemically administered anti-carcinoma ITs in nude rat models, simulating micrometastatic disease. The monoclonal antibodies MOC31, BM7 and 425.3, which recognize epithelial glycoprotein 2, MUC-1 mucin and the epidermal growth factor receptor, chemically conjugated to Pseudomonas exotoxin A (PE), inhibited protein synthesis of the 2 breast cancer cell lines at concentrations of 0.3-0.4 ng/ml, except for BM7-PE, which was less efficacious (65 ng/ml). In the MA-11 model in nude rats, a single i. v. dose of 20 microg MOC31-PE prevented development of metastasis in the spinal cord in 11/19 (58%) of the animals. Similarly, 425.3-PE treatment gave 6/9 (66%) long-term survivors. In rats injected intracardially or intratibially with MT-1 cells, treatment with 425. 3-PE prevented metastasis in 4/10 (40%) and intratibial tumor growth in 17/18 (94%) of the rats. Importantly, an equimolar dose of free 425.3 (antibody) was ineffective, whereas PE alone was toxic. With BM7-PE, 5/17 (29%) cures were obtained in the intratibial model. The results demonstrate that systemic short-term treatment with non-toxic doses of the 3 ITs tested can effectively inhibit the development of experimental breast cancer metastasis and/or local tumor growth in bone. The results support the development of the ITs towards clinical evaluation for possible use as short-term adjuvant therapy in patients at high risk of early relapse.

Photochemical internalisation increases the cytotoxic effect of the immunotoxin MOC31-gelonin.

Photochemical internalisation (PCI) was recently demonstrated as a unique procedure for site-specific delivery of several types of membrane impermeable macromolecules from endocytotic vesicles to the cytosol (Berg et al., 1999). The technology is based on the cytosolic release of endocytosed macromolecules from endosomes and lysosomes upon exposure of cells to photosensitising compounds, which became localised to these vesicles, and light. In our study the possibility to increase the cytotoxic effect of the immunotoxin MOC31-gelonin by PCI was examined. The type I ribosome-inactivating protein gelonin was covalently linked to the monoclonal IgG1 antibody MOC31, directed against epithelial glycoprotein-2 (EGP-2), an antigen expressed on most carcinoma cells. Five different cell lines, of which 4 expressed EGP-2, were treated with MOC31-gelonin and endosomal and lysosomal localising photosensitisers, followed by exposure to light. Insignificant cytotoxicity of the MOC31-gelonin was observed when the cells were incubated with the immunotoxin alone. However, in combination with endosomal and lysosomal localising photosensitizers, we demonstrate synergistic toxic effect of the MOC31-gelonin conjugate in a light-dependent manner. Our results indicate that PCI is a promising tool for increasing the cytotoxicity of immunotoxins, which is important for further improvement of the PCI concept towards possible use in cancer therapy.

Helicobacter pylori--in vitro susceptibility to garlic (Allium sativum) extract.

Gastric cancer is the major cancer in the developing world and one of the top two worldwide. Helicobacter pylori is a bacterium implicated in the etiology of stomach cancer. The incidence of stomach cancer is lower in individuals and populations with high Allium vegetable intakes. Allium vegetables, particularly garlic, have antibiotic activity. Standard antibiotic regimens against H. pylori are frequently ineffective in high-risk populations. As part of our study of the role of Allium vegetable intake on cancer prevention, we wished to investigate its antimicrobial activity against H. pylori. An aqueous extract of garlic cloves was standardized for its thiosulfinate concentration and tested for its antimicrobial activity on H. pylori grown on chocolate agar plates. Minimum inhibitory concentration was 40 micrograms thiosulfinate per milliliter. Staphylococcus aureus tested under the same conditions was not susceptible to garlic extract up to the maximum thiosulfinate concentration tested (160 micrograms/ml). To our knowledge, this is the first report of H. pylori's susceptibility to garlic extract of known thiosulfinate concentration. It is plausible that the sensitivity of H. pylori to garlic extract at such low concentration may be related to the reported lower risk of stomach cancer in those with a high Allium vegetable intake. Furthermore, it may identify a strategy for a low-cost intervention, with few side effects, in populations at high risk for stomach cancer, particularly where antibiotic resistance and the risk of reinfection are high.

Therapeutic efficacy of a doxorubicin immunoconjugate in a preclinical model of spontaneous metastatic human melanoma.

Doxorubicin (DOX) was conjugated to a monoclonal antibody (mAb 425) directed against the human epidermal growth factor receptor. The immunoreactivity of these conjugates, with an average of six to eight molecules of DOX per antibody, was largely conserved, and their in vitro cytotoxicity against metastatic human melanoma cells (M24 met) was improved over that of free DOX. An evaluation of the therapeutic efficacy of mAb 425-DOX indicated that this immunoconjugate suppressed the growth of primary and secondary M24 met tumors in mice with severe combined immunodeficiency and prolonged the life span of these animals, whereas an equivalent dose of free DOX was ineffective. Conjugation of DOX to an irrelevant mAb also increased its antitumor effect over that of equivalent amounts of free drug but to a lesser extent than that achieved by the mAb 425-DOX conjugate. These results demonstrate targeted delivery and striking antitumor activity of DOX immunoconjugates in a preclinical model of spontaneous, metastatic human melanoma that was insensitive to free DOX.

Immunoconjugates of Pseudomonas exotoxin A: evaluation in mice, monkeys, and man.

Immunotoxins of PE were constructed with stable thioether linkages using two monoclonal antibodies to ovarian cancer, OVB-3 and NR-LU-10. Antigens recognized by both antibodies have limited normal tissue distribution and are expressed on virtually all ovarian cancers. Both antibodies form highly potent conjugates (ID50 = 100 pg/ml) with high selectivity (greater than or equal to 4 logs) and can eliminate greater than or equal to 5 logs of tumor cells in vitro. The conjugates have been evaluated for efficacy in both ovarian and colon carcinoma ascites xenografts. In the ovarian model, the conjugates produce an increase in life span (ILS) of 200 to 300 with some cures against established but low tumor burden ascites. Increasing the tumor burden decreases efficacy and duration of responses. A lower ILS of 150 to 200 is achieved in the more aggressive colon model. However, the combination of immunotoxin with chemotherapy, which is ineffective on its own, demonstrated enhanced activity (ILS = 300). Toxicity of the conjugates is hepatic and easily monitored by liver function tests (LDH). Antitoxin responses are highly variable, but typically have a rapid onset and appear to be predicted by preexisting levels. Pilot clinical evaluation in ovarian cancer (intraperitoneal) is ongoing.

Enhanced therapeutic efficacy against an ovarian tumor xenograft of immunotoxins used in conjunction with recombinant alpha-interferon.

The antitumor effects of two immunotoxins were evaluated in vitro and in vivo against the human ovarian carcinoma cell line, OVCAR-3. The immunotoxins used were composed of recombinant ricin A chain (rRTA) covalently attached to a monoclonal antibody directed toward the human transferrin receptor (45412/rRTA, also called 454A12 MAB-rRTA by Cetus Corporation) or Pseudomonas exotoxin coupled to an anticarcinoma monoclonal antibody (NR-LU-10/PE). Preliminary characterization of the NR-LU-10 antigen by immunoprecipitation and cellular fluorescence demonstrated two dominant cell surface polypeptide moieties with molecular weights of 40,000 and 45,000 and a minor component with a molecular weight of 33,000. The immunotoxins were used alone or in combination with recombinant human alpha-interferon (rhIFN-alpha). Protein synthesis was inhibited in a dose-dependent manner in OVCA-3 cells incubated in vitro with either NR-LU-10/PE or 454A12/rRTA (50% inhibitory concentrations, 1 and 75 ng/ml, respectively). Unconjugated NR-LU-10 or 454A12 abrogated the activity of the relevant immunotoxins. Concomitant incubation in vitro of OVCAR-3 cells with NR-LU-10/PE or 454A12/rRTA and a noncytotoxic concentration of rhIFN-alpha potentiated the inhibitory activity of the immunotoxins via a mechanism independent of antigenic upregulation. This potentially synergistic combination was then tested in vivo. The median survival time (MST) of mice given injections i.p. of 4 x 10(6) OVCAR-3 cells was 46 days. Cohorts of mice that received intracavitary treatment beginning 5 days posttumor cell inoculation with either 0.25 or 0.5 microgram of NR-LU-10/PE every other day for a total of 10 treatments exhibited a significantly increased MST of 63 and 104 days, respectively (P less than 0.0001). Likewise, the i.p. injection of either 2.5 or 10 micrograms of 454A12/rRTA given in an identical schedule resulted in a MST of 89 and greater than 120 days, respectively (P less than 0.0001). When rhIFN-alpha was administered i.p. in conjunction with those doses of either immunotoxin, a significant increase in the MST was observed in comparison with mice given immunotoxin alone. The combination of 5 x 10(4) units of rhIFN-alpha and 0.25 microgram of NR-LU-10/PE resulted in 67% long-term survivors (greater than 120 days) compared with only 13% survival of mice given the immunotoxin alone. Similarly, 2.5 micrograms of 454A12/rRTA plus rhIFN-alpha resulted in an enhanced therapeutic response (89% long-term survivors) when compared with 454A12/rRTA alone (29%).(ABSTRACT TRUNCATED AT 400 WORDS)

Selective elimination of breast cancer cells from human bone marrow using an antibody-Pseudomonas exotoxin A conjugate.

A pancarcinoma monoclonal antibody (NR-LU-10), homogeneously reactive with human breast cancer cells, was conjugated to Pseudomonas exotoxin A. The immunotoxin was evaluated for its potential for purging breast cancer cells from human bone marrow. The immunotoxin NR-LU-10 antibody did not react with normal bone marrow preparations yet readily detected 1% contamination of bone marrow by MCF-7 breast cancer cells added to normal bone marrow without significantly inhibiting the colony-forming ability of bone marrow progenitor cells. NR-LU-10-Pseudomonas exotoxin A has potential for purging bone marrow of breast cancer cells without impairing the growth of bone marrow progenitor cells.

Immunotoxins of Pseudomonas exotoxin A (PE): effect of linkage on conjugate yield, potency, selectivity and toxicity.

Conjugates of monoclonal antibodies and Pseudomonas exotoxin A (PE) were formed with disulfide or thioether bonds. Thioether conjugates which formed with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) modified PE and reduced antibody formed with an 80% yield of equimolar conjugate within 30 min with an offering of one to one (toxin:antibody). The efficiency and kinetics of thioether formation were much higher with SMCC than with other maleimide reagents as well as more efficient than disulfide linkers. Thioether linkage resulted in immunotoxin consistently more potent and more selective in vitro than disulfide bonded conjugate. Thioether bonded conjugates also proved to have other favorable in vivo properties compared to disulfide conjugates: (1) a longer half-life in serum; (2) increased tumor localization; and (3) reduced toxicity. Toxicity of thioether linked holotoxin conjugates was directed at the liver hepatocyte but was easily monitored by serum liver enzymes. The conjugates are currently undergoing clinical evaluation for treatment of ovarian cancer with intraperitoneal administration. Research is ongoing to further decrease residual toxicity without reducing the potency of the conjugate.

Enhanced therapeutic efficacy of an immunotoxin in combination with chemotherapy against an intraperitoneal human tumor xenograft in athymic mice.

A mouse IgG2b anti-pan carcinoma monoclonal antibody, NR-LU-10, was shown to bind homogeneously to ascites xenografts of both ovarian and colon carcinoma. Following linkage to a highly potent holotoxin, Pseudomonas exotoxin A (PE), NR-LU-10 demonstrated high potency and selectivity in vitro (ID50 = 100 pg/ml; elimination of greater than or equal to 4.5 logs of cells). The conjugate was evaluated for therapeutic efficacy against a human colon tumor (HT-29) transplantable in the peritoneal cavity of nude mice. Beginning 3 days after HT-29 injection, mice received either three or six i.p. injections of 0.5 micrograms of unconjugated NR-LU-10 or immunotoxin conjugate (NR-LU-10/PE) every other day. Mice that received three or six treatments of NR-LU-10 alone had median survival times (MSTs) of 39 and 40 days, respectively, which did not differ significantly from the MST observed for the untreated control groups (MST = 35 days). In contrast, treatment with three or six injections of 0.5 micrograms NR-LU-10/PE exhibited significantly increased MSTs (P = 0.002) of 50 and 60 days, respectively. Coinjection of unconjugated NR-LU-10 (20 micrograms) and 0.5 micrograms of NR-LU-10/PE blocked the therapeutic effect of the immunotoxin (MST = 33 days). The therapeutic efficacy of NR-LU-10/PE was further enhanced against HT-29 when administered i.p. during and after cytoreductive chemotherapy. The i.p. administration of 300 mg/lg of cyclophosphamide plus 100 mg/kg of the chemoprotective drug, WR-2721, 10 and 17 days posttumor cell inoculation induced a significant increase in MST from 36 days to 59 days (P = 0.002). Interestingly, groups of mice that received either two, four, or seven treatments of NR-LU-10/PE following cytoreductive therapy exhibited a further significant increase (P = 0.001) in MSTs of 89, 97, and 105 days, respectively. Therefore, the use of immunotoxin therapy following cytoreductive chemotherapy significantly prolonged survival time of mice bearing the HT-29 colon tumor over that observed with chemotherapy or NR-LU-10/PE alone.

Oxygen metabolism of the HL-60 cell line: comparison of the effects of monocytoid and neutrophilic differentiation.

HL-60 cells are promyelocytic leukemia cells that respond to culture conditions with differentiation into granulocytelike or macrophagelike phagocytes. O2 metabolism is critical to the microbicidal function of phagocytic cells. O2 metabolism was studied in HL-60 cells differentiated with dimethylsulfoxide (Me2SO) and 1,25(OH)2D3, with the objective of 1) determining the validity of these cells as models for human neutrophils and monocytes, respectively, and 2) determining whether these cells are capable of forming hydroxyl radical. Me2SO-treated cells had morphology consistent with human neutrophils. O2 consumption by these cells in response to phorbol myristate acetate (PMA; 100 ng/ml) or opsonized zymosan (3 mg/ml) was less than that by neutrophils, as was superoxide formation. O2 metabolism was not inhibited by KCN or antimycin A. Myeloperoxidase (MPO) activity decreased during differentiation but remained greater than that of human neutrophils. Cytochalasin B enhanced recovery of superoxide secreted in response to zymosan, implying its release from the phagosome. 1,25(OH)2D3-treated cells had morphology consistent with monocytes. O2 consumption and superoxide release were less than with Me2SO-treated cells. Unlike the case with human monocytes, O2 consumption was not inhibited by KCN or antimycin A. MPO activity was minimally reduced by differentiation. Cytochalasin B inhibited recovery of superoxide. Luminol-dependent luminescence was greater among 1,25(OH)2D3-treated cells than among Me2SO-treated cells. Free radicals were also measured with a spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Spin trapping allows direct, simultaneous detection of superoxide and hydroxyl radicals. Regardless of the mechanism of differentiation, only superoxide was formed by HL-60 cells. These results show that Me2SO-treated HL-60 cells represent an excellent model for the study of human neutrophil oxidative function. However, 1,25(OH)2D3-treated cells are quite different in their O2 metabolism from peripheral blood monocytes.

The metabolite 1 alpha,25-dihydroxyvitamin D3 enhances lymphokine-mediated activation of the oxidative metabolism of the U937 cell line and phosphorylation of a 48-kD respiratory burst-associated protein.

U937 cells respond to a variety of stimuli with increased differentiation as manifested by reduced growth, increased adherence, increased expression of several surface receptors, and increased capacity for phagocytosis and formation of reactive oxygen intermediates. In the present study the effects of lymphocyte conditioned media, recombinant interferon-gamma (IFN-gamma), and 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the ability to form reactive oxygen intermediates by U937 cells were measured by using the luminol-dependent luminescence (LDL) assay. Neither 1,25(OH)2D3 alone nor IFN-gamma alone enhanced competence for phorbol myristate acetate-stimulated LDL. Cells were capable of moderate LDL after exposure to lymphocyte conditioned media, and this was enhanced by 1,25(OH)2D3 (10(-8) mol/L) and other vitamin D metabolites at higher concentrations. This effect was not secondary to accelerated production of myeloperoxidase, which is important in the LDL assay. Enhanced phorbol myristate acetate-stimulated phosphorylation of a 48-kd substrate was observed in 32P-labeled intact cells treated with 1,25(OH)2D3 alone or in combination with IFN-gamma. Treatment of cells with IFN-gamma or lymphocyte conditioned media did not alter phosphorylation. These results support the concept that 1,25(OH)2D3 plays a role in phagocyte differentiation and activation beyond the effects of lymphokines. Protein kinase C-mediated phosphorylation reactions may be necessary for the ability of U937 cells to reduce O2 and required for maximal activity under some conditions of incubation.

Biological activity of 19-nor, 10-keto, 25-hydroxyvitamin D3.

19 nor, 10 keto, 25-hydroxyvitamin D3 (19/10-25OHD3) is a metabolite of 25-OHD3 produced in vitro by various phagocytes including normal human blood monocytes and transformed cell lines, U937 and HL-60. We recently reported that 19/10-25OHD3, induced differentiation of U937 cells. In these studies, 19/10-25OHD3 alone produced no detectable effect on the growth rates, surface adherence, and oxidative metabolism of U937 and HL-60 cells. When combined with lymphocyte-conditioned medium (LCM), 19/10-25OHD3 reduced proliferation, increased surface adherence and stimulated luminol-dependent luminescence (LDL) of the U937 cells. In contrast, the combination of 19/10-25OHD3 and LCM had no effect on the growth of HL-60 cells but did increase the surface adherence and the expression of a complement receptor component. 19/10-25OHD3 competed for tritium-labeled 1,25(OH)2D3 binding to receptors extracted from cultured human skin fibroblasts. This displacement capacity was 600 times weaker than that of unlabeled 1,25(OH)2D3. Incubation of human skin fibroblasts for 24 hr with 19/10-25OHD3 induced 25OHD3-24-hydroxylase activity in the fibroblasts. The inductive potency of 19/10-25OHD3 was 1/50 that of 1,25(OH)2D3. These results demonstrate bioactivity of 19/10-25OHD3 in several systems. At least one of these responses, the induction of 25OHD3-24-hydroxylase, is a receptor-mediated event. Some of the other responses may be independent of the cellular receptor for 1,25(OH)2D3. Interestingly, the potency of 19/10-25OHD3 was highest in the receptor-mediated response (1:50) and lower in the other parameters, ranging from 1:100 to 1:600 compared to 1,25(OH)2D3. This range of bioactivity in phagocytes and fibroblasts is presently explained.

17 Beta-estradiol inhibits the oxidative metabolism of U937 cells indirectly via lymphocytes.

The effect of 17 beta-estradiol (E) on the oxidative metabolism of U937 cells was studied. E had no direct effect on the proliferation, surface adherence, or luminol-enhanced luminescence (LEL) of the U937 cells. Exposure of U937 cells to lymphocyte-conditioned medium (LCM) and 1,25-dihydroxyvitamin D3 allowed a maximal LEL response by cells stimulated with phorbol myristate acetate. In contrast, LCM from lymphocytes exposed to E (LCM-E) did not stimulate LEL to the same extent as did an equal volume of LCM. Increasing the E concentration in the lymphocyte medium was associated with a dose-dependent reduction in the LEL response of the U937 cells. Mixing equal quantities of LCM and LCM-E significantly decreased LEL levels. LEL stimulated by LCM, gamma-interferon, or differentiation-inducing factor was reduced by the presence of LCM-E. The inhibitory action of LCM-E was reversible and metabolite specific. 17 alpha-E produced an effect that was only one tenth the magnitude of the E effect. These findings indicate that E can modulate the differentiation of phagocytes indirectly by altering the synthesis and/or secretion of lymphokines.

Immunotoxins to a human melanoma-associated antigen: comparison of gelonin with ricin and other A chain conjugates.

Gelonin, a ribosome-inactivating protein from the seeds of Gelonium multiflorum, has been conjugated to antibodies. Previous reports have indicated variable potency of such immunotoxins. The lack of toxicity of gelonin, however, makes it attractive for immunoconjugate production. The ribosome-inactivating protein was covalently linked (using N-succinimidyl-3-(2-pyridyldithio)propionate) to monoclonal antibody, 9.2.27, directed to a human melanoma-associated glycoprotein/proteoglycan. The immunoconjugate showed high selectivity with dose-dependent cytotoxic activity to cultured human melanoma cells (50% inhibitory dose; 1-3 X 10(-11) M versus antigen-positive cells; 1-3 X 10(-7) M versus antigen-negative cells). Specificity and immunoreactivity of the conjugate were similar to those of unconjugated antibody. Biodistribution studies with iodine trace-labeled conjugate in nude mice indicated that tumor localization of the gelonin conjugate was decreased compared to unconjugated antibody. However, a significant therapeutic effect of the conjugate was found with multiple but not single dose i.v. treatment in nude mice bearing established palpable melanoma. These in vivo experiments showed that gelonin conjugates are not toxic up to 2 mg total dose/mouse and significantly retarded the growth of established s.c. tumor. Comparison of gelonin conjugates in vitro and in vivo with other A-chain conjugates of 9.2.27 (abrin and ricin) indicated that gelonin had similar potency, better selectivity, better tumor localization, and more significant therapeutic effects.

Immunotoxins to a human melanoma-associated antigen: resistance to pokeweed antiviral protein conjugates in vitro.

Pokeweed antiviral protein (PAP) from the summer leaves of Phytolacca americana was purified and conjugated via N-succinimidyl-3-(2-pyridyldithio)propionate to 9.2.27 anti-melanoma antibody to a glycoprotein-proteoglycan complex. The conjugate was highly potent (50% inhibition dose of 5 X 10(-11)-10(-13) M on antigen-positive melanoma) and highly selective (5 X 10(-8) on antigen-negative melanoma). Human melanoma cells were selected for resistance to in vitro killing of the PAP conjugate by cycling through a killing and recovery sequence. Resultant cultures were shown to be more than 2 logs less sensitive to the killing of the PAP conjugate than untreated cultures. Isolation of clones by limiting dilution and reanalysis indicated that the resistant polyclonal culture contained clones with a range of sensitivities. Resistant cultures were also resistant to other A-chain conjugates of 9.2.27, but not to intact toxins like ricin and abrin. Resistant cultures showed no change in antigen expression after selection with the PAP conjugate of 9.2.27. Thus, just as with many other chemotherapeutic agents, tumor cells can become resistant to agents inhibiting protein synthesis even when targeted with monoclonal antibody. The mechanisms of this resistance and modalities to minimize resistance are currently being explored.