Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

Endocytosis and intracellular localisation of type 1 ribosome-inactivating protein saporin-s6.

Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from abasic sites in HeLa nuclei after intoxication with saporin-S6.

Reductive activation of type 2 ribosome-inactivating proteins is promoted by transmembrane thioredoxin-related protein.

Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.

Spiroindolones, a potent compound class for the treatment of malaria.

Recent reports of increased tolerance to artemisinin derivatives--the most recently adopted class of antimalarials--have prompted a need for new treatments. The spirotetrahydro-beta-carbolines, or spiroindolones, are potent drugs that kill the blood stages of Plasmodium falciparum and Plasmodium vivax clinical isolates at low nanomolar concentration. Spiroindolones rapidly inhibit protein synthesis in P. falciparum, an effect that is ablated in parasites bearing nonsynonymous mutations in the gene encoding the P-type cation-transporter ATPase4 (PfATP4). The optimized spiroindolone NITD609 shows pharmacokinetic properties compatible with once-daily oral dosing and has single-dose efficacy in a rodent malaria model.

Time course and efficiency of protein synthesis inhibition following intracerebral and systemic anisomycin treatment.

Since its discovery in the 1960s, anisomycin has been used for studying the impact of protein synthesis for manifold cerebral processes such as long-term plastic changes after learning. The common limitation of nearly all pharmacological experiments, including anisomycin treatment, is to precisely verify the affected brain regions. Here we illustrate anisomycin effects on protein synthesis in distinct brain regions of mice (C57BL/6JOlaHsd), revealing differences between three modes of anisomycin application (subcutaneous, s.c.; intraperitoneal, i.p.; local microinfusions into the hippocampus). Our method is based on inhibition of the incorporation of the radioactively-labelled amino acids [(35)S]-Methionine/Cysteine into newly synthesised proteins. Washing the brain slices before autoradiography removes pools of amino acids, which have not been incorporated into newly synthesised proteins, thus, illustrating pure protein synthesis. By comparing different routes of systemic anisomycin application (i.p. versus s.c.; 150 mg/kg) it became evident that the effect of i.p. injection of anisomycin is fully reversed after 6 h, whereas s.c. injection is inhibiting protein synthesis in the hippocampus even 9 h after application. Local microinfusions of anisomycin into the hippocampus were shown to have long-lasting effects as well, which reversed as late as 9 h after injection. The diffusion of anisomycin was maximal at 3 h after injection and more precisely confined to the intended area using a lower dose (20 microg/site) instead of the commonly used dose of 62.5 microg/site. The broad time window of anisomycin action, as revealed in our study, has to be considered, if it comes to the interpretation of time course studies within the context of protein synthesis-dependent processes.

Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway.

Facilitating the uptake of molecules into living cells is of substantial interest for basic research and drug delivery applications. Arginine-rich peptides have been shown to facilitate uptake of high molecular mass cargos into cells, but the mechanism of uptake is complex and may involve multiple receptors. In this report, we show that a derivative of the aminoglycoside antibiotic neomycin, in which all of the ammonium groups have been converted into guanidinium groups, can carry large (>300 kDa) bioactive molecules across cell membranes. Delivery occurs at nanomolar transporter concentrations and under these conditions depends entirely on cell surface heparan sulfate proteoglycans. Conjugation of guanidinoneomycin to the plant toxin saporin, a ribosome-inactivating agent, results in proteoglycan-dependent cell toxicity. In contrast, an arginine-rich peptide shows both heparan sulfate-dependent and -independent cellular uptake. The high selectivity of guanidinoneomycin for heparan sulfate suggests the possibility of exploiting differences in proteoglycan compositions to target delivery to different cell types.

Drug evaluation: PTC-124--a potential treatment of cystic fibrosis and Duchenne muscular dystrophy.

PTC-124, a 1,2,4-oxadiazole compound, is in development by PTC Therapeutics Inc as an orally active small molecule that can override nonsense stop translation signals to produce full-length proteins. PTC-124 is currently being evaluated in phase II clinical trials against cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD). The functional properties of PTC-124 are similar to the aminoglycoside antibiotic gentamicin, but the two compounds are chemically distinct and PTC-124 does not exhibit any antibiotic characteristics. In vitro experiments showed PTC-124 to be superior to gentamicin at ribosomal read-through of nonsense mutations. In vivo investigations revealed that PTC-124 was effective in restoring the production of full-length protein in animal models of CF and DMD. Phase I clinical trials reported that PTC-124 was well tolerated in healthy patients. The author concludes that the encouraging results observed to date make PTC-124 an attractive option for further well-designed, long-term human studies on larger sample populations. The author also predicts that if results continue to be positive, PTC-124 could also be trialed in other single gene disorders with nonsense mutations such as hemophilia, neurofibromatosis, retinitis pigmentosa, bullous skin diseases and lysosomal storage disorders.

Transgene delivery and gelonin cytotoxicity enhanced by photochemical internalization in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis patients.

The objective of this study was to determine if photochemical internalization (PCI) of gelonin can improve the treatment outcome as compared to photodynamic therapy (PDT) and gene transduction of fibroblast-like synoviocytes (FLS)in vitro. For this purpose synovial tissue was obtained under synovectomy of rheumatoid arthritis (RA) patients. Primary single cell suspensions were treated with the photosensitizer meso-tetraphenylporphine (TPPS2a) and light exposure (PDT) followed by evaluation of the cell survival by flow cytometry. PCI of gelonin was performed on FLS in passages 4 and 5 after removal from patients followed by measurements of protein synthesis 24 h after treatment. Additionally FLS were transduced with an adenovirus encoding the E.coli. lacZ gene and treated with PCI to evaluate the effect on the transduction rate. As a result all the cells in the primary cell suspension were susceptible to PDT but CD 106- (FLS) and CD14-positive (monocytes) cells were more sensitive to inactivation by PDT than CD2- (T-cells) and CD19-positive (B-cells) cells. With respect to protein synthesis FLS became up to 4-fold more sensitive to light when combining the photochemical treatment with the gelonin incubation. The fraction of virally transduced FLS was approximately doubled by means of PCI. In conclusion our experiments showed that PCI increased the cytotoxic effect of gelonin and adenoviral transduction of FLS derived from RA patients.

Reminder effects: the molecular cascade following a reminder in young chicks does not recapitulate that following training on a passive avoidance task.

Memory traces, once established, are no longer sensitive to disruption by metabolic inhibitors. However, memories reactivated by reminder are once again vulnerable, in a time-dependent manner, to amnestic treatment. To determine whether the metabolic events following a reminder recapitulate those following initial training we examined the temporal dynamics of amnesia induced by the protein synthesis inhibitor anisomycin and the glycosylation inhibitor 2-deoxygalactose. The effects of both were transient and dependent on time of reminder post-training and time of injection relative to reminder, and differed from those following initial training. 2-[(14)C]-deoxyglucose uptake increased in two brain regions, the intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) following reminder as it did following training, but the increase was bilateral rather than confined to the left hemisphere and was more marked in LPO than IMHV. C-fos expression after reminder was increased only in the LPO, the chick brain region associated with a late phase of memory processing and recall. Thus although, like initial consolidation, memory processing after reminder is sensitive to inhibitors of protein synthesis and glycosylation, the temporal and pharmacological dynamics indicate differences between these two processes.

Aqueous and vitreous penetration of linezolid (Zyvox) after oral administration.

OBJECTIVE: To investigate the penetration of linezolid, a synthetic oxazolidinone antibiotic, into the aqueous and vitreous humor after oral administration. DESIGN: Noncomparative interventional, prospective case series study, randomized into group 1 (dose, one 600-mg tablet) or group 2 (2 doses of 600 mg given 12 hours apart). PARTICIPANTS: Patients undergoing pars plana vitrectomy between March 2001 and August 2002 at the University of Illinois at Chicago Eye Center who had not had prior vitrectomy surgery. METHODS: Aqueous, vitreous, and plasma samples were obtained and analyzed from 29 patients after oral administration of 1 dose (group 1A, 13 patients [13 eyes] sampled less than 2 hours after administration; group 1B, 9 patients [9 eyes] sampled more than 2 hours after administration) or 2 doses 12 hours apart (group 2, 7 patients [7 eyes]) before surgery. MAIN OUTCOME MEASURES: Aqueous, vitreous, and plasma concentrations of linezolid (micrograms per milliliter). RESULTS: Group 1A achieved mean aqueous, vitreous, and plasma levels of 0.77+/-0.6 microg/mL, 0.3+/-0.3 microg/mL, and 5.0+/-3.3 microg/mL, respectively. Group 1B achieved mean aqueous, vitreous, and plasma levels of 3.8+/-1.2 microg/mL, 2.3+/-1.4 microg/mL, and 7.6+/-2.7 microg/mL, respectively. Group 2 achieved mean aqueous, vitreous, and plasma levels of 6.6+/-2.7 microg/mL, 5.7+/-2.7 microg/mL, and 10.3+/-4.1 microg/mL, respectively. CONCLUSIONS: Mean inhibitory aqueous and vitreous minimum inhibitory concentrations for 90% of isolates (MIC(90)) were achieved against all gram-positive bacteria, including vancomycin-resistant enterococcus, methicillin-resistant Staphylococcus aureus, and streptococcal species after 2 doses given 12 hours apart. Mean MIC(90) were achieved for many gram-positive pathogens after only one dose in many patients after approximately 4 hours.

Phase I and pharmacokinetic study of KRN5500, a spicamycin derivative, for patients with advanced solid tumors.

BACKGROUND: KRN5500, a novel spicamycin derivative, shows an inhibitory effect on protein synthesis. This phase I study was aimed at investigating the toxicity, maximum tolerated dose (MTD) and pharmacokinetics of this compound. PATIENTS AND METHODS: Patients with solid tumors not amenable to standard forms of treatment were eligible. KRN5500 was administered as a 2 h intravenous infusion every 4 weeks at doses of 3, 6, 10, 15 and 21 mg/m(2). Pharmacokinetic evaluation was performed at the first cycle. RESULTS: Eighteen patients with advanced solid tumors were enrolled. A total of 26 cycles of KRN5500 were administered. The major toxicities were nausea, vomiting, diarrhea, fatigue and a mild reversible prolongation of prothrombin time. Grade 4 pulmonary toxicity (interstitial pneumonitis) was observed in one patient at a dose level of 15 mg/m(2). Severe fatigue was observed in one patient at a dose level of 21 mg/m(2) and the duration of fatigue tended to increase with the dose of KRN5500. Nausea and vomiting were frequently observed and became prolonged with increasing dose of KRN5500. These toxicity profiles were identified as unacceptable and further dose escalation above 21 mg/m(2) was withheld. The MTD was therefore determined as 21 mg/m(2). The peak plasma concentration and the area under the concentration-time curve of KRN5500 increased proportionally to the dose, suggesting linear pharmacokinetics. No objective antitumor response was observed. CONCLUSION: KRN5500, a structurally novel protein synthesis inhibitor, warrants further investigation to overcome these toxicity profiles and improve its efficacy.

Dual blockade of mitogen-activated protein kinases ERK-1 (p42) and ERK-2 (p44) and cyclic AMP response element binding protein (CREB) by neomycin inhibits glioma cell proliferation.

Several growth factors and their receptors are expressed in inappropriately high abundance in gliomas and are further upregulated during the transition from low- to high-grade malignancy. In glioma cells growth factors induce expression of mitogen-activated protein kinase (MAPK) pathways. Here we report that neomycin restrained glioma cell proliferation in vitro by inhibition of p42/44 MAPK and the cyclic AMP element binding protein (CREB)-directed transcription pathways. Since alteration of gene transcription by inhibition of specific transcriptional regulatory proteins has important therapeutic potential, neomycin offers great promise for treating cancer and other diseases associated with a sustained MAPK activity.

Fatores hepatotróficos e regeneraçäo hepática. Parte I: o papel dos hormônios / Hepatotrophic factors and liver regeneration. Part I: the role of hormones

No complexo processo de proliferaçäo celular, os hormônios agem de diferentes maneiras ao atingirem seus receptores nos tecidos-alvo. Os principais fatores ligados ao crescimento hepático säo HGF, TGF-alpha, IL-6, TNF-alpha, norepinefrina, EGF e insulina. O GH estimula tanto o fígado a produzir fatores de crescimento, como a expressäo genética do HGF e a síntese de DNA. Hormônios tireoideanos aumentam a capacidade proliferativa dos hepatócitos. A insulina age sinergicamente com GH e glucagon. Näo tem potencial mitogênico primário mas intensifica o estímulo regenerativo iniciado pela epinefrina e norepinefrina. Esta amplifica os sinais mitogênicos do EGF e HGF, induz a secreçäo de EGF e antagoniza os efeitos inibitórios do TGF-beta 1. O glucagon isoladamente näo produz efeitos mas provavelmente participa na síntese de DNA e da resposta homeostásica pela qual a glicemia é mantida estável durante a regeneraçäo. Também há indícios de açäo hepatotrófica da gastrina.

Activities of sordarins in experimental models of candidiasis, aspergillosis, and pneumocystosis.

Sordarin derivatives represent a new class of antifungal agents that act as potent inhibitors of fungal protein synthesis and possess a broad spectrum of activity. The in vivo activity of GM193663 and GM237354 was studied in mouse models of disseminated candidiasis and aspergillosis and in a rat model of pneumocystosis. The pharmacokinetic behavior of both sordarin derivatives was studied in mice and rats. In all studies, compounds were administered by the subcutaneous route. After a subcutaneous dose of 50 mg/kg of body weight to mice, the maximum level in serum, area under the concentration-time curve, half-life, and clearance for GM193663 and GM237354 were 51.8 and 23 microg/ml, 79.5 and 46 microg. h/ml, 0.8 and 0.85 h, and 21 and 25 ml/h, respectively. Systemic candidiasis and aspergillosis were established in CD-1 male mice infected with Candida albicans or Aspergillus fumigatus. For systemic candidiasis, compounds were given three times per day for seven consecutive days at 15, 30, 60, or 120 mg/kg/day. GM193663 and GM237354 showed dose-related efficacy against C. albicans, with 50% effective doses, 1 month after infection, of 25.2 and 10.7 mg/kg/dose, respectively. In experimental infections with A. fumigatus, GM237354 was given three times per day at 30, 60, or 120 mg/kg/day for five consecutive days. Animals treated with GM237354 demonstrated irregular responses. The survival of animals treated with GM237354 was 0, 30, and 0% at 30, 60, and 120 mg/kg/day, respectively. The therapeutic efficacy of GM193663 and GM237354 against Pneumocystis carinii was studied in an experimental P. carinii pneumonia (PCP) rat model. After a subcutaneous dose of 10 mg/kg given to rats, the maximum level in serum, area under the concentration-time curve, half-life, and clearance for GM193663 and GM237354 were 6.6 and 7.2 microg/ml, 8.5 and 11.8 microg. h/ml, 0.7 and 0.8 h, and 230 and 133 ml/h, respectively. To induce spontaneous PCP, rats were chronically immunosuppressed with dexamethasone. Infected animals were treated twice daily for 10 days at 0.2, 2, or 10 mg/kg/day. The therapeutic effect was estimated by the reduction in the number of cysts in the lungs of treated versus untreated animals. GM193663 and GM237354 significantly reduced the mean (+/- standard deviation) log number of cysts from 7.6 +/- 0.2 in the untreated group to 4.7 +/- 0.2 and 4.6 +/- 0.1, respectively, when the drugs were administered at a dose of 2 mg/kg/day. The log number of cysts was also reduced in infected animals given lower doses of the compounds (0.2 mg/kg/day). In summary, GM193663 and GM237354 are new sordarin derivatives that may potentially play a major role in the treatment of candidiasis and PCP. Further testing with Aspergillus in other animal models is warranted.

Comparative cytotoxicity and pharmacokinetics of antimelanoma immunotoxins containing either natural or recombinant gelonin.

Immunotoxins are a class of targeted therapeutic agents under development by various research groups. The murine monoclonal antibody designated ZME-018 recognizes a high molecular weight glycoprotein present on most human melanoma cells and biopsy specimens and has been utilized for clinical imaging studies in patients with melanoma. The plant toxin gelonin is a ribosome-inactivating protein (RIP) with n-glycosidase activity similar to that of ricin A chain. In previous studies by our group, the gelonin toxin was sequenced, cloned and expressed in E. coli. The purified recombinant gelonin (RG) was found to have identical protein synthesis inhibitory activity to that of natural gelonin (NG). For comparative purposes, chemical conjugates of antibody ZME and either RG or NG were produced using the heterobifunctional crosslinking reagents SPDP and SMPT. The ZME-NG and ZME-RG immunotoxins were found to be 10(4)- to 10(5)-fold more cytotoxic to antigen-positive human melanoma cells than free toxin. NG toxin alone was cytotoxic to intact cells (IC(50) = 100 nM) while RG was nontoxic to cells at doses up to 1 microM. Both ZME-NG and ZME-RG immunoconjugates were nontoxic to antigen-negative (Me-180) cells. ZME-RG immunotoxins constructed with the more stable SMPT reagent were slightly more effective in culture than conjugates made with SPDP. Tissue distribution studies in tumor-bearing nude mice demonstrated that tumor uptake of the ZME-RG immunotoxin was similar to that of the intact ZME antibody with reduced distribution to normal organs compared to an immunoconjugate produced with NG. Pharmacokinetic studies showed that the terminal-phase plasma half-life of ZME-RG was similar to that of ZME itself (42 h vs 50 h) and almost threefold higher than that of ZME-NG (11.5 h). The area under the concentration curve (Cxt) for ZME-RG was 50% lower than that for ZME due to an increased apparent volume of distribution (Vd(a)) but was almost tenfold higher than the Cxt for ZME-NG. These studies suggest that immunoconjugates comprising RG demonstrate identical in vitro cytotoxic effects to immunoconjugates produced with NG and immunotoxins with RG display improved in vivo pharmacodynamics and tissue distribution compared to immunotoxins containing NG.

Uptake of tetracycline by aortic aneurysm wall and its effect on inflammation and proteolysis.

BACKGROUND: Proteolytic degradation of the aortic wall by matrix metalloproteinases (MMPs) is considered important in the pathogenesis of abdominal aortic aneurysms (AAAs). Many of these MMPs are inhibited by tetracycline derivatives, which may have the potential to retard aneurysm growth. METHODS: Patients undergoing elective repair of an AAA (n = 5) received an intravenous bolus of tetracycline (500 mg) on induction of anaesthesia and levels of tetracycline in serum, aneurysm wall and mural thrombus were assessed by microbiological assay. In a separate series of patients (n = 7) aneurysm biopsies were placed into explant culture (with and without tetracyline) and the accumulation of protein, hydroxyproline, MMP-9, interleukin (IL) 6 and monocyte chemoattractant protein (MCP) 1 in the medium was assessed by colorimetric assay or immunoassay. RESULTS: At aortic cross-clamping the median concentration of tetracycline was 8.3 microg/ml in serum, 2.9 microg per g tissue in aortic wall and zero in mural thrombus. Tetracycline inhibited, in a concentration-dependent manner, both MMP-9 and MCP-1 secretion (P = 0.022 and P = 0.018 respectively), but did not alter hydroxyproline or IL-6 secretion. At the highest concentration of tetracycline (100 microg/ml) median MMP-9 secretion was reduced from 27 to 5 ng/ml (P = 0.007) and median MCP-1 secretion was reduced from 50 to 10 ng/ml (P = 0.008). CONCLUSION: Tetracycline rapidly penetrates the aortic wall, but the concentration achieved may be insufficient to alter collagen turnover through limitation of MMP production or activity.

Coupled cellular trafficking and diffusional limitations in delivery of immunotoxins to multicell tumor spheroids.

Immunotoxins have the potential to be powerful tools for selective cell killing, but their lack of clinical success against solid tumors indicates a need to better understand factors which limit immunotoxin transport in three-dimensional systems. In this work, a previously developed model which related immunotoxin toxicity to cellular trafficking in a single cell was coupled with a term accounting for diffusive transport of immunotoxin in a solid tumor sphere. This created a mathematical model which is capable of simulating the biological response of multicell tumor spheroids (MTS) to immunotoxin treatment. The model was used to predict the kinetics of protein synthesis inhibition in MTS treated with transferrin receptor-targeted immunotoxins as a function of immunotoxin concentration and toxin choice. HeLa cells were grown as MTS and treated with immunotoxins constructed from the anti-transferrin receptor antibody OKT9 and the toxins gelonin or CRM107, and the average protein synthesis inhibition and growth rates were measured. With no fitted parameters, the mathematical model quantitatively predicted the experimental observations. Immunotoxins were generally less effective against MTS than monolayer cells at equivalent conditions; for OKT9-gelonin at high concentrations this decrease in efficacy was attributed primarily to heterogeneous receptor distribution in MTS whereas for OKT9-CRM107 the decrease was caused primarily by a large barrier to penetration of the immunotoxin into the spheroid. The experimentally verified model was used to define the conditions which lead to large penetration barriers. In general, transport barriers in MTS become more important as immunotoxins become more effective against cells grown as monolayers. The proposed model is unique in its ability to predict toxicity in MTS directly, and is an important step toward understanding immunotoxin effect on tumors in vivo.

Kinetics of cellular trafficking and cytotoxicity of 9.2.27-gelonin immunotoxins targeted against the high-molecular-weight melanoma-associated antigen.

The high-molecular-weight melanoma-associated antigen (HMW-MAA) is expressed on a large majority of melanoma tissues but not on most normal or other neoplastic tissues. Monoclonal antibody 9.2.27 binds with high affinity and specificity to the HMW-MAA, making it an attractive choice as an agent for targeting toxins or chemotherapeutic agents specifically towards melanomas. To characterize the interactions between 9.2.27 and melanoma cells more carefully, data on the kinetics of binding, internalization, and degradation of 9.2.27 by SK-MEL-2 cells were collected. Binding of 9.2.27 to SK-MEL-2 cells was rapid, and followed by slow loss of surface-bound antibody, probably because of loss of surface antigen caused by degradation and/or shedding. A small fraction (approx. 5%) of surface-bound 9.2.27 was internalized and degraded. A mathematical model describing these interactions was developed, and equilibrium and kinetic constants were fitted to the data. To evaluate the utility of 9.2.27 as a toxin-targeting agent, 9.2.27-gelonin immunotoxins were constructed and tested in protein synthesis inhibition assays. The dependence of the toxicity data for 9.2.27-gelonin on time and concentration was quantitatively related to the accumulated intracellular exposure to 9.2.27-gelonin by a relatively simple equation. This equation had been previously validated for immunotoxins targeted against the transferrin receptor, for which the trafficking kinetics are quite dissimilar from those of the HMW-MAA. The success of the approach here suggests that this method may be widely applicable for analysis of immunotoxin efficacy.