Ribosome-inactivating Protein Gelonin Research Articles

Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization.

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Circumvention of resistance to photodynamic therapy in doxorubicin-resistant sarcoma by photochemical internalization of gelonin

A wide range of anti-cancer therapies have been shown to induce resistance upon repetitive treatment and such adapted resistance may also cause cross-resistance to other treatment modalities. We here show that MES-SA/Dx5 cells with adapted resistance to doxorubicin (DOX) are cross-resistant to photodynamic therapy (PDT). A DOX-induced increased expression of the reactive oxygen species (ROS)-scavenging proteins glutathione peroxidase (GPx) 1 and GPx4 in MES-SA/Dx5 cells was indicated as the mechanism of resistance to PDT in line with the reduction in PDT-generated ROS observed in this cell line. ROS-induced p38 activation was, in addition, shown to be reduced to one-third of the signal of the parental MES-SA cells 2h after PDT, and addition of the p38 inhibitor SB203580 confirmed p38 activation as a death signal after PDT in the MES-SA cells. The MES-SA/Dx5 cells were also cross-resistant to ionizing radiation in agreement with the increased GPx1 and GPx4 expression. Surprisingly, PDT-induced endo/lysosomal release of the ribosome-inactivating protein gelonin (photochemical internalization (PCI)) was more effective in the PDT-resistant MES-SA/Dx5 cells, as measured by synergy calculations in both cell lines. Analysis of death-inducing signaling indicated a low activation of caspase-3 and a strong PARP I cleavage after PDT and PCI in both cell lines. The PARP I activation was, however, stronger after PCI than after PDT in the MES-SA cells, but not in the MES-SA/Dx5 cells, and therefore cannot explain the strong PCI effect in the MES-SA/Dx5 cells. In conclusion PCI of recombinant gelonin circumvents ROS resistance in an apoptosis-independent manner.

Abstract 4399: EphA2-targeting antibody-gelonin conjugate specifically binds and kills target-expressing tumor cells, including multi-drug resistant (MDR) lines

Abstract The EphA2 receptor is over-expressed on the surface of many different human tumors, and has been shown to internalize upon ligand or antibody binding. The fully human monoclonal antibody anti-EphA2 antibody was developed by phage display methods and specifically binds both human and rodent EphA2 receptors. The type II ribosome-inactivating protein gelonin was genetically engineered with an extra cysteine residue to facilitate chemical cross-linking with targeting proteins. The fully human anti-EphA2 antibody, anti-EphA2 antibody, was conjugated to the recombinant gelonin (rGel) using the heterobifunctional cross-linker SPDP and partially reduced rGel. The conjugated antibody (anti-EphA2 antibody/rGel) was separated from free antibody and rGel by gel-permeation and affinity chromatography. The final material was composed of antibody+1 rGel (predominant) and antibody+2 rGel (minor) and was essentially free of contaminating rGel or unreacted anti-EphA2 antibody. The anti-EphA2 antibody/rGel conjugate was assessed for cell-free protein synthesis inhibitory activity and found to be similar to free rGel. The conjugate proved specifically cytotoxic to the EphA2 expressing PC-3, MDA-MB-231 and CT-26 cell lines, with IC50 values of 3.8, 6.1 and 11 ng/ml, respectively. The Targeting Index for the anti-EphA2 antibody/rGel conjugate (vs. rGel) was high, with values of 1909, 306 and 75 for PC-3, PC-3-MDR and MDA-MB-231, respectively. Confocal microscopy showed that the anti-EphA2 antibody/rGel conjugate was specifically and rapidly internalized. The maximal intracellular concentration was achieved 4 hours after exposure. Minimal contact time studies showed specific cytotoxicity with only 1 hour exposure while optimal cytotoxicity was achieved with an exposure time of 24 hours. Of special interest, the anti-EphA2 antibody/rGel conjugate demonstrated no significant loss in specific cytotoxicity against Dox-resistant (MDR expressing) cell lines derived from either MDA-MB-231 or PC-3 cells compared to their parental cell lines. These data suggest that the development of MDR-resistant phenotypes may not impair the successful utilization of rGel-based targeted therapeutics. These results support the continued evaluation of the rGel antibody conjugate technology. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4399.

Photochemical Internalization of Therapeutic Macromolecular Agents: A Novel Strategy to Kill Multidrug-Resistant Cancer Cells

Drug resistance is a major problem for chemotherapy. Entrapment of anticancer drugs in endolysosomal compartments or active extrusions by plasma membrane proteins of the ATP-binding cassette (ABC) superfamily are important resistance mechanisms. This study evaluated photochemical internalization (PCI) of membrane-impermeable macromolecules that are not the target of ABC drug pumps for treating multidrug-resistant (MDR) cancer cells. We used the drug-sensitive uterine fibrosarcoma cell line MES-SA and its MDR, P-glycoprotein (P-gp)-overexpressing derivative MES-SA/Dx5 with the photosensitizer disulfonated meso-tetraphenylporphine (TPPS(2a)) and broad spectrum illumination. The PCI of doxorubicin, the ribosome-inactivating protein gelonin and adenoviral transduction were assessed in both cell lines, together with the uptake and excretion of TPPS(2a) and of two fluid phase markers easily detectable by fluorescence [lucifer yellow (LY) and fluorescein isothiocyanate (FITC)-dextran], as a model of gelonin uptake. Both cell lines were resistant to PCI of doxorubicin, but equally sensitive to PCI of gelonin, even though the endocytosis rates of LY and FITC-dextran were significantly lower in the MDR cells. In control studies, MES-SA/Dx5 cells were more resistant to photodynamic therapy (TPPS(2a) + light only). This was not mediated by P-gp, as there were no differences in the uptake and efflux of TPPS(2a) between the cell lines. After adenoviral infection, PCI enhanced gene delivery in both cell lines. In conclusion, PCI of macromolecular therapeutic agents that are not targets of P-gp is a novel therapeutic strategy to kill MDR cancer cells.

Studies on the anti-mitogenic, anti-phage and hypotensive effects of several ribosome inactivating proteins

An investigation was conducted to compare the anti-mitogenic, anti-phage and hypotensive activities of several ribosome inactivating proteins (RIPs) in order to ascertain whether the RIPs differed in their potencies in the various bioassays. Agrostin, luffin and saporin elicited a dose-dependent suppression of the mitogenic response of murine splenocytes to concanavalin A. The three RIPs were approximately equipotent in this regard, with near maximal inhibition attained at a dose of 83 nM and approximately 50% inhibition at 830 pM. Trichosanthin was slightly more potent than the three aforementioned RIPs. All of these RIPs were capable of inhibiting the replication of phage M13 in the bacterium Escherichia coli, the ranking of potencies being luffin>trichosanthin>agrostin when tested at a concentration of 3.5 μM. The RIPs gelonin and saporin did not exert a conspicuous antiviral effect at the same dose. After intravenous administration into normotensive rats via the external jugular vein, the RIPs saporin, trichosanthin, gelonin and momordin evoked a mild hypotensive response while luffin and agrostin were inactive. The hypotensive response, however, lacked dose dependence. The RIPs trichosanthin, momordin and gelonin did not affect the blood pressure response to angiotensin I. Chemical modification of the arginine residues of the RIPs brought about a reduction in their ability to inhibit cell-free translation. It appears that the ranking of potency of RIPs in one bioassay was different from the rankings in other assays.

Polynucleotide:Adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA.

Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose((R)). Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG.

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.

Toxin and gene transfer into cells by extracorporeal shock waves

Shock wave application to cells in vitro causes a transient increase of the permeability of the cell membrane which does not lead to cell death. It was hypothesized that shock waves might be a new method of transferring therapeutic agents directly into cells. To test this, biological effects resulting from the acoustical transfer of proteins and nucleic acids into cells were examined. Protein transfer was examined with the ribosome inactivating proteins gelonin and saporin which inhibit the cellular protein synthesis. Dose response curves were established with three tumor cell lines in the presence and absence of shock waves. Compared to the controls, shock waves enhanced the action of gelonin and saporin from 300 to 40 000 fold. In vivo experiments with an animal tumor model established that acoustic transfer of these agents into cells also occurred in vivo. Gene transfer by shock waves was examined in a number of cell lines by plasmid vectors carrying standard reporter genes. Transfer succeeded in many cases yet the transduction efficiency was lower than with other established methods of gene transfer. In summary shock wave permeabilization is a new method for tumour therapy and gene transfer.

Direct evidence for the deoxyribonuclease activity of the plant ribosome inactivating protein gelonin

Several plant ribotoxins, including gelonin, were reported to have additional weak nuclease activities on supercoiled DNA. The potential contribution of this activity to their cytotoxicity has not been given serious consideration due to concerns about contaminating nucleases in the protein preparations. We now report the degradation of single-stranded DNA by preparations of native plant gelonin and recombinant gelonin produced in E. coli. The DNase activity of both preparations is similarly modulated by zinc. An SDS-PAGE DNase assay identifies gelonin as the polypeptide responsible for deoxyribonuclease activity.

T-cell-targeted immunofusion proteins from E. coli.

Antibodies and antibody domains are ideal agents for targeting the surface of cells, and fusion proteins between cell-targeting domains and cytotoxic proteins may be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking the humanized Fab, F(ab')2, or single-chain antibody form of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. To maximize the product yield and simplify the production process, each fusion protein was linked to a bacterial signal sequence for expression in E. coli as a secreted protein. More than 30 fusion genes were assembled with antibody domains and gelonin in various physical orientations. Each immunofusion accumulated in the bacterial culture supernatant in a properly folded, active form. Bacteria transformed with each fusion gene were then grown in a fermentor, and product was recovered from the cell-free fermentation broth by column chromatography. All of the immunofusion proteins were purified by a single process and each was tested for cytotoxicity toward antigen-positive human cells. A compact cGMP fermentation area was built to manufacture these fusion proteins. Our integrated approach to microbial protein production, including molecular genetics, bacterial fermentation, and initial isolation, is described in detail.

T Cell-targeted Immunofusion Proteins from Escherichia coli

Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking humanized Fab, F(ab')2, or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A. H. C., and Carroll, S. F. (1994) J. Biol. Chem. 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found. Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form. All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells. A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates. Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome-inactivating protein.

Actions of selected proteins, peptides and amino acid derivatives on mouse embryonic development In Vitro

1. 1. The actions of various compounds with antiproliferative and/or immunomodulatory activities including the ribosome-inactivating protein gelonin, the tetrapeptide tuftsin, the opioid peptide leucine-enkephalin, the antireproductive tripeptide Thr-Ser-Lys, the melatonin analog 6-methoxy-2-benzoxazolinone, and the amino acid derivatives 5-hydroxytryptamine and oxalysine on mouse embryonic development and organogenesis in vitro were investigated. 2. 2. The various compounds were tested up to a concentration of 300 μg/ml. It was found that only 6-methoxy-2-benzoxazolinone, a derivative of tryptophan, produced an increase in the number of abnormal embryos and reductions in the final somite numbers and axial lengths of embryos. 3. 3. 6-Methoxy-2-benzoxazolinone treatment also resulted in an increased incidence of abnormal morphogenesis of various organ primordia, including abnormal yolk sac circulation, twisting of body axis, absence of forelimb bud and opening of cranial neural tube.

Preparation and characterization of interleukin-2-gelonin conjugates made using different cross-linking reagents.

Conjugates of IL-2 with the ribosome-inactivating protein gelonin were prepared using heterobifunctional reagents to link the proteins via disulfide, acid-labile, and noncleavable linkers. In each case, one protein was modified using 2-iminothiolane. The sulfhydryl groups so introduced were then reacted either with 2-nitro-5-dithiobenzoate groups or with iodoacetamido groups which had been introduced into the second protein. In the case of the acid-labile linkage, a reagent which forms a labile bond upon reaction with amino groups, 4-(iodoacetamido)-1-cyclohexene-1,2-dicarboxylic acid anhydride (its synthesis is described in this paper) was used to modify the toxin. The conjugates were separated from nonconjugated proteins by gel filtration on Sephadex G100 (SF). Each was analyzed with respect to its ribosome-inactivating activity, its ability to bind to the IL-2 receptor, and its in vitro cytotoxicity. The ribosome-inactivating activity of gelonin was unaffected by modification with 2-iminothiolane and was retained in conjugates prepared using this reagent. Modification of the toxin with 4-(iodoacetamido)-1-cyclohexene-1,2-dicarboxylic acid anhydride to form the acid-labile link drastically reduced the activity of the toxin. However, the activity of the toxin was recovered following acid treatment to release the native protein. Conjugates containing each type of linkage exhibited both specific binding and selective cytotoxicity toward cells expressing the IL-2 receptor. The most potent of these toxins, that containing the disulfide linkage, exhibited a cytotoxicity which was 2 orders of magnitude greater than that of unconjugated gelonin.

Bryodin, a single-chain ribosome-inactivating protein, selectively inhibits the growth of HIV-1-infected cells and reduces HIV-1 production.

Bryodin, a single-chain ribosome-inactivating protein (RIP) isolated from Bryonia cretica ssp dioica (cucurbitaceae), was found to selectively inhibit the growth of persistently HIV-1-infected T lymphoma cells (KE37/1) and human lung fibroblast when used in concentrations from 2-20 micrograms/ml. Uninfected KE37/1 cells remained unaffected at the same doses of bryodin. In addition, bryodin reduced HIV production in the surviving infected cells. Two isoforms of bryodin were purified by dye ligand chromatography. Both isoforms exerted the growth-inhibiting influence and reduced HIV production. Trichosanthin, another member of the RIP family, had similar inhibitory effects on the growth of HIV-1 infected cells and on HIV-1 production. Bryodin and trichosanthin were effective in about the same dose range. No selective effects for HIV-infected cells were observed with the RIPs gelonin and ricin.

Purification of immunotoxins containing the ribosome-inactivating proteins gelonin and momordin using high performance liquid immunoaffinity chromatography compared with Blue Sepharose CL-6B affinity chromatography

Comparable amounts of the ribosome-inactivating proteins (RIPs) ricin A chain, gelonin and momordin were allowed to bind to Blue Sepharose CL-6B (immobilised Cibacron Blue F3GA) in phosphate buffer, pH 7.5, and were then eluted quantitatively with buffer containing 0.5 M NaCl. Differences in the elution profiles indicated that the RIPs possessed different affinities for the Cibacron Blue F3GA dye. • Conjugation of the RIPs to the monoclonal antibody Fib75 resulted in decreased affinity for Blue Sepharose. Under conditions allowing the complete separation of the Fib75-ricin A chain immunotoxin from unconjugated Fib75, the Fib75 immunotoxins made with gelonin and momordin failed to bind completely to the Blue Sepharose column. The Fib75-gelonin and Fib75-momordin fractions that eluted from the column with 0.5 M NaC1 were free of unconjugated Fib75 but were enriched in multiply substituted conjugate molecules. • A high performance liquidd immunoaffinity chromatography procedure based on the selective binding of conjugated RIP to immobilised affinity-purified anti-RIP antibody permitted the complete separation of the gelonin and momordin immunotoxins from unconjugated Fib75 without altering the composition, molecular integrity or cytotoxic activity of the immunotoxins.

The heavy chain of tetanus toxin can mediate the entry of cytotoxic gelonin into intact cells

An artificial conjugate of the heavy chain of tetanus toxin linked by a disulphide bond to the impermeant ribosome-inactivating protein gelonin is cytotoxic to intact HT29 cells by inhibiting intracellular protein synthesis. Neither toxin nor gelonin alone has any significant effect. This shows that the heavy chain has the ability to mediate internalization of a protein to which it is bound by a disulphide bond. Thus the normal role of the tetanus toxin heavy chain may be to allow entry of the light chain into a cell.

Hormonotoxins: conjugation of human choriogonadotropin with the ribosome inactivating protein gelonin and comparison with a lutropin conjugate

The properties of two hormonotoxins, human choriogonadotropin and ovine pituitary lutropin, each conjugated to the ribosome inactivating protein gelonin, have been compared. In both cases the linkage is effected through a disulfide bond provided by the cross-linker N- succinimidyl-3-(2- pyridyldithio) propionate . In each conjugate the hormone and toxin were calculated to be present in a ratio of 1:1. Immunological reactivity, receptor binding activity and steroidogenic activity of the hormonotoxins in radioimmunoassay or in membranes or cells containing LH receptors were reduced but not abolished in comparison with the thiolated hormones. Purified hormonotoxins were characterized by immunoassays specific for both interaction components, viz. the hormone (LH/hCG) and gelonin. Binding of the toxic component (gelonin) to the mouse Leydig tumour cells (MA-10 cells) was shown to occur via the hormone part of the hormonotoxins. This was effectively competed by the native hormone but not free gelonin. Both hormonotoxins were cytotoxic to MA-10 cells as protein synthesis was inhibited in their presence. This was associated with destruction of tumor cells in culture.

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. V. Evidence that humoral immune response to monoclonal antibodies and immunotoxin conjugates abrogates their cytotoxic activity.

Monoclonal antibodies, either alone or conjugated to toxins, hold promise as important therapeutic agents. However, the immune response to these foreign protein agents may markedly limit their therapeutic utility in vivo. We have administered both an interleukin-2 receptor-specific monoclonal antibody (anti-IL-2R) and a CD2-specific monoclonal antibody linked to the ribosome-inactivating protein gelonin to macaque monkeys. The monkeys developed high-titer antibody responses to mouse Ig and, when immunotoxin was administered, to the toxin gelonin. Their antimouse Ig antibody responses were broadly reactive with mouse Ig of differing idiotypes and isotypes. Furthermore, sera from these monkeys blocked the in vitro cytotoxic effect of anti-IL-2R or immunotoxin. This blocking was mediated by both the antimouse Ig and the antigelonin antibodies. Serum from a monkey infused with one CD2-specific monoclonal antibody blocked the in vitro cytotoxicity of two other isotypically different CD2-specific monoclonal antibody conjugates. In addition, this serum blocked the in vitro cytotoxicity of a gelonin-monoclonal antibody conjugate of an unrelated specificity. These data indicate that the immune response to some monoclonal antibodies and toxins might preclude the later use of this class of substances in an individual. Therefore, strategies for the parental therapeutic use of monoclonal antibodies and immunotoxins must take into consideration the possible limiting effects of the humoral immune response to these agents.

Effects of thiolation on the immunoreactivity of the ribosome-inactivating protein gelonin.

Gelonin purified from the seeds of Gelonium multiflorum using cation-exchange and gel-filtration chromatography was characterized for its purity, homogeneity and Mr by reverse-phase h.p.l.c. and SDS/polyacrylamide-gel electrophoresis analysis and judged to be 98% pure. As the cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has been used for linking gelonin via its epsilon-NH2 group to its carrier antibodies or hormones for immunotoxin or hormonotoxin respectively, an attempt was made to study the effect of this modification of gelonin on its immunoreactivity. A radioimmunoassay was developed for this purpose. By sequential modification, four categories of amino group modifications on immunoreactivity were observed. Even one or two modifications, representing one-twentieth to one-tenth of available epsilon-NH2 groups in the protein caused about 75% loss in immunoreactivity, with additional reactions contributing to further deteriorations. By using a gelonin radioimmunoassay, the immunoreactivity of gelonin in three hormonotoxins was determined with gelonin and modified gelonin as standards. The gelonin equivalent in our hormonotoxins was in agreement with the values determined by spectrophotometric and gel-electrophoresis methods. As the immunoreactivity of gelonin-SPDP was not further altered after conjugation to its carrier protein ovine lutropin, a specific radioimmunoassay of gelonin could be used to evaluate the molar ratio of the conjugates prepared by using SPDP as cross-linker and gelonin-SPDP as a standard.

Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis

Conjugates of the single-chain ribosome-inactivating protein gelonin with ligands that bind to cell surface molecules vary greatly in their cytotoxicity. Conjugates that are not endocytosed after binding to cells exhibit low cytotoxicity similar to that of free gelonin, while conjugates that are endocytosed demonstrate enhanced cytotoxicity relative to free gelonin. However, the number of internalized gelonin molecules needed to intoxicate cells to the same degree has been found to be similar for all conjugates and for free gelonin. The intracellular concentration of gelonin has to be between 2,000-10,000 molecules/cells to achieve a surviving fraction of 0.37. Our studies revealed the presence of three distinct categories of cell surface molecules, those that are efficient in mediating endocytosis of immunotoxins, those that are only moderately efficient, and those that seem not to cause internalization of bound immunotoxins.