Significance of antigen, drug, and tumor cell targets in the preclinical evaluation of doxorubicin, daunorubicin, methotrexate, and mitomycin-C monoclonal antibody immunoconjugates

We tested drug monoclonal antibody immunoconjugates in vitro in 72 h 3H-thymidine assays and in vivo in athymic mice bearing human tumor xenografts of the same target cells. Experimental arms included control, monoclonal antibody, drug, drug + antibody, the test immunoconjugate, and a negative control immunoconjugate with an equivalent molar amount of drug for in vitro experiments, and the amount of drug conjugated to 500 micrograms of antibody in the animal experiments. Monoclonal antibodies included T101, an IgG2a that reacts with a rapidly modulating antigen, 9.2.27, an IgG2a that reacts with a slowly modulating antigen, and ME7, an IgG1 that reacts with a slowly modulating antigen. Cells used in testing included MOLT-4 (T lymphoma), 8392 (B lymphoma), and M21 (melanoma). Drugs tested were doxorubicin, daunorubicin, methotrexate, and mitomycin-C. M21 cells were resistant to daunorubicin in vitro but were inhibited by the 9.2.27 daunorubicin immunoconjugate. T101, 9.2.27, and ME7 cis-aconitate anthracycline immunoconjugates and mitomycin-C-glutarate immunoconjugates were specifically cytotoxic only for antigen positive cells in vitro and were superior to free drug in vivo. These results confirm that antigen specific-cytotoxic drug immunoconjugates can be produced that are superior to the same dose of free drug. However, each monoclonal antibody drug target system is unique and must be well-characterized for appropriate interpretation of data.     

Unidirectional potentiation of binding between two anti-FBP MAbs: Evaluation of involved mechanisms

The monoclonal antibody MOv19 directed to a folate binding protein shows temperature-dependent potentiation of binding of the noncompeting monoclonal antibody MOv18 to the relevant antigen, but the mechanism involved in this phinomenon had remained unclear. Use of chimeric versions of both monoclonal antibodies and the F(ab′)₂ and fan fragments of MOv19 revealed an increment in MOv18 binding in all combinations irrespective of the orgin of the Fc portin of the monoclonal antibody. The potentiating effect of bivalent MOv19 fragments on ¹²⁵l-MOv18 binding was similar to that of the entire monoclonal antibody and occurred at saturating concentrations of both reagents at which monovalent binding prevails. Similarly, the monovalent fragment also induced a significant increase in MOv18 bunding. Howener, the potentiation sccurred only at very high concentrations of antibody fragment. Homologous inhibition was drastically reduced using MOv19 Fab fragment, suggesting a low binding stability of the monovalent reagent. Immunoblotting analysis and binding in the presence of exogenous purified folate binding protein indicated a cross-linking between soluble and cell surface molecules mediated by the bivalent monoclonal antibodies. The extentof the increase in MOv18 binging at O°C with high amounts of exogenous folate binding protein was lower than that obtained at 37⁰C in the absence of added molecule. Release of ¹²⁵l-MOv18 from the cell surface was significantly higher in the absence of MOv19 than in its presence. Affinity constant values of ¹²⁵l-MOv18 binding evaluated in the presence of MOv19 or control monoclonal antibody MINT5 were comparable, whereas the number of binding sites per cell detected by ¹²⁵l-MOv18 was significantly higher in the presence of MOv19 than MINT5. Together, the data suggest that monoclonal antibody MOv19 induces a conformational change of the molecule it binds that increases the number of antigenic sites anvailable for MOv18 binding and, in turn, the binding stability of the latter, MOv19 bivalency also contributes to the MOv18 binding increment by cross-linking released and cell surface–anchored folate binding protein molecules. © Wiley-Liss, Inc.     

Presence of H type 3/4 chains of ABO histo-blood group system in serous cells of human submandibular gland and regulation of their expression by the secretor gene (FUT2).

We have investigated by immunochemistry the distribution of H Type 3/4 chains of the ABO histo-blood group system in human submandibular gland using a monoclonal anti-H MBr1 antibody specific for H Type 3/4 chains, and have found the expression of H Type 3/4 chains was mainly in the serous cells. Serous cells from secretors were stained by MBr1 but not by anti-A and anti-B antibodies, whereas serous cells from nonsecretors exhibited a negative reaction with MBr1. Mucous cells were not stained by MBr1. Only a few striated duct cells showed a weak reaction with anti-H MBr1. These results suggested that the H Type 3/4 chains were distributed predominantly in the serous cells of the human submandibular gland and that secretor Type alpha(1,2)fucosyltransferase (Se enzyme) controlled the synthesis of H Type 3/4 chains in vivo. Saliva also contained H Type 3/4 chains, which were controlled by the secretor gene (FUT2). The differences in the distributions of H Type 1, H Type 2, and H Type 3/4 chains of the ABO histo blood group system in the submandibular gland are discussed.     

Immunoconjugates of doxorubicin and murine antihuman breast carcinoma monoclonal antibodies prepared via an N-hydroxysuccinimide active ester intermediate of cis-aconityl-doxorubicin: preparation and in vitro cytotoxicity

Doxorubicin (DXR) conjugated to murine monoclonal antibodies (MoAb) raised against human breast tumor cells demonstrated a MoAb-specific, molar ratio-dependent in vitro cytotoxicity. These conjugates were prepared on a scale sufficient to allow for subsequent clinical trials (1 to 3 g of MoAb per conjugation reaction). The conjugation reaction proceeded via an N-hydroxysuccinimide (NHS) active ester intermediate of cis-aconityl-DXR (CA-DXR), resulting in a cis-aconitate acid-sensitive linker between the DXR and MoAb. Molar ratios of DXR to MoAb ranged from 40 to 45. The immunoreactivity of conjugated MoAb was only slightly decreased from naked MoAb. When immunoconjugates were incubated with MoAb-reactive tumor cells for 3 hours, specific cell-killing was observed. If the exposure time was lengthened to 18 hours, however, nonspecific killing resulted. Incubation of the immunoconjugate with the nonspecific adsorbant Amberlite XAD-2 caused an average 30% decrease in the DXR-to-MoAb molar ratio, suggesting a population of drug that is tightly but noncovalently associated with MoAb.     

Human carcinoma cell lines xenografted in athymic mice: biological and antigenic characteristics of an intraabdominal model

Summary In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6–8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBrl, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.     

Immunoconjugates that neutralize HIV virions kill T cells infected with diverse strains of HIV-1

Two murine monoclonal antibodies, G3.519, recognizing the CD4-binding region, and BAT123, a variable region of gp120 of human immunodeficiency virus, were chemically coupled to pokeweed antiviral protein isolated from seeds (PAP-S). The immunoconjugates were purified by Sephacryl S-200 gel filtration and Mono S ion exchange chromatography. Immunoconjugate G3.519-PAP-S specifically killed human T cells, H9, infected with three diverse HIV-1 strains, HTLV-IIIB, -IIIMN, and -IIIRF. Inhibition of thymidine incorporation by the immunoconjugate was concentration-dependent, with the ID50 ranging from 1.4 x 10(-10) M to 1.7 x 10(-9) M. Immunoconjugate BAT123-PAP-S was effective in killing H9 cells infected with HTLV-IIIB (ID50 = 4.3 x 10(-11) M) and -IIIMN (ID50 = 4.7 x 10(-10) M), but not -IIIRF. Both immunoconjugates did not inhibit thymidine incorporation in uninfected H9 cells up to a concentration of 5.3 x 10(-8) M, and their cytotoxic activities could be competitively blocked by the respective unconjugated antibodies. The immunoconjugates retained the ability to neutralize HIV virions to infect T cells and to prevent the syncytium formation. These in vitro studies suggest that the use of immunoconjugates capable of killing HIV-infected T cells and neutralizing virus may provide an alternative treatment for HIV-infected persons.     

Role of cis prolines 112 and 126 in the functional activity of ribonucleolytic toxin restrictocin

Restrictocin is a 149 amino acid ribonucleolytic toxin produced by the fungus Aspergillus, which specifically cleaves a single phosphodiester bond within 28S rRNA resulting in a potent inhibition of protein synthesis in eukaryotic cells. Restrictocin has 12 prolines out of which three at positions 48, 112, and 126 are cis. Prolines at position 112, 118, and 126 were individually mutated to alanine to investigate their role in the catalytic and membrane interaction activity of restrictocin. All mutants were expressed in Escherichia coli, and recombinant proteins purified to homogeneity. Mutation of P112 resulted in a remarkable 50- and 100-fold reduction, respectively, in the ribonucleolytic and cytotoxic activities of restrictocin, whereas the interaction of P112A with phospholipid membranes increased. Mutants P118A and P126A exhibited 3-5-fold decreased ribonucleolytic and cytotoxic activities, however, their membrane interaction activity was marginally reduced compared to restrictocin. The study demonstrates that P112 is absolutely essential to maintain the functionally active conformation of restrictocin. Also, prolines 112, 118, and 126 do not appear to be directly involved in the membrane interaction activity of restrictocin.     

Cancer-stroma targeting therapy by cytotoxic immunoconjugate bound to the collagen 4 network in the tumor tissue

Some cytotoxic immunoconjugates have been approved for malignant lymphoma, a representative of hypervascular and stroma-poor tumors. However, many human solid tumors possess abundant intercellular stromata that prevent diffusion of cancer cell-specific monoclonal antibodies (mAb) and become a barrier preventing immunoconjugates from directly attacking cancer cells. Here we show the successful development of a new strategy that overcomes this drawback and achieves a highly localized concentration of a topoisomerase I inhibitor, SN 38, by conjugating it via an ester bond to a mAb targeted against collagen 4, a plentiful component of the tumor stroma. Poly(ethylene glycol) (PEG) was utilized as a spacer, close to each bond, to maintain stability in the blood. Immunoconjugates selectively extravasated from leaky tumor vessels and minimally from normal vessels because the immunoconjugates are too large to pass through normal vessel walls. Stroma-targeting immunoconjugates bound to the stroma to create a scaffold, from which sustained release of cytotoxic agent occurred and the agent subsequently diffused throughout the tumor tissue to damage both tumor cells and vessels. Cancer-stroma-targeting immunoconjugate therapy was thus validated as a new modality of oncological therapy, especially for refractory, stromal-rich cancers.     

Idiotypic replica of an anti-human tumor-associated antigen monoclonal antibody. Analysis of monoclonal Ab1 and Ab3 fine specificity

CaMBr1 is a tissue-specific and tumor-associated saccharidic epitope, defined by mAb MBr1 (Ab1), expressed on glycoconjugates of the human mammary carcinoma cell line MCF-7 and of normal and neoplastic mammary epithelial cells. An anti-anti-idiotypic monoclonal Ab3, 2G-3, identifying a human breast tumor associated antigen, was raised by using as immunogen a mouse anti-idiotypic monoclonal Ab2, A3B10, which behaves as the internal image of CaMBr1. mAb 2G-3, as well as MBr1, defines a saccharidic epitope on glycoconjugates extracted from MCF-7 cells and shows MBr1-like reactivity on normal and neoplastic-tissues. Experimental evidence, however, suggests that the fine immunoreactivity of the two antibodies is not identical, because MBr1 has a preferential reactivity with glycolipids and 2G-3 with glycoproteins. We suggest that a possible biologic explanation for our findings could reside in the nature of the immunogens used to raise the two mAb (glycolipid vs protein "internal image").     

Anti-human tumor antibodies induced in mice and rabbits by "internal image" anti-idiotypic monoclonal immunoglobulins

MBr1 is a murine monoclonal antibody, defining a saccharidic epitope [CaMBr1] of a human tissue-specific, tumor-associated globoside, present on the mammary carcinoma cell line MCF-7. The same epitope is shared by glycoproteins present on normal and neoplastic mammary epithelial cells, and by mucins from some ovarian cyst fluids. We have used MBr1 as the monoclonal antitumor antibody in an idiotypic sequence of immunizations in order to obtain and characterize "internal images" of the original epitope to be used as substitutes of the nominal antigen in serologic immunoassays. Two monoclonal anti-idiotypic antibodies (beta-1 and beta-2), which reacted with paratope-related idiotopes on MBr1, were obtained. The analysis of the antigenic and immunogenic properties of these molecules by both "antigen" and "antibody" competition assays provided evidence that both beta-1 and beta-2 bear "internal images" of the MBr1-defined epitope. Moreover, when injected in mice and rabbits both beta-1 and beta-2 induced anti anti-idiotypic antibodies, which mimicked MBr1 in binding MCF-7 as well as normal and neoplastic mammary gland epithelial cells. These data are discussed in terms of their possible application to the production of tumor-associated antigen substitutes and their use in serologic immunoassays.     

Fractionation of the ribosome inactivating protein preparations with triazine dyes

Aspergillins are ribosome-inactivating proteins (RIPs), isolated from several strains of Aspergillus. The interaction between Cibacron Blue F3GA and two members of this family, alpha sarcin and mitogillin, and other RIPs of type I, was studied. Alpha sarcin retention depended on pH and ionic strength. By chromatography on Affi-Gel Blue in mild experimental conditions, mitogillin and PAP-I did not interact with the dye, whereas 40% of alpha sarcin and 70-90% of briodin, RTA and gelonin were recovered in the bound fraction. In all cases, the major fraction showed a higher toxicity level in protein synthesis inhibition assays. The unbound alpha sarcin, conjugated with the anti-ovarian carcinoma monoclonal antibody MOv17, showed on OVCA 432 a cytotoxicity which was 900 times higher than that exerted by the alpha sarcin alone.     

Isolation and nucleotide sequence of the Aspergillus restrictus gene coding for the ribonucleolytic toxin restrictocin and its expression in Aspergillus nidulans: the leader sequence protects producing strains from suicide

We describe the cloning and characterization of the gene coding for the ribotoxin restrictocin, from Aspergillus restrictus (gene res, EMBL accession Number X56176). This toxin is a potent inhibitor of protein synthesis in eucaryotes and is of potential interest as a component of immunotoxins. To analyze the mechanism of self-protection in the producing organism, the res gene was cloned into the vector pFB39 and introduced into Aspergillus nidulans. The secretion of active restrictocin from transformants suggests that the pro-toxin is not an active nuclease but is activated during the process of secretion.     

Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin.

Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.     

Selective cytotoxicity of antibody-ricin-A-chain conjugate for human tumor B cells. II. Comparison of activity of immunotoxins conjugated with poly- and monoclonal antibodies

The A-chain of a plant toxin ricin has been coupled to poly- and monoclonal antibodies specific to the L-chains of human IgG. The inhibitory effect of the conjugates has been compared with the ability of the antibodies to bind to target cells. Cytotoxicity of the conjugates has been monitored following incorporation of 14C-leucine radioactivity into Burkitt lymphoma cells with surface Ig. The 50% inhibition of protein synthesis is observed 18 h after treatment of cells with immunotoxins, when the concentration of the conjugates with poly- and monoclonal antibodies is 1.2.10(-9) M and 0.7.10(-9) M, respectively. The data take into account that only part of the polyclonal antibodies molecules is able to react with target cells. The control conjugates containing either monoclonal antibodies that do not react with the lymphoma cells surface L-chains or nonimmune serum IgG proved to have no effect on target cells even at the level of 10(-7) M. The immunotoxins with poly- and monoclonal antibodies produce almost the same kinetics of protein synthesis inhibition, when incubated with lymphoma cells for 60 min. However, a 30 min treatment reveals a considerably higher cytotoxicity of the conjugate with monoclonal antibodies.     

Mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic toxin restrictocin

Restrictocin, a member of the fungal ribotoxin family, specifically cleaves a single phosphodiester bond in the 28S rRNA and potently inhibits eukaryotic protein synthesis. Residues Tyr47, His49, Glu95, Phe96, Pro97, Arg120, and His136 have been predicted to form the active site of restrictocin. In this study, we have individually mutated these amino acids to alanine to probe their role in restrictocin structure and function. The role of Tyr47, His49, Arg120, and His136 was further investigated by making additional mutants. Mutating Arg120 or His136 to alanine or the other amino acids rendered the toxin completely inactive, whereas mutating Glu95 to alanine only partially inactivated the toxin. Mutation of Phe96 and Pro97 to Ala had no effect on the activity of restrictocin. The Tyr47 to alanine mutant was inactive in inhibiting protein synthesis, and had a nonspecific ribonuclease activity on 28S rRNA similar to that shown previously for the His49 to Ala mutant. Unlike the His136 to Ala mutant, the double mutants containing Tyr47 or His49 mutated to alanine along with His136 did not compete with restrictocin to cause a significant reduction in the extent of cleavage of 28S rRNA. In a model of restrictocin and a 29-mer RNA substrate complex, residues Tyr47, His49, Glu95, Arg120, and His136 were found to be near the cleavage site on RNA. It is proposed that in restrictocin Glu95 and His136 are directly involved in catalysis, Arg120 is involved in the stabilization of the enzyme-substrate complex, Tyr47 provides structural stability to the active site, and His49 determines the substrate specificity.     

Crystal structures of restrictocin-inhibitor complexes with implications for RNA recognition and base flipping.

The cytotoxin sarcin disrupts elongation factor binding and protein synthesis by specifically cleaving one phosphodiester bond in ribosomes. To elucidate the molecular basis of toxin action, we determined three cocrystal structures of the sarcin homolog restrictocin bound to different analogs that mimic the target sarcin/ricin loop (SRL) structure of the rat 28S rRNA. In these structures, restrictocin contacts the bulged-G motif and an unfolded form of the tetraloop of the SRL RNA. In one structure, toxin loops guide selection of the target site by contacting the base critical for recognition (G4319) and the surrounding S-shaped backbone. In another structure, base flipping of the tetraloop enables cleavage by placing the target nucleotide in the active site with the nucleophile nearly inline for attack on the scissile bond. These structures provide the first views of how a site-specific protein endonuclease recognizes and cleaves a folded RNA substrate.     

Understanding structural/functional properties of immunoconjugates for cancer therapy by computational approaches

Monoclonal antibodies coupled to highly toxic molecules (immunoconjugates) are currently being developed for cancer therapy. We have used an in silico procedure for evaluating some physicochemical properties of two tumor-targeting anti-HER2 immunoconjugates: (a) the single-chain antibody scFv(FRP5) linked to a bacterial toxin, that has been recently progressed to phase I clinical trial in human cancer; (b) the putative molecule formed by the intrinsically stable scFv(800E6), which has been proposed as toxin carrier to cancer cells in human therapy, joined to the same toxin of (a). Structural models of the immunoconjugates have been built by homology modeling and assessed by molecular dynamics simulations. The trajectories have been analyzed to extract some biochemical properties and to assess the potential effects of the toxin on the structure and dynamics of the anti-HER2 antibodies. The results of the computational approach indicate that the antibodies maintain their correct folding even in presence of the toxin, whereas a certain stiffness in correspondence of some structural regions is observed. Furthermore, the toxin does not seem to affect the antibody solubility, whereas it enhances the structural stability. The proposed computational approach represent a promising tool for analyzing some physicochemical properties of immunoconjugates and for predicting the effects of the linked toxin on structure, dynamics, and functionality of the antibodies.     

Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli

Pseudomonas Exotoxin A (PE) and truncated PE have been used to prepare immunotoxin with monoclonal antibodies. Truncated Pseudomonas Exotoxin A (PE38KDEL) was expressed with the pET-32a(+) vector in Escherichia coli under control of a T7 promoter. The recombinant protein was purified by His-Ni(2+) metal affinity chromatography and gel filtration. The biological activity of PE38KDEL was evaluated by the inhibition assay of protein synthesis in rabbit reticulocyte lysate system, and the cytotoxicity was tested in Hut 102 and hepatocellular cell lines by the MTS assay. PE38KDEL can significantly inhibit luciferase synthesis in cell-free protein synthesis assay and was slightly cytotoxic in the Hut 102 and hepatocellular cell lines. The results suggest that PE38KDEL would be useful for the preparation of more potent immunotoxins.     

Serotherapy of ovarian cancer

The development of monoclonal antibodies has permitted the identification of several ovarian-tumor-associated antigens which might serve as targets for serotherapy in vivo. With the exception of antibodies directed against growth factor receptors, unmodified monoclonal reagents must activate complement (C') components or bind effector cells to destroy tumor targets. Antibody-dependent cell-mediated cytotoxicity (ADCC) may be particularly important for eliminating tumor cells in vivo. A shortage of functionally active effector cells can limit the efficacy of serotherapy with heteroantisera or monoclonal reagents. The use of immunostimulants such as Corynebacterium parvum has increased the number and activity of effector cells for ADCC within the peritoneal compartment of mice and of patients with ovarian cancer. Intraperitoneal serotherapy can achieve direct contact between antibody and microscopic deposits of ovarian tumor cells which persist following cytoreductive operations and cytotoxic chemotherapy. Conjugation of monoclonal antibodies with radionuclides, drugs or toxins might increase the potency of serotherapy and circumvent the effector shortage. Clinical studies to date have evaluated radionuclide conjugates for imaging and for therapy. Patients with a small volume of disease have responded to treatment. Preclinical models suggest that drug and toxin conjugates might also prove active. Recent studies have demonstrated a synergistic interaction between different immunotoxins. Ovarian carcinoma is likely to be a valuable clinical model for evaluating immunoconjugates which react with epithelial tumor cells.     

Involvement of loops L2 and L4 of ribonucleolytic toxin restrictocin in its functional activity

Restrictocin, a member of the fungal ribotoxin family, specifically cleaves a single phosphodiester bond in the 28S rRNA and potently inhibits eukaryotic protein synthesis. The long loops in restrictocin molecule have been shown structurally to be involved in target RNA recognition. In this study we have investigated the role of some putative substrate-interacting residues in loops L2 and L4, spanning residues 36-48 and 99-117, respectively in restrictocin catalysis. The residues Lys42, Ser46, Pro48 and Lys111 were individually mutated to alanine to probe their role in restrictocin function. The mutation of Lys111 to alanine, although did not affect the ribonucleolytic activity, rendered the toxin completely inactive in inhibiting translation in HeLa cells as well in an in vitro cell free system. The loop L4 in restrictocin appears to be more critical compared to loop L2 for its interaction with the specific substrate.