Naoji Umemoto

Localization of human airway trypsin-like protease in the airway: an immunohistochemical study

Abstract. Human airway trypsin-like protease (HAT) has been isolated from mucoid sputum of patients with chronic airway diseases. In order to clarify the cellular source of this novel protease in the human airway, we examined the localization of immunoreactive HAT in bronchial tissues obtained at surgery and fixed in 4% paraformaldehyde using an extremely sensitive immunohistochemical technique called a catalyzed signal amplification method and a monoclonal antibody against recombinant HAT. HAT immunoreactivity was demonstrated in cytoplasm of ciliated cells of bronchial epithelium and/or at the basal part of cilia. No positive reaction was found in submucosal glands or mast cells. The heterogeneous distribution of HAT immunoreactivity within the bronchial epithelium indicates that its expression might be changeable and that it might be closely related to the physiological status of the airway epithelium. Non-specific but intense reaction caused by endogenous avidin-binding activity (EABA) was selectively detected in submucosal glands, but was effectively blocked by successive treatments with avidin and biotin. These results indicate that HAT may be synthesized in the ciliated cells and that it may play some physiological roles within the epithelial layer and on the airway surface. It is necessary to keep in mind that some cells show strong EABA, especially when a highly sensitive immunohistochemical technique is applied.

A novel covalent modification of antibodies at their amino groups with retention of antigen-binding activity

A novel method of covalent modification of antibodies at their amino groups with retention of antigen-binding activity is described. The procedure is as follows: (a) blockade of those amino groups of antibodies whose integrity is essential to their antigen-binding activity with 2,3-dimethylmaleic anhydride, a reversible amino group-blocking reagent; (b) modification of residual amino groups with reagents reactive with the amino groups; and (c) removal of dimethylmaleyl groups by hydrolysis. This procedure was used for covalent conjugation of methotrexate (MTX) with two monoclonal antibodies against human melanoma-associated antigens using MTX N-succinimidyl ester. MTX attached to the antibodies at sites other than the amino groups via less stable bond(s) was removed by treatment with hydroxylamine.

Target-selective cytotoxicity of methotrexate conjugated with monoclonal anti-MM46 antibody.

SummaryIn studies on antitumor antibody-cytotoxic drug conjugates as potential tumor-selective cytotoxic agents, methotrexate (MTX) was conjugated via its active ester derivative with a murine monoclonal antibody (aMM46) to a mouse mammary tumor antigen (MM antigen) on syngeneic, ascitic C3H/He mouse mammary tumor MM46 cells. The conjugate retained full antibody activity, as assayed by complement-dependent cytolysis. The target-selective cytotoxicity of aMM46-MTX was verified by the observations that this conjugate showed greater cytotoxicity than the corresponding normal mouse immunoglobulin (nIg) conjugate to MM46 cells, neither aMM46 nor nIg being cytotoxic, and that it showed less cytotoxicity to MM antigen negative mouse mammary tumor MM48 cells than to MM46 cells, its cytotoxicity to MM48 cells being similar to that of the nIg conjugate. From the results of assays of cell binding and uptake of 131I-labeled aMM46 and aMM46-3H-MTX, aMM46 and aMM46-MTX were internalized after their binding to MM46 cell surface antigen. Leupeptin, an inhibitor of the lysosomal endopeptidase cathepsin, decreased the cytotoxicity of aMM46-MTX, supporting the involvement of lysosomal degradation of the conjugate in its action.

Selective in vitro and in vivo growth inhibition against human yolk sac tumor cell lines by purified antibody against human α-fetoprotein conjugated with mitomycin C via human serum albumin

SummaryThe anticancer drug mitomycin C (MMC) was conjugated with an affinity-purified horse antibody to human α-fetoprotein (aAFP) with human serum albumin (HSA) as the intermediate drug carrier. The conjugate (aAFP:HSA:MMC molar ratio, 1:1:30) retained full antibody binding activity as determined by a competitive binding radioimmunoassay. In a cytotoxicity test in which the AFP-producing human yolk sac tumor TG-1 cells were preincubated with test materials for 2 h followed by an additional 48-h culture in fresh medium, the conjugate was 20-fold more cytotoxic than free MMC at an equivalent MMC concentration of 100 ng/ml. The in vivo antitumor effect of the conjugate was tested against the human yolk sac tumor JOG-9 growing in athymic nude mice. When the tumor-bearing mice were treated with a total of 6 injections given on 2 consecutive days and then every other day starting 8 days after SC tumor inoculation [2 (equivalent MMC) μg/head per injection], the conjugate retarded tumor growth more effectively than free MMC and normal horse immunoglobulin conjugate.

Review : Molecular Design of Methotrexate-Antibody Conjugates for Targeted Cancer Treatment

urrently available cytotoxic drugs have only limited performance C in the treatment of cancer largely because of their insufficient tumor selectivity. With increasing number of monoclonal antibodies (Ab’s) to tumor associated antigens in the last decade [1,2], attention has been drawn to an approach of drug targeting with drug-Ab conjugates [3-6]. A wide range of cytotoxic drugs such as chlorambucil [7], daunomycin [8], adriamycin [9], mitomycin C [10], actinomycin D [11], neocarzinostatin [12], vindesine [13], desacetyl-vinblastine [14], and methotrexate have been studied for conjugation. Although some clinical trials of drug-Ab conjugates have begun, this approach is still largely at an experimental stage where various strategies are being examined in order to construct improved modalities. This article focuses on the molecular design of methotrexate (MTX) conjugates, their rationale, and methods of synthesis. MTX has been recommended as a drug suitable for conjugation based on several considerations. Clinical effectiveness of MTX has been well-

Cytotoxicities of two disulfide-bond-linked conjugates of methotrexate with monoclonal anti-MM46 antibody

SummaryIn studies on (antitumor antibody)-drug conjugates as potential antitumor agents, the amide derivatives of methotrexate (MTX) with cysteine and with 2-mercaptoethylamine (cysteamine) (MTX-Cys and MTX-MEA, respectively) were linked via a disulfide bond with a monoclonal antibody (αMM46) to a mouse mammary tumor MM46 with attached 3-(2-pyridyldithio) propionyl groups to give conjugates of MTX with αMM46 (MTX-Cys-SS-αMM46 and MTX-MEA-SS-αMM46, respectively). These two conjugates are both linked by a disulfide bond and are very similar in structure, but MTX-MEA-SS-αMM46 showed only weak in vitro cytotoxicity against MM46 cells, whereas MTX-Cys-SS-αMM46 had strong cytotoxicity. The cytotoxicity of the latter was comparable to that of the conventional direct MTX-αMM46 conjugate prepared with an MTX-active ester. However, this conjugate had a greater selectivity than that of the direct conjugate, calculated as the IC50 (concentration of a conjugate by MTX equivalence required for suppression of the number of viable MM46 cells to 50% of that of the untreated control) for the corresponding nonspecific conjugate divided by the IC50 for the αMM46 conjugate. The inhibitory activities of MTX-Cys and MTX-MEA on dihydrofolate reductase were similar. The cytotoxicity of MTX-Cys-SS-αMM46 was not affected by thiamine pyrophosphate, an inhibitor of the active transport of MTX across the cell membrane, but was decreased significantly by ammonium chloride, a lysosomotropic amine. However, the cytotoxicity was decreased only to a small extent by leupeptin, an inhibitor of lysosomal cysteine proteases cathepsins B, H, and L. These results suggest that the cytotoxicity is mediated by lysosomes, and may involve lysosomal enzymes other than cathepsins B, H, and L.

Cytotoxicity of a hybrid prepared by coupling diphtheria toxin A-chain with immunoglobulin Fab′ with N,N′-o-phenylenedimaleimide

Abstract A hybrid protein was prepared by coupling the A-chain of diphtheria toxin with the Fab′ fragment of immunoglobulin with N , N′ - o -phenylenedimaleimide (PDM). Although in this hybrid, the two components were linked with each other with bonds which could not be reductively cleaved with 2-mercaptoethanol as in a hybrid cross-linked with a disulfide bond (e.g. Fab′-S-S-A-chain), it exhibited a potent cytotoxicity in vitro , one-third of that of Fab′-S-S-A-chain, against the target L1210 cells.

Drug/Antibody Conjugates. In Vitro Cytotoxicity of a Human Serum Albumin‐Mediated Conjugate of Methotrexate with Anti‐MM46 Monoclonal Antibody

In studies on antitumor antibody-drug conjugates as potential antitumor agents with improved tumor-selectivity [ 1, 21 , we prepared a conjugate of methotrexate (MTX) with a monoclonal antibody (aMM46) to an antigen on ascitic C3H/He mouse mammary tumor MM46 cells (MM antigen) with human serum albumin (HSA) as an intermediary [3 ] . As shown in Fig. 1, using an active ester of MTX, we first linked MTX to HSA which had been conditioned to have about 1 mol of thiol group per mol of HSA. The resulting HSA-MTX was then reacted with aMM46 with the maleimide group introduced (MTX/HSA/IgG molar binding ratio, 28.0/ 1.16/ 1 .O). We examined the in vitro cytotoxicity of the conjugate aMM46-HSA-MTX by culturing MM46 cells or MM antigennegative MM48 cells, another C3H/He mouse mammary tumor cell line, with the conjugate and controls for two to three days followed by enumeration of the viable cells. MTX conjugated with