Biotherapy 3: 65-76, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Immunotoxins Oliver W. Press Departments of Medicine and Biological Structure, University of Washington and the Fred Hutchinson Cancer Research Center, Seattle, WA, USA Received 6 March 1990

Key words: immunotherapy, immunotoxins, monoclonal antibodies, ricin, tumor immunology Abstract

Immunotoxins (ITs) are chimeric molecules constructed by covalently conjugating monoclonal antibodies (MoAbs) to plant or bacterial toxins (e.g. ricin or pseudomonas exotoxin). The antibody moiety allows specific targeting of ITs to tumor-associated antigens, while the toxin moiety is responsible for cell killing by irreversible inactivation of protein synthesis. Since ITs must reach the cytosol to kill cells, the rates of endocytosis, the pathways of intracellular routing, and the rates of translocation to the cytoplasm are important determinants of the efficacy of an IT. Promising in vitro and in vivo IT results have been reported by many groups, and phase I clinical trials in cancer patients are currently underway. Abbreviations: dgA: deglycosylated A-chain; GVHD: graft versus host disease; HAMA: human anti-mouse antibodies; 2IT: 2-iminothiolane; IT: immunotoxin; IT-A: A-chain immunotoxin; IT-B: B-chain immunotoxin; MoAb: monoclonal antibody; RME: receptor mediated endocytosis; SPDP: N-succinimidyl-3-(2-pyridyldithio)-propionate; SMPT: N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)-toluene;SPDB: N-succinimidyl-3-(2-pyridyldithio)-butyrate.

I. Introduction

Immunotoxins (ITs) are chimeric antineoplastic agents constructed by covalently conjugating monoclonal antibodies (MoAbs) to plant or bacterial toxins [ 1-4]. The antibody moiety allows specific targeting of ITs to tumor-associated antigens, while the toxin moiety is responsible for cell killing. Many plant toxins (e.g., ricin, abrin, modeccin) are heterodimers consisting of an A-chain which This research was supported in part by a First Independent Research and Training award from the National Institutes of Health (R29 CA 46134-03).

causes irreversible inactivation of 60S ribosomes, and a B-chain which binds to galactose residues and facilitates transport of the Achain from endocytic vesicles to the cytosol. Other plant toxins are single chain ribosome inactivating proteins (RIPs) which are devoid of galactose binding subunits and therefore are minimally toxic unless conjugated to a monoclonal antibody (MoAb) or other binding moiety. Endo et al. [5] has recently shown that ricin A-chain and related RIPs are specific N-glycosidases which catalytically hydrolyze the glycosidic bond of adenosine residue 4324 in the 23S rRNA of eukaryotic 60S ribosomal subunits, resulting in the re-

66 lease of this adenine residue from the phosphoribose backbone. Ribosomes modified in this manner are no longer capable of binding elongation factors normally, and render cells incapable of protein synthesis. Kinetic studies have estimated that one ricin A-chain can inactivate 1500 ribosomes per minute, and suggest that a single A-chain molecule in the cytosol can kill a cell. Although diphtheria toxin also has A and B chains analogous to those of ricin, its mode of action is quite different. Diphtheria

toxin enzymatically ADP-ribosylates a unique amino acid (called diphthamide) of elongation factor-2 (EF-2) resulting in its irreversible inactivation and the termination of cellular protein synthesis [3]. Pseudomonas extotoxin also ADP-ribosylates EF-2, although it is a single polypeptide chain containing a binding domain, a translocation domain, and a toxin domain [3, 4]. A list of the most commonly used toxins and their properties is presented in Table 1.

Table L Toxins used in immunotoxin synthesis. Toxin

Plant holotoxins Ridn Abrin Modeccin Viscumin

Source

Structure

Molecular weight

Function

Special features

Ricinus communis Abrus precatorius Adenia digitata Viscum album

A-B Dimer

A-30 k D B-32 kD A-30 k D B-35 kD A-28 k D B-38 kD A-32 k D B-32 k D

Inactives 60S ribosomes Binds to Galactose as above

1,2 2,3

as above

5

as above

2

Plant ribosomal inactivating proteins (RIPs) Gelonin Gelonian~ multiflorum Saporin Saponaria o~cinalis Momordin Momort~ca charantia Bryodin Bryonia diocia Pokeweed Phytolacca Antiviral americana Protein Tricosanthin

A-B Dimer A-B Dimer A-B Dimer

Monomer

30 k D

Inactivates 60S ribosomes

4

"

30 kD

"

3,4

"

31 kD

"

4

"

30 kD

"

4

"

28 kD

"

3,4

Trikosanthes kirolowii

"

30 kD

"

4

Corynebacterium diptheriae Pseudomonas aeruginosa

Nicked A-B Dimer

A-22 k D B-40 k D

A D P ribosylates EF-2 binds to D T receptor

5

Polypeptide (3 domains)

72 kD

A D P ribosylates EF-2

5

Bacterial toxins Diptheria Toxin Pseudomonas Exotoxin 1= 2= 3= 4= 5=

glycosylated with mannose and fucose residues translocates to cytosol well at neutral or slightly alkaline p H no glycosylation no natural cell-binding moiety translocates to cytosol at p H 4 - 5

67

II. Immunotoxin synthesis Many different IT constructs have been prepared (see Figure 1). In all cases, a covalent linkage is employed to conjugate the antibody or ligand to the toxin component. This linkage must not interfere with antibody

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Immunotoxins.

Immunotoxins (ITs) are chimeric molecules constructed by covalently conjugating monoclonal antibodies (MoAbs) to plant or bacterial toxins (e.g. ricin...
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