Monovalent Immunotoxin Containing Truncated Form of Pseudomonas Exotoxin as Potent Antitumor Agent1

Recombinant truncated forms of Pseudomonas exotoxin A that lack the cell binding domain of Pseudomonas exotoxin A were coupled to an F(ab') fragment of a monoclonal antibody HB21 directed against the human transferrin receptor. One of these was NlysPE40. The other, NlysPE38QQRi has two amino groups on residues near the NH2-terminus and has no amino groups near the COOH-terminus. The proteins were linked by a stable thioether bond that connected the sulfhydryl group present in the hinge region of the antibody fragment to an amino group on the toxin. The F(ab')-PE40 immunotoxin, containing NlysPE40, exhibited potent cytotoxic activity on human carcinoma cell lines with a concentration of immunotoxin at which isotope incorpora tion falls by 50% when compared to nontreated cells (IlUn) of 5.3 PM (0.5 ng/ml) on both the epidermoid carcinoma A431 and on the colon carcinoma Colo205. Immunotoxins made with whole antibody were con siderably less active, with an ID50 of 15.9 PM (3.1 ng/ml) on these cell lines. F(ab')-PE38QQR, the immunotoxin containing NlysPE38QQR> was found to be the most active agent with an UK,, of 1.05 PM (0.1 ng/ml) on A431 cells. The greater cytotoxicity of immunotoxins con taining fragmented antibody was probably due to the higher binding affinity of F(ab' ) conjugates in comparison to whole antibody conjugates to the transferrin receptor. The increase in cytotoxic activity of the immunotoxin made with NlysPE38QQR than that with NlysPE40 may reflect selective coupling of the toxin through NH2-terminal amino groups. The monovalent and divalent immunotoxins had dose-depen dent antitumor effects on human epidermoid carcinoma xenografts in nude mice. A431 tumors completely regressed in all animals at a total dose of 105 pmol (10 *ig)of F(ab')-PE38QQR and of 154 pmol (30 UK) of IgG-PE38QQR. Furthermore, the f-(ah ) immunotoxin was less toxic to mice than the conjugate containing IgG (840 pmol or 80 «ig of total dose causing measurable adverse effects versus 208 pmol or 40 MR, respectively). Thus, a truncated Pseudomonas exotoxin A molecule cou pled to the l-(ab ) fragment of an antibody is more active and less toxic in mice than an immunotoxin made with a whole antibody. Therefore, the therapeutic index for the monovalent immunotoxin is about four times better than that for the divalent immunotoxin. INTRODUCTION MAbs,4 by virtue of their affinities toward defined molecules, are the subject of intense interest in the field of targeted therapy (1,2). MAbs which bind to antigens that are expressed prefer entially on cancer cells or certain differentiated cells (1,3) have been utilized either without modification or as F(ab')2 or F(ab') fragments (2). MAbs have been linked to chemicals, enzymes, radioisotopes, or toxins in order to visualize tumors or cause cell death (1, 2). Antibodies conjugated to toxins are termed Received 6/3/92; accepted 7/17/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Part of this work has been presented in a poster discussion session. "Immu nology 8," at the 83rd Annual Meeting of the American Association for Cancer Research, San Diego, CA, May 20-23, 1992. 2 Recipient of a postdoctoral fellowship from the Medical Research Council of Canada. 3 To whom requests for reprints should be addressed. 4 The abbreviations used are: MAb, monoclonal antibody; IT, immunotoxin; PE, Pseudomonas exotoxin A; SMCC, succinimidyl 4(yv-maleimidomethyl)cyclohexan 1-carboxylate; 211. 2-iminothiolane. ITs, and these agents are now being evaluated for their useful ness in the treatment of cancer and other diseases (4). Our laboratory has been developing immunotoxins contain ing PE or mutant forms of this toxin (5). PE is a M, 66,000 single-chain protein containing three disulfide bonds (6). PE is composed of three structural domains (I, II, III), and each of these domains performs at least one specific function (7, 8). Domain la binds to the PE receptor (8, 9); domain II is required for translocation (10, 11); and domain III catalyzes the ADPribosylation of elongation factor 2 which arrests protein syn thesis in eukaryotic cells causing cell death (8). Prior to translocation, PE is cleaved after arginine 279 to produce a A/r 37,000 COOH-terminal fragment, which comprises all of do main III and a portion of domain II, that reaches the cytosol ( 10). ITs containing PE, or forms of PE that lack its cell binding domain (PE40), have been shown to possess high cytotoxic activities /// vitro and to display prominent antitumor effects on solid tumor xenografts growing in nude mice (12-14). An ad ditional lysine residue has been introduced near the amino ter minus of PE40 (lysPE40, Ref. 12; NlysPE40, Ref. 13) which is believed to facilitate the reactivity of the toxin with cross-link ing reagents in the conjugation reaction. Despite the fact that the ITs containing PE or its derivatives are very active on cultured cells, they often bind to antigens with 3to 10-fold less avidity than the unconjugated MAb (13-15). Diminished binding may be due to chemical alteration of the antigen binding site or the presence of the toxin at a site which, by steric hindrance, diminishes the contact between the anti body and the antigen. We have previously found that lysPE40 or NlysPE40 may be linked to either the heavy or light chain of a MAb in an IT (12).5 Therefore, we support the idea that the physical presence of the toxin conjugated to an antibody makes binding of an IT less efficient than an antibody alone. In an attempt to make an immunotoxin with higher activity, we have prepared an immunotoxin with a defined structure in which an amino group at the amino terminal of a truncated form of PE is coupled to a thiol group in a F(ab') fragment of an antibody. In this immunotoxin, (a) the toxin is linked to an antibody at a specific location on the heavy chain; {/>)the site of coupling between these two proteins is removed as far as pos sible from the antibody combining site; and (c) the toxin is coupled to an antibody through residues on the toxin preceding the proteolytic cleavage site in domain II at position 279 (16) (Fig. 1). To ensure that coupling occurs at the amino end of the toxin, we also used a derivative of NlysPE40, NlysPE38QQR, in which all three lysines in domain III have been mutated to other residues (16).6-7 Thus, in NlysPE38QQR, the two pri mary amino groups available for modification are located ex clusively in domain II at or near the NH2-terminal and up stream of the processing site (Fig. 1). NlysPE38QQR also has a deletion of amino acids 365-380 of PE which removes a disulfide bond in domain Ib that is not essential for activity and 5 Unpublished observation. 6 W. Debinski and I. Pastan, manuscript in preparation. 7 Y. Jinno, D. FitzGerald, and I. Pastan, unpublished observation.