1. Field of the Invention
The present invention relates to recombinant active single chain immunotoxin fusion proteins which selectively kill cells bearing appropriate antigens or receptors. In particular, the present invention relates to a genetically modified form of diphtheria toxin (DT) and a single chain antibody [anti-TFR(Fv)] that binds to the human transferrin receptor.
2. Background Information
Immunotoxins made by chemically coupling tumor-specific monoclonal antibodies to bacterial or plant toxins including Pseudomonas exotoxin (PE) and Diphtheria (DT) have been shown to possess in vitro and in vivo antitumor activity (Pastan, I. et al., 1986, Cell 47, 641-648; Vitetta, E. S. et al., 1987, Science 238, 1098-1104; Fitzgerald, D. and I. Pastan, 1989, J. Natl. Cancerhzst. 81, 1455-1463). Both PE and DT kill the cells by ADP-ribosylating elongation factor 2 thereby inhibiting protein synthesis. X-ray crystallographic and mutational analysis show the PE molecule is made up of three distinct domains, namely; an amino terminal binding domain, a central translocation domain and a carboxyl activity domain (Allured, V. S. et al., 1986, Proc. Natl. Acad. 2 Sci. USA, 83, 1320-1324; Hwang, J. et al., 1987, Cell, 48, 129-136). Diphtheria toxin however is mainly divided into two chains, A and B, linked by a disulfide bridge. In contrast to PE, chain B of DT which is on the carboxyl end is responsible for receptor binding and chain A which is present on the amino end contains the enzymatic activity (Uchida, T. et al., 1972, Science, 175, 901-903; Uchida, T. et al., 1973, J. Biol. Chem. 248, 3838-3844). Apparently the last 150 amino acids of DT are responsible for its cell binding activity (Uchida, T. et al., 1972, Science, 175, 901-903; Uchida, T. et al., 1973, J. Biol. Chem. 248, 3838-3844; Rolf, J. M. et al., 1990, J. Biol. Chem., 265, 7331-7337).
Immunotoxins have been constructed by attaching antibodies either to native Pseudomonas exotoxin (PE) that has a Mr of 66 kDa or to truncated forms of PE that lack its cell binding domain and have a Mr of about 40 kDa (referred to as PE40). Immunotoxins made with PE40 or LysPE40, a PE40 derivative with an extra lysine residue at its amino terminus that facilitates coupling to antibodies, have a more specific action on target cells because PE cannot bind to its cellular receptor (Pirker, R. et al., 1985, Cancer Res. 45, 751-757; Kondo, T. et al., 1988, J.Biol. Chem., 263, 9470-9475; Batra, J. K. et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 8545-8549). One such immunotoxin, anti-transferrin receptor (TFR)-LysPE40, was shown to be highly cytotoxic to cells expressing the human transferrin receptor and when given intraperitoneally to mice caused regression of A431 tumors growing as subcutaneous xenografts (Batra, J. K. et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 8545-8549). Immunoconjugates with low nonspecific toxicity have also been made by using modified DT with mutations in its cell binding domain which, unlike PE, is present on the carboxyl end of the toxin (Youle, R. J. et al., 1988, In Immunotoxins, Frankel, A. E. (ed.), pp.113-122; Murphy, J. R. F., 1988, In Immunotoxin, Frankel, A. E. (ed), pp.123-140).
Immunotoxins constructed by chemical cross linking are heterogeneous and are difficult to produce in large amounts because the antibody and toxin must be made separately and then conjugated together in a reaction that often has a low yield.
To overcome this problem, a recombinant single-chain immunotoxin has recently been described directed at the P55 subunit of the human (IL-2) interleukin 2 (IL-2) receptor termed anti-Tac(Fv)PE40. It consist of the variable domains of a monoclonal antibody directed at the interleukin-2 receptor arranged in a single chain form linked to the amino terminus of PE40 (Chaudhary, V. K. et al., 1989, Nature, 339, 394-397; Batra, J. K. et al., 1990, J. Biol. Chem. 265, 15198-15202). Recently, Chaudhary et al. constructed anti-Tac (Fv) PE40 from cDNA but such constructs may also be made from RNA. The single chain immunotoxin is constructed starting with hybridoma RNA and using polymerase chain reaction (PCR) to isolate DNA encoding the variable heavy and variable light chains of the antibodies and inserting this DNA into an expression vector containing PE40 sequence (Chaudhary, V. K. et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1066-1070). Anti-Tac(Fv)-PE40 selectively killed cell lines that express the p55 subunit of the human IL-2 receptor. This chimeric protein was also very effective in killing cells activated in a mixed leukocyte reaction (MLR) as well as human phytohemagglutinin-stimulated blasts (Batra, J. K. et al., 1990, J. Biol. Chem., 265, 15198-15202).
Murphy and his coworkers (Williams et al., 1990, J. Biol. Chem 265, 11885-11889) has previously shown that recombinant proteins could be made by fusing the carboxyl end of a mutant form of diphtheria toxin (DT) to the lymphokine, IL-2. In other studies, a fusion protein called DT-388-IL-2 has been prepared that consists of the first 388 amino acids of DT fused to the human IL-2 (V. K. Chaudhary, D. J. Fitzgerald and Ira Pastan, unpublished results). This protein was also very cytotoxic to cells bearing the human as well as the mouse high affinity IL-2 receptor.
Thus, it is clear that a need exists to create recombinant chimeric immunotoxins because of their potential usefulness as antitumor agents. To circumvent the problem of high titer antibodies to PE40 that may arise upon repeated treatment with single chain antibodies linked to PE40, construction of other active single chain immunotoxins are required having different toxin moieties that place the antigen binding portion at either the amino terminus, the carboxyl terminus, or at both termini. The present invention provides the construction of such immunotoxins.