Abstract
Immunotoxin therapy for cancer utilizes hybrid proteins composed of a cell targeting moiety which is a monoclonal antibody reactive with the cancer cell surface, and a cytotoxic agent which is either a bacterial or plant toxin, to specifically attack cancer cells. The immunotoxin B3LysPE38, composed of the modified Pseudomonas aeruginosa exotoxin A (LysPE38) chemically linked to a murine monoclonal antibody (B3), was effective in killing various forms of cancer in mice. To test the therapeutic value in human, a phase I clinical study was designed in which 0.1–1.0 mg of B3LysPE38/Kg of body weight would be administered to each patient. Early estimates of the expected enrolled patient population for this study indicated a need for gram amounts of highly purified immunotoxin. Therefore, a fermentation and purification procedure was developed to isolate 10 grams of LysPE38 (>99% pure) with clearance levels of pyrogen at 10E.U. and bacterial DNA at 6pg per mg of LysPE38. The high density fermentation process was based on an adaptive control strategy and the purification process was composed of ion exchange and hydrophobic interaction column chromatography.
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References
Pastan, I.; FitzGerald, D.: Recombinant toxin for cancer treatment. Science 254 (1991) 1173–1177
Velu, T. J. et al.: Epidermal growth factor-dependent transformation by a human EGF receptor proto-oncogene. Science 238 (1987) 1408–1410
Kawano, M. et al.: Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332 (1988) 83–85
Hellstrom, K. E.; Hellstrom, I.: Oncogene-associated tumor antigens as targets for immunotherapy. FASEB J. 3 (1989) 1715–1722
Hendler, F.J.; Ozanne, B.W.: Human squamous cell lung cancers express increased epidermal growth factor receptors. J. Clin. Invest. 74 (1984) 647–651
Jones, N.R. et al.: Epidermal growth factor receptor expression in 72 meningiomas. Cancer 66 (1990) 152–155
Lau, J.L.T.; Fowler, J. E. J.; Ghosh, L.: Epidermal growth factor in the normal and neoplastic kidney and bladder. J. Urol. 139 (1988) 170–175
Dunn, W. A.; Connolly, T. P.; Hubbard, A. L.: Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. J. Cell Biol. 102 (1986) 24–36
Ghetie, M.-A. et al.: Evaluation of Racin A chain-containing immunotoxins directed against CD19 and CD22 antigens on normal and malignant human B-cells as potential reagent for in vivo therapy. Cancer Res. 48 (1988) 2610–2617
Muraro, R. et al.: Definition by monoclonal antibodies of a repertoire of epitopes on carcinoembryonic antigen differentially expressed in human colon carcinomas versus normal adult tissues. Ibid. 45 (1985) 5769–5780
Krolick, K. A. et al.: In vivo therapy of a murine B cell tumor (BCLI) using antibody-Racin A chain immunotoxins. J. Exp. Med. 155 (1982) 1797–1809
Willingham, M. C.; FitzGerald, D. J.; Pastan, I.: Pseudomonas exotoxin coupled to a monoclonal antibody against ovarian cancer inhibits the growth of human cancer in a mouse model. PNAS U.S.A. 84 (1987) 2474–2478
Batra, J. K. et al.: Antitumor activity in mice of an immunotoxin made with anti-transferrin receptor and a recombinant form of Pseudomonas exotoxin. Ibid. 86 (1989) 8545–8549
Pai, L. H. et al.: Antitumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin. Ibid. 88 (1991) 3358–3362
Brinkman, U. et al.: B3 (Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice. Ibid. 88 (1991) 8616–8620
Allured, V. S. et al.: Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Ibid. 83 (1986) 1320–1324
Hwang, J. et al.: Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell 48 (1987) 129–136
Siegall, C. B. et al.: Functional analysis of domains II, Ib and III of Pseudomonas exotoxin. J. Biol. Chem. 264 (1989) 14256–14261
Chung, D. W.: Collier, R. J.: Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect. Immun. 16 (1977) 832–841
Sambrook, J.; Fritsch, E. F.; Maniatis, T.: Molecular Cloning, A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press 1989
Debinski, W.; Pastan, I.: An immunotoxin with increase activity and homogenicity produced by reducing the number of lysine residues in recombinant pseudomonas exotoxin. Bioconjug. Chem. 5 (1994) 40–46
Studier, F. W. et al.: Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185 (1990) 60–89
Fass, R. et al.: Use of high density culture of Escherichia coli for high level production of recombinant Pseudomonas aeruginosa exotoxin A. Appl. Microbiol. Biotechnol. 36 (1991) 65–69
Hsiao, J. et al.: Adaptive control strategy for maintaining dissolved oxygen concentration in high density growth of recombinant E. coli. Ann. N.Y. Acad. Sci. 665 (1992) 320–333
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Tsai, A., Gallo, M., Petterson, T. et al. Large-scale production and purification of clinical grade pseudomonas aeruginosa exotoxin A from E. coli. Bioprocess Engineering 12, 115–118 (1995). https://doi.org/10.1007/BF00369587
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DOI: https://doi.org/10.1007/BF00369587