This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bang, S. Articles by Pastan, I. Search for Related Content PubMed PubMed Citation Articles by Bang, S. Articles by Pastan, I.

HA22 (R490A) Is a Recombinant Immunotoxin with Increased Antitumor Activity without an Increase in Animal Toxicity

Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland

Requests for reprints: Ira Pastan, Laboratory of Molecular Biology, National Cancer Institute, NIH, Building 37, Room 5106, 37 Convent Drive, MSC 4264, Bethesda, MD 20892-4264. Phone: 301-496-4797; Fax: 301-402-1344; E-mail: pastani{at}mail.nih.gov.

Purpose: RFB4 (dsFv)-PE38 (BL22) is a recombinant immunotoxin containing an anti-CD22 (Fv) fused to truncated Pseudomonas exotoxin A, which induces a high complete remission rate in patients with purine analogue–resistant hairy cell leukemia. HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. Our goal was to improve the activity of HA22.

Experimental Design: Arg⁴⁹⁰, which is located in the catalytic domain (III) of the immunotoxin HA22, was mutated to alanine. Purified immunotoxins were produced and tested for cytotoxic activity in cell culture and for antitumor activity and nonspecific toxicity in mice. ADP-ribosylation activity was also measured.

Results: HA22 (R490A) is 2-fold more cytotoxic than HA22 on several CD22-positive cell lines. When injected i.v., HA22 (R490A) has more potent antitumor activity than HA22 against CA46 tumors in mice. HA22 and HA22 (R490A) have similar LD₅₀s (1.3 mg/kg) and similar plasma half-lives. The R490A mutation also improved the cytotoxicity of the antimesothelin recombinant immunotoxin SS1 (dsFv)-PE38 (SS1P). In vitro ADP-ribosylation assays show that HA22 R490A has increased activity. Increased cytotoxic activity is probably related to this increase in ADP-ribosylation activity.

Conclusion: Protein engineering can be used to increase the efficacy of recombinant immunotoxins. Because HA22 (R490A) has increased antitumor activity without increased animal toxicity, immunotoxins with this mutation are candidates for clinical development.

Key Words: Cytotoxicity • Pharmacokinetics • ADP-ribosylation

This article has been cited by other articles:

				J. E. Weldon, L. Xiang, O. Chertov, I. Margulies, R. J. Kreitman, D. J. FitzGerald, and I. Pastan A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity Blood, April 16, 2009; 113(16): 3792 - 3800. [Abstract] [Full Text] [PDF]

				R. F. Alderson, R. J. Kreitman, T. Chen, P. Yeung, R. Herbst, J. A. Fox, and I. Pastan CAT-8015: A Second-Generation Pseudomonas Exotoxin A-Based Immunotherapy Targeting CD22-Expressing Hematologic Malignancies Clin. Cancer Res., February 1, 2009; 15(3): 832 - 839. [Abstract] [Full Text] [PDF]

				K. Matsushita, I. Margulies, M. Onda, S. Nagata, M. Stetler-Stevenson, and R. J. Kreitman Soluble CD22 as a tumor marker for hairy cell leukemia Blood, September 15, 2008; 112(6): 2272 - 2277. [Abstract] [Full Text] [PDF]

				M. Onda, S. Nagata, D. J. FitzGerald, R. Beers, R. J. Fisher, J. J. Vincent, B. Lee, M. Nakamura, J. Hwang, R. J. Kreitman, et al. Characterization of the B Cell Epitopes Associated with a Truncated Form of Pseudomonas Exotoxin (PE38) Used to Make Immunotoxins for the Treatment of Cancer Patients J. Immunol., December 15, 2006; 177(12): 8822 - 8834. [Abstract] [Full Text] [PDF]

				Z. Li, S. P. Mahesh, D. F. Shen, B. Liu, W. O. Siu, F. S. Hwang, Q.-C. Wang, C.-C. Chan, I. Pastan, and R. B. Nussenblatt Eradication of Tumor Colonization and Invasion by a B Cell-Specific Immunotoxin in a Murine Model for Human Primary Intraocular Lymphoma Cancer Res., November 1, 2006; 66(21): 10586 - 10593. [Abstract] [Full Text] [PDF]

				Y. Zhang, L. Xiang, R. Hassan, C. H. Paik, J. A. Carrasquillo, B.-s. Jang, N. Le, M. Ho, and I. Pastan Synergistic Antitumor Activity of Taxol and Immunotoxin SS1P in Tumor-Bearing Mice Clin. Cancer Res., August 1, 2006; 12(15): 4695 - 4701. [Abstract] [Full Text] [PDF]