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: Arg(490), 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 approximately 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 LD(50)s (approximately 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.
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:Arg(490), 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 approximately 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 LD(50)s (approximately 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.
Authors: Waleed Haso; Daniel W Lee; Nirali N Shah; Maryalice Stetler-Stevenson; Constance M Yuan; Ira H Pastan; Dimiter S Dimitrov; Richard A Morgan; David J FitzGerald; David M Barrett; Alan S Wayne; Crystal L Mackall; Rimas J Orentas Journal: Blood Date: 2012-12-14 Impact factor: 22.113
Authors: Alisa B Lee-Sherick; Rachel M A Linger; Lia Gore; Amy K Keating; Douglas K Graham Journal: Br J Haematol Date: 2010-08-31 Impact factor: 6.998
Authors: Zhuqing Li; Sankaranarayana P Mahesh; De Fen Shen; Baoying Liu; Willie O Siu; Frank S Hwang; Qing-Chen Wang; Chi-Chao Chan; Ira Pastan; Robert B Nussenblatt Journal: Cancer Res Date: 2006-11-01 Impact factor: 12.701