A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity

Abstract

Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins. (Blood. 2009; 113:3792-3800) IntroductionMonoclonal antibodies, either alone or as immunoconjugates linked to other agents, have become valuable therapies for the targeted treatment of cancer. In recent years, several antibodybased therapies have progressed through regulatory approval by the Food and Drug Administration, and it is expected that many more will follow. 1 Immunotoxins are a category of immunoconjugate in which antibodies are joined to protein toxins. They exploit the precision of antibodies and the lethality of protein toxins to target and kill cancer cells expressing specific cell surface proteins. Any tumor-associated cell-surface antigen is a potential target for immunotoxins.A variety of plant, fungal, and bacterial toxins have been adapted for use with immunotoxins, including ricin, diphtheria toxin, and Pseudomonas exotoxin A (PE). 2,3 PE-based immunotoxins are currently in clinical trials for the treatment of CD22-expressing lymphomas and leukemias, as well as mesothelinexpressing solid tumors. 4,5 A phase 1 trial of the anti-CD22 PE immunotoxin BL22 had a high overall response rate of 81% but was particularly effective against drug-resistant hairy cell leukemia (HCL). 6 A phase 1 trial of the anti-CD25 PE immunotoxin LMB-2 showed a 23% response rate in patients with hematologic malignancies refractory to standard chemotherapy. 7 A phase 1 trial of the antimesothelin PE immunotoxin SS1P demonstrated minor but encouraging responses for treating solid tumors in patients with mesothelioma or ovarian cancer who had failed standard therapies....

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

“…The direct genetic modification used in this study eliminates the additional step of PEGylation and retains the full cytotoxic capability of parental molecule. Other studies have shown that using genetic engineering to mutate T-cell-specific epitopes and the proteolytic cleavage site can improve the pharmacokinetic profile and in vivo efficacy of the biological drug (Tangri et al, 2005;Weldon et al, 2008). Additional studies may show that incorporation of similar mutations into EGF4KDEL 7Mut can further enhance its already potent antitumour activity.…”

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes

“…HA22-LR (molecular weight 50.5 kDa) is smaller than HA22 (molecular weight 63 kDa) and consequently has a shorter half-life in mice (15 vs. 8 min) (17). To assess the half-life of HA22-LR-LO10, BALB/c mice were injected with 10 μg of either HA22-LR or HA22-LR-LO10 and bled at intervals between 2 and 60 min.…”

“…To assess the half-life of HA22-LR-LO10, BALB/c mice were injected with 10 μg of either HA22-LR or HA22-LR-LO10 and bled at intervals between 2 and 60 min. We measured the concentration of RIT in mice sera by ELISA (14,17). Data were fit to a single exponential decay function.…”

Functional analysis of a survivin-like gene in Bombyx mori

“…Bm-svv was then expressed in non-permissive or permissive cells to determine its effect on cell activity using the Cell Counting Kit-8 (CCK-8), which has been frequently employed in mammalian cells (Weldon et al 2009;Ding et al 2010;Kunz et al 2009;Tamura et al 2010), and found that this method is also useful for the determination of insect cell activity.…”