Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes

Abstract

Many nonhuman proteins have useful pharmacological activities, but are infrequently effective in humans because of their high immunogenicity. A recombinant immunotoxin (HA22, CAT8015, moxetumomab pasudotox) composed of an anti-CD22 antibody variable fragment fused to PE38, a 38-kDa portion of Pseudomonas exotoxin A, has produced many complete remissions in drugresistant hairy-cell leukemia when several cycles of the agent can be given, but has much less activity when antibodies develop. We have pursued a strategy to deimmunize recombinant immunotoxins by identifying and removing B-cell epitopes. We previously reported that we could eliminate most B-cell epitopes using a combination of point mutations and deletions. Here we show the location and amino acid composition of all of the B-cell epitopes in the remaining 25-kDa portion of Pseudomonas exotoxin. Using this information, we eliminated these epitopes to produce an immunotoxin (HA22-LR-

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

“…Our hypothesis that B-cell epitopes are mainly comprised of bulky hydrophilic amino acids was confirmed by the results of this and the previous study (9). In both studies we were able to silence epitopes by mutating such residues to Ala.…”

“…1). HA22-LR-8M has excellent antitumor activity, yet does not induce the formation of antitoxin antibodies when injected repeatedly into mice, and does not induce a recall response in preimmunized mice (9). These results show that, using a mouse model, it is possible to produce an active nonimmunogenic RIT.…”

“…Binding of HA22 or HA22 mutants to antibodies in human sera was measured in a displacement assay as previously described (9). Briefly, human sera were obtained under National Institutes of Health Institutional Review Board-approved protocols.…”

“…We previously described the design and construction of the RIT HA22-LR-8M in which the mouse B-cell epitopes were identified and silenced by mutations (9). To do this, domain II of PE was deleted (except for the furin processing site) and eight point mutations were introduced into domain III to remove large hydrophilic residues (HA22-LR-8M in Fig.…”

Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein

“…85,86 Efforts to increase the circulation time of these agents include the design of fusion proteins in which the immunotoxins are linked to Fabs 87 or to full-length immunoglobulins. 88 The immunogenicity problem is being addressed by identifying and eliminating B cell epitopes using site-directed mutagenesis 89 or by choosing human-derived proteins as the cytotoxic moieties. 90,91 In considering the application of any of these specific cell-killing modalities, treatment of viral infections has the advantage of targeting a viral-encoded antigen, thereby avoiding side-effects frequently encountered in cancer therapy owing to killing of normal cells expressing the targeted human antigen; on the other hand, a single virus-infected cell MCD patients; the results were interpreted as reflecting mainly (in addition to the possible direct anti-KSHV activity of valganciclovir) the ability of the products of the KSHV genes ORF21 (encoding thymidine kinase) and ORF36 (encoding a phosphotransferase) to activate the conversion of zidovudine and ganciclovir, respectively, into cytotoxic compounds.…”