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Reducing immunotoxin immunogenicity


Figure 1(C) shows the location of the epitopes, some of them discontinuous and separated by more than 50 amino acids.


Mutation of the immunogenic epitopes of PE38


Once the seven epitopes were identified, the next step was to evaluate their activity and combine them into a single molecule. Moxetumomab pasudotox, an affinity-optimized anti-CD22 recombinant immuno- toxin previously known as HA22 or CAT-8015, was chosen for these experiments. Mutation of key residues within each epitope to alanine, serine, or glycine produced seven point mutants, each with490% cytotoxic activity: R313A, Q332S, R432G, R467A, R490A, R513A, and K590S [7]. These mutants corresponded to epitopes 3, 1, 4, 2, 5, 6, and 7, respectively. An additional point mutant, E548A, also part of epitope 6, was 17% active. However, when all eight of these mutations were combined in a single molecule, cytotoxic activity was preserved at *100% [7]. This mutant, termed


HA22-8X, was tested against a human CD22þBur- kitt lymphoma CA46 subcutaneous xenograft model, and found to have antitumor activity similar to that of moxetumomab pasudotox. Additional experiments observing the generation of anti-PE antibodies in mice dosed with moxetumomab pasudotox or HA22-8X demonstrated that HA22-8X was signifi- cantly less immunogenic than moxetumomab pasu- dotox [7].


Removal of multiple epitopes by domain deletion


Concurrent with research identifying and eliminating epitopes in PE38, experiments were also investigat- ing the susceptibility of recombinant immunotoxins to lysosomal protease digestion. Research suggests that only a small number of toxin molecules reach the cytosol, and a significant proportion of internalized recombinant immunotoxins traffic unproductively into lysosomes for degradation. If lysosomal degra- dation of recombinant immunotoxins could be minimized, it might be possible to increase the proportion of recombinant immunotoxins that reach the cytosol. Multiple protease cleavage sites were identified in domains II and Ib, and targeted for removal [8]. To determine whether the sites could be removed en bloc, the entire region, comprised of residues 251–394, was removed from moxetumomab pasudotox and replaced only by amino acids 274– 284, which includes the furin cleavage site between Arg279 and Gly280. This strategy has the additional benefit of removing immunogenic epitopes along with protease cleavage sites. The resulting mutant,


89


HA22-LR, had between 22 and 212% of the cytotoxic activity of moxetumomab pasudotox on six different cell lines, with a median relative cytotoxic activity of 102% [8]. When tested on freshly obtained primary chronic lymphocytic leuke- mia (CLL) cells, however, HA22-LR was more cytotoxic than moxetumomab pasudotox by a median of 416.8-fold [8]. A possible explanation is that in CLL cells, lysosomal proteases are a key impediment for cytotoxic activity, which is circum- vented by recombinant immunotoxins lacking pro- tease sites. Alternatively, it is possible that the molecule, because of its smaller size, translocates more efficiently into the cytosol. Another surprising finding from this work was the lack of non-specific toxicity of HA22-LR in mice. Compared to mox- etumomab pasudotox, which was 100% lethal to mice at 2 mg/kg, HA22-LR failed to kill any of 10 mice at 20 mg/kg [8]. Antitumor studies in SCID (severe combined immunodeficiency) mice bearing CA46 xenograft tumors confirmed that HA22-LR was much more effective than moxetumomab pasu- dotox in inducing complete regressions, due to higher doses allowed by its low toxicity.


Summary


In summary, several immunogenic epitopes have been identified on the surface of PE38 that are thought to be targets for neutralizing antibodies. Two methods that have reduced the immunogenicity of recombinant immunotoxins include point muta- tions and large deletions of epitope-containing regions. The latter method yielded particularly exciting results in view of the greatly increased cytotoxicity toward CLL cells and the greatly diminished animal toxicity, although it raises sig- nificant questions about the function of domain II in PE. Further work is proceeding to combine both of these methods to obtain optimally active and non- immunogenic recombinant immunotoxins [9]. Such agents would be particularly appropriate for targeting solid tumors, where immunogenicity is a major obstacle to successful therapy with recombinant immunotoxins.


Acknowledgement


This work was supported in part by the National Cancer Institute, Intramural Program, and MedIm- mune, LLC.


Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.


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