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....

Recombinant immunotoxins are hybrid proteins composed of an Fv that binds to a tumor antigen fused to a bacterial or plant toxin. Immunotoxin BL22 targets CD22 positive malignancies and is composed of an anti-CD22 Fv fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38). BL22 has produced many complete remissions in drug-resistant Hairy cell leukemia, where many treatment cycles can be given, because neutralizing antibodies do not form. In marked contrast, only minor responses have been observed in trials with immunotoxins targeting solid tumors, because only a single treatment cycle can be given before antibodies develop. To allow more treatment cycles and increase efficacy, we have produced a less immunogenic immunotoxin by identifying and eliminating most of the B cell epitopes on PE38. This was accomplished by mutation of specific large hydrophilic amino acids (Arg, Gln, Glu, Lys) to Ala, Ser, or Gly. The new immunotoxin (HA22-8X) is significantly less immunogenic in three strains of mice, yet retains full cytotoxic and anti-tumor activities. Elimination of B-cell epitopes is a promising approach to the production of less immunogenic proteins for therapeutic purposes.antibody engineering ͉ BL22 ͉ HA22 ͉ immunotherapy ͉ Pseudomonas exotoxin A I mmunotoxins (ITs) are hybrid proteins that are composed of a cancer-specific antibody attached to a bacterial or plant toxin (1). Initially ITs were made by chemically coupling toxins to whole antibodies. Now they are made using a combination of antibody and protein engineering (2, 3). ITs kill cells by binding to a cell surface protein, being internalized by endocytosis and eventually reaching the cytosol, where they arrest protein synthesis by inactivating EF2 or ribosomes (4, 5). Our laboratory has developed recombinant immunotoxins (RITs) in which the Fv portion of an antibody is directly fused to a 38-kDa portion of the bacterial toxin Pseudomonas exotoxin A (PE). Three RITs are currently in clinical trials and all three have shown anti-tumor activity in phase 1 trials. LMB-2 [anti-Tac-(Fv)-PE38] targets CD25 expressed on many T cell malignancies and some B cell malignancies (6). BL22 [anti-CD22-(Fv)-PE38] targets CD22 expressed on most B cell malignancies (7), and SS1P antimesothelin-(Fv)-PE38 targets the mesothelin antigen expressed on mesotheliomas and on ovarian, lung, pancreatic, and gastric cancers (8). Because these ITs contain a portion of a bacterial protein, they can induce the formation of neutralizing antibodies, hindering their efficacy. In patients with B-and T-cell malignancies the formation of neutralizing antibodies is infrequent because of the immune-suppressed state of patients with these malignancies (6, 7). However, in patients with solid tumors treated with SS1P and other ITs, antibody formation was very frequently detected 21 days after the first treatment cycle, preventing readministration of the IT (9).Previous studies have shown that the formation of antibodies to foreign proteins can be prevented by coupling the protein to high-mol...

Recombinant immunotoxins (RITs) are hybrid proteins used to treat cancer. These proteins are composed of an Fv that reacts with cancer cells joined to a portion of Pseudomonas exotoxin A, which kills the cell. Because the toxin is a foreign protein, it can induce neutralizing antibodies and thereby limit the number of doses a patient can receive. We previously identified seven major mouse B-cell epitopes in the toxin, and subsequently silenced them using point mutations that converted large hydrophilic amino acids to alanine, yet retained full antitumor activity. Here we present results in which we identify and silence human B-cell epitopes in the RIT HA22. We obtained B cells from patients with antibodies to RITs, isolated the corresponding variable fragments (Fvs), and constructed a phage-display library containing Fvs that bind to the RITs. We then used alanine scanning mutagenesis to locate the epitopes. We found that human and mouse epitopes frequently overlap but are not identical. Most mutations that remove mouse epitopes did not remove human epitopes. Using the epitope information, we constructed a variant immunotoxin, HA22-LR-LO10, which has low reactivity with human antisera, yet has high cytotoxic and antitumor activity and can be given to mice at high doses without excess toxicity. The toxin portion of this RIT (LR-LO10) can be used with Fvs targeting other cancer antigens and is suitable for clinical development.cancer treatment | immunotherapy | leukemia | mesothelioma | protein engineering