An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes

Abstract

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

“…Fifteen of the 56 strongly binding phage did not react with HA22-LR, indicating that the epitopes recognized by these Fvs are located on domain II. We used the remaining 41 phage to identify the residues that make up the B-cell epitopes in domain III by measuring their binding to 36 different mutant proteins in which individual amino acids on the surface of domain III were mutated from a large polar amino acid to alanine or glycine, as previously described (14,15). The results are shown in Fig.…”

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

“…One important issue is whether the mouse is a valid model for patient anti-toxin responses. Onda and Pastan have tested sera from patients receiving non-mutated PE38-targeted toxins and found that their anti-PE38 antibodies bind to the same seven B-cell epitopes defined by the mouse studies and eliminated in EGF4KDEL 7mut (Onda et al, 2008). This implies that the mouse model correlates directly to the human antibody response and is highly useful for immunogenicity studies.…”

“…To address this issue, Onda and Pastan used serum samples from patients receiving a BL22, a PE38-containing anti-leukemia agent, to map out regions of the molecule that elicited the strongest antibody response . Upon identifying seven major reactive epitopes, they proved that mutations could be made to these regions without compromising tumour cell killing (Onda et al, 2008). Thus, we eliminated the same seven epitopes on our EGF4KDEL molecule using site-directed mutagenesis.…”

“…DNA sequencing analysis (Biomedical Genomics Center, University of Minnesota) was used to verify that the gene was correct in sequence and had been cloned in frame. To create an EGF4KDEL molecule with decreased immunogenicity, eight amino acids representing the seven major epitopes on PE38 (Onda et al, 2008) were mutated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene. La Jolla CA, USA).…”

“…A potential solution to this problem was recently published by Onda and Pastan who showed that there are only seven major B-cell epitopes on the PE38 molecule ). Subsequently, they described a series of mutations that did not alter the catalytic activity of PE38 yet nearly eliminated the immune response of mice (Onda et al, 2008). In these studies, they show that the mouse is a valid model of antitoxin immunogenicity.…”