Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes

Abstract

Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.deimmunization | protein engineering I mmunotoxins are chimeric proteins that combine the "magic bullet" specificity of an antibody with the high potency of a toxin. The high specificity of recombinant immunotoxins (RITs) leads to a dramatic decrease in side effects compared with chemotherapy. Moxetumomab Pasudotox (MP) is an RIT that consists of PE38, a fragment of Pseudomonas exotoxin A, fused to an anti-CD22 Fv (1). In a phase I trial for refractory hairy-cell leukemia (HCL), MP had an 86% response rate (2), with 46% complete remissions, and is now in phase III clinical trials (3).Immunogenicity is a stumbling block in the clinical success of many therapeutic proteins (4). Formation of neutralizing antidrug antibodies (5) inactivates the therapeutic agent and can cause serious adverse effects. Although MP had low immunogenicity in the immune-suppressed patients of the HCL trial, some patients did eventually develop antibodies. Consequently, fewer doses could be given to these patients, leading to a reduced response rate. Additionally, RITs targeting solid tumors are less effective than MP because of their high immunogenicity in patients with normal immune systems (6, 7).The role of helper T cells in mounting an immune response is well-established (8, 9). It was previously shown that elimination of murine T-cell epitopes reduced neutralizing antibody formation in mice (10), leading us to the hypothesis that reduction of human T-cell epitopes in the bacterial moiety of RITs would diminish its immunogenicity in humans, allowing more treatment cycles and better antitumor responses, as previously attempted for other therapeutic proteins like erythropoietin (11).To circumvent the immunogenicity of PE38, we previously used peptide pools to map the approximate location of the T-cell epitopes and found an immunodominant and promiscuous epitope that stimulated T cells in 42% of all donors (12). Here, we have done high-resolution mapping of the epitopes ...

In Vitro Evaluation of Humanized/De-immunized Anti-PSMA Immunotoxins for the Treatment of Prostate Cancer

“…Mazor et al described that deletion of domain II can silence the immunodominant B-and T-cell epitopes, and thereby reduce the immunogenicity of the toxin domain and diminish the production of ADA (11,35). Furthermore, it has been shown that structural and functional changes within the translocation domain II of PE result in enhanced stability (36,37).…”

Targeting a Cancer-Specific Epitope of the Epidermal Growth Factor Receptor in Triple-Negative Breast Cancer

“…However, co-administration of immunotoxins and selective immunosuppressive agents has resulted in statistically significant reductions in antitoxin antibody formation in mice and humans (27,44). Alternately, identification and removal of major B-and T-cell epitopes from the immunotoxin has proven successful at reducing immune responses (45,46). Another limitation centers on the xenograft model itself.…”

Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity

“…Protein immunogenic potential was quantified by measuring the activation of human immune cells from 19 donors (Table S3) using an established protocol (6,30). Briefly, 2 × 10 6 live PBMCs were cultured in 24-well plates using 1 mL of RPMI 1640 medium supplemented with 5% human AB serum and either 5 μg/mL wild-type P99βL or 5 μg/mL 30:50:E9 deimmunized β-lactamase.…”