Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A

Abstract

Recombinant immunotoxins (RITs) are chimeric proteins that are being developed for cancer treatment. We have produced RITs that contain PE38, a portion of the bacterial protein Pseudomonas exotoxin A. Because the toxin is bacterial, it often induces neutralizing antibodies, which limit the number of treatment cycles and the effectiveness of the therapy. Because T cells are essential for antibody responses to proteins, we adopted an assay to map the CD4(+) T-cell epitopes in PE38. We incubated peripheral blood mononuclear cells with an immunotoxin to stimulate T-cell expansion, followed by exposure to overlapping peptide fragments of PE38 and an IL-2 ELISpot assay to measure responses. Our observation of T-cell responses in 50 of 50 individuals correlates with the frequency of antibody formation in patients with normal immune systems. We found a single, highly immunodominant epitope in 46% (23/50) of the donors. The immunodominant epitope is DRB1-restricted and was observed in subjects with different HLA alleles, indicating promiscuity. We identified two amino acids that, when deleted or mutated to alanine, eliminated the immunodominant epitope, and we used this information to construct mutant RITs that are highly cytotoxic and do not stimulate T-cell responses in many donors.

Fig. 1.

Structural model of RIT variants. (A) Structural model of Moxetumomab Pasudotox. The V_L is in cyan, and the V_H is in magenta. Domain II of the toxin is in gray, and domain III is in yellow. (B) HA22-LR. The linker containing the furin cleavage sequences is in gray. Image courtesy of Byungkook Lee (National Cancer Institute, NIH, Bethesda, MD).

Fig. 2.

Representative pattern of CD4⁺ T-cell IL-2 secretion in response to PE38-derived peptides in a single donor. (A) IL-2 secretion in response to stimulation with 22 peptide pools from PE38. (B) IL-2 response to the five peptides that comprise pool 3. (C) IL-2 response of isolated CD4⁺ T cells to the five peptides that comprise pool 3.

Fig. 3.

CD4⁺ T-cell IL-2 secretion in response to PE38-derived peptide pools. Samples from 50 normal donors were stimulated with PE38-containing immunotoxin for 14 d and were restimulated with the overlapping peptides pools. Each pool was tested in quadruplicate, and SFC/1E6 cells were calculated. (A) Visual illustration of the strongest (black), weak (gray), and negative (white) responses. Donors were clustered using an automatic sorting based on the responsiveness of the pools. (B) Relative responses to 22 pools (n = 50).

Fig. 4.

CD4⁺ T-cell response to single peptides that compose pool 3 and HLA II restriction. (A) Response to overlapping peptides that compose pool 3 (n = 23). (B) HLA class II restriction. Expanded PBMC were incubated with antibodies against HLA DR, DP, and DQ, a combination of the three (All), or class I (Pan). The responses to stimulation with peptide 15 were compared with the positive control (peptide 15 with no antibody), and the relative response was calculated for each donor (n = 9).

Fig. 5.

Purity and activity of mutant immunotoxins. (A) Tris⋅glycine gel (4–20%) run under nonreducing conditions shows the purity of the variant immunotoxins. Lane 1, HA22-L297A; lane 2, HA22-Y298A; lane 4, WT HA22; lane 5, HA22-LR. (B) Representative WST8 cytotoxicity assay in a CD22⁺ cell line (Daudi) after 3-d incubation with either HA22 or mutant immunotoxins.

Fig. 6.

Mutant immunotoxins do not create new T-cell epitopes. Representative responses to 22 peptide pools after stimulation with (A) HA22, (B) HA22-LR, (C) HA22-L297A, or (D) HA22-Y298A and restimulation with appropriate mutant peptides.

Fig. P1.

Mutation or deletion of T-cell epitopes produces highly cytotoxic RITs that do not activate T cells. (Left) Structural models of RITs with V_L (cyan) and V_H (magenta) domains are shown on the left. Domain II of the toxin is in gray, and domain III is in yellow. (Top Left) HA22 (Moxetumomab Pasudotox). (Middle Left) HA22-L297A with the epitope (red) and mutation L297A (blue). (Bottom Left) HA22-LR with deletion of almost all of domain II. (Center) Cytotoxic activity curves indicating the protein potency obtained using CD22⁺ cells (Daudi) with HA22 (black line, Top Center), HA22-L297A (blue line, Middle Center), and HA22-LR (red line, Bottom Center). (Right) Results of T-cell activation obtained by IL-2 ELISpot assays showing stimulation by HA22 and very little stimulation by HA22-L297A, HA22-LR, or no peptide (control). Images (Left) courtesy of Byungkook Lee (National Cancer Institute, NIH, Bethesda, MD).