Identification and Elimination of an Immunodominant T-Cell Epitope in Recombinant Immunotoxins Based on Pseudomonas Exotoxin A
Total Page：16
File Type：pdf, Size：1020Kb
Identification and elimination of an immunodominant PNAS PLUS T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A Ronit Mazora,b, Aaron N. Vassalla,1, Jaime A. Eberlea,2, Richard Beersa, John E. Weldona, David J. Venzonc, Kwong Y. Tsangd, Itai Benharb, and Ira Pastana,3 aLaboratory of Molecular Biology, cBiostatistics and Data Management Section, Center for Cancer Research, and dLaboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and bDepartment of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel Contributed by Ira Pastan, October 17, 2012 (sent for review August 9, 2012) Recombinant immunotoxins (RITs) are chimeric proteins that are RITs to be given, we initially focused on identifying and re- being developed for cancer treatment. We have produced RITs moving B-cell epitopes. We initially identified the B-cell epitopes that contain PE38, a portion of the bacterial protein Pseudomonas in PE38 that are responsible for the mouse immune response exotoxin A. Because the toxin is bacterial, it often induces neutral- and used this information to make mutant immunotoxins that izing antibodies, which limit the number of treatment cycles and can be given to mice for many cycles without inducing antibody the effectiveness of the therapy. Because T cells are essential for production (11, 12). We have extended these studies to identify antibody responses to proteins, we adopted an assay to map the and remove human B-cell epitopes (13). T cells play a pivotal CD4+ T-cell epitopes in PE38. We incubated peripheral blood mono- role in the ability to elicit an antibody response. One of the early nuclear cells with an immunotoxin to stimulate T-cell expansion, events in the development of antibodies is the antigen-specific followed by exposure to overlapping peptide fragments of PE38 activation of CD4+ T-helper cells (14). CD4+ T-cell support is and an IL-2 ELISpot assay to measure responses. Our observation of initiated by antigen-presenting cells (APCs), which display peptide T-cell responses in 50 of 50 individuals correlates with the frequency fragments derived from foreign proteins on MHC class II mole- of antibody formation in patients with normal immune systems. We cules that bind T-cell receptors (14, 15). Several studies have found a single, highly immunodominant epitope in 46% (23/50) of identified human-specific T-cell epitopes in therapeutic proteins the donors. The immunodominant epitope is DRB1-restricted and (16–18), and in some cases proteins were produced by mutating was observed in subjects with different HLA alleles, indicating pro- amino acids within the protein and were shown to be less immu- miscuity. We identified two amino acids that, when deleted or mu- nogenic using mouse models (19–22). tated to alanine, eliminated the immunodominant epitope, and we The goal of the current study was to identify and remove human used this information to construct mutant RITs that are highly cy- T-cell epitopes in immunotoxins containing PE38. Depending on totoxic and do not stimulate T-cell responses in many donors. the type of assay used, it is possible to identify peptides that result in T-cell activation, but these peptides might never be deimmunization | immunogenicity | protein engineering | formed in vivo. To ensure that the epitopes we identified were antidrug antibodies naturally produced by APCs, we adapted an assay developed by Sette and colleagues (23) in which we first incubated donor pe- ecombinant immunotoxins (RITs) are chimeric proteins that ripheral blood mononuclear cells (PBMCs) with RITs for 14 d to Rare being developed for the targeted therapy of cancer. Our allow the immunotoxin to be processed by donor APCs and rel- laboratory has produced RITs composed of the variable frag- evant peptides to be presented to T cells. We subsequently ex- ment (Fv) of an antibody specific for a tumor-associated cell- posed the activated T cells to overlapping synthetic peptides from MEDICAL SCIENCES surface antigen, joined to a 38-kDa fragment of Pseudomonas the sequence of PE38 and used an ELISpot assay for IL-2 to exotoxin A (PE38) that kills the target cell (1). We are currently measure T-cell activation. We analyzed samples from 50 normal developing RITs that target CD22 for B-cell malignancies donors with no recorded previous exposure to PE38 and with (HA22, also known as “Moxetumomab Pasudotox”)andmes- a broad distribution of HLA alleles and found that all donors othelin for mesothelioma and other epithelial malignancies (SS1P). showed a significant response to at least one peptide, as would be In a recently completed phase 1 trial in refractory hairy cell expected from a highly immunogenic foreign protein. We also leukemia, HA22 had a response rate of 86%, with 46% complete found one immunodominant epitope that was HLA class II remissions (2). HA22 also has produced complete remissions in DRB1-restricted and promiscuous because of the diversity of several children with acute lymphoblastic leukemia (3). Although donors that responded to it. Using alanine-scanning mutagenesis PE38 is a bacterial protein, HA22 does not frequently induce the we identified single amino acids within PE38 that were re- formation of neutralizing antibodies in patients with hematologi- sponsible for the epitope and constructed highly active mutant cal malignancies, because their immune systems are suppressed by chemotherapy and by the malignant cells, which proliferate in the bone marrow. This suppression usually allowed HA22 to be Author contributions: R.M., A.N.V., K.Y.T., and I.P. designed research; R.M., A.N.V., J.A.E., given for many cycles, contributing to the high response rate (4). and R.B. performed research; R.M., J.E.W., D.J.V., I.B., and I.P. analyzed data; and R.M. and In contrast, the response rate to SS1P was much lower in patients I.P. wrote the paper. with mesothelioma, who have normal immune systems that rapidly The authors declare no conflict of interest. produce antibodies to PE38. Therefore, most patients only re- 1Present address: Yale Medical School, New Haven, CT 06511. ceived a single cycle of treatment (4, 5). 2Present address: Columbia University Medical Center, College of Physicians and Sur- The formation of neutralizing antibodies is a common occur- geons, New York, NY 10032. rence when foreign proteins are used as therapeutic agents in 3To whom correspondence should be addressed. E-mail: [email protected]. humans (6, 7), and the more foreign the protein, the more likely See Author Summary on page 20790 (volume 109, number 51). – it is that a rapid immune response will be generated (8 10). To This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. deimmunize PE38 and thus allow more treatment cycles with 1073/pnas.1218138109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1218138109 PNAS | Published online December 3, 2012 | E3597–E3603 Downloaded by guest on September 28, 2021 RITs targeting CD22 in which the T-cell response was abolished in 34% of donors and diminished in an additional 42%. Results The structure of an RIT is shown in Fig. 1A. It is composed of an Fv fused to a 38-kDa portion of PE38. The Fv binds to the cancer cell and brings PE38 into the cell. PE38 is made up of two domains; domain II (amino acids 253–364) carries out toxin processing, and domain III (amino acids 395–613) contains the ADP ribosylating activity. Amino acids 365–394 are not needed for activity and are not present in PE38. Because of the bacterial origin of PE38, we focused on that portion of the RIT. Identification of T-Cell Epitopes. To identify T-cell epitopes in the PE38, we used an assay originally developed to identify peptides responsible for human T-cell responses to a hay fever allergen (23). We stimulated PBMCs from 50 donors with intact RIT, followed by restimulation with 111 15-mer overlapping peptides spanning the entire sequence of PE38 (Table S1) and an ELI- Spot assay for IL-2 to measure T-cell response (Fig. S1). Fig. 2A shows an example of a screen of pools for one of the 50 donors. Only a single pool (pool 3) had a response that met the three criteria we established for a positive response [≥85 spot- forming cells (SFCs) per 106 cells, more than three times the number in the negative control, and ≥10% of all of the spots for that donor]. The response to pool 14 fulfilled two of the three criteria (the response was ≥85 SFC per 106 cells and was greater than three times that of the negative control); however, the spots made up only 8% of the total spots, and therefore the response not considered positive. We subsequently performed a fine screen 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. of pool 3 with the individual peptides (Fig. 2B) and found that only peptides 14 and 15 gave positive responses. Pool 3 contains an immunodominant epitope. We screened a total of 50 donors and found that all 50 gave positive responses, as would be expected for a highly immunogenic protein such as PE38 (4). Fig. 3A presents the positive and negative responses of the 50 donors in a heatmap format. The strongest positive re- sponse for each donor is shown in black, weaker responses in gray, and the absence of a response in white.