Identification and elimination of an immunodominantT-cell epitope in recombinant immunotoxins basedon Pseudomonas exotoxin ARonit 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 andBiology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and bDepartment of Molecular Microbiology and Biotechnology, TheGeorge 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 arebeing developed for cancer treatment. We have produced RITsthat contain PE38, a portion of the bacterial protein Pseudomonasexotoxin A. Because the toxin is bacterial, it often induces neutral-izing antibodies, which limit the number of treatment cycles andthe effectiveness of the therapy. Because T cells are essential forantibody responses to proteins, we adopted an assay to map theCD4+ T-cell epitopes in PE38. We incubated peripheral blood mono-nuclear cells with an immunotoxin to stimulate T-cell expansion,followed by exposure to overlapping peptide fragments of PE38and an IL-2 ELISpot assay to measure responses. Our observation ofT-cell responses in 50 of 50 individuals correlates with the frequencyof antibody formation in patients with normal immune systems. Wefound a single, highly immunodominant epitope in 46% (23/50) ofthe donors. The immunodominant epitope is DRB1-restricted andwas observed in subjects with different HLA alleles, indicating pro-miscuity. We identified two amino acids that, when deleted or mu-tated to alanine, eliminated the immunodominant epitope, and weused this information to construct mutant RITs that are highly cy-totoxic and do not stimulate T-cell responses in many donors.

deimmunization | immunogenicity | protein engineering |antidrug antibodies

Recombinant immunotoxins (RITs) are chimeric proteins thatare being developed for the targeted therapy of cancer. Our

laboratory has produced RITs composed of the variable frag-ment (Fv) of an antibody specific for a tumor-associated cell-surface antigen, joined to a 38-kDa fragment of Pseudomonasexotoxin A (PE38) that kills the target cell (1). We are currentlydeveloping RITs that target CD22 for B-cell malignancies(HA22, also known as “Moxetumomab Pasudotox”) and mes-othelin for mesothelioma and other epithelial malignancies (SS1P).In a recently completed phase 1 trial in refractory hairy cellleukemia, HA22 had a response rate of 86%, with 46% completeremissions (2). HA22 also has produced complete remissions inseveral children with acute lymphoblastic leukemia (3). AlthoughPE38 is a bacterial protein, HA22 does not frequently induce theformation of neutralizing antibodies in patients with hematologi-cal malignancies, because their immune systems are suppressed bychemotherapy and by the malignant cells, which proliferate inthe bone marrow. This suppression usually allowed HA22 to begiven for many cycles, contributing to the high response rate (4).In contrast, the response rate to SS1P was much lower in patientswith mesothelioma, who have normal immune systems that rapidlyproduce antibodies to PE38. Therefore, most patients only re-ceived a single cycle of treatment (4, 5).The formation of neutralizing antibodies is a common occur-

rence when foreign proteins are used as therapeutic agents inhumans (6, 7), and the more foreign the protein, the more likelyit is that a rapid immune response will be generated (8–10). Todeimmunize PE38 and thus allow more treatment cycles with

RITs to be given, we initially focused on identifying and re-moving B-cell epitopes. We initially identified the B-cell epitopesin PE38 that are responsible for the mouse immune responseand used this information to make mutant immunotoxins thatcan be given to mice for many cycles without inducing antibodyproduction (11, 12). We have extended these studies to identifyand remove human B-cell epitopes (13). T cells play a pivotalrole in the ability to elicit an antibody response. One of the earlyevents in the development of antibodies is the antigen-specificactivation of CD4+ T-helper cells (14). CD4+ T-cell support isinitiated by antigen-presenting cells (APCs), which display peptidefragments derived from foreign proteins on MHC class II mole-cules that bind T-cell receptors (14, 15). Several studies haveidentified human-specific T-cell epitopes in therapeutic proteins(16–18), and in some cases proteins were produced by mutatingamino acids within the protein and were shown to be less immu-nogenic using mouse models (19–22).The goal of the current study was to identify and remove human

T-cell epitopes in immunotoxins containing PE38. Depending onthe type of assay used, it is possible to identify peptides thatresult in T-cell activation, but these peptides might never beformed in vivo. To ensure that the epitopes we identified werenaturally produced by APCs, we adapted an assay developed bySette and colleagues (23) in which we first incubated donor pe-ripheral blood mononuclear cells (PBMCs) with RITs for 14 d toallow the immunotoxin to be processed by donor APCs and rel-evant peptides to be presented to T cells. We subsequently ex-posed the activated T cells to overlapping synthetic peptides fromthe sequence of PE38 and used an ELISpot assay for IL-2 tomeasure T-cell activation. We analyzed samples from 50 normaldonors with no recorded previous exposure to PE38 and witha broad distribution of HLA alleles and found that all donorsshowed a significant response to at least one peptide, as would beexpected from a highly immunogenic foreign protein. We alsofound one immunodominant epitope that was HLA class IIDRB1-restricted and promiscuous because of the diversity ofdonors that responded to it. Using alanine-scanning mutagenesiswe identified single amino acids within PE38 that were re-sponsible for the epitope and constructed highly active mutant

Author contributions: R.M., A.N.V., K.Y.T., and I.P. designed research; R.M., A.N.V., J.A.E.,and R.B. performed research; R.M., J.E.W., D.J.V., I.B., and I.P. analyzed data; and R.M. andI.P. wrote the paper.

The authors declare no conflict of interest.1Present address: Yale Medical School, New Haven, CT 06511.2Present address: Columbia University Medical Center, College of Physicians and Sur-geons, New York, NY 10032.

3To whom correspondence should be addressed. E-mail: pastani@mail.nih.gov.

See Author Summary on page 20790 (volume 109, number 51).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218138109/-/DCSupplemental.

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RITs targeting CD22 in which the T-cell response was abolishedin 34% of donors and diminished in an additional 42%.

ResultsThe structure of an RIT is shown in Fig. 1A. It is composed of anFv fused to a 38-kDa portion of PE38. The Fv binds to the cancercell and brings PE38 into the cell. PE38 is made up of twodomains; domain II (amino acids 253–364) carries out toxinprocessing, and domain III (amino acids 395–613) contains theADP ribosylating activity. Amino acids 365–394 are not neededfor activity and are not present in PE38. Because of the bacterialorigin of PE38, we focused on that portion of the RIT.

Identification of T-Cell Epitopes. To identify T-cell epitopes in thePE38, we used an assay originally developed to identify peptidesresponsible 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 peptidesspanning 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 thethree criteria we established for a positive response [≥85 spot-forming cells (SFCs) per 106 cells, more than three times thenumber in the negative control, and ≥10% of all of the spots forthat donor]. The response to pool 14 fulfilled two of the threecriteria (the response was ≥85 SFC per 106 cells and was greaterthan three times that of the negative control); however, the spotsmade up only 8% of the total spots, and therefore the responsenot considered positive. We subsequently performed a fine screen

of pool 3 with the individual peptides (Fig. 2B) and found thatonly peptides 14 and 15 gave positive responses.

Pool 3 contains an immunodominant epitope. We screened a totalof 50 donors and found that all 50 gave positive responses, aswould be expected for a highly immunogenic protein such asPE38 (4). Fig. 3A presents the positive and negative responses ofthe 50 donors in a heatmap format. The strongest positive re-sponse for each donor is shown in black, weaker responses ingray, and the absence of a response in white. We found 108positive responses and 992 negative responses (white). Pools 4, 5,13, 21, and 22 gave no responses. In 16 of the 50 donors a singlepool induced a response. Fifteen donors had two responses, 14donors had three responses, and 5 donors had four responses.Fig. 3A also shows that pool 3 produced the most responses (23of 50), and for nine of these donors it was the only pool thatinduced a response. We also found that the second most fre-quent responses were in pool 16 in domain III, where 16 of 50donors responded.Fig. 3B summarizes the responses of the 50 donors to stimu-

lation by the 22 pools. As previously shown (24, 25), individualdonors responded variably; to correct for these variations, thevalues were normalized to the total number of spots for eachdonor. Pool 3 elicited a positive response in 23 donors, which isextraordinarily strong. The pool 3 responses were statisticallygreater than all other pools (P < 0.0001 in Friedman’s test).Out of the entire screen for all of the donors and all of the

Fig. 1. Structural model of RIT variants. (A) Structural model of Mox-etumomab Pasudotox. The VL is in cyan, and the VH is in magenta. Domain IIof the toxin is in gray, and domain III is in yellow. (B) HA22-LR. The linkercontaining the furin cleavage sequences is in gray. Image courtesy ofByungkook Lee (National Cancer Institute, NIH, Bethesda, MD).

Fig. 2. Representative pattern of CD4+ T-cell IL-2 secretion in response toPE38-derived peptides in a single donor. (A) IL-2 secretion in response tostimulation with 22 peptide pools from PE38. (B) IL-2 response to the fivepeptides that comprise pool 3. (C) IL-2 response of isolated CD4+ T cells tothe five peptides that comprise pool 3.

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pools (106,998 SFC per106 cells), pool 3 in domain II had morethan 21% of all of the spots in the screen (22,850 SFC per106

cells), and pool 16 in domain III produced the second largestnumber of responses (8.2% of all of the spots in the screen).To identify individual peptides within pool 3 that caused a

response in 23 donors, samples were screened with individualpeptides (Fig. 4A). Peptides 14 and 15 (which have a core sequenceof LVALYLAARLSW) had a significantly stronger response thanthe other peptides in pool 3 (P < 0.0001, Mann–Whitney–Wilcoxontest). This epitope does not correspond to any human B-cellepitopes (13).In addition to a fine screen of the peptides in pool 3, we iden-

tified the HLA-DRB1 allotypes of the donors. The samples usedfor the fine screen of pool 3 contained donors with many differentHLA-DRB1 allotypes, including homozygosity for DRB1_04 and

DRB1_15 (Table S2), indicating that the two immunodominantpeptides comprise a promiscuous epitope.

Response is CD4+ T-cell specific. To confirm that the epitopes arespecific for CD4+ T cells, we performed the ELISpot assay usingCD4+ cells isolated from the expanded PBMCs. Following theidentification of peptides containing an epitope in a particulardonor, CD4+ cells were isolated by negative MACS selectionand were combined with irradiated (4,000 rad), freshly thawedautologous PMBCs in an ELISpot plate. Irradiated PBMCs wereplated alone to confirm that those cells did not contribute to theresponse, and the CD4-fractionated cells also were plated toconfirm that CD8+ or other PBMCs were not responsible for theresponse. A typical result from one of the 10 donors tested isshown in Fig. 2C. We found that only peptides 14 and 15 pro-duced a significant response and that the strongest response wasobserved with CD4+ T cells. The response from CD4-depletedcells was very low and probably was attributable to contaminatingCD4+ cells in the depleted population. There was no responsefrom irradiated PBMCs. We conclude that the T-cell responsesboth are specific for peptides and are limited to the CD4+ subset.

HLA restriction. To determine which HLA molecules are requiredfor the T-cell response, we measured the ability of antibodiesspecific for HLA-DR, DP, or DQ to inhibit the response (Fig.4B). The responses of all nine donors were inhibited by the anti-DR antibody but not by antibodies against either DP or DQ. Amixture of all three antibodies likewise inhibited the response.Antisera to HLA class I did not block the response. These datashow that the responses are restricted by class II HLA-DR.

HLA types within the donor pool. To determine if the responsesoccurred in donors with different HLA alleles, we analyzed theHLA-DRB1 status of all of the donors and compared this resultwith the frequency of HLA-DRB1 alleles in the human pop-ulation. We found that the representation of HLA-DRB1 allelesin our cohort is not dramatically different from the worldwidedistribution of human HLA (www.allelefrequencies.net) (26)(Fig. S2). Our cohort is enriched for HLA-DR08 and HLA-DR15 and lacks DR09, but the frequencies of these alleles arewell within the reported ranges of some major ethnic groups (27).We conclude that the distribution of our donors is representativeof the population that will be treated with RITs containing PE38.

Identification of amino acids required for T-cell activation. Toidentify amino acid variants that reduced the CD4+ T cellresponses in the immunodominant peptide, we synthesized a setof variant peptides for the sequence shared by peptides 14 and15 (LVALYLAARLSW). To keep the peptide in a 15-mer for-mat, we used the scaffold of peptide 15, because more donorsresponded to peptide 15 (21/23) than to peptide 14 (20/23). Eachvariant had one amino acid replaced with alanine, and in theplace of alanine we used glycine. The sequences for the var-iants are shown in Table 1. The peptides were tested on 21donors who were positive for peptide 15 in the IL-2 ELISpot as-say. The number of spots relative to the variant peptides wasnormalized to the parent peptide, so that peptide 15 hada response of 100% for each run.Representative responses of 10 of the 21 donors to each variant

peptide are shown in Table 1. The average relative responses ofthe 21 donors to the mutant peptides L294A, L297A, Y298A,and R302A were below 20%. The mutant peptides L297A andY298A had even more pronounced effects, with average relativeresponses below 11%. The responses to other variant peptides,such as S304A and W305A, were not greatly affected by thealanine mutations, with average relative scores of 110.5% and119.4%, respectively.

Fig. 3. CD4+ T-cell IL-2 secretion in response to PE38-derived peptide pools.Samples from 50 normal donors were stimulated with PE38-containingimmunotoxin for 14 d and were restimulated with the overlapping peptidespools. Each pool was tested in quadruplicate, and SFC/1E6 cells were calcu-lated. (A) Visual illustration of the strongest (black), weak (gray), and negative(white) responses. Donors were clustered using an automatic sorting based onthe responsiveness of the pools. (B) Relative responses to 22 pools (n = 50).

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Mutant immunotoxins. Because the responses to peptides con-taining L297A and Y298A in domain II of PE38 were very low,we incorporated these mutations into HA22, making HA22-L297A and HA22-Y298A. We also were able to delete the entireregion containing the epitope. In a previous study focused oneliminating B-cell epitopes in immunotoxin HA22-LR, wedeleted most of domain II of PE38 (amino acids 253–364, cor-responding to peptides 1–38) but maintained excellent cyto-toxic activity. HA22-LR retains the 11 amino acids making upthe furin site (in pool 2) (Fig. 1B); based on the in silico pre-diction algorithm (28) these 11 residues were not predicted tomake up a T-cell epitope. We were able to purify the mutantRITs to near homogeneity, as shown in Fig. 5B. The proteinswith point mutations and HA22 have the expected size of 63 kDa(lanes 1, 2 and 4), whereas HA22-LR has the expected mo-lecular weight of 51 kDa (lane 5).

Properties of mutant immunotoxins. The cytotoxic activities of themutant andWTRITs were measured in four cell lines, as previouslydescribed (29). Fig. 5A shows a representative assay using Daudicells, and Table 2 shows the average EC50 values for each cell line.All three mutant immunotoxins (HA22-L297A, HA22-Y298A,and HA22-LR) were very cytotoxic, although somewhat less sothan the parent molecule, HA22. Compared with HA22, HA22-L297A showed relative activities >50% in all four cell lines andwas more active than the other two mutants in three of the fourcell lines. We would not expect the differences in the relative

activities to be clinically significant, because all mutants werevery active.The stability of the mutant immunotoxins was investigated by

heating them for 15 min at various temperatures. We found thatall four proteins were completely stable up to 50 °C. We foundthat L297A lost 50% of its activity at 51 °C, LR at 56 °C, Y298Aat 57 °C, and HA22 at 58 °C (Fig. S3). Thus, the introduction ofthe point mutations had very little effect on thermal stability.To test the capacity of the mutant RITs HA22-L297A and

HA22-Y298A to stimulate a T-cell response, we synthesizedmutant peptides containing the alanine mutations present in themutant immunotoxins for all of the overlapping peptides thatcontain position L297A and Y298A (peptides 12, 13, 14, 15, and16). We also examined the HA22- LR deletion mutant using theWT peptides. We pooled the variant peptides and used them toevaluate the immunogenicity of the variant RITs. PBMCS fromdonors known to have a positive response in pool 3 were culturedwith HA22, HA22-L297A, HA22-Y298A, or HA22-LR and wererestimulated with the appropriate peptide pools or the wholeprotein. A representative response to the four proteins is shownin Fig. 6. PBMCs that underwent in vitro expansion with WTHA22 and were restimulated with WT peptides on day 14 had aresponse of 713 SFC per106 cells in pool 3, which is statisticallysignificant over the no-peptide control (P < 0.0001 in a Student ttest). The same donors’ PBMCs were stimulated with the mutantproteins and then were restimulated with the mutant peptidepools (or WT peptides for LR). These cells had an extremely low

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). Theresponses to stimulation with peptide 15 were compared with the positive control (peptide 15 with no antibody), and the relative response was calculated foreach donor (n = 9).

Table 1. CD4+ T-cell response to alanine-variant peptides

Peptide Sequence Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 Donor 7 Donor 8 Donor 9 Donor 10 Average

No peptide 1 13 2 4 3 3 1 3 1 5 3.7WT 15 LVALYLAARLSWNQV 100 100 100 100 100 100 100 100 100 100 100L294A AVALYLAARLSWNQV 6 4 8 9 4 25 3 7 19 27 11.2V295A LAALYLAARLSWNQV 5 103 2 27 19 13 63 14 1 5 25.2A496G LVGLYLAARLSWNQV 11 118 10 24 16 19 57 27 31 34 34.5L297A LVAAYLAARLSWNQV 6 2 8 8 4 10 6 19 19 17 9.8Y298A LVALALAARLSWNQV 6 2 5 9 6 16 29 14 8 15 10.9L299A LVALYAAARLSWNQV 49 11 21 11 13 3 17 19 39 22 20.4A300G LVALYLGARLSWNQV 89 64 35 82 91 16 135 32 223 72 83.9A301G LVALYLAGRLSWNQV 65 87 54 67 142 6 107 53 167 88 83.6R302A LVALYLAAALSWNQV 20 49 0 22 22 2 31 86 5 18 25.6L303A LVALYLAARASWNQV 96 74 49 108 108 79 66 78 209 77 94.4S304A LVALYLAARLAWNQV 101 109 83 106 92 121 92 68 233 99 110.5W305A LVALYLAARLSANQV 70 114 72 102 124 54 116 59 305 178 119.4

Data are shown as percent of response to WT peptide. Gray shading indicates a response of <10% to WT peptide. Boldface and underlining indicate theamino acid residues that were changed.

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response of <15 SFC per 106 cells, similar to that in the negativecontrol. PBMCs that were stimulated with mutant proteins andrestimulated with WT peptides showed no significant response.Furthermore, the deletion and removal of the amino acids in do-main II (pools 1–8) did not induce the formation of new epitopescorresponding to the PE38 peptides. Restimulation using the wholeprotein did not give any response.

DiscussionIn this study we report the identification, characterization, andelimination of an immunodominant T-cell epitope in PE38. Ourapproach was to stimulate donor PBMCs with intact RIT, fol-lowed by restimulation with overlapping peptides and an ELISpotassay for IL-2 to measure T-cell response. We identified twooverlapping peptides that contain an immunodominant, DR-restricted, promiscuous epitope and several subdominant epitopes.The immunodominant epitope, located in domain II of PE, stim-ulated substantial T-cell responses in 23 of 50 normal donors. Wethen used alanine-scanning mutagenesis to identify the aminoacids responsible for establishing the epitope and used this in-formation to construct mutant RITs that did not stimulate T cellsbut retained excellent cytotoxic activity.

In Vitro Expansion. Previously, human helper T-cell epitopes havebeen identified by exposure of PBMCs or dendritic cells topeptides, followed by measurement of T-cell proliferation bythymidine incorporation. We initially tried variations of this as-say and found, as previously described (30), that many donorsdid not respond to peptide stimulation and that often the mag-nitude of the response was low. The lack of a response in somedonors was unexpected, because immunotoxins containing PE38are highly immunogenic in humans with a normal immune sys-tem (4, 5). In an attempt to improve the number of responses, weadopted the assay developed by Sette and colleagues (23) toidentify epitopes in a hay fever allergen. In this approach PBMCsare incubated with whole protein to allow the generation and

presentation of normally processed peptides that initiate theexpansion of responsive T cells. This first incubation is followedby a second one in which cells are stimulated with syntheticpeptides and by an ELISpot assay to measure IL-2 production.The addition of the in vitro expansion step resulted in very strongand reproducible responses, ranging from hundreds to thousandsof SFC per 106 cells in 100% of the donors. The backgroundresponse level was very low, with a median of only 10 SFC per106 cells. Because of the wide interest in deimmunizing proteins,we believe this approach could be of general use.We initially evaluated IL-4, IFN-γ, and IL-2 to measure T-cell

stimulation and found that they gave very similar responses. Weused IL-2 because it supports T-cell activation, differentiation,and memory and is a less specialized cytokine than IL-4 or IFN-γ(31). In addition, it was shown previously in vaccination studiesthat IL-2 is a reliable indicator of CD4 T-cell activation, whereasIFN-γ was more variable (32).In vitro expansion with whole protein is useful for evaluating

the immunogenicity of mutant RITs, in which specific T-cellepitopes have been abolished. Evaluating T-cell activation withpeptides alone is useful for showing that a known epitope isabolished (20), but it is insufficient for demonstrating that newepitopes have not been created. For instance, mutations couldalter antigen processing of the protein and potentially gen-erate new epitopes. This assay also allows one to evaluate theemergence of subdominant epitopes after the removal of animmunodominant epitope.

Previous Efforts to Map PE38 T-Cell Epitopes. An earlier study in-vestigating PE38 for the presence of CD4+ T-cell epitopes useda 3H-thymidine incorporation assay to monitor peptide activa-tion of dendritic cells (30). This study identified three epitopes;the major one corresponds to peptides 75 and 76 (in pools 15and 16), and the two others correspond to peptides 15 and 65 (inpools 3 and 13, respectively). The response rates were 23% forthe major epitope and 14% for the other two. Peptides 75, 76,and 15 also were identified in our assay (peptides 75 and 76 wereparticularly responsive peptides in the fine screens of pools 15and 16), although with markedly different dominance. Peptide65, however, which was found to be equally as immunogenic aspeptide 15 by dendritic cell presentation and T-cell proliferation,was not found in our study. We predict that this discrepancy isa result of antigen processing and presentation of the full RIT,such that peptide 65 is irrelevant in a native setting.

HLA II Restriction and Promiscuity. To investigate the molecularbasis of the immunodominant epitope, we evaluated the HLArestriction of the responsive donors and found that responseswere restricted to class II and specifically to HLA DR. Examina-tion of the HLA DRB1 composition of the donors that respon-ded to peptides 14 and 15 shows a wide range of haplotypes,including homozygosity and numerous combinations of differentDR haplotypes (Table S2). This result indicates that this epitopeis promiscuous among several HLA class II haplotypes. More-over, in silico prediction using Propred (28), which predicts thebinding of peptides to HLA class II molecules, supports our

Table 2. Summary of EC50 and relative activity of parent and variant molecules on four CD22+ cell lines

Protein

CA46 (n = 5) Raji (n = 5) Daudi (n = 4) HAL-01 (n = 4)

EC50 ± SD(ng/mL)

Relativeactivity (%)

EC50 ± SD(ng/mL)

Relativeactivity

EC50 ± SD(ng/mL)

Relativeactivity (%)

EC50 ± SD(ng/mL)

Relativeactivity (%)

HA22 0.18 ± 0.09 100 0.15 ± 0.09 100 0.1 ± 0.05 100 1.14 ± 0.43 100HA22-LR 0.41 ± 0.27 45 0.45 ± 0.18 33 0.11 ± 0.05 85 1.5 ± 0.45 76HA22-L297A 0.32 ± 0.15 58 0.27 ± 0.13 54 0.12 ± 0.05 83 2.25 ± 0.61 51HA22-Y298A 0.79 ± 0.52 23 0.47 ± 0.34 32 0.34 ± 0.15 28 6.7 ± 1.16 17

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

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conclusion. The peptides corresponding to the immunodominantepitope are predicted to bind in the top third percentile to 48of the 51 DR molecules covered by the software, indicatinga strong, promiscuous epitope. We therefore expect that removalof this single epitope will affect a broad and diverse populationand will allow many patients to benefit from the deimmunizationof the RIT.Southwood et al. (33) described an HLA-DR supertype that

is characterized by a large group of DR molecules that pro-miscuously bind to an overlapping peptide repertoire. Thesupertype includes DRB1_0101, DRB1_0401, and DRB1_0701,which share a binding motif characterized by a large aromatic orhydrophilic residue in position 1 and a small noncharged residuein position 6. The promiscuous epitope identified in our study,LVALYLAARLSW, matches this description, with the aromatictyrosine being at P1 and the small leucine at P6.

Deletion of the Immunodominant Epitope. In this study we dem-onstrate that the epitope can be destroyed by point mutationsthat presumably interfere with binding to the HLA molecule orto the T-cell receptor. Alternatively, it can be destroyed bya deletion of the epitope as part of a major deletion of a largeportion of PE38 (Fig. 1A) (34). Weldon et al. (29) have reportedthat most of the amino acids in domain II, except for an 11-residue sequence comprising the furin cleavage site, can be de-leted from HA22 without major loss of cytotoxic activity (Fig.1B). Deletion of the entire epitope should be more beneficialthan the point mutations, because it eliminates both the immu-nodominant epitope in the pool and the subdominant epitopes inpools 1 and 2. Based on the heatmap in Fig. 3A that shows that

18 of the 50 donors had epitopes only in domain II, the LRdeletion should eliminate 36% of responses completely.

New Epitopes Are Not Created. One potential concerns surround-ing the elimination of T-cell epitopes are that new epitopes willappear as a consequence of the mutation or that cryptic epitopeswill emerge that were suppressed by the stronger epitopes (35).We used alanine mutagenesis because alanine substitutions re-duce the binding of a peptide to an HLA molecule (36, 37). Weexamined the mutant immunotoxins and did not find new epit-opes, probably because the alanine substitution destroyed theepitope, either by disrupting peptide–HLA binding or, less likely,by disrupting the binding to the T-cell receptor. This result is inagreement with findings by Yeung et al. (38), who identifiedmurine T-cell epitopes in human IFN-β and found that elim-inating the immunodominant epitopes did not result in a re-sponse directed at the subdominant epitope.In summary, we have identified the T-cell epitopes in PE38,

including a promiscuous immunodominant epitope that can beeliminated without a major loss in activity or stability. Immu-notoxin HA22-LR, with a deletion of almost all the amino acidsin domain II, was not immunogenic in 34% of donors and wasless immunogenic in an additional 42% of the donors. We nowplan to combine the domain II deletion with point mutationsthat remove epitopes in domain III. These deletions shouldproduce an RIT with very low immunogenicity that can be in-vestigated in animal models. We anticipate that immunotoxinscontaining mutations that eliminate T-cell epitopes can be givenfor several cycles to patients with normal immune systems,allowing better antitumor responses to be achieved.

Materials and MethodsPeptide Synthesis. A series of 15-mer peptides, which overlap by 12 aminoacids and span the entire sequence of PE38, were synthesized by AmericanPeptides. Peptides were purified to >95% homogeneity by HPLC, and theircomposition was confirmed by mass spectrometry. Peptides were dissolvedin DMSO at 10 mM and stored at −20 °C. For initial screening, peptides werepooled into groups of five consecutive peptides, with the exception of pool22, which contained six peptides (Table S1).

Human Donor PBMC Samples. Apheresis samples from healthy volunteerdonors at the National Institutes of Health (NIH) blood bank were used for allexperiments. Specimenswere collected under research protocols approved bythe NIH Institutional Review Board (99-CC-0168) and were obtained afterinformed consent. PBMCs were isolated by Ficoll-Hypaque (GE Healthcare)density-gradient separation according to manufacturer’s instructions, andthe buffy coat was collected and washed three times with Dulbecco’s PBSwithout Ca and Mg. PBMCs were cryopreserved in liquid nitrogen ata concentration of 1–3 × 107 cells/mL. Class I and II HLA typing was per-formed using PCR sequence-specific primers/sequence-specific oligonucleo-tides–based tissue typing by the HLA typing unit in the Warren G. MagnusonClinical Center, NIH.

In Vitro Expansion of PE38-Specific T Cells. In vitro expansion was carried out aspreviously described (23). PBMCs were stimulated with an RIT containingPE38 (typically LMB9) (39), because we had a large amount of this proteinavailable in a highly purified state at a final concentration of 5 μg/mL or75 mM. Medium alone was used as a negative control, and CEFT (5 μg/mL ofeach peptide in the peptide mixture) (Axxura) was used as a positive control.On day 14, cells were harvested, washed once, and screened for reactivityagainst peptide pools. On day 17, the screen was repeated along with finescreens for the individual peptides.

ELISpot Assays. The secretion of IL-2 following stimulation with RIT wasanalyzed in an IL-2 ELISpot assay according to manufacturer’s recom-mendations (Mabtech). Negative controls were incubated with medium, andpositive controls were incubated with CEFT peptides or phytohemagglutinin(Sigma). Spots were counted by computer-assisted image analysis (Immu-nospot 5.0; Cellular Technology Limited). Each assay was performed inquadruplicate. Positive pools were fine screened to identify the individualimmunogenic peptides by testing individual peptides from the pool. The

Fig. 6. Mutant immunotoxins do not create new T-cell epitopes. Repre-sentative responses to 22 peptide pools after stimulation with (A) HA22, (B)HA22-LR, (C) HA22-L297A, or (D) HA22-Y298A and restimulation with ap-propriate mutant peptides.

E3602 | www.pnas.org/cgi/doi/10.1073/pnas.1218138109 Mazor et al.

threshold for a positive response was based on an initial analysis of the resultsfrom the screening of five donors and included three factors: A response wasconsidered positive if (i) the value was ≥85 SFCs per 106 cells, (ii) the value wasmore than three times that of the negative control, and (iii) the spots in thepool made up more than 10% of all of the spots for that donor. This thresholdgave reproducible responses for all donors.

CD4+ T-Cell Isolation. CD4+ T cells were purified by negative selection fromfrozen aliquots of PBMCS using the MACS CD4+ T-cell isolation kit andLS separation column (Miltenyi Biotec) according to the manufacturer’sprotocol. CD4+ T-cell populations were >80% pure, as determined by flowcytometry, and were >90% viable as judged by Trypan blue exclusion.

HLA Class II Restriction. Antibody inhibition assays were performed to de-termine the HLA restriction of the immunodominant epitope as previouslydescribed (23). The responses of nine donors from various HLA groups topeptide 15 when inhibited with HLA antibodies was measured. The responseswere normalized to the untreated control.

Construction, Expression, and Purification of RIT. HA22 and mutant RITsthereof are composed of the heavy-chain Fv fused to PE38 (VH-PE38) disul-fide-linked to the light-chain Fv (VL). The mutations L297A and Y298A wereintroduced into the parent expression plasmid (HA22 VH-PE38) using PCRoverlap extension (40). The resulting PCR products were cloned back into the

parent plasmid, and the mutations were confirmed by DNA sequencing. AllRITs were purified by a standard protocol (41).

WST8 Assay. Cytotoxicity assays were performed on CD22+ human Burkittlymphoma cell lines (CA46, Raji, and Daudi) and an acute lymphoblastic leu-kemia cell line (HAL-01). The assay was performed as previously described (29).

Thermal Stability. RITs were heated for 15 min at various temperatures aspreviously described (42).

Statistical Analysis. A nonparametric Friedman’s test was used to comparethe screen results of the 22 pools for 50 donors. P < 0.01 was considered sta-tistically significant. The Mann–Whitney–Wilcoxon test was used to comparethe alanine-variant peptides and positive single peptides within the pools.

ACKNOWLEDGMENTS. We thank Dr. Byungkook Lee for providing thefigures showing the structures of the immunotoxins, Dr. Paul Robins forimmunological advice, Susan L. Strobl for help in technical troubleshooting,Dr. Alexandro Sette and Carla Ossehof for sharing their epitope-mappingprotocol, Dr. Onda Massanori for providing useful reagents, and Dr. JayBerzofsky, Dawn Walker and Dr. Jon Yewdell for providing helpful com-ments. This research was supported by the Intramural Research Programof the Center for Cancer Research, National Cancer Institute, National Insti-tutes of Health and by a Cooperative Research and Development Agreementwith MedImmune, LLC.

1. FitzGerald DJ, Wayne AS, Kreitman RJ, Pastan I (2011) Treatment of hematologicmalignancies with immunotoxins and antibody-drug conjugates. Cancer Res 71(20):6300–6309.

2. Kreitman RJ, et al. (2012) Phase I trial of anti-CD22 recombinant immunotoxinmoxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia.J Clin Oncol 30(15):1822–1828.

3. Wayne AS, et al. (2011) A novel anti-CD22 immunotoxin, moxetumomab pasudotox:Phase I study in pediatric acute lymphoblastic leukemia. Blood 118:1317a.

4. Hassan R, et al. (2007) Phase I study of SS1P, a recombinant anti-mesothelin immunotoxingiven as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma,ovarian, and pancreatic cancers. Clin Cancer Res 13(17):5144–5149.

5. Kreitman RJ, Hassan R, Fitzgerald DJ, Pastan I (2009) Phase I trial of continuous infusionanti-mesothelin recombinant immunotoxin SS1P. Clin Cancer Res 15(16):5274–5279.

6. Wolbink GJ, Aarden LA, Dijkmans BA (2009) Dealing with immunogenicity ofbiologicals: Assessment and clinical relevance. Curr Opin Rheumatol 21(3):211–215.

7. Krieckaert CL, Bartelds GM, Wolbink GJ (2010) Therapy: Immunogenicity of biologictherapies-we need tolerance. Nat Rev Rheumatol 6(10):558–559.

8. Nagata S, Pastan I (2009) Removal of B cell epitopes as a practical approach forreducing the immunogenicity of foreign protein-based therapeutics. Adv Drug DelivRev 61(11):977–985.

9. van den Berg HA, Rand DA (2004) Foreignness as a matter of degree: The relativeimmunogenicity of peptide/MHC ligands. J Theor Biol 231(4):535–548.

10. Shankar G, Shores E, Wagner C, Mire-Sluis A (2006) Scientific and regulatoryconsiderations on the immunogenicity of biologics. Trends Biotechnol 24(6):274–280.

11. Onda M, et al. (2006) Characterization of the B cell epitopes associated witha truncated form of Pseudomonas exotoxin (PE38) used to make immunotoxins forthe treatment of cancer patients. J Immunol 177(12):8822–8834.

12. Hansen JK, et al. (2010) A recombinant immunotoxin targeting CD22 with lowimmunogenicity, low nonspecific toxicity, and high antitumor activity in mice.J Immunother 33(3):297–304.

13. Liu W, et al. (2012) Recombinant immunotoxin engineered for low immunogenicityand antigenicity by identifying and silencing human B-cell epitopes. Proc Natl AcadSci USA 109(29):11782–11787.

14. Parker DC (1993) T cell-dependent B cell activation. Annu Rev Immunol 11:331–360.15. Parker DC (1993) The functions of antigen recognition in T cell-dependent B cell

activation. Semin Immunol 5(6):413–420.16. Harding FA, et al. (2005) A beta-lactamase with reduced immunogenicity for the

targeted delivery of chemotherapeutics using antibody-directed enzyme prodrugtherapy. Mol Cancer Ther 4(11):1791–1800.

17. Tangri S, et al. (2005) Rationally engineered therapeutic proteins with reducedimmunogenicity. J Immunol 174(6):3187–3196.

18. Cantor JR, et al. (2011) Therapeutic enzyme deimmunization by combinatorialT-cell epitope removal using neutral drift. Proc Natl Acad Sci USA 108(4):1272–1277.

19. De Groot AS, Knopp PM, Martin W (2005) De-immunization of therapeutic proteinsby T-cell epitope modification. Dev Biol (Basel) 122:171–194.

20. Moise L, et al. (2012) Effect of HLA DR epitope de-immunization of Factor VIII in vitroand in vivo. Clin Immunol 142(3):320–331.

21. Jones TD, et al. (2004) The development of a modified human IFN-alpha2b linked tothe Fc portion of human IgG1 as a novel potential therapeutic for the treatment ofhepatitis C virus infection. J Interferon Cytokine Res 24(9):560–572.

22. Warmerdam PA, et al. (2002) Elimination of a human T-cell region in staphylokinaseby T-cell screening and computer modeling. Thromb Haemost 87(4):666–673.

23. Oseroff C, et al. (2010) Molecular determinants of T cell epitope recognition to thecommon Timothy grass allergen. J Immunol 185(2):943–955.

24. Deenadayalan A, et al. (2010) Immunoproteomic identification of human T cell

antigens of Mycobacterium tuberculosis that differentiate healthy contacts fromtuberculosis patients. Mol Cell Proteomics 9(3):538–549.

25. Khan N, et al. (2007) T cell recognition patterns of immunodominant cytomegalovirus

antigens in primary and persistent infection. J Immunol 178(7):4455–4465.26. Gonzalez-Galarza FF, Christmas S, Middleton D, Jones AR (2011) Allele frequency net:

A database and online repository for immune gene frequencies in worldwidepopulations. Nucleic Acids Res 39(Database issue):D913–D919.

27. Marsh SGE, Parham P, Barber LD (2000) The HLA Factsbook (Academic, San Diego).28. Singh H, Raghava GP (2001) ProPred: Prediction of HLA-DR binding sites. Bioinformatics

17(12):1236–1237.29. Weldon JE, et al. (2009) A protease-resistant immunotoxin against CD22 with greatly

increased activity against CLL and diminished animal toxicity. Blood 113(16):

3792–3800.30. Harding FA (2008) MedImmune L US 2008/0193976.31. Tassignon J, et al. (2005) Monitoring of cellular responses after vaccination against

tetanus toxoid: Comparison of the measurement of IFN-gamma production by ELISA,ELISPOT, flow cytometry and real-time PCR. J Immunol Methods 305(2):188–198.

32. De Rosa SC, et al. (2004) Vaccination in humans generates broad T cell cytokine

responses. J Immunol 173(9):5372–5380.33. Southwood S, et al. (1998) Several common HLA-DR types share largely overlapping

peptide binding repertoires. J Immunol 160(7):3363–3373.34. Allured VS, Collier RJ, Carroll SF, McKay DB (1986) Structure of exotoxin A of

Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci USA 83(5):

1320–1324.35. Liu Z, Williams KP, Chang YH, Smith JA (1993) Immunodominance: A single amino

acid substitution within an antigenic site alters intramolecular selection of T cell

determinants. J Immunol 151(4):1852–1858.36. Allen PM, et al. (1987) Identification of the T-cell and Ia contact residues of a T-cell

antigenic epitope. Nature 327(6124):713–715.37. Johansen BH, Vartdal F, Eriksen JA, Thorsby E, Sollid LM (1996) Identification of

a putative motif for binding of peptides to HLA-DQ2. Int Immunol 8(2):177–182.38. Yeung VP, et al. (2004) Elimination of an immunodominant CD4+ T cell epitope in

human IFN-beta does not result in an in vivo response directed at the subdominant

epitope. J Immunol 172(11):6658–6665.39. Kuan CT, Pastan I (1996) Improved antitumor activity of a recombinant anti-Lewis(y)

immunotoxin not requiring proteolytic activation. Proc Natl Acad Sci USA 93(3):

974–978.40. Horton RM, Cai ZL, Ho SN, Pease LR (1990) Gene splicing by overlap extension: Tailor-

made genes using the polymerase chain reaction. Biotechniques 8(5):528–535.41. Pastan I, Beers R, Bera TK (2004) Recombinant immunotoxins in the treatment of

cancer. Methods Mol Biol 248:503–518.42. Liu W, et al. (2012) A recombinant immunotoxin engineered for increased stability

by adding a disulfide bond has decreased immunogenicity. Protein Eng Des Sel 25

(1):1–6.

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