of April 13, 2017.This information is current as

Heterogeneous ImmunogenicityRestricted Peptide Ligands with−Class II

Structure-Based Design of Altered MHC

L. TopalianRosenberg, Donald F. Hunt, Roy A. Mariuzza and Suzanne Ferdynand Kos, John Sidney, Alessandro Sette, Steven A.McMiller, A. Michelle English, Jeffrey Shabanowitz, Shuming Chen, Yili Li, Florence R. Depontieu, Tracee L.

ol.1300467http://www.jimmunol.org/content/early/2013/10/08/jimmun

published online 9 October 2013J Immunol 

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The Journal of Immunology

Structure-Based Design of Altered MHC Class II–RestrictedPeptide Ligands with Heterogeneous Immunogenicity

Shuming Chen,*,1 Yili Li,†,1 Florence R. Depontieu,* Tracee L. McMiller,*

A. Michelle English,‡ Jeffrey Shabanowitz,‡ Ferdynand Kos,x John Sidney,{

Alessandro Sette,{ Steven A. Rosenberg,‖ Donald F. Hunt,‡ Roy A. Mariuzza,† and

Suzanne L. Topalian*

Insights gained from characterizing MHC–peptide–TCR interactions have held the promise that directed structural modifications

can have predictable functional consequences. The ability to manipulate T cell reactivity synthetically or through genetic engineer-

ingmight thus be translated into new therapies for common diseases such as cancer and autoimmune disorders. In the current study,

we determined the crystal structure of HLA-DR4 in complex with the nonmutated dominant gp100 epitope gp10044–59, associated

with many melanomas. Altered peptide ligands (APLs) were designed to enhance MHC binding and hence T cell recognition of

gp100 in HLA-DR4+melanoma patients. IncreasedMHC binding of several APLs was observed, validating this approach biochemi-

cally. Nevertheless, heterogeneous preferences of CD4+ T cells from several HLA-DR4+ melanoma patients for different gp100

APLs suggested highly variable TCR usage, even among six patients who had been vaccinated against the wild-type gp100 peptide.

This heterogeneity prevented the selection of an APL candidate for developing an improved generic gp100 vaccine in melanoma.

Our results are consistent with the idea that even conservative changes in MHC anchor residues may result in subtle, yet crucial,

effects on peptide contacts with the TCR or on peptide dynamics, such that alterations intended to enhance immunogenicity may be

unpredictable or counterproductive. They also underscore a critical knowledge gap that needs to be filled before structural and

in vitro observations can be used reliably to devise new immunotherapies for cancer and other disorders. The Journal of Immu-

nology, 2013, 191: 000–000.

Melanoma is an aggressive form of skin cancer that iscurable in its early stages but carries a poor prognosisfollowing distant organ metastasis (1). It is also highly

immunogenic, as evidenced by endogenous antimelanoma T andB cell responses and the susceptibility of melanoma to drugs with

a purely immunological mode of action, such as IL-2 (2), anti–CTLA-4 (3), anti–programmed cell death-1 (4), and anti–pro-

grammed cell death ligand-1 (5). Efficient vaccination with tu-mor-specific Ags can redirect the antitumor immune response and

provide synergistic treatment effects when combined with sys-

temic immune-enhancing agents (6–8). Thus, there is a need to

develop optimal cancer vaccines and tumor Ag-specific detectionmethods for monitoring treatment outcomes in vitro. Rational

chemical modification of tumor-specific peptide Ags to increase

their immunogenicity, based on structural models, may facilitate

this approach.Gp100, a melanocyte lineage-specific transmembrane glyco-

protein, is expressed in most melanomas and is involved in a

multiple-step process of pigment production (9). Gp100 has been a

widely used target for melanoma immunotherapy since the dem-

onstration that tumor-infiltrating lymphocytes and circulating Tcells from melanoma patients commonly recognize this Ag

(10, 11). Despite the fact that the most gp100-directed melanoma

therapies have focused on stimulating CD8+ T cell responses,

CD4+ T cells play a central role in inducing and maintainingtumor-specific CD8+ T cells (12). Devising immunotherapies that

can efficiently raise specific CD4+ T cell responses is therefore an

important goal.A gp100 MHC class II (MHC II)–restricted peptide, gp10044–59,

was identified from HLA-DRB1*0401 (hereafter HLA-DR4)–

positive melanoma cell lines (13) and was subsequently validated

as a dominant epitope in a transgenic animal model (14). This

peptide can generate melanoma-specific CD4+ T cells from theperipheral blood of melanoma patients following repetitive

in vitro stimulation (14, 15). Nevertheless, in a clinical trial using

gp10044–59 as a vaccine, no enhancement of gp100-specific reactivity

*Department of Surgery, Johns Hopkins University School of Medicine and the SidneyKimmel Comprehensive Cancer Center, Baltimore, MD 21287; †W.M. Keck Laboratoryfor Structural Biology, University of Maryland Institute for Bioscience and Biotechnol-ogy Research, Rockville, MD 20850; ‡Department of Biochemistry, University of Vir-ginia, Charlottesville, VA 22908; xDepartment of Oncology, Johns Hopkins UniversitySchool of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore,MD 21287; {La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037;and ‖Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda,MD 20892

1S.C. and Y.L. contributed equally to this work.

Received for publication February 15, 2013. Accepted for publication September 10,2013.

This work was supported by National Institutes of Health (NIH)-National Instituteof Allergy and Infectious Diseases Grant AI073654 (to R.A.M. and S.L.T.), NIH-National Institute of Allergy and Infectious Diseases Contract HHSN272200900044C(to A.S.), and NIH Grant AI033993 (to D.F.H.).

The atomic coordinates and structure factors for the gp10044-59-HLA-DR4 complexhave been submitted to the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do)under accession number 4IS6.

Address correspondence and reprint requests to Dr. Suzanne L. Topalian or Dr. RoyA. Mariuzza, Department of Surgery, Johns Hopkins University School of Medicine,1550 Orleans Street, Cancer Research Building 2, Room 508, Baltimore, MD 21287(S.L.T.) or W.M. Keck Laboratory for Structural Biology, University of MarylandInstitute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rock-ville, MD 20850 (R.A.M.). E-mail addresses: stopali1@jhmi.edu (S.L.T.) and rmariuzz@umd.edu (R.A.M.)

Abbreviations used in this article: APL, altered peptide ligand; CII, collagen II; CM,complete medium; ETD, electron transfer dissociation; HA, influenza hemagglutinin;IVS, in vitro stimulation; LTQ, linear quadrupole ion trap; MART-1, melanoma Agrecognized by T cells 1; MBP, myelin basic protein; MHC II, MHC class II; MS/MS,tandem mass spectrometry; WT, wild-type.

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was detected in the peripheral blood of patients following vac-cination, dampening enthusiasm for its therapeutic potential (16).Because gp10044–59 is a nonmutated self-Ag with intermediate

binding affinity for HLA-DR4 (15), we hypothesized that alteredpeptide ligands (APLs) with single amino acid substitutions couldbe designed to confer higher MHC binding affinity and henceimproved immunogenicity. Such APLs derived from gp100 MHCclass I–restricted epitopes have been employed as melanoma vac-cines (17–20). Whereas unmodified HLA-A2–restricted gp100209–217and gp100280–288 peptides induced melanoma-reactive CTLs fromlimited numbers of melanoma patients in vitro, and numerousrestimulations were required, the APLs gp100209–217(210M) andgp100280–288(288V) with enhanced MHC affinity showed superiorimmunogenicity in vitro and in vivo (17). Similarly, in mice,a variant of gp100 that bound H-2Db with increased affinity in-duced high frequencies of melanoma-specific CTLs in the en-dogenous CD8+ repertoire (21).APLs based on MHC II–restricted epitopes have rarely been

explored, because these peptides are heterogeneous in length andmore degenerate in MHC binding specificity than class I–re-stricted peptides (22), making it difficult to precisely define MHCII–specific peptide binding motifs. However, combined informa-tion from MHC II–peptide crystal structures, ligand sequencing,and binding affinity determinations has enriched our knowledge ofthe general chemical properties permitting optimal peptide bindingto HLA-DR4. A dominant large hydrophobic or aromatic residuein the P1 binding position, a hydroxylated residue at P6, and ahydrophobic or polar residue at P9 appear to be favored (22–25).To obtain precise information about the binding characteristics

of gp10044–59 to HLA-DR4 as a basis for designing optimalmelanoma vaccines and immunomonitoring tools, we determinedthe crystal structure of this MHC–peptide complex. APLs basedon structural data were compared with the wild-type (WT) peptidefor their ability to detect gp100-specific reactivity in melanomapatients vaccinated against the WT peptide or to raise melanoma-specific T cells from prevaccination PBMCs.

Materials and MethodsIsolation and sequencing of native gp100 peptides complexedto HLA-DR4

Peptide–HLA-DR complexes were isolated from cultured 1102-mel mel-anoma cells on an anti–HLA-DR affinity column and peptides were elutedas described (26). Peptides were then analyzed by nanoflow HPLC-microelectrospray ionization coupled to either a hybrid linear quadrupoleion trap (LTQ)–Fourier-transform ion cyclotron resonance mass spec-trometer or an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific)modified to perform electron transfer dissociation (ETD). Data were ac-quired as previously described (27). In brief, a precolumn loaded with 1e7cell equivalents of MHC peptides was connected with polytetrafluoro-ethylene tubing (0.06-inch outer diameter and 0.012-inch inner diameter;Zeus Industrial Products) to the end of an analytical HPLC column (360-mm outer diameter and 50 mm inner diameter) containing 7-cm C18reverse-phase packing material (5-mm particles; YMC). Peptides wereeluted into the mass spectrometer at a flow rate of 60 nl/min with a gra-dient: A = 0.1 M acetic acid (Sigma-Aldrich) in water; B = 70% aceto-nitrile (Malinckrodt) and 0.1 M acetic acid in water; and 0–60% B in 40min and 60–100% B in 5 min. Parameters used to acquire ETD/tandemmass spectrometry (MS/MS) spectra in a data-dependent mode on themodified LTQ instrument have been described previously (28).

Sequence analysis was performed by searching MS/MS spectral dataagainst a database consisting only of the gp100 protein using the Open MassSpectrometry Search Algorithm software to generate a list of candidatespectra (29). A precursor mass tolerance of 60.01 Da was used; MS/MSspectra were searched using a monoisotopic fragment ion mass toleranceof 60.5 Da. Data were searched allowing variable modifications forphosphorylation of serine, threonine, and tyrosine residues and oxidationof methionine, with a total of 1024 variable modifications per peptidebeing allowed. Other parameters used were: peptide charge range from +2

to +5, +2 charge state products allowed for peptides of charge +3 andabove, and peptide size range 4–25 aa with no enzyme restriction. Peptidesequence assignments were validated by manual interpretation of thecorresponding ETD and/or collisionally activated dissociation MS/MSspectra. Approximate copy numbers per melanoma cell for each peptidewere determined by comparing peak areas of the observed parent ions tothat of angiotensin I and vasoactive intestinal peptide (DRVYIHPFHLHSDAVFTDNYTR, 100 fmol; Sigma-Aldrich) spiked into the samplemixture.

Synthesis of HLA-DR4–restricted peptides

Synthetic peptides used in this study were made with Fmoc chemistry,isolated by HPLC to $90% purity, and validated with mass spectrometry(Global Peptides or Pi Proteomics). Peptides included: gp100 overlappingpeptides (Table I); WT gp10044–59 (WTWNRQLYPEWTEAQRLD);gp10044–59 APLs (Table III); influenza hemagglutinin (HA)307–319(PKYVKQNTLKLAT); and CDC27758–772 (MNFSWAMDLDPKGAN)(30).

MHC–peptide binding affinity assay

Competition assays to quantitatively measure the binding affinities ofnative and altered gp100 peptides for purified HLA-DR4 were performedessentially as detailed elsewhere (31–33). Briefly, purifiedHLA-DR4molecules(5–500 nM) were incubated with various concentrations of unlabeled peptideinhibitors and 0.1–1 nM 125I-radiolabeled probe peptide (human myelinbasic protein [MBP]85–101, sequence PVVHFFKNIVTPRTPPY) for 48 hin PBS containing 0.05–0.15% Nonidet P-40 in the presence of a pro-tease inhibitor mixture. MHC binding of the radiolabeled peptide wasdetermined by capturing MHC–peptide complexes on L243 (anti–HLA-DRA) Ab–coated Lumitrac 600 plates (Greiner Bio-one, Frickenhausen,Germany) and measuring bound cpm using the TopCount microscintillationcounter (Packard Instrument, Meriden, CT). Peptides were typically testedat six different concentrations covering a 100,000-fold dose range inthree or more independent assays. Under the conditions used, in which[label] , [MHC] and IC50 $ [MHC], the measured IC50 values are rea-sonable approximations of the KD values (34, 35).

MHC–peptide protein preparation, crystallization, datacollection, and structure determination

The gp10044–59–HLA-DR4 complex was assembled in two steps to max-imize the yield of recombinant protein. First, HLA-DR4 bearing CLIP87–101peptide (PVSKMRMATPLIMQA) was prepared by in vitro folding frombacterial inclusion bodies. Second, gp10044–59 was loaded into CLIP–HLA-DR4 using the peptide-exchange catalyst HLA-DM. Briefly, theextracellular portions of the HLA-DR4 a- and b-chains (residues 1–181and 1–192, respectively) were expressed separately as inclusion bodies inEscherichia coli BL21 (DE3) cells (Novagen). Inclusion bodies weredissolved in 8 M urea, 50 mM Tris-HCl (pH 8), and 10 mM DTT, followedby purification on a Poros HQ20 anion exchange column (PerSeptiveBiosystems) in 50 mM Tris-HCl, 8 M urea, and 1 mM DTT at pH 8 (DRa)or pH 8.5 (DRb), using a linear NaCl gradient (36). For in vitro folding,the purified subunits were diluted to a final concentration of 40 mg/l eachin a folding solution containing 50 mM Tris-HCl, 30% (w/v) glycerol, 0.5 mMEDTA, 3 mM reduced glutathione, and 0.9 mM oxidized glutathione (pH8). CLIP peptide (GenScript) was added to a final concentration of 5 mM,and the folding mixture was kept for 2 wk at 4˚C. The final folding solutionwas concentrated and dialyzed against 50 mM Mes (pH 6). Purification wascarried out with sequential Superdex S-200 and Mono Q FPLC columns (GEHealthcare). The CLIP–HLA-DR4 complex was concentrated to 0.8 mg/mland loaded with gp10044–59 by overnight incubation at 37˚C in 100 mMsodium citrate–HCl (pH 5.8) containing 200 mM gp10044–59 and 0.2 mg/mlsoluble HLA-DM.

The gp10044–59–HLA-DR4 complex was crystallized at room temper-ature in hanging drops by mixing equal volumes of the protein solution at5 mg/ml and a reservoir solution of 14% (w/v) polyethylene glycol 8000,0.2 M magnesium acetate, and 0.1 M HEPES (pH 7). For data collection,crystals were transferred to a cryoprotectant solution (mother liquor con-taining 30% [w/v] polyethylene glycol 8000), prior to flash-cooling in anitrogen stream. X-ray diffraction data to 2.5 A resolution were recorded atbeamline X29 of the Brookhaven National Synchrotron Light Source withan ADSC Quantum-315 CCD detector (Area Detector Systems). All datawere indexed, integrated, and scaled with the program HKL 2000 (37).Data collection statistics are shown in Table II.

The structure was solved by molecular replacement with the programPhaser (38) using HLA-DR4 (Protein Data Bank accession code 1D5Z)

2 DESIGN OF MHC II–RESTRICTED ALTERED PEPTIDE LIGANDS

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(39) as a search model. Refinement was carried out using CNS1.2 (40),including iterative cycles of simulated annealing, positional refinement,and B factor refinement, interspersed with model rebuilding into dA-weightedFo 2 Fc and 2Fo 2 Fc electron density maps using XtalView (41). Refinementstatistics are summarized in Table II. Stereochemical parameters were evalu-ated with PROCHECK (42). Atomic coordinates and structure factors havebeen deposited in the Protein Data Bank (accession code 4IS6 http://www.rcsb.org/pdb/home/home.do).

Patients

Patients with unresectable stage IV melanoma who expressed HLA-DRB1*0401 were treated with a synthetic gp100 peptide vaccine. Patientswere vaccinated s.c. with 5 mg gp10044–59 peptide emulsified in IFA every3 wk for a series of four inoculations (one treatment cycle), as described(16). PBMCs were collected by leukapheresis before treatment and 3 wkafter every two vaccinations for in vitro immunologic monitoring studies.Patients underwent radiologic restaging after each treatment cycle. Allpatients were treated in the Surgery Branch of the National Cancer Insti-tute, National Institutes of Health (Bethesda, MD), on a clinical trial ap-proved by the Institutional Review Board of the National Cancer Institute,after signing informed consent (16). In addition to DRB1*0401, HLA-DRalleles expressed by the six patients reported in this study included thefollowing: DRB1*0404, patient 6; DRB1*0701, patients 1, 3, and 5;DRB1*1104, patient 2; DRB1*1501, patient 4; DRB3*02, patient 2;DRB4*01, all patients; and DRB5*01, patient 4.

gp100-specific CD4+ T cell clone 100.1.G7

The gp10044–59–specific CD4+ T cell clone 100.1.G7 (G7 clone here-after) was raised by repetitive in vitro peptide stimulation of PBLsfrom an HLA-DR4+ melanoma patient whose tumor expressed gp100protein. Briefly, T cell cultures were initiated in 24-well plates in thepresence of IL-2. GM-CSF (200 U/ml) and IL-4 (100 U/ml) were addedon day 0 to generate dendritic cells as APCs, along with 25 mM pep-tide. T cells were restimulated every 10 to 14 d with irradiated peptide-pulsed autologous PBMCs or HLA-DR4+ EBV-transformed B cells andcloned after 28 d by limiting dilution culture, as previously described(26). Long-term CD4+ T cell clones were cultured in RPMI 1640 mediumsupplemented with 10% heat-inactivated human AB serum, IL-2, -7, and-15, and 20% conditioned medium from lymphokine-activated killer cellcultures.

T cell functional assays: ELISAs

Peptide-specific CD4+ T cells at 1e5–3e5 cells per well were coculturedovernight in flat-bottom 96-well plates with 1e5 peptide-pulsed HLA-DR4+ allogeneic EBV-B cells or autologous PBMCs. Culture supernatantswere harvested for measurement of GM-CSF and IFN-g secretion by T cellsusing ELISA with commercially available kits (R&D Systems).

In vitro stimulation and ELISPOT assay

Cryopreserved PBMCs from vaccinated patients were thawed and sus-pended in complete medium (CM; RPMI 1640 plus 10% heat-inactivatedhuman AB serum, 2 mM glutamine, 10 mM HEPES buffer, and antibiotics)at 1e6 cells/ml in 24-well plates with 20 mM peptide, 200 U/ml GM-CSF,and 100 U/ml IL-4. Parallel cultures were grown with IL-7 and IL-15 (25ng/ml each) instead of GM-CSF and IL-4. Cells were incubated at 37˚C.After 3 d, IL-2 was added at a final concentration of 10 IU/ml. At 9–12 d,some cells were harvested for ELISPOT assay, and the remaining cellswere restimulated with peptide-pulsed irradiated autologous PBMCs andcultured in CM containing 150 IU/ml IL-2. At day 20, cells were harvestedagain for ELISPOT assay. One day prior to ELISPOT assays, approxi-mately half of the culture volume was replaced with fresh CM (withoutIL-2).

ELISPOT assays were conducted in MultiScreen-IP Filter Plates coatedovernight at 4˚C with 50 ml/well 20 mg/ml mouse anti-human IFN-g Ab(clone 1D1K; Mabtech). On the following day, the plates were washed andblocked with AB medium for 2 h at 37˚C. For some assays, fresh cry-opreserved PBMCs were thawed into ELISPOT media (RPMI 1640 with10% heat-inactivated human AB serum, 2 mM glutamine, and 10 mMHEPES buffer) at 4e6 cells/ml. After 2 h incubation at 37˚C and 5% CO2,

2e5 PBMCs were plated directly to each IFN-g Ab-coated well. ForELISPOT assays using peptide-stimulated T cell cultures, 1e5 T cells wereplated into each IFN-g Ab-coated well. Then, 1e5 irradiated (5000 rad)autologous PBMCs were added to T cells in each well as APCs. Gp100peptides were added at 20 mM in 100 ml total volume. An unrelated HLA-DR4–restricted peptide, CDC27758–772, was used as a negative control.PMA/ionomycin stimulation provided a positive control. After overnightincubation at 37˚C, cells were discarded, and the plates were washed withPBS/0.05% Tween 20 followed by PBS. A total of 100 ml biotinylatedmouse anti-human IFN-g mAb (clone 7-B6-1; Mabtech) diluted at 2 mg/mlin PBS with 0.5% BSA was added to each well and incubated at 37˚C for2 h. The plates were washed, developed with avidin–peroxidase complex(Vector Laboratories), and stained with AEC substrate (Sigma-Aldrich).Spots were counted by a KS ELISPOT automated reader system (CarlZeiss). The number of spots was averaged from triplicate wells. Peptide-specific T cells were defined as showing $20 spots per 1e6 fresh PBMCsor cultured T cells and greater than or equal to twice the numbers of spotsobserved in the negative control wells.

ResultsT cell recognition of native DR4-restricted gp100 peptides

The DR4-restricted peptide gp10044–59 was originally describedby Halder et al. (13) as a dominant peptide displayed on the sur-face of melanoma cells. The immunogenicity of this peptide wasconfirmed in DR4-transgenic mice and human in vitro studies

FIGURE 1. The CD4+ T cell clone 100.1.G7 (G7) is gp10044–59 specific and HLA-DR4 restricted. (A) G7 T cells were cultured overnight with allogeneic

HLA-DR4+ EBV-B cells prepulsed with gp10044–59 or HA307–319 peptides (25 mM). GM-CSF secreted by T cells into culture supernatants was measured by

ELISA. Results representative of four experiments. (B) Recognition of gp10044–59 peptide-pulsed EBV-B cells by G7 is specifically blocked by an anti–

HLA-DR mAb. G7 cells are homozygous for HLA-DR4. (C) Specific recognition of HLA-DR4+ melanoma cells by G7 T cells, which were coincubated

with HLA-DR4+ (1359 and 1102) or HLA-DR42 (1363) EBV-B or melanoma cells overnight. HA, HA307–319, an HLA-DR4–restricted peptide that was

used as a negative control; mel, melanoma cell lines.

Table I. Naturally processed HLA-DR4–restricted overlapping gp100peptides

Residues Copy Number/Cell Amino Acid Sequence

44–59 4500 WNRQLYPEWTEAQRLD44–57 2000 W------------R45–57 900 N-----------R45–59 500 N-------------D46–57 300 R----------R46–59 800 R------------D40–57 100 RTKAW------------R

All peptides have the same core binding region (in bold) as WT gp10044–59.

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(14, 15), leading to its clinical testing as a melanoma vaccine.Although many MHC II–restricted peptides occur as nested peptidesets, naturally occurring sequences overlapping gp10044–59 havenot been previously identified. To identify native nested gp100peptides with potentially enhanced immunogenicity, we searchedpeptides eluted from DR4 molecules displayed on cultured mela-noma cells. A set of seven nested gp100 peptides spanning residues40–59 was identified. Peptide abundance ranged from 100–4500copies/cell (Table I). We assessed the recognition of these peptidesby measuring specific cytokine secretion from the CD4+ gp100-specific DR4-restricted G7 clone (Fig. 1). As shown in Fig. 2,recognition of gp10044–59 exceeded all other peptides. Truncatedpeptides lacking the N-terminal residues Trp44 and Asn45, outsidethe peptide’s MHC core binding sequence, were not well recog-nized by T cells; this is reminiscent of our findings with anotherMHC II–restricted melanoma-associated peptide, phospho–mela-noma Ag recognized by T cells 1 (MART-1), and highlights the

Table II. Data collection and refinement statistics

gp10044–59–HLA-DR4

Data collection statisticsSpace group P21212Unit cell (A, ˚) a = 90.3, b = 117.6, c = 41.9Resolution (A) 30.0–2.50Observations 144,536Unique reflections 14,952Completeness (%)a 93.0 (71.2)Mean I/s(I)a 37.6 (6.2)Rsym (%)a,b 6.1 (24.4)

Refinement statisticsResolution range (A) 30.0–2.50Rwork (%)c 26.9Rfree (%)c 29.8Protein atoms 3,133

Rms deviations from idealityBond lengths (A) 0.009Bond angles (˚) 1.47

Ramachandran plot statisticsMost favored (%) 86.2Additionally allowed (%) 12.6Generously allowed (%) 0.9Disallowed 0.3

aValues in parentheses are statistics for the highest resolution shell (2.50–2.54 A).bRsym = +|Ij 2 , I.|/+Ij, where Ij is the intensity of an individual reflection and

,I. is the average intensity of that reflection.cRwork = +||Fo| 2 |Fc||/+|Fo|, where Fc is the calculated structure factor. Rfree is as

for Rwork but calculated for a randomly selected 10.0% of reflections not included inthe refinement.

FIGURE 2. G7 clone recognition of gp10044–59 exceeds recognition of

other members of a nested set of naturally processed gp100 peptides. APCs

were pulsed with gp10044–59 or nested peptides at the indicated concen-

trations overnight. G7 cells were added and cultured overnight. Super-

natants were harvested and tested for IFN-g secretion by ELISA. Similar

results were obtained for GM-CSF secretion (not shown). HA, HA307–319,

an HLA-DR4–restricted peptide that was used as a negative control.

FIGURE 3. Structure of the gp10044–59–HLA-DR4 complex. (A) Elec-

tron density map for the bound gp10044–59 peptide. The 2Fo 2 Fc map at

2.5 A resolution is contoured at 1d. The peptide is drawn in stick repre-

sentation with carbon atoms in yellow, oxygen atoms in red, and nitrogen

atoms in blue. (B) Top view of the gp10044–59–HLA-DR4 complex,

looking down on the peptide-binding groove. The molecular surface of

HLA-DR4 is cyan (MHC a-chain) and green (MHC b-chain). (C) Con-

formation of high- and low-affinity peptides bound to HLA-DR4. The

conformation of gp10044–59 (yellow) was compared with those of CII1168–1180(green), HA307–319 (blue), and MBP111–129 (pink) by superimposing the

a1b1 domains of HLA-DR4 in the gp10044–59–HLA-DR4 and CII1168–1180–

HLA-DR4 (Protein Data Bank accession code 2SEB) (47), HA307–319–

HLA-DR4 (1J8H) (45), and MBP111–129–HLA-DR4 (3O6F) (48) com-

plexes. The peptides are viewed from the side of the b1 helix; gp10044–59,

CII1168–1180, and HA307–319 bind HLA-DR4 with higher affinity than

MBP111–129.

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important effects that N-terminal residues outside the bindinggroove may have on nonmutated (self) epitope recognition (43).Thus, we were unable to identify a naturally processed over-lapping peptide that was better recognized by the G7 clone thangp10044–59. Furthermore, this peptide was the most abundantlyexpressed member of the nested peptide set. Therefore, all sub-sequent experiments were based on the gp10044–59 native peptidesequence.

Structure of gp10044–59 bound to HLA-DR4

The crystal structure of gp10044–59 complexed to HLA-DR4 wasdetermined to 2.5 A resolution (Table II). Continuous and un-ambiguous electron density was observed for gp10044–59 residuesArg46 to Leu58; however, N-terminal residues Trp44 and Asn45 andC-terminal residue Asp59 were not defined in the electron densitymap (Fig. 3A), despite their importance for recognition by cloneG7 (Fig. 2).In the structure, the primary anchor residues for gp10044–59

bound to HLA-DR4 are Leu48 (P1) and Thr53 (P6); the secondaryanchor residues are Glu51 (P4), Glu54 (P7), and Gln56 (P9). Thisresult is consistent with the sequence motif for HLA-DR4 deducedfrom phage display and synthetic peptide libraries (24, 33, 44).Leu48 fulfills the requirement for a large nonpolar residue at P1 forefficient binding to HLA-DR4. Thr53 at P6 is a suitable primaryanchor as well. By contrast, none of the secondary anchors (P4Glu51, P7 Glu54, and P9 Gln56) conform to the optimal bindingmotif for HLA-DR4. Thus, the P4 pocket of HLA-DR4, whichis lined by b-chain residues Phe26, Lys71, Ala74, and Tyr78, ishydrophobic and prefers nonpolar residues (24, 33, 44). In thegp10044–59–HLA-DR4 complex, the charged C-group of the P4Glu51 side chain points toward the surface of HLA-DR4 insteadof into the P4 pocket (Fig. 3B). The P7 pocket of HLA-DR4,formed by b-chain residues Try47, Trp61, Leu67, and Lys71, islikewise hydrophobic, such that the side chain of P7 Glu54 adoptsa conformation very similar to that of P4 Glu51. The side chain ofP9 Gln56 projects deep into the primarily nonpolar P9 pocket ofHLA-DR4 (Fig. 4, left panel), which is formed by a-chain resi-dues Asn69, Ile72, and Met73 and b-chain residues Tyr37, Asp57,and Trp61. P9 Gln56 makes extensive van der Waals contacts withthese residues, in addition to two side-chain–side-chain hydrogenbonds: P9 Glu56 Oε1–Oh DR4 Tyr37b and P9 Glu56 Nε2–Od1DR4 Asp57b (Fig. 4, right panel).The conformation of gp10044–59, which binds HLA-DR4 with

intermediate affinity (15), was directly compared with those ofthree other peptides that bind HLA-DR4: 1) a self-peptide fromcollagen II (CII1168–1180) that binds HLA-DR4 with relatively highaffinity (24, 44); 2) a foreign peptide from influenza virus HA307–319

that also binds HLA-DR4 with high affinity (45); and 3) a self-peptide from MBP111–129, which binds weakly to HLA-DR4 (46).The main chain of gp10044–59 superposes very closely onto thoseof CII1168–1180 and HA307–319 in complex with HLA-DR4 (45, 47),from residues P2 to P9 (Fig. 3C). The C terminus of gp10044–59(residues P10 and P11), which is positioned ∼1 A higher than thatof CII1168–1180 or HA307–319, is located outside the peptide-bindinggroove. However, all three peptides sit deeply in the bindinggroove. By contrast, the low-affinity MBP111–129 peptide divergesfrom gp10044–59, CII1168–1180, and HA307–319 at anchor residuesP6 and P7 (Fig. 3C), due to a longer side chain at P6 (MBP111–129:Gln; gp10044–59: Thr; CII1168–1180: Ala; and HA307–319: Thr) (48).The consequent elevation of MBP111–129 results in fewer contactsto HLA-DR4 compared with gp10044–59, CII1168–1180, or HA307–319.

APLs and their affinity for DR4

According to the crystal structure of the DR4–gp10044–59 complexas well as published HLA-DR4 peptide binding motifs (22–24),we designed several APLs with substituted MHC anchor residues(Table III). The amino acid substitutions L48F (P1), E51Q andE51A (P4), E54L and E54T (P7), and Q56A (P9) are locatedat MHC anchor residues revealed by crystal structure and weredesigned to enhance MHC–peptide affinity. Indeed, these anchor-modified APLs were found to have 2–10-fold higher affinities forDR4 than the WT peptide. Y49M (P2), P50A (P3), W52A (P5),and A55G (P8), in which the substituted amino acids are posi-tioned at potential TCR contact residues, had higher or similarMHC-binding affinities compared with WT peptide. As a negativecontrol, Q56I was designed to reduce binding affinity to DR4 dueto the large isoleucine residue in P9, incompatible with the cor-responding shallow binding groove in HLA-DR4; the MHC bind-ing affinity of this modified peptide was ∼50% lower than WT(Table III).

Recognition of gp10044–59 APLs by gp10044–59–specific CD4+

T cell clone G7

T cell recognition of gp10044–59 APLs was first characterized bycytokine secretion from the G7 clone. Peptides modified at puta-tive TCR contact residues—Y49M (P2), P50A (P3), and W52A(P5)—were not recognized by the G7 clone, confirming the im-portance of these residues for T cell specificity (Fig. 5). In addi-tion, a replacement of threonine by valine in anchor position 6 alsoabrogated T cell recognition (not shown), suggesting that P6 hashigh stringency for a hydroxylated residue. However, G7 secretedtwo to three times more IFN-g and GM-CSF in response to Q56Athan to WT peptide (Fig. 5), nominating the Q56A APL for furtherstudy. Other APLs showed equivalent or lower recognition by G7

FIGURE 4. Interaction of P9 Gln56 with

HLA-DR4. Left panel: molecular surface of

HLA-DR4 (MHC a-chain, cyan; MHC b-chain,

green) showing the P9 pocket that accommodates

the side chain of P6 Gln56. Right panel: contact

residues of HLA-DR4 are drawn and labeled.

Hydrogen bonds are indicated by broken black

lines.

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compared with WT peptide (Fig. 5). The ability of G7 to cross-react with gp10044–59 and Q56A APL is easily understood instructural terms, because these peptides differ by only a singleresidue at anchor position P9. The Gln56 side chain of gp10044–59(and presumably the Ala56 side chain of Q56A APL) is seques-tered in the P9 pocket of HLA-DR4 and is therefore not exposedto TCR G7 (Fig. 3B).

Comparison of gp100 WT peptide versus the Q56A APL indetecting gp100-specific immunity in PBLs from vaccinatedpatients

In a prior study of patients with melanoma receiving the gp100 WTvaccine, no detectable immunity against the vaccinating peptidewas detected in posttreatment PBMCs using a standard IFN-gELISPOT assay. Because the Q56A APL had higher affinity forDR4 and provoked greater recognition by the G7 clone, weassessed Q56A for its ability to detect gp100-specific reactivity inpre- and posttreatment PBLs from six patients previously reportedin this clinical trial (16) (Fig. 6). Uncultured PBLs were assessedfor reactivity against WT peptide or the Q56A APL using an IFN-gELISPOT assay. In addition, PBLs were in vitro stimulated (IVS)for 10–20 d with WT or Q56A peptide to amplify gp10044–59–specific T cells, prior to ELISPOT. With IVS, the CD4+ T cellpopulation increased to 60–90% of cultured T cells (data notshown). We found reproducible evidence that new specific anti-gp100 responses developed after vaccination in two of six patientsusing uncultured or 10–20 d IVS T cells (patients 1 and 2). Inpatient 3, IVS cultures showed greater gp100-specific activity

after four compared with two vaccines; pretreatment T cells didnot proliferate in IVS culture and thus could not be assessed.There was pre-existing immunity against gp10044–59 that persistedafter vaccination in uncultured PBMCs and IVS cultures in patient4. Finally, two patients did not manifest evidence of a pre-existingor vaccination-induced anti-gp100 response (data not shown).Among the four patients with anti-gp100 immunity, only patient 3had improved detection of responses using the Q56A APL, whilealso recognizing WT peptide. IVS cultures raised against WT orQ56A peptides showed equivalent recognition of the reciprocalpeptide in patients 1, 2, and 4 (Fig. 6). In summary, IVS-enhancedELISPOT techniques enabled the detection of gp100-specificimmunity in four of six patients with melanoma (in three of fourpatients, immunity increased following vaccination). Unlike resultswith the G7 T cell clone, the Q56A APL was not consistentlysuperior to WT peptide in revealing these responses.

T cells raised against WT peptide prefer a variety of APLs

We proceeded to evaluate a panel of gp100 APLs for their abilityto detect gp100-specific CD4+ T cell responses in vitro by testingtheir recognition by T cells from vaccinated melanoma patientsthat were stimulated in vitro with WT peptide. Fig. 7A showsresults from three patients, demonstrating that IVS T cells fromeach patient had a unique preference for recognizing variousAPLs. These results suggest heterogeneous TCR usage by gp100WT-specific T cells from individual patients. Several APLs thatwere preferred by individual patients (L48F, E54L, and E54T)were assessed for their ability to generate gp100-specific CD4+

T cells from the prevaccination PBLs of the six patients withmelanoma. None of these APLs was superior to WT or Q56Apeptide in stimulating gp100-specific CD4+ T cells, and in somecases, APLs provoked APL-specific responses that did not cross-react with WT peptide (not shown). Finally, a peptide poolcomprised of WT peptide plus the APLs L48F, E54L, E54T, andQ56A was evaluated for its ability to raise gp100-specific im-munity in prevaccination T cells or to detect postvaccinationgp100-specific immunity in melanoma patients 1, 2, and 4, similarto experiments shown in Fig. 6. The peptide pool was not superiorto the WT peptide in raising or detecting gp100-specific immunity(Fig. 7B).

DiscussionInsights gained from the characterization of MHC–peptide–TCRinteractions hold the promise that directed structural modificationscan have predictable functional consequences. The ability tomanipulate T cell reactivity synthetically or through genetic en-

FIGURE 5. Q56A substitution at the P9 anchor residue of gp10044–59 confers enhanced recognition by the G7 clone. HLA-DR4+ 1102-EBV cells were

pulsed with gp100 peptides at the indicated concentrations for 24 h. Then G7 cells were added and cultured overnight. Supernatants were harvested and

tested for IFN-g (left panel) and GM-CSF (right panel) secretion by ELISA. T cell recognition of the Q56A was enhanced ∼5-fold compared with WT

gp100 peptide. APLs not shown in this figure did not stimulate detectable cytokine secretion, including Y49M (P2 substitution), P50A (P3), W52A (P5),

and T53V (P6). Results representative of three experiments. HA, HA307–319, an HLA-DR4–restricted peptide that was used as a negative control; WT, WT

gp10044–59.

Table III. gp10044–59 APLs and their affinity for HLA-DR4

gp10044–59and APLs Amino Acid Sequence

SubstitutedPosition

IC50

(nM)

WT WNRQLYPEWTEAQRLD NA 627L48F ----F----------- P1* 280Y49M -----M---------- P2 521P50A ------A--------- P3 189E51A -------A-------- P4* 199E51Q -------Q-------- P4* 200W52A --------A------- P5 372T53V ---------V------ P6* 489E54L ----------L----- P7* 65E54T -----------T---- P7* 104A55G ------------G--- P8 553Q56A -------------A-- P9* 276Q56I -------------I-- P9* 926

*Indicates MHC anchor residues according to crystal structure.NA, Not applicable.

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gineering might thus be translated into new therapies for commondiseases such as cancer and autoimmune disorders. In an earlyexample with the HLA-A2–restricted gp100 epitope 209–217, anMHC anchor residue substitution improved MHC–peptide bindingaffinity, which translated into enhanced CD8 T cell recognitionin vitro and increased immunogenicity in the clinic as a melanomavaccine (8, 17). However, subsequent investigations on the sametheme using MHC I–restricted melanoma-associated peptides andmelanoma-specific TCRs have yielded variable results, demon-strating that structural modifications of MHC–peptide–TCR in-teractions may have unpredictable or undesirable functionalconsequences (49, 50), which may involve collateral damage tonormal tissues when tolerance to nonmutated tumor Ags such asgp100 is alleviated. Furthermore, in vitro functional testing may

not always be predictive of in vivo effects (51). These diffi-culties are amplified in the context of MHC II interactions, forwhich peptide binding motifs are permissive and not rigid,enabling binding of a single peptide to multiple MHC II alleles(52). An APL of an HLA-DR2–restricted MBP epitope con-taining modified TCR contact residues, designed to antagonizeautoreactive T cells in patients with multiple sclerosis, causeddisease exacerbation rather than alleviation in some vaccinatedpatients (53).In the current study, APLs from a dominant HLA-DR4–re-

stricted gp100 epitope were designed to enhance MHC bindingand hence T cell recognition in patients with melanoma. APLdesign was based on precise structural definition of the WTpeptide–MHC II complex, and increased MHC binding wasvalidated by direct affinity measurements. Nevertheless, we ob-served heterogeneous preferences of CD4+ T cells from severalHLA-DR4+ melanoma patients for different gp100 APLs, sug-gesting highly variable TCR usage, even among patients whohad been vaccinated against the WT gp100 peptide. This couldreflect heterogeneous responses of TCRs from different patientsto APL–DRB1*0401 complexes, but might also reflect immuneresponses to APLs bound to alternative DR alleles expressed byindividual patients. This heterogeneity prevented the selection ofan APL candidate for developing an improved generic melanomavaccine.Although we did not determine crystal structure of Q56A APL

bound to HLA-DR4, it is unlikely that the conformation of thisanchor-modified peptide differs significantly from that of WTgp10044–59, which could have explained loss of TCR reactivity.Q56A APL and gp10044–59 differ by only a single residue, atanchor position P9. The possible effect on the peptide backboneof replacing P9 Gln by Ala may be assessed by examining twoother peptide–HLA-DR4 structures, one involving CII1168–1180, inwhich P9 is Gly (47), and the other involving HA307–319, in whichP9 is Leu (45). As shown in Fig. 3C, the main chain of gp10044–59superposes very closely onto those of CII1168–1180 and HA307–319,even though these three unrelated peptides have different residuesat P9 (Gln, Gly, and Leu, respectively). This suggests that the P9anchor modification in Q56A APL exerts subtle effects on peptidecontacts with TCR or on peptide dynamics, such that an alterationintended to enhance immunogenicity may be counterproductive.Indeed, there is growing evidence for an important role of peptidedynamics in modulating TCR recognition (54–56). In one espe-cially relevant study (55), replacing suboptimal anchor residues ofthe HLA-A2–restricted MART-127–35 melanoma Ag unexpectedlyabolished recognition by most MART-1–specific T cell clones,despite a 40-fold improvement in peptide binding to MHC. More-over, crystal structures of WT and anchor-modified MART-127–35peptides bound to HLA-A2 showed that the anchor modificationsdid little to alter the conformation of the peptide or the MHCbinding groove (49). Instead, NMR and molecular dynamicssimulations revealed that the anchor modifications increased theflexibility of both MART-127–35 and HLA-A2 (55). Although theresulting entropic effects improved binding of the peptide toMHC, they also increased the entropic penalty for TCR binding toprohibitive levels, resulting in loss of TCR affinity for peptide–MHC. A similar mechanism may explain the loss of T cell rec-ognition that we observed for Q56A APL. In a related study, TCRrecognition of an APL of lymphocytic choriomeningitis viruspeptide p33 was found to be entropically driven, whereas recog-nition of WT p33 was enthalpically driven (57). If different pep-tides can alter peptide–MHC dynamics in ways that affect TCRrecognition, the commonly used approach of optimizing anchorresidues to enhance peptide binding to MHC may be insufficient

FIGURE 6. Detection of gp100 specific immunity in four vaccinated

patients with melanoma. Reactivity against gp10044–59 in uncultured

PBMCs or in 10–20 d IVS cultures was determined by IFN-g ELISPOT

assays. 20 mM WTor Q56A peptide was used to stimulate CD4+ T cells in

both IVS cultures and ELISPOT assays. Data from uncultured PBMCs

from patient 3 are not shown, because all values were ,20 spots/106 cells.

CDC27, an unrelated HLA-DR4–restricted control peptide. Mean 6 SEM.

*p , 0.02, one-tailed t test compared with CDC27. P2, P4, and P8,

PBMCs collected from patients post two, four, or eight vaccinations; Pre,

prevaccine; SFC, spot-forming colonies; WT, WT gp10044–59.

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to reliably generate improved vaccine candidates, as we found inthe current study.In the gp10044–59–HLA-DR4 structure, N-terminal residues

Trp44 and Asn45 lie outside the peptide-binding groove and are notdefined in the electron density, suggesting flexibility. Nevertheless,these two N-terminal residues are required for efficient recogni-tion by TCR G7, which is reminiscent of the way some autore-active TCRs engage self- or altered self-peptides, including tumorAgs, presented by MHC II molecules (43, 58). For example, x-raycrystallographic studies of tumor-specific TCRs that recognize asomatically mutated human melanoma Ag (mutant triosephosphateisomerase [mutTPI]) have revealed substantial alterations in the to-pology of TCR binding to peptide–MHC compared with antiforeignTCRs (59, 60). In these antitumor TCR–peptide–MHC complexes,the TCR is skewed toward the peptide N terminus relative to itscentral position in antiforeign TCR–peptide–MHC complexes,resulting in low-affinity binding (KD .200 mM) that likely enabledescape from negative thymic selection. In another example, humanautoimmune TCR Ob.1A12, which recognizes a self-peptide fromMBP85–99 bound to HLA-DR2b, was found to only contact the N-terminal half of MBP85–99 (61). TCR Ob.1A12 cross-reacts with anE. coli peptide having limited sequence identity with MBP85–99.Cross-reactivity is due to structural mimicry of a binding hotspot atthe N-terminal portion of the bacterial and self-peptide (62).We attempted to measure the binding of TCR G7 to gp10044–59–

HLA-DR4 by surface plasmon resonance (data not shown).However, we could not detect an interaction, even after injectinga high concentration (up to 200 mM) of rTCR G7 over immobi-lized gp10044–59–HLA-DR4, which precluded further analysis.This result is consistent with previous findings that autoreactiveTCRs generally bind self-Ags, including tumor Ags, with very low

affinity (58). For example, the human melanoma-specific TCR E8binds mutTPI–HLA-DR1 with a KD .200 mM (59). The lowaffinities of G7 and E8 for their self-peptide–MHC ligands likelyenabled these autoreactive T cells to escape negative thymic se-lection (58).Although an explosion of information in the field of molecular

immunology over the past two decades has yielded extraordinarilyprecise and extensive data on molecular interactions and structure-function relationships in the immune system, there remainsa critical gap in knowledge that hinders reliable clinical translationfrom structural and in vitro observations. As demonstrated in thecurrent study, peptide modifications that improve binding to MHCII molecules do not necessarily translate to increased antigenicity.Confounding this strategy is the heterogeneity of human CD4+

T cell responses, as well as possible dynamical effects of peptidemodifications. Predicting these effects will require the use ofmolecular dynamics simulations in conjunction with structuralinformation. Such structure-guided computational design mayeventually allow successful reorientation of immune responsesagainst cancer with vaccines and other immunomodulatory ther-apies.

AcknowledgmentsWe thank H. Robinson (Brookhaven National Synchrotron Light Source)

for x-ray data collection. Support for beamline X29 comes from the Offices

of Biological and Environmental Research and Basic Energy Sciences of

the Department of Energy and the National Center for Research Resources

of the National Institutes of Health.

DisclosuresThe authors have no financial conflicts of interest.

FIGURE 7. T cells stimulated in vitro with

gp10044–59 WT peptide show selective recognition

of a variety of APLs. (A) T cells were derived from

melanoma patients receiving a gp100 WT peptide

vaccine. Results of IFN-g ELISAs are shown.

Background values for IFN-g secretion by T cells

stimulated with APC + HA307–319 have been sub-

tracted from data shown, as follows: patient 2, 213

(1 mM peptide stimulation) and 208 pg/ml (10 mM

peptide stimulation); patient 4, 187, and 191 pg/ml;

and patient 5, 346 and 388 pg/ml. (B) A peptide

pool including APLs was not superior to the WT

peptide in raising or detecting gp100-specific im-

munity. Reactivity against gp10044–59 in 10-d IVS

cultures was determined by IFN-g ELISPOT

assays. CD4+ T cells were stimulated with 20 mM

WT gp100 or peptide pool (WT, L48F, E54L E54T,

and Q56A) in both IVS cultures and ELISPOT

assays. CDC27, an unrelated HLA-DR4–restricted

peptide, was used as a negative control. Mean 6SEM. *p , 0.02, 3p , 0.05, one-tailed t test

compared with HA or CDC27 control peptides. P4,

P6, and P8, PBMCs collected from patients post

four, six, or eight vaccinations; SFC, spot-forming

colonies; WT, WT gp10044–59.

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