Although it has been demonstrated that CTLs can be raised against tumor-associated self-antigens, achieving consistent and effective clinical responses has proven challenging. Superagonist altered peptide ligands (APLs) can often elicit potent antitumor CTL responses where the native tumor-associated epitope fails. Current methods have identified a limited number of superagonist APLs, including the prototypic 27L mutant of MART-1. However, more comprehensive screening strategies would be desirable. In this study, we use a novel genetic screen, involving recombinant technology and class I Ag cross-presentation, to search for supraoptimal superagonists of the 27L MART-1 mutant by surveying the effectiveness of virtually every single amino acid substitution mutant of 27L to activate human Ag-specific CTL clones recognizing the wild-type MART-126–35 epitope. We identify three novel mutant epitopes with superagonist properties that are functionally superior to 27L; however, the ability of a given analogue to act as superagonist varies among patients and suggests that a given superagonist APL may be ideally suited to different patients. These findings endorse the use of comprehensive methods to establish panels of potential superagonist APLs to individualize tumor peptide vaccines among patients.

It has been well established that CTLs (1) have the potential to directly kill malignant cells, which express and display specific antigenic peptides in the context of specific class I MHC molecules (2, 3). These antigenic peptides, often referred to as CTL epitopes, are peptides of unique amino acid sequences, usually 9–11 aa in length. The tumor-associated antigenic peptide that is being targeted can be used as a peptide-based vaccine to promote the antitumor CTL response (4, 5). However, when the target peptide is derived from nonmutated differentiation Ags, as is often the case (e.g., melanosomal proteins), it can be insufficient to engender robust and sustained antitumor CTL responses (6, 7). This is a result of immune tolerance mechanisms that generally suppress or eliminate high-avidity autoreactive T cells (8). As a result of these mechanisms, the vast majority of tumor-specific CTLs, specifically those that recognize nonmutated tumor-associated Ags, are eliminated in the thymus and in the periphery. What remains is a low frequency of tumor-specific CTLs, CTLs that bear low-avidity TCRs, or both for the cognate tumor Ag (912).

It has been shown that one way to activate and mobilize these rare and low-avidity tumor-specific CTLs is with the use of superagonist altered peptide ligands (APLs) (13, 14). These are mutant peptide ligands that deviate from the native peptide sequence by one or more amino acids and that activate specific CTL clones more effectively than the native epitope. Generally, these alterations either allow the peptide to bind better to the restricting class I MHC molecule (13, 14) or interact more favorably with the TCR of a given tumor-specific CTL subset (1). Importantly, superagonist APLs have demonstrated favorable responses in clinical studies (15, 16).

To date, the study and utilization of APLs remains limited due to a lack of comprehensive methods to identify them. A common method to identify superagonist APLs involves comparing the amino acid sequence of the tumor-associated CTL epitope to the so-called consensus binding motif for the restricting class I MHC allotype (13, 14). Where the tumor-associated epitope deviates from the consensus sequence, the appropriate amino acids can be substituted, allowing the peptide to bind better to the class I MHC molecule. However, not all of the poorly stimulatory CTL epitopes deviate from the consensus motif and thus render this approach less feasible. Some researchers take more random approaches. One of these involves substituting one or more specific amino acids into every position of the epitope; an example of this type of approach includes alanine scanning (17, 18). Another of these random approaches includes making every single amino acid substitution at one or two positions—positions either predicted to play a role in class I MHC secondary binding or to be directly involved in engaging the TCR (1, 19). Although the above approaches have identified a number of superagonist APLs of clinically relevant Ags, they are severely limited in scope and poten-tially overlook a large number of superagonist APLs.

In this study, we use a novel comprehensive genetic approach to identify superagonist APLs. Using saturation mutagenesis, we construct recombinant mutant peptide libraries that theoretically contain every single amino acid substitution of the cognate ligand. This approach takes advantage of the speed and simplicity of molecular biology and exploits the unique ability of dendritic cells (DCs) to cross-present extracellular Ags on class I MHC molecules.

The oligonucleotides used in this study were purchased from Operon Biotechnologies (Huntsville, AL). They were designed to have a complimentary 5′ KpnI site and a complimentary 3′ PstI site. The sequences of the saturation mutagenesis sense strands of the MART-126–35 positional oligonucleotides are shown in Table I (each sense strand has a corresponding mutant antisense strand).

Table I.
Sense strands of MART-126–35 positional saturation mutagenesis oligonucleotides
MART-126–35 LibrarySense Strand
P1 CATCGAGGGAAGGNNNCTCGCCGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA 
P2 CATCGAGGGAAGGGAGNNNGCCGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA 
P3 CATCGAGGGAAGGCAGCTCNNNGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA 
P4 CATCGAGGGAAGGCAGCTCGCCNNNATCGGCATTCTGACCGTTTAATGAATTCTGCA 
P5 CATCGAGGGAAGGCAGCTCGCCGGANNNGGCATTCTGACCGTTTAATGAATTCTGCA 
P6 CATCGAGGGAAGGCAGCTCGCCGGAATCNNNATTCTGACCGTTTAATGAATTCTGCA 
P7 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCNNNCTGACCGTTTAATGAATTCTGCA 
P8 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCATTNNNACCGTTTAATGAATTCTGCA 
P9 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCATTCTGNNNGTTTAATGAATTCTGCA 
MART-126–35 LibrarySense Strand
P1 CATCGAGGGAAGGNNNCTCGCCGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA 
P2 CATCGAGGGAAGGGAGNNNGCCGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA 
P3 CATCGAGGGAAGGCAGCTCNNNGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA 
P4 CATCGAGGGAAGGCAGCTCGCCNNNATCGGCATTCTGACCGTTTAATGAATTCTGCA 
P5 CATCGAGGGAAGGCAGCTCGCCGGANNNGGCATTCTGACCGTTTAATGAATTCTGCA 
P6 CATCGAGGGAAGGCAGCTCGCCGGAATCNNNATTCTGACCGTTTAATGAATTCTGCA 
P7 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCNNNCTGACCGTTTAATGAATTCTGCA 
P8 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCATTNNNACCGTTTAATGAATTCTGCA 
P9 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCATTCTGNNNGTTTAATGAATTCTGCA 

N represents any one of the four chemical bases.

NNN represents totally randomized codons. This can be likened to a slot machine with three positions and four possibilities at each position. Each pull of the machine’s lever should yield one of 64 possibilities. Thus, NNN represents any one of the 64 codons. In a given positional library consisting of 100 mutant oligonucleotide pairings, each codon has high likelihood of being represented.

Synthetic peptides were purchased from Sigma-Genosys (The Woodlands, TX) at >90% purity and resuspended in DMSO (Sigma-Aldrich, St. Louis, MO). Peptides were ELAGIGILTV (A27L), GLAGIGILTV (E26G), SLAGIGILTV (E26S), and ELAGIGIMTV (L33M).

DNA sequence analysis was carried out at ACGT (Wheeling, IL).

Saturation mutagenesis oligonucleotides were cloned into the expression vector pQE40 (Qiagen, Valencia, CA). The plasmids were transformed into Escherichia coli (M15 pREP) and cultivated in Luria-Bertani broth. Expression of the minigene was induced using isopropyl β-d-thiogalactoside. Minigene products were expressed as fusion proteins containing hexahistidine tags. After recombinant protein induction, bacteria were lysed with 8 M urea (pH 8). Lysate was harvested and applied to Mg2+-coated paramagnetic beads (Talon beads; Invitrogen/Dynal, Carlsbad, CA), which bind specifically to hexahistidine.

For saturation mutagenesis libraries, bacterial clones were cultured individually in wells of 96-well plates. The genetic approach described above and used in this study is based on a method to identify APLs in a murine model initially developed by C.S.A.-A. while a student in the Frelinger Lab (University of Rochester, Rochester, NY).

Melanoma cell lines A375 and Mel 526, CTL clones, and the TAP-deficient cell line T2 were maintained in RPMI 1640 containing 25 mM HEPES, 2 mM l-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA), and 10% human serum from normal donors. DCs were prepared from adherent monocytes, isolated from the PBMCs of HLA-A2+ healthy donors. IL-4 (500 U/ml; R&D Systems, Minneapolis, MN) and GM-CSF (800 U/ml; Amgen, Thousand Oaks, CA) were added to the monocytes to promote their differentiation into DCs. MART-126–35–specific CTL clones were generated as described by Li et al. (20). PBMCs used in this study were obtained from HLA-A2+ melanoma patients.

After the isolation of the recombinant minigene APL products on Talon beads, the bead-bound products were “fed” to 1 × 105 immature DCs in 96-well plates. After 4 h of incubation at 37°C, 1 × 105 of MART-126–35–specific CTL clones were added to DC/bead preparations. After 12 h of incubation at 37°C, the supernatant was harvested and assayed for the concentration of IFN-γ induced by the APL clones.

Anti–IFN-γ Abs (Endogen, Cambridge, MA) used in the sandwich ELISA were used at a concentration of 1 μg/ml in PBS/0.1% BSA.

On day 0, monocyte-derived DCs were pulsed with 1 μM of each MART-126–35 analogue peptide for 2 h at 37°C. The DCs were washed and added to 5 × 106 HLA-A2+ PBMCs from melanoma patients at a 1:20 ratio in 24-well plates. On day 2, 12.5 U/ml IL-2, 5 ng/ml IL-7, 1 ng/ml IL-15, and 10 ng/ml IL-21 were added to each culture. Cytokines were replenished every 2–3 d for 1 wk. After the 1 wk primary stimulation, cultures were restimulated with 1 × 106 irradiated monocytes pulsed with 10 μM of the peptide used in the primary stimulation. IL-2, IL-7, and IL-15 were added to secondary stimulations on day 2. Cytokines were replenished every 2–3 d. A total of 5 × 105 cells from each culture were stained with allophycocyanin-labeled anti-CD8 Ab (Caltag Laboratories, Burlingame, CA) and PE-labeled MART-126–35 HLA-A2.1 tetramers (Immune Monitoring Laboratory, Fred Hutchinson Cancer Research Center, Seattle, WA). Stained cells were analyzed using a FACScalibur flow cytometer and CellQuest (BD Pharmingen, San Diego, CA) and analyzed using FlowJo software, version 8.5 (Tree Star, San Carlos, CA). Cells were stained with tetramers in 25 μl 2% FCS/BSA for 1 h at room temperature, followed by anti-CD8 Ab for 15 min at 4°C.

After in vitro peptide stimulation of PBMCs from MelPt-B, MelPt-C, MelPt-D, MelPt-F and a healthy donor (Healthy-1) MART-126–35 tetramer- and CD8-positive cells were sorted and isolated on a FACSAria (BD Biosciences, Franklin Lakes, NJ). Isolated cells were replicated using 30 ng/ml anti-CD3 Ab (OKT3) and IL-2 at a concentration 50 U/ml in the presence of irradiated feeder PBMCs and EBV-transformed lymphoblastoid cells for 2 wk. IL-2 was replenished every 2–3 d. After the stimulation, cultures were stained for the generation of MART-126–35 tetramer- and CD8-positive cell populations. The polyclonal cell lines were tested for lytic activity and TCR Vβ usage (MelPt-C only) (described below).

Target cells were labeled with 100 μCi 51Cr and cocultured with effector cells for 4 h at 37°C plus 5% CO2. Targets were melanoma cell lines A375 (HLA-A2+/NY-ESO-1+) and Mel 526 (HLA-A2+/MART-1+) and T2 cells pulsed with 1 μM MART-126–35 (positive control) or NY-ESO-1157–165 (negative control). Effector cells were MART-126–35 tetramer-positive polyclonal cell lines generated with A27L, E26S, or L33M peptides. Assays were performed in triplicate at a 50:1, 25:1, or 12.5:1 E:T ratio. Released 51Cr was measured with a γ scintillation counter, and percentage specific lysis was determined by using the formula: percentage specific release = (experimental release − spontaneous release)/(maximum release − spontaneous release). Spontaneous release was <10% of the maximum release.

TCR Vβ spectratype analysis was carried out by the Immune Monitoring Laboratory at Fred Hutchinson Cancer Research Center. Briefly, cDNA was generated from 1 × 106 MART-126–35 tetramer-staining polyclonal cell lines. Multiplex Vβ PCR primers were then used to amplify the variable regions of CDR3 of the TCR β-chain. Sequence analysis to determine the Vβ usage of the TCRs was conducted using the online program GenScan (http://genes.mit.edu/GENSCAN.html).

To identify superagonist APLs in this study, we use a novel genetic system. This system employs saturation mutagenesis of agonist peptide-encoding oligonucleotides (Table I), which when expressed in E. coli will contain position-specific single amino acid substitutions. The positional libraries are designed such that the codon of interest is totally randomized (NNN), resulting in a pool of oligonucleotides that contains every given codon sequence. This mutagenesis approach might be likened to a slot machine that contains three positions (a codon) where each position has the same four possibilities (A, C, G, or T). When pulled, there is a 1 in 64 chance of getting any combination of the. If pulled 100 times, then there is a high probability that every sequence will be represented (80% certainty, according to a Poisson distribution). In this instance, the 100 pulls represent 100 bacterial colonies, each containing a different mutant agonist peptide-encoding oligonucleotide. When cloned and expressed, each amino acid should be represented in a library of 100, with 80% certainty, according to a Poisson distribution. A positional library can be generated for each position (amino acid) of the target peptide. The APL minigene constructs are fused to a hexahistidine tag and can easily be separated from bacterial proteins on Co2+-coated paramagnetic beads (Dynal). APLs are screened for their abilities to activate epitope-specific CTL clones following cross-presentation of the bead-bound ligand on class I MHC molecules by immature DCs.

To validate this system and to verify that it was sensitive enough to detect our model tumor-associated HLA-A2–restricted antigenic peptide, MART-126–35, as well as an APL superagonist epitope of MART-126–35, called MART-126–35 A27L (henceforward referred to as A27L), oligonucleotides encoding the appropriate peptide sequences were cloned, expressed, and assayed for the ability to activate Ag-specific CTL clones as described in 1Materials and Methods. The CTL clone used in this assay, called M26-H1, is specific for MART-126–35 and expresses IFN-γ in response to HLA-A2/MART-126–35 complexes. In this study, the IFN-γ response elicited by the recombinant unmodified MART-126–35 cross-presented construct is significantly higher than that elicited by the HLA-A2–restricted negative control, NYESO-1157–165 (Fig. 1). Further, the IFN-γ response elicited by the recombinant superagonist APL, A27L, was >2-fold higher than that elicited by the recombinant wild-type construct. Yet, the activation of M26-H1 by the unmodified MART-126–35 construct was clearly distinguishable from that elicited by the HLA-A2–restricted negative control construct, NYESO1157–165. These results suggest that the HLA-A2 cross-presented recombinant ligands are sufficient to elicit detectable Ag-specific responses from CTL clones and also that superagonist APLs can be distinguished based on an increase in IFN-γ expression relative to that of the wild-type CTL ligand.

FIGURE 1.

Native and superagonist CTL determinants can be distinguished in a bead-based cross-presentation assay. Oligonucleotides encoding MART-126–35, NY-ESO-1157–65, or MART-126–35 A27L were cloned into and expressed by pQE40 expression vectors in 5 ml bacterial cultures. The minigene products were isolated and fed to immature DCs as described in 1Materials and Methods. MART-126–35–specific CTL clones were used to detect the presence of the cross-presented minigene products. Induced IFN-γ expression was determined by standard sandwich ELISA. A27L synthetic peptide at 1 μM was used a positive control. Experiment was performed in triplicate.

FIGURE 1.

Native and superagonist CTL determinants can be distinguished in a bead-based cross-presentation assay. Oligonucleotides encoding MART-126–35, NY-ESO-1157–65, or MART-126–35 A27L were cloned into and expressed by pQE40 expression vectors in 5 ml bacterial cultures. The minigene products were isolated and fed to immature DCs as described in 1Materials and Methods. MART-126–35–specific CTL clones were used to detect the presence of the cross-presented minigene products. Induced IFN-γ expression was determined by standard sandwich ELISA. A27L synthetic peptide at 1 μM was used a positive control. Experiment was performed in triplicate.

Close modal

The saturation mutagenesis APL library screen depends on 200 μl bacterial expression cultures in 96-well plates. Fig. 1 clearly shows that cross-presented recombinant ligands can be detected by Ag-specific CTL. However, in that experiment, recombinant proteins were produced at high concentrations in 5 ml cultures. To determine whether the recombinant protein produced in these significantly smaller cultures would be sufficient to reflect detectable and varying degrees of activation, we constructed a position 2 (P2) library of MART-126–35 (EXAGIGILTV). By screening this library, in addition to determining whether 200 μl cultures produce sufficient concentrations of recombinant protein, we would be able to determine whether previously identified superagonist APLs, including A27L, could be identified from among 88 unique mutant APL clones. The P2 library screen (Fig. 2), using the CTL clone M26-H1, clearly shows that the wild-type recombinant ligand MART-126–35 (in green on the chart) elicits significantly more IFN-γ than the negative control (in red on the chart). Furthermore, the APL clones from the library that contained leucine residues at P2 (A27L) elicited significantly more IFN-γ expression in comparison with that of the wild-type ligand. Amino acid content was determined from replicated glycerol stock of the P2 bacterial library. Interestingly, APL clones containing methionine residues at P2 also elicited greater IFN-γ expression than wild-type MART-126–35, although not as great as that elicited by the leucine-containing APLs, A27L. Like A27L, A27M has also been described as a superagonist APL of MART-126–35. Thus, 200 μl bacterial cultures produce sufficient concentrations of the recombinant ligands to be detected in this screen. Also, superagonist APLs can be identified in a library of at least 88 unique APL clones.

FIGURE 2.

Previously described superagonists identified in a MART-126–35 P2 saturation mutagenesis APL screen. Eighty-eight P2 saturation mutagenesis clones were screened as described in 1Materials and Methods. A MART-126–35 control construct is depicted in light gray (first bar on left), whereas the NY-ESO-1157–165 negative control construct is depicted in a checkered pattern (second bar from left). DNA sequence analysis was performed for APL clones eliciting comparable IFN-γ expression as the native construct. The amino acid is indicated above clones that were sequenced for codon content.

FIGURE 2.

Previously described superagonists identified in a MART-126–35 P2 saturation mutagenesis APL screen. Eighty-eight P2 saturation mutagenesis clones were screened as described in 1Materials and Methods. A MART-126–35 control construct is depicted in light gray (first bar on left), whereas the NY-ESO-1157–165 negative control construct is depicted in a checkered pattern (second bar from left). DNA sequence analysis was performed for APL clones eliciting comparable IFN-γ expression as the native construct. The amino acid is indicated above clones that were sequenced for codon content.

Close modal

On the basis of previous experiments demonstrating that superagonist APLs can be uncovered using the saturation mutagenesis screen, remaining positional libraries of MART-126–35 (with the exception of P10, which already contains an anchor residue that conforms to the HLA-A2 C-terminal consensus binding motif) were screened using similar methods. Because a potent superagonist APL of MART-126–35 has already been identified in A27L, we chose to use A27L as the basis for our mutational strategy. That is, we chose to fix leucine in P2, while mutating other positions independently. We reasoned that this would allow us to identify superagonist APLs more effective than A27L. The APL libraries were screened with two different high-avidity MART-126–35–specific CTL clones (screening results are presented for M26-H1 only). A high-avidity TCR is defined here as having the ability to recognize tumor cells that express both MART-1 and HLA-A2 class I molecules. The vast majority of the MART-126–35 derivative mutant peptide clones screened from each of the positional libraries were not as effective as A27L at activating the MART-126–35–specific CTL clone (Fig. 3). However, several clones from the P1, P3 (data not shown), and P8 libraries appeared to work similarly as well as the A27L recombinant construct. It should be noted that the initial screen was conducted by screening two unique APL library clones simultaneously in a single well. Although this approach allows us to screen twice as many APL clones, the potency of any agonist APL in the pool is potentially underestimated in the initial screen.

FIGURE 3.

Eight positional libraries of A27L were screened using the saturation mutagenesis technique. A, Eighty-eight mutant clones were screened for each of eight positional libraries of A27L P1, P3 (data not shown), P4, P5 (data not shown), P6, P7, P8, and P9 as described in 1Materials and Methods. Two clones are screened simultaneously for each library. Activation was assessed by IFN-γ expression. IFN-γ was measured by sandwich ELISA. A positive control (A27L) is illustrated in light gray (far left bar), whereas the negative control (NYESO-1157–165) is illustrated in a checkered pattern (second from left). APL clonal wells indicated with an arrow were deconvoluted, and each mutant APL was rescreened separately. B, The table shows the IFN-γ activity elicited by individual clones relative to the activity elicited by A27L. The clones that were initially assayed together are indicated by shading. A bold number indicates the APL clone that is most responsible for the activation of the screening CTL clone. DNA sequence analysis was used to determine the amino acid encoded.

FIGURE 3.

Eight positional libraries of A27L were screened using the saturation mutagenesis technique. A, Eighty-eight mutant clones were screened for each of eight positional libraries of A27L P1, P3 (data not shown), P4, P5 (data not shown), P6, P7, P8, and P9 as described in 1Materials and Methods. Two clones are screened simultaneously for each library. Activation was assessed by IFN-γ expression. IFN-γ was measured by sandwich ELISA. A positive control (A27L) is illustrated in light gray (far left bar), whereas the negative control (NYESO-1157–165) is illustrated in a checkered pattern (second from left). APL clonal wells indicated with an arrow were deconvoluted, and each mutant APL was rescreened separately. B, The table shows the IFN-γ activity elicited by individual clones relative to the activity elicited by A27L. The clones that were initially assayed together are indicated by shading. A bold number indicates the APL clone that is most responsible for the activation of the screening CTL clone. DNA sequence analysis was used to determine the amino acid encoded.

Close modal

Agonist candidates were selected and rescreened based on their abilities to elicit more or comparable levels of IFN-γ from M26-H1 in the initial screen (Fig. 3B). When tested independently, both of the clones from the P3 libraries elicited less IFN-γ expression from the MART-126–35–specific CTL clone relative to that from A27L (data not shown). When rescreened independently, it was apparent that only one of the two mutant peptide clones from the P1 and P8 wells was responsible for the increased IFN-γ expression. The DNA encoding these putative MART-126–35 agonist peptides was prepared from the duplicated bacterial glycerol stocks. We found that the enhancing mutations for the P1 putative agonists contained either glycine (E26G) or serine (E26S) residues at P1 instead of the naturally occurring glutamate residue. The P8 putative agonist contained a methionine residue (L33M) at P8 rather than the naturally occurring leucine residue. No additional putative agonists were identified from the library screens using the second CTL clone, M26-H2 (data not shown).

To analyze the putative superagonist APLs on a molar basis, individual peptides were synthesized at >90% purity. To determine whether these APLs would be similarly recognized by unique MART-126–35–specific CTL clones, the APLs were tested against four clones bearing unique TCRs. These included two high-avidity CTL clones (M26-H1 and M26-H2) and two low-avidity CTL clones (M26-L1 and M26-L2) (Fig. 4). We define a low-avidity TCR as having the ability to respond to HLA-A2–positive peptide-pulsed target cells but not to cells displaying naturally processed and presented determinants from HLA-A2/MART-1–positive tumors. It has been suggested that low-avidity T cells have the potential to mediate Ag-specific cell and tissue destruction (10, 12).

FIGURE 4.

APLs identified in a saturation mutagenesis screen activate unique MART-126–35–specific CTL clones differently. Two unique high-avidity MART-126–35–specific CTL clones, M26-H1 (A) and M26-H2 (B), and two unique low-avidity MART-126–35–specific CTL clones, M26-L1(C) and M26-L2 (D), were assayed against the agonist peptides A27L (square), E26G (circle), E26S (triangle), L33M (diamond), and NY-ESO-1157–165 (asterisk). Peptides were titrated on T2 target cells. IFN-γ expression was measured by standard ELISA. Assay was performed in triplicate.

FIGURE 4.

APLs identified in a saturation mutagenesis screen activate unique MART-126–35–specific CTL clones differently. Two unique high-avidity MART-126–35–specific CTL clones, M26-H1 (A) and M26-H2 (B), and two unique low-avidity MART-126–35–specific CTL clones, M26-L1(C) and M26-L2 (D), were assayed against the agonist peptides A27L (square), E26G (circle), E26S (triangle), L33M (diamond), and NY-ESO-1157–165 (asterisk). Peptides were titrated on T2 target cells. IFN-γ expression was measured by standard ELISA. Assay was performed in triplicate.

Close modal

Fig. 4A shows that each of the newly identified agonist peptides is similarly effective in activating M26-H1—the high-avidity CTL clone used in the initial screen (Fig. 3)—as compared with the MART-126–35 superagonist peptide, A27L. A similar pattern of activation is found when the identified agonist peptides are used to stimulate the CTL clone M26-H2. In contrast to the above results, the low-avidity MART-126–35–specific CTL clones yielded widely divergent results in response to different agonist peptides. For example, although the CTL clone M26-L1 recognizes the peptide E26S >100-fold better than A27L (based on half-maximal activation), the CTL clone M26-L2 recognizes A27L better than it does E26S. Similarly, although L33M is scarcely recognized by the CTL clone M26-L1, it is the most effective agonist for activating M26-L2. Thus, these analogues might be considered “conditional” agonists, because they do not elicit generalized patterns of activation among unique Ag-specific clonotypes.

To determine how well the identified agonist APLs could prospectively generate MART-126–35–specific CTL populations from melanoma patient PBMC preparations, the APLs were used to stimulate eight different patient PBMC samples under standard in vitro conditions (Table II). One week following the second in vitro stimulation, cultures were stained with the wild-type MART-126–35/HLA-A2 tetramer. Similar to the observations made using different MART-126–35–specific CTL clones, none of the peptide ligands was universally effective in generating MART-126–35–specific CTL populations from all of the patient PBMC samples (Fig. 5). Any given APL was more or less effective in generating Ag-specific CTLs from any given patient PBMC sample. For example, although the agonist peptide E26S is the least effective at generating MART-126–35–specific CD8-positive populations from the PBMCs of MelPt-C (3-fold less than A27L), it is the most effective APL for generating such T cell populations from MelPt-D (5-fold more than A27L). Similarly, whereas the agonist peptide L33M is 14-fold more effective than A27L in generating of MART-126–35–specific CD8 positive populations from the PBMCs of MelPt-E, it is 14-fold less effective than A27L in generating MART-126–35–specific CD8 populations from the PBMCs of MelPt-G. These findings demonstrate that any one CTL ligand may or may not be effective at generating Ag-specific CTL populations from the PBMCs of any given patient and suggest the importance of establishing a panel of potential superagonist APLs.

Table II.
Identified MART-126–35 APLs exhibit differential capacities to generate MART-126–35–specific CTL populations from the PBMCs of different melanoma patient donors
% M26 Tetramer Positive
PatientA27LE26GE26SL33M
MelPt-A 3.14 (1) 1.68 (0.53) 3.36 (1.07) 0.98 (0.31) 
MelPt-B 2.97 (1) 1.31 (0.44) 4.3 (1.45) 7.7 (2.6) 
MelPt-C 40.6 (1) 45.6 (1.12) 15.6 (0.38) 41.1 (1.02) 
MelPt-D 0.65 (1) 1.73 (2.66) 3.43 (5.27) 2.07 (3.1) 
MelPt-E 1.77 (1) 8.42 (4.75) 6.88 (3.88) 24.2 (13.67) 
MelPt-F 5.45 (1) 3.35 (0.61) 3.72 (0.68) 3.07 (0.56) 
MelPt-G 33.4 (1) 1.89 (0.06) 1.75 (0.05) 2.37 (0.07) 
MelPt-H 1.24 (1) 2.03 (1.63) 1.31 (1.06) 2.77 (2.2) 
% M26 Tetramer Positive
PatientA27LE26GE26SL33M
MelPt-A 3.14 (1) 1.68 (0.53) 3.36 (1.07) 0.98 (0.31) 
MelPt-B 2.97 (1) 1.31 (0.44) 4.3 (1.45) 7.7 (2.6) 
MelPt-C 40.6 (1) 45.6 (1.12) 15.6 (0.38) 41.1 (1.02) 
MelPt-D 0.65 (1) 1.73 (2.66) 3.43 (5.27) 2.07 (3.1) 
MelPt-E 1.77 (1) 8.42 (4.75) 6.88 (3.88) 24.2 (13.67) 
MelPt-F 5.45 (1) 3.35 (0.61) 3.72 (0.68) 3.07 (0.56) 
MelPt-G 33.4 (1) 1.89 (0.06) 1.75 (0.05) 2.37 (0.07) 
MelPt-H 1.24 (1) 2.03 (1.63) 1.31 (1.06) 2.77 (2.2) 

APLs were used to stimulate PBMC cultures in vitro. After a 1 wk secondary stimulation, cells were stained with FITC-labeled anti-CD8 Abs and allophycocyanin-labeled HLA-A2/MART-126–35 tetramers and analyzed by flow cytometry. Percentage tetramer-positive is indicated. Fold difference relative to A27L is indicated in parentheses. Differences of >2-fold are indicated in bold.

FIGURE 5.

APLs generate different CTL responses from the PBMCs of different melanoma patients. Identified APLs were used to stimulate the PBMCs of different melanoma patients in vitro. After 1 wk primary and 1 wk secondary peptide stimulation, cultures were stained with FITC-labeled anti-CD8 Ab and allophycocyanin-labeled HLA-A2/MART-126–35 tetramer and analyzed by flow cytometry. Data are representative of at least three different experiments.

FIGURE 5.

APLs generate different CTL responses from the PBMCs of different melanoma patients. Identified APLs were used to stimulate the PBMCs of different melanoma patients in vitro. After 1 wk primary and 1 wk secondary peptide stimulation, cultures were stained with FITC-labeled anti-CD8 Ab and allophycocyanin-labeled HLA-A2/MART-126–35 tetramer and analyzed by flow cytometry. Data are representative of at least three different experiments.

Close modal

In recent vaccination studies, it has been demonstrated that the use of APLs poses the risk of generating Ag-specific T cells that display relatively low antitumor functional avidity (7, 21). To determine whether the MART-126–35–specific CTLs that were generated with these novel MART-126–35 agonist peptides were of sufficient functional avidity to kill HLA-A2/MART-1–positive tumor targets, polyclonal lines of CD8-positive MART-126–35 tetramer-staining cells were established from the PBMCs of MelPt-B, MelPt-C, MelPt-D, MelPt-F, or a healthy donor (Healthy-1), stimulated with either A27L, E26S, or L33M agonist peptides. These cell lines were screened for reactivity to unmodified MART-126–35 peptide-pulsed HLA-A2–positive targets and to HLA-A2/MART-1–positive tumor targets at varying E:T ratios in a standard chromium release assay (Table III). The results illustrate that the CTL populations that were generated from each PBMC source with either of the APLs can kill targets that display wild-type MART-126–35 in the context of HLA-A2 and recognize the epitope with sufficient affinity to kill tumors expressing MART-1.

Table III.
MART-126–35 and tumor-specific lysis by CTLs generated with MART-126–35 peptide analogues
% Specific Lysis from Polyclonal CTL Lines Generated with the Indicated Peptide
MART-126–35 A27L
MART-126–35 E26S
MART-126–35 L33M
PatientE:TT2aT2 + M26bA375Mel 526T2T2 + M26A375Mel 526T2T2 + M26A375Mel 526
MelPt-B 50     0c 40 58 63 13 80 
 25     31 45 52 12 70 
 12.5     21 30 41 58 
MelPt-C 50 73 35 87 41 91 58 
 25 10 52 30 66 32 12 84 52 
 12.5 42 25 54 25 11 76 45 
MelPt-D 50 50 10 32 87 41 14 90 57 
 25 42 10 30 65 32 11 84 54 
 12.5 35 25 54 25 75 45 
MelPt-F 50 92 65     23 70 44 
 25 81 50     21 72 42 
 12.5 73 43     22 65 38 
Healthy-1 50 52 58 34 28 57 13 65 
 25 42 42 26 15 49 13 57 
 12.5 31 35 18 10 38 10 44 
% Specific Lysis from Polyclonal CTL Lines Generated with the Indicated Peptide
MART-126–35 A27L
MART-126–35 E26S
MART-126–35 L33M
PatientE:TT2aT2 + M26bA375Mel 526T2T2 + M26A375Mel 526T2T2 + M26A375Mel 526
MelPt-B 50     0c 40 58 63 13 80 
 25     31 45 52 12 70 
 12.5     21 30 41 58 
MelPt-C 50 73 35 87 41 91 58 
 25 10 52 30 66 32 12 84 52 
 12.5 42 25 54 25 11 76 45 
MelPt-D 50 50 10 32 87 41 14 90 57 
 25 42 10 30 65 32 11 84 54 
 12.5 35 25 54 25 75 45 
MelPt-F 50 92 65     23 70 44 
 25 81 50     21 72 42 
 12.5 73 43     22 65 38 
Healthy-1 50 52 58 34 28 57 13 65 
 25 42 42 26 15 49 13 57 
 12.5 31 35 18 10 38 10 44 

Where values are not shown, polyclonal cell lines were not established for that condition.

a

T2 is a TAP-deficient cell line that expresses peptide-unbound HLA-A2 molecules unless pulsed extracellularly. T2 has been pulsed with NYESO-1157–165 unless indicated otherwise.

b

M26, unmodified MART-126–35 peptide.

c

Numbers represent the percentage specific lysis obtained from each target. T375 is HLA-A2–positive and MART-1–negative

To determine whether unique or shared MART-126–35–specific CTL clonotypes were generated with each of the peptide ligands (A27L, E26S, and L33M), spectratype analysis was performed on CTL lines derived from MelPt-C PBMCs to determine their Vβ TCR usage. We found that the agonist peptides A27L, E26S, and L33M generated CTL populations that primarily (>90%) used TCR Vβ24, Vβ8, and Vβ3, respectively. This suggests that the different analogue peptides preferentially generate specific TCR-utilizing CTL subsets. Taken together, these results demonstrate the ability of the identified APLs to elicit MART-126–35–specific CTL responses that are capable of directly killing MART-1–expressing tumors and suggest that unique MART-126–35–specific TCR subpopulations are being preferentially generated by the different MART-126–35 analogue peptides.

In this report, we use a novel genetic technique to screen for poten-tial superagonist APLs of a clinically relevant tumor-associated Ag, MART-1. Rather than screening a limited subset of possible agonists, this technique allowed us to screen virtually every single amino acid mutant of MART-126–35 in a rapid and cost-effective manner. A comprehensive approach to identifying APLs is perhaps the most ideal, given the tremendous ramifications that even subtle amino acid substitutions can induce on specific CTL responses (2224). Borbulevych et al. (22) have demonstrated somewhat of a disconnect between the overall structure of a given CTL epitope and the CTL response that it elicits. In a 2007 study, the authors demonstrated that structurally dissimilar analogue peptides of MART-127–35 could more effectively induce MART-127–35–specific CTL responses than analogue peptides of MART-127–35 with very similar HLA-A2–associated conformations. This presents a challenge for the ability to predict superagonist APLs and highlights the necessity of utilizing thoroughly comprehensive screening techniques.

Other approaches include the use of a positional scanning synthetic combinatorial library (PS-SCL), which represents a powerful technique involving a totally comprehensive method for screening peptide libraries and allows for the identification of undefined CTL epitopes as well as superagonist ligands (25, 36). In contrast to the genetic screen described in this paper, which identifies single amino acid substitution APLs, PS-SCL screens peptides that are randomized at every position except one. Although PS-SCL scanning has been shown to be capable of identifying superagonist analogues (26, 27), the need to deduce the sequence of the putative superagonists and the subsequent testing of large numbers of potentially nonproductive APLs that are non-cross-reactive for the native eptiope present additional hurdles.

In this study, from a screen of nine positional APL libraries, we identify three novel agonists of MART-126–35. Because our goal was to identify APLs of MART-126–35 more effective than the most effective APL described to date, MART-126–35 A27L, we chose to use A27L as the basis of our screen and fixed leucine into P2 of all of the APLs screened. Two of the identified agonist peptides contained P1 substitutions of glutamate for glycine and serine. Previous studies have demonstrated that glutamate at P1 of MART-126–35 is not ideal for the most effective binding of the peptide to HLA-A2 (14, 18). In 1998, Valmori et al. demonstrated that substituting an alanine residue for the glutamate residue at P1 of MART-126–35 resulted in significantly higher affinity of the peptide for HLA-A2 (18). Importantly, as compared with the native sequence, the alanine-containing agonist peptide was recognized significantly better by MART-126–35–specific CTL clones. The same group demonstrated that a substitution of glutamate at P1 of MART-126–35 with tyrosine or phenylalanine residues also increased both the binding of these agonists to HLA-A2 and their recognition by MART-126–35–specific CTL clones. Given these findings, it is not surprising that we would identify enhancing mutations at P1. However, in the studies just described, it was not made clear what effect that the P1 mutants would have when leucine simultaneously occupies the N-terminal anchor position.

In this report we identified an agonist of MART-126–35 that contains a methionine substitution at P8 for leucine (L33M). Preliminary data suggest that this agonist binds better to HLA-A2 relative to A27L but, like the other agonists, is not recognized by all of the MART-126–35–specific CTL clones used in this study. The crystal structures of MART-126–35 A27L generated by Sliz et al. (28) and Borbulevych et al. (22) do not suggest that L33 is closely associated with binding to HLA-A2.

The finding that different agonist peptides could be recognized by different CTL clones suggested to us that any given agonist APL may be more or less effective for different patients. We confirmed this hypothesis by performing in vitro stimulations with the identified agonist peptides on the PBMCs of multiple melanoma patients. We found that although a given agonist APL might have a high stimulatory capacity for one patient, it could be relatively ineffective for another patient. This would suggest that different clones are being mobilized with different agonist peptides. Indeed, we went on to show that, at least for one patient, unique MART-126–35–specific CTL clones are being generated with different agonist peptides. Importantly, all of these clones were of sufficient affinity to recognize endogenously processed and presented epitopes of MART-1 and kill MART-1+, HLA-A2+ tumors. This is not the first demonstration that different agonists can activate different Ag-specific CTL populations. Recently, Hou et al. (7) demonstrated that the superagonist epitope of carcinoembryonic Ag (CEA), called Cap1-6D, generated unique CEA-specific CTL clones in comparison with Cap1, the native epitope. In that report, however, the superagonist proved to generate CTLs of lower “functional avidity,” which were significantly less effective at killing CEA-positive tumor targets. Similarly, in a recent vaccination study by Speiser et al. (21) utilizing unmodified MART-126–35 or the superagonist MART-126–35 A27L in conjunction with the potent adjuvant CpG, the authors found that although the superagonist MART-126–35 A27L routinely generated a higher percentage of Ag-specific CTLs, the CTLs that were generated displayed considerably lower antitumor functional avidity than the Ag-specific CTLs that were generated using the unmodified antigenic peptide. In 1998, Rivoltini et al. (16) demonstrated that a superagonist of MART-126–35, called 1L, could generate unique MART-1–specific CTL clonotypes relative to the native peptide. In that study, the authors showed that 1L could more effectively elicit MART-1–specific CTL responses from the PBMCs of five different melanoma patients.

Our attempts to directly compare the functional avidity of MART-126–35–specific CTLs generated via unmodified MART-126–35 peptide with CTLs generated via the analogues described here have been hampered by the inability to generate MART-126–35 tetramer- and CD8-positive T cell populations using the unmodified peptide (data not shown). Although we do not directly address the functional avidity of the CTLs generated in this study via agonist peptides relative to those generated with the unmodified peptide, we show that these newly identified agonist MART-126–35 peptides elicit different Ag-specific CTL responses from patient to patient and that the generated CTL populations are capable of effectively killing tumors. Thus, these agonist APLs might be considered “conditional” superagonist ligands. It is probable that by using several unique MART-126–35–specific CTL clones in the initial screen that we can identify other potential superagonist peptides. Given that naive MART-1–specific CTL populations are uniquely abundant and highly diverse in healthy individuals, we will plan to determine how well our findings translate to other Ags that generally have significantly lower specific precursor CTL populations.

We believe the findings of this study suggest the promise of identifying panels of potential tumor-associated superagonist pep-tides, any of which may be most effective at generating potent antitumor CTL responses from a given patient. Further, we suggest that the novel genetic APL screen described here is a rapid, robust, and inexpensive approach for identifying superagonist APLs.

We thank Jianhong Cao in the Immune Monitoring Core Lab (Fred Hutchinson Cancer Research Center, Seattle, WA) for generating the peptide-MHC tetramers used in this study and Rebecca Rodmyre, Ivy Lai, and Kaye Dowdy for assistance in the preparation of T cell clones.

Disclosures The authors have no financial conflicts of interest.

This work was supported by Grants 3 P50 CA083636 and R33 CA122904 from the National Institutes of Health, the National Cancer Institute, and The J. Orin Edson Foundation. C.Y. is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.

Abbreviations used in this paper:

APL

altered peptide ligand

CEA

carcinoembryonic Ag

DC

dendritic cell

P

position

PS-SCL

positional scanning synthetic combinatorial library.

1
Zaremba
S.
,
Barzaga
E.
,
Zhu
M.
,
Soares
N.
,
Tsang
K. Y.
,
Schlom
J.
.
1997
.
Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
Cancer Res.
57
:
4570
4577
.
2
Boon
T.
,
van der Bruggen
P.
.
1996
.
Human tumor antigens recognized by T lymphocytes.
J. Exp. Med.
183
:
725
729
.
3
Knuth
A.
,
Wölfel
T.
,
Klehmann
E.
,
Boon
T.
,
Meyer zum Büschenfelde
K. H.
.
1989
.
Cytolytic T-cell clones against an autologous human melanoma: specificity study and definition of three antigens by immunoselection.
Proc. Natl. Acad. Sci. USA
86
:
2804
2808
.
4
Espinoza-Delgado
I.
2002
.
Cancer vaccines.
Oncologist
7
(
Suppl 3
):
20
33
.
5
Overwijk
W. W.
,
Restifo
N. P.
.
2001
.
Creating therapeutic cancer vaccines: notes from the battlefield.
Trends Immunol.
22
:
5
7
.
6
Dunn
G. P.
,
Old
L. J.
,
Schreiber
R. D.
.
2004
.
The immunobiology of cancer immunosurveillance and immunoediting.
Immunity
21
:
137
148
.
7
Hou
Y.
,
Kavanagh
B.
,
Fong
L.
.
2008
.
Distinct CD8+ T cell repertoires primed with agonist and native peptides derived from a tumor-associated antigen.
J. Immunol.
180
:
1526
1534
.
8
Kazansky
D. B.
2007
.
Intrathymic selection: new insight into tumor immunology.
Adv. Exp. Med. Biol.
601
:
133
144
.
9
McMahan
R. H.
,
Slansky
J. E.
.
2007
.
Mobilizing the low-avidity T cell repertoire to kill tumors.
Semin. Cancer Biol.
17
:
317
329
.
10
Morgan
D. J.
,
Kreuwel
H. T.
,
Fleck
S.
,
Levitsky
H. I.
,
Pardoll
D. M.
,
Sherman
L. A.
.
1998
.
Activation of low avidity CTL specific for a self epitope results in tumor rejection but not autoimmunity.
J. Immunol.
160
:
643
651
.
11
Ohashi
P. S.
,
Oehen
S.
,
Buerki
K.
,
Pircher
H.
,
Ohashi
C. T.
,
Odermatt
B.
,
Malissen
B.
,
Zinkernagel
R. M.
,
Hengartner
H.
.
1991
.
Ablation of “tolerance” and induction of diabetes by virus infection in viral antigen transgenic mice.
Cell
65
:
305
317
.
12
Zehn
D.
,
Bevan
M. J.
.
2006
.
T cells with low avidity for a tissue-restricted antigen routinely evade central and peripheral tolerance and cause autoimmunity.
Immunity
25
:
261
270
.
13
Chen
J. L.
,
Dunbar
P. R.
,
Gileadi
U.
,
Jäger
E.
,
Gnjatic
S.
,
Nagata
Y.
,
Stockert
E.
,
Panicali
D. L.
,
Chen
Y. T.
,
Knuth
A.
, et al
.
2000
.
Identification of NY-ESO-1 peptide analogues capable of improved stimulation of tumor-reactive CTL.
J. Immunol.
165
:
948
955
.
14
Valmori
D.
,
Fonteneau
J. F.
,
Lizana
C. M.
,
Gervois
N.
,
Liénard
D.
,
Rimoldi
D.
,
Jongeneel
V.
,
Jotereau
F.
,
Cerottini
J. C.
,
Romero
P.
.
1998
.
Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.
J. Immunol.
160
:
1750
1758
.
15
Fong
L.
,
Hou
Y.
,
Rivas
A.
,
Benike
C.
,
Yuen
A.
,
Fisher
G. A.
,
Davis
M. M.
,
Engleman
E. G.
.
2001
.
Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy.
Proc. Natl. Acad. Sci. USA
98
:
8809
8814
.
16
Rivoltini
L.
,
Kawakami
Y.
,
Sakaguchi
K.
,
Southwood
S.
,
Sette
A.
,
Robbins
P. F.
,
Marincola
F. M.
,
Salgaller
M. L.
,
Yannelli
J. R.
,
Appella
E.
, et al
.
1995
.
Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.
J. Immunol.
154
:
2257
2265
.
17
Singh
R. A.
,
Zang
Y. C.
,
Shrivastava
A.
,
Hong
J.
,
Wang
G. T.
,
Li
S.
,
Tejada-Simon
M. V.
,
Kozovska
M.
,
Rivera
V. M.
,
Zhang
J. Z.
.
1999
.
Th1 and Th2 deviation of myelin-autoreactive T cells by altered peptide ligands is associated with reciprocal regulation of Lck, Fyn, and ZAP-70.
J. Immunol.
163
:
6393
6402
.
18
Valmori
D.
,
Gervois
N.
,
Rimoldi
D.
,
Fonteneau
J. F.
,
Bonelo
A.
,
Liénard
D.
,
Rivoltini
L.
,
Jotereau
F.
,
Cerottini
J. C.
,
Romero
P.
.
1998
.
Diversity of the fine specificity displayed by HLA-A*0201-restricted CTL specific for the immunodominant Melan-A/MART-1 antigenic peptide.
J. Immunol.
161
:
6956
6962
.
19
McCue
D.
,
Ryan
K. R.
,
Wraith
D. C.
,
Anderton
S. M.
.
2004
.
Activation thresholds determine susceptibility to peptide-induced tolerance in a heterogeneous myelin-reactive T cell repertoire.
J. Neuroimmunol.
156
:
96
106
.
20
Li
Y.
,
Bleakley
M.
,
Yee
C.
.
2005
.
IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8 T cell response.
J. Immunol.
175
:
2261
2269
.
21
Speiser
D. E.
,
Baumgaertner
P.
,
Voelter
V.
,
Devevre
E.
,
Barbey
C.
,
Rufer
N.
,
Romero
P.
.
2008
.
Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen.
Proc. Natl. Acad. Sci. USA
10504
:
3849
3854
.
22
Borbulevych
O. Y.
,
Insaidoo
F. K.
,
Baxter
T. K.
,
Powell
D. J.
 Jr.
,
Johnson
L. A.
,
Restifo
N. P.
,
Baker
B. M.
.
2007
.
Structures of MART-126/27-35 peptide/HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition.
J. Mol. Biol.
372
:
1123
1136
.
23
Ding
Y. H.
,
Baker
B. M.
,
Garboczi
D. N.
,
Biddison
W. E.
,
Wiley
D. C.
.
1999
.
Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical.
Immunity
11
:
45
56
.
24
Kersh
G. J.
,
Miley
M. J.
,
Nelson
C. A.
,
Grakoui
A.
,
Horvath
S.
,
Donermeyer
D. L.
,
Kappler
J.
,
Allen
P. M.
,
Fremont
D. H.
.
2001
.
Structural and functional consequences of altering a peptide MHC anchor residue.
J. Immunol.
166
:
3345
3354
.
25
Nino-Vasquez
J. J.
,
Allicotti
G.
,
Borras
E.
,
Wilson
D. B.
,
Valmori
D.
,
Simon
R.
,
Martin
R.
,
Pinilla
C.
.
2004
.
A powerful combination: the use of positional scanning libraries and biometrical analysis to identify cross-reactive T cell epitopes.
Mol. Immunol.
40
:
1063
1074
.
26
Rubio-Godoy
V.
,
Pinilla
C.
,
Dutoit
V.
,
Borras
E.
,
Simon
R.
,
Zhao
Y.
,
Cerottini
J. C.
,
Romero
P.
,
Houghten
R.
,
Valmori
D.
.
2002
.
Toward synthetic combinatorial peptide libraries in positional scanning format (PS-SCL)-based identification of CD8+ tumor-reactive T-cell ligands: a comparative analysis of PS-SCL recognition by a single tumor-reactive CD8+ cytolytic T-lymphocyte clone.
Cancer Res.
62
:
2058
2063
.
27
Venturini
S.
,
Allicotti
G.
,
Zhao
Y.
,
Simon
R.
,
Burton
D. R.
,
Pinilla
C.
,
Poignard
P.
.
2006
.
Identification of peptides from human pathogens able to cross-activate an HIV-1-gag-specific CD4+ T cell clone.
Eur. J. Immunol.
36
:
27
36
.
28
Sliz
P.
,
Michielin
O.
,
Cerottini
J. C.
,
Luescher
I.
,
Romero
P.
,
Karplus
M.
,
Wiley
D. C.
.
2001
.
Crystal structures of two closely related but antigenically distinct HLA-A2/melanocyte-melanoma tumor-antigen peptide complexes.
J. 2Immunol.
167
:
3276
3284
.