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The Journal of Immunology, 1998, 161: 877-889.
Copyright © 1998 by The American Association of Immunologists

Stringent Allele/Epitope Requirements for MART-1/Melan A Immunodominance: Implications for Peptide-Based Immunotherapy

Maria P. Bettinotti*, Christina J. Kim{dagger}, Kang-Hun Lee*, Matthew Roden{dagger}, Janice N. Cormier{dagger}, Monica Panelli{dagger}, Kenneth K. Parker{ddagger} and Francesco M. Marincola1,*,{dagger}

* HLA Laboratory, Department of Transfusion Medicine, Clinical Center, {dagger} Surgery Branch, Division of Clinical Sciences, National Cancer Institute, and {ddagger} Biological Resources Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The exclusiveness of the relationship between peptide and HLA alleles, combined with their extensive polymorphism, emphasizes the need for immunization strategies based on endogenous processing of full length proteins (containing multiple epitopic determinants) for presentation to T cells. This could allow vaccination regardless of the patient’s HLA phenotype, assuming that individual molecules can be efficient T cell Ags in association with various HLA alleles. An endogenous system of Ag presentation was developed using dendritic cells infected with recombinant viral vectors expressing the melanoma-associated Ag MART-1/Melan A. CD8+ T cells from melanoma patients were activated in vitro by coincubation with infected dendritic cells and tested for recognition of HLA-A-matched melanoma targets. This allowed the analysis of T cell induction in association with any HLA-A allele of a given patient’s phenotype. In this system, MART-1/Melan A could not efficiently immunize in association with HLA-A alleles other than A*0201, including the one residue variant from A*0201: HLA-A*0226. Clonal analysis of MART-1/Melan A-specific CTL confirmed that MART-1/Melan A immunodominance is strongly restricted to the AAGIGILTV/HLA-A*0201 combination. The stringent epitope/allele requirements for MART-1/Melan A/TCR interactions were not associated with limitations in the TCR repertoire. In conclusion, autologous induction of MART-1/Melan A CTL by whole Ag processing and presentation is restricted to a unique allele/ligand combination and is excluded by minimal changes in HLA structure. Thus, whole protein vaccination for small m.w. Ags may provide no further advantage over a peptide-based approach.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cellular immune responses against melanoma have recently been exploited for the development of active immunization strategies for the immunotherapy of cancer. Among the several melanoma-associated Ags (MAA)2 so far identified, MART-1/Melan A has received particular attention because of its "immunodominance" in patients carrying the HLA-A*0201 phenotype (1). Indeed, ~90% of tumor-infiltrating lymphocytes (TIL) originated from HLA-A*0201 patients recognize the MART-127–35 (AAGIGILTV) sequence (2), suggesting its usefulness for the development of immunotherapeutic strategies. Since HLA-A*0201 is the predominant allele in the North American Caucasian population, which is most frequently affected by this disease (3, 4), peptide-based vaccinations restricted to HLA-A2 patients were initiated by administering the MART-127–35 peptide with the purpose of enhancing T cell reactivity against melanoma in vivo.

Peptide-based vaccinations have several potential advantages: simplicity and low cost of administration, minimal toxicity, and evidence of in vivo effectiveness in eliciting T cell activation (5). We recently monitored T cell activation in the PBMC of patients undergoing the first anti-melanoma, peptide-based vaccination trial. This trial was based on the s.c. administration of the MART-127–35 peptide in conjunction with IFA. A more than threefold increase in IFN-{gamma} release by MART-127–35-specific CTL was noted in 15 of 18 patients treated with this regimen (6). Similar results were reproduced in a cohort of patients undergoing gp100/Pmel 17 epitope-based vaccination (7). Our unpublished observations (available upon request) have shown that the powerful activation of T cell reactivity observed after peptide administration could not be achieved by other modalities of vaccination, based on the in vivo induction of expression, processing, and presentation of whole MAA (MART-1/Melan A or gp100/Pmel 17) using recombinant technology.

One disadvantage of epitope-based vaccination is the exclusiveness of the relationship between peptide and HLA alleles. Because of the intrinsic limitations posed by HLA polymorphism (8, 9, 10, 11), a vaccination strategy based on endogenous processing and presentation of whole T cell Ag (containing the maximum epitopic potential) could broaden the application of MAA vaccines. An ideal vaccine might be engineered that could be offered regardless of the patient’s HLA phenotype and without identification of immunogenic epitopes. Therefore, the concept of whole Ag vaccination has gained significant popularity recently as an alternative to peptide-based vaccination. To be successful, this strategy requires that individual molecules function with efficiency comparable to T cell Ags in association with various HLA alleles. Few lines of evidence, however, show that MAA can comparably induce T cell reactivity in association with different HLA class I restriction elements. For example, there is no evidence that MART-1/Melan A can be recognized in association with HLA alleles other than A*0201. For larger MAA recognized by HLA-A*0201-restricted CTL, epitopic determinants associated with other HLA-A class I alleles have been identified, such as HLA-A3 for gp100/pMel 17 (12), HLA-A24 and -B44 for tyrosinase (13, 14), and HLA-A1 for MAGE-3 (15). However, responses to these epitopes are generally observed less frequently. For example, tyrosinase, which is predominantly recognized by HLA-A24-restricted TIL (13), is probably not a dominant immunogen in association with HLA-A*0201, as none of 10 HLA-A*0201-restricted TIL cultures analyzed recognized this MAA (16).

Although we are aware that the ease with which a MAA can elicit T cell reactivity in vitro may not directly correlate with clinical significance (17), we hypothesized, for the purpose of this study, that differences in induction of T cell reactivity under comparable conditions of stimulation could, in principle, be suggestive of different immunogenic potential. In this study, therefore, we compared the ability of MART-1/Melan A to elicit non-HLA-A*0201-restricted T cell-specific CTL responses using an autologous Ag presentation system consisting of dendritic cells (DC) infected with recombinant vaccinia virus (rVV) encoding for MART-1/Melan A followed by a second stimulation with DC infected with recombinant fowl pox virus (rFP) encoding for MART-1/Melan A (18). When stimulated under conditions in which HLA-A*0201-restricted anti-melanoma reactivity could be frequently observed, no reactivity was detected in association with other HLA-A class I alleles no matter how structurally similar these alleles are to HLA-A*0201.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture medium (CM)

Iscove’s medium (Life Technologies, Grand Island, NY) with 10% heat-inactivated human AB serum, 10 mM HEPES buffer, 100 U/ml penicillin-streptomycin, 0.5 mg/ml amphotericin B, 0.03% glutamine was used for all experiments.

Peptide synthesis: MART-1, G9-209-2 M, and Flu M1 peptides

A panel of 110 overlapping nonamer peptides (MART-11–9 through MART-1110–118) encompassing the MART-1/Melan A sequence (118 amino acids) was synthesized by a solid phase method using a multiple peptide synthesizer and purified by HPLC, as previously described (2). Several size-variant analogues of MART-127–35 encompassing this nonamer-flanking sequences (AEEAAGIGILTVILV) were also synthesized. Each peptide was HPLC purified, and the presence of truncated peptides was excluded by mass spectrometry. gp100–209 (ITDQVPFSV) and gp100-209-2 M peptides (IMDQVPFSV) were produced by Chiron Mimotopes Peptide Systems (San Diego, CA). The G9-209-2 M peptide is a nine-amino acid sequence modified at position 2 (T -> M) from the natural gp100–209 epitope (16). This modification enhances binding to HLA-A*0201 and induction of T cell reactivity (19). Flu-M158–66 (GILGFVFTL) from the influenza matrix protein was synthesized by Multiple Peptide Systems (San Diego, CA). All peptides were used at a final concentration of 1 µM (unless otherwise stated) from aliquots dissolved in 100% DMSO (Sigma Chemical, St. Louis, MO) and stored at -70°C.

T2, EBV-B, melanoma, and TIL cultures

T2 cells were used as targets in cytokine release assays for HLA-A*0201-restricted epitopes. This cell line expresses HLA-A*0201 and is defective in endogenous processing, which enhances the effectiveness of exogenous peptide loading (20, 21). B lymphoblastoid cells derived from peripheral blood were transformed with exogenous EBV (22) or were obtained from the European Collection of Animal Cell Cultures (23). In particular, the following EBV-B cell lines were used: LCL.721 (HLA-A*0201), RML (HLA-A*0204), AM (HLA-A*0205), and CLA (HLA-A*0206). Other EBV-B cell lines were developed from patients, and typing of relevant HLA class I alleles was done by sequencing.

HLA-matched tumor targets. To assess recognition of naturally processed and presented MAA, cell lines generated from patients at the National Cancer Institute and possessing different HLA types were fully characterized for HLA class I, MART-1/Melan A, and gp100/Pmel 17 expression. Furthermore, the following cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA): the breast cancer cell line MDA-231 (ATCC HTB 26), SK23-MEL (ATCC HTB 71), and A375-MEL (ATCC CRL 1619). The tumor lines were maintained in monolayer culture in CM (24).

TIL cultures. TIL 1235 and TIL 1520 were generated from metastases of HLA-A*0201 melanoma patients. These cultures were >99% CD8+. The CD8+ T cell clone A42 was established from a TIL culture (1, 2). Both TIL 1235 and clone A42 recognize MART-127–35 (25). TIL 1520 recognizes the gp100–209 sequence of gp100/pMel 17. All TIL recognize HLA-A*0201/MAA-expressing tumors. TIL were propagated in CM plus 6000 IU/ml of IL-2 (Chiron Corporation, Emeryville, CA).

HLA typing

HLA class I typing and HLA-A*02 subtyping was done by sequence-specific primer PCR of genomic DNA (4) isolated from PBL or, when relevant, from EBV-B and tumor lines (26). When indicated, HLA alleles were typed by direct sequencing of PCR products amplified from genomic (27) or complementary DNA (28). The name A*0226 has been officially assigned by the World Health Organization Nomenclature Committee (29); the GenBank accession number is AF008933.

Development of DC and in vitro sensitization of CD8+ T cells

Preparation of CD8+ T cells and DC. Leukaphereses were performed in patients with metastatic melanoma referred for treatment to the Surgery Branch, National Cancer Institute. All PBL collections were done before enrollment of patients into a MAA-specific vaccination protocol, and therefore CTL reactivity reflected the natural predisposition to react against MAA. PBMC were separated by Ficoll-Hypaque gradient and used for preparation of DC and T cells. CD8+ enrichment was achieved by positive selection on biomagnetic beads (Dynal Corporation, Great Neck, NY). In all experiments, the T cell population was >95% CD8+ (anti-CD8 mAb, Becton Dickinson, San Jose, CA). After Ficoll-Hypaque separation, 1 to 3 x 108 PBMC were processed for preparation of DC by culture in 75-cm2 flasks for 2 to 3 h at 37°C as previously described (18, 30). The nonadherent cells were removed, and the adherent cells were cultured for 5 to 7 days in 10 ml of CM supplemented with human recombinant granulocyte/macrophage-CSF (hrGM-CSF, 2000 IU/ml; PeproTech, Rocky Hill, NJ) and human rIL-4 (hrIL-4, 2000 IU/ml; PeproTech) (18).

Infection of DC with pox viruses. To induce endogenous expression of Ag, DC were infected with rVV or rFP encoding for MART-1/Melan A (rVV-MART-1, rFP-MART-1), gp100 (rVV-gp100, rFP-gp100), or Flu (rVV-Flu) (18, 31). DC (1 x 106) were incubated in a minimal volume of CM at 37°C with rVV (1 x 107 plaque-forming units/ml) for 1 h. The DC were then washed twice, and one aliquot was used to quantify the amount of virally driven expression of MAA (M2-7C10, MART-1/Melan A-specific (32), and HMB45, gp100/Pmel 17-specific mAb; Biogenex Laboratories, San Ramon, CA) by intracellular FACS as previously described (33). DC preparations showing a >50% rate of expression of the relevant MAA were used for in vitro sensitization.

In vitro sensitization of PBL. Cells (4–5 x 106/well CD8+ lymphocytes) were coincubated with 1 x 106 rVV-infected DC in 24-well plates. CD8+ T cell cultures were restimulated after 1 wk with rFP-infected DC. rFP vectors were used for the restimulation because they are not immunologically cross-reactive with rVV and therefore are least likely to enhance a virally (rVV)-driven CTL expansion. IL-2 (300 IU/ml) was added 24 h after stimulation and every 2 to 3 days thereafter. Effector cells were tested 7 to 10 days after restimulation.

Peptide pulsing of DC. In some instances, in vitro sensitization was carried with DC presenting exogenously provided peptides. Recovered DC were pulsed for 2 h at 37°C (1 x 106 cells/ml and 1 µg/ml peptide).

Development of T cell clones. Bulk CTL cultures were cloned by limiting dilution according to Riddell’s technique (34, 35). Cultures were plated in 96-well plates at 1 or 10 cells/well and maintained in the presence of OKT3 (30 ng/ml), 50,000/well irradiated allogeneic PBMC (3,000 rads), and 10,000/well irradiated EBV-B cells (10,000 rads). IL-2 (120 IU/ml) was added every other day. Clones were selected according to their reactivity against HLA-matched, MAA-expressing tumor targets.

Assessment of CTL reactivity. T cells (105) were coincubated with 105 stimulator cells for 24 h at 37°C in 200 µl of CM. IL-2, IL-4, IL-12, IFN-{gamma}, GM-CSF, and TNF-{alpha} in the supernatants was measured by Quantikine ELISA kits (R&D Systems, Minneapolis, MN). Data are presented as picograms per milliliter. Cytokine release was significant when threefold higher in test than in control conditions and above 100 pg/ml.

Reconstitution assay and determination of ß2-microglobulin (ß2m) dissociation rates

Peptide binding was analyzed by HLA class I heavy chain reconstitution assay and quantified by ß2m dissociation rates (36). Heterotrimers containing 125I-labeled ß2m, soluble HLA class I heavy chains, and peptides of interest were purified by gel filtration, and aliquots were analyzed at various time points to determine of the rate of ß2m dissociation by injection over a second gel filtration column. All peptides were assessed at a 10-µM concentration. Reconstitution values are given as the percentage of 125I-ß2m in the heterotrimer fraction over the total 125I-ß2m: (125I-ß2m-HLA/125I-ß2m-HLA + 125I-ß2m) x 100. Dissociation rates represent time required for 50% reduction in heterotrimers/125I-ß2m radioactivity.

Vß-specific analysis of T cell clonality

T cell clonality was analyzed by Vß-specific PCR amplification. Direct heteroduplex analysis and sequencing were used to screen and characterize T cell clones belonging to the same Vß family (37). Vß-specific primer sequences were selected based on the complete DNA sequence of the human ß-TCR locus (38).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
rVV- and rFP-driven induction of anti-MART-1/Melan A reactivity

An autologous system of rVV- or rFP-driven expression, processing, and presentation of endogenous Ag was engineered. This system theoretically allows equal opportunity of CTL induction in association with various HLA/epitope combinations. MART-1/Melan A protein expression was induced in DC by infection with rVV-MART-1 (primary in vitro stimulation) followed, a week later, by infection with rFP-MART-1 (secondary in vitro stimulation). Preliminary data had shown that this binary system of stimulation was highly successful in inducing MART-1/Melan A-specific, HLA-A*0201-restricted T cells in melanoma patients not previously exposed to immunologic manipulation (18). We also addressed the effect of viral Ag expression as a possible source of misleading culture results. Surprisingly, no pox virus-specific reactivity was observed in the absence of recent in vivo exposure to rVV. This finding was independent of an individual’s previous juvenile exposure to small pox vaccine. In contrast, CD8+ cells induced from patients recently exposed to either rVV-MART-1 or rVV-gp100 for vaccination purposes demonstrated strong anti-viral reactivity, which concealed the MAA-specific T cell response (Table IGo). Neither humoral nor cellular cross-reactivity was observed between rVV and rFP (data not shown). Thus, the expression of viral molecules did not interfere with the assay results in the naive population analyzed in this study.


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  		Table I. Effect of recent in vivo exposure to rVV-gp100 on the development of MAA and pox virus-specific T cell reactivity in vitroa

 
Induction of anti-MART-1/Melan A and antitumor reactivity in HLA-A*0201-expressing and -nonexpressing patients

We attempted to induce MART-1/Melan A-specific CTL from PBMC obtained from 17 individuals carrying the HLA-A*0201 phenotype and not previously exposed to MAA-specific vaccination. We observed HLA-A*0201-restricted MART-1 reactivity in 15 of the 17 patients (88%). In all cases, MART-1 reactivity was directed against the immunodominant epitope MART-127–35 (Tables II and III). Among the 15 MART-1-reactive cultures, 13 could also recognize HLA-A*0201-matched tumors (76%). The two CTL cultures unresponsive to MART-127–35 could not recognize HLA-matched tumors. In only one case was non-HLA-A*0201-restricted recognition of tumor noted (patient 5, Tables II, IV, and V). Thus, in paired conditions of stimulation, HLA-A alleles different from HLA-A*0201 failed to present MART-1/Melan A to autologous CTL with an efficiency comparable to HLA-A*0201 (Fisher’s exact test, p < 0.001). Possible recognition of MAA associated with HLA-B and -C locus alleles was also tested when autologous EBV-B lines were available (6 patients). The EBV-B lines were infected with rVV-expressing relevant (rVV-MART-1) or irrelevant (rVV-gp100) Ag and tested for recognition. In this fashion, a panel of other common HLA-B (including HLA-B7, B8 B44 and B57) and HLA-C alleles could be tested. In no case was specific recognition of rVV-MART-1-infected autologous EBV-B lines noted. The dominant effect of the HLA-A*0201/MART-127–35 combination on CTL expansion and activation could have concealed cryptic CTL responses. We therefore analyzed PBL from 12 additional melanoma patients not expressing HLA-A*0201 (Table IIGo). In no case was antitumor reactivity recognized independently from the level of structural divergence from HLA-A*0201 of the HLA allele analyzed. An extreme was represented by patient 21 in which a new HLA-A*02 allele was identified as HLA-A*0226. This allele is characterized by a point mutation (T -> A) with respect to HLA-A*0201, at nucleotide 527, which corresponds to a substitution at position 152 from a hydrophobic valine to a hydrophilic glutamic acid.


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  		Table II. Induction of MART-1/Melan A and tumor-specific reactivity in HLA-A*0201 expressing or nonexpressing subjectsa

 
Screening of MART-1/Melan A-specific CTL for alternate epitope/HLA reactivities

Tumor lines may be unpredictable in MAA expression, processing, or presentation. Therefore, CTL cultures generated either from HLA-A*0201-expressing or -nonexpressing individuals were screened by incubation with HLA-A matched EBV-B cell lines separately pulsed with each of 110 overlapping MART-1 nonamer peptides (1 µM). Six strongly MART-127–35-reactive, HLA-A*0201-restricted (patients 1, 2, 4, 5, 9, and 14), four MART-127–35-nonreactive or poorly reactive HLA-A*0201-restricted (patients 3, 6, 7, 12), and six non-HLA-A*0201 cultures (patient 19, 22, 24, 25, 26, 27) were tested. Although reactivity could be consistently detected by pulsing HLA-A*0201-expressing targets with MART-127–35 in MART-1-reactive cultures from HLA-A*0201 patients, no reactivity toward other HLA-A allele/peptide combination was noted (Table IIIGo). Thus, these experiments failed to induce demonstrable anti-MART-1/Melan A specificity associated with HLA alleles different from HLA-A*0201, underscoring the high stringency of the MART-1/HLA-A*0201 combination. It is, however, important to note that for Ags different from MART-1/Melan A, exceptions to the epitope/allele stringency demonstrated by MART-1 were noted as already reported by others (39). For example, in patient 15 (HLA-A*0201, A*0205) stimulation of CD8+ cultures with DC infected with rVV-Flu induced Flu M158–66 reactivity in the context of HLA-A*0201 and A*0205.


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  		Table III. Reactivity of CTL toward consecutive overlapping nine-mer peptides (MART-11–9 through MART-1110–118) pulsed on EBV-B cell linesa

 
Analysis of tumor recognition by CTL clones in HLA-A*0201 and non-A*0201 patients

In patients bearing the HLA-A*0201 phenotype, a weak, subdominant non-HLA-A*0201-restricted MART-1 reactivity could have been concealed by nonspecific proliferation of CTL in the presence of IL-2 or by predominant expansion of HLA-A*0201-restricted MART-1-specific CTL. This possibility was tested by clonal analysis of early bulk CTL cultures. In three patients who showed good HLA-A*0201-restricted recognition of melanoma, CTL clones were developed by limiting dilution. All three patient cultures efficiently recognized MART-127–35 in the context of HLA-A*0201- and HLA-A*0201-expressing tumors (Table IVGo). In addition, the CTL of patient 5 (HLA-A*0201, -*01) suggested the recognition of HLA-A*01-matched targets. Cloning was performed at limiting dilution of 100, 10, and 1 cell/well. Because of the good yield of reactive clones at the higher dilution, the 100 cell/well cultures were not analyzed (Table VGo).


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  		Table IV. IFN-{gamma} release by bulk CTL cultures used for the development of clones recognizing MART-1a

 

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  		Table V. Analysis of HLA-A restriction of anti-MART-1 reactivity in CTL clonesa

 
Screening was performed by analyzing IFN-{gamma} release upon incubation of the clones with SK23-MEL (HLA-A*01, 0201/MART-1/Melan A positive) or with 888-MEL (HLA-A*01, *24/MART-1/Melan A positive). Several HLA-A*0201-restricted CTL clones were identified in all patients. However, only in patient 5 could we identify non-HLA-A*0201-restricted tumor recognition by one of 40 melanoma- specific clones studied (Table VIGo). This clone was noted to recognize three different HLA-A*01 melanoma targets but not other HLA-A*01 target cells expressing little or no MART-1/Melan A. The HLA-A*01 restriction was formally proven by recognition of 624.38-MEL (HLA-A*0201,*03) transduced with HLA-A*01 cDNA (624.38/A1-MEL). An extensive search for the identification of the epitope recognized by this clone was performed by pulsing HLA-A*01-positive 888-EBV with the 110 overlapping nonamer MART-1 peptides; however, none of them induced IFN-{gamma} release. It is possible that this CTL recognizes a non-nonamer or a posttranslationally modified epitope. In addition to this exception, clonal analysis of three CTL cultures failed to unveil subdominant MART-1 specificities associated with HLA-A alleles other than HLA-A*0201.


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  		Table VI. Characterization of HLA-A1-restricted recognition of tumor by a CTL clone generated from a bulk culture (patient 5)a

 
Restriction of HLA-A*0201-associated MART-1/Melan A immunodominance to MART-127–35

The high prevalence of HLA-A*0201-restricted anti MART-1 reactivity noted in melanoma patients was further characterized to assess whether epitopes other than MART-127–35 contributed to the immunodominant T cell response. Preliminary analyses had shown that none of the MART-1/Melan A-specific CTL cultures recognized peptide sequences other than MART-127–35. To further characterize the uniqueness of this recognition, the CTL clones from the three patients described in the previous section were tested for epitope specificity. In addition to MART-127–35, four MART-1/Melan A peptide sequences were selected (MART-131–39, MART-132–40, MART-135–43, MART-156–64) that had been proposed by others as alternative T cell epitopes in the context of HLA-A*0201 (40, 41). Without exception, all CTL clones analyzed recognized uniquely MART-127–35. An example is illustrated for patient 4 (Table VIIGo). The specificity of epitope recognition was not associated with limitations in TCR repertoire. The TCR usage contrasts with the strict specificity of the MART-127–35 response and supports the concept that immunodominance correlates with capacity to maintain a broad TCR repertoire (42).


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  		Table VII. Epitope immunodominance of HLA-A*0201-restricted MART-1/Melan A-specific clones from patient 4 (IFN-{gamma} release shown in pg/ml/24 h)

 
In contrast to MART-1/Melan A, it was easy to demonstrate HLA-A*0201-restricted responses to multiple gp100 epitopes using the rVV/rFP system. Two of four gp100-specific CTL cultures could recognize more than one of the known gp100 epitopes (Table VIIIGo). The uniqueness of the dominance of HLA-A*0201/MART-127–35 combination could not be extended, therefore, to other MAA, particularly those of larger size such as gp100 (600 amino acids). Furthermore, the identification of multiple epitopes for gp100 correlated with the multiplicity of HLA-A*0201-restricted TIL specificities (16), suggesting that this in vitro system provides culture results comparable with natural CTL reactivity in vivo and indirectly supports the validity of the negative results obtained with MART-1/Melan A.


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  		Table VIII. Epitopes recognized by HLA-A*0201-restricted, gp100-specific CTL from three different melanoma patients induced with DC infected with rVV-gp100a

 
Size restriction of anti-MART-1/Melan A recognition in the context of HLA-A*0201 to the ... AEEAAGIGILTV peptide sequence (MART-124–35)

The specificity of the interaction between TCR and the immunodominant region of the MART-1/Melan A molecule has never been extensively analyzed in terms of peptide size requirements. To evaluate this question, we synthesized size analogues encompassing overlapping portions of the MART-127–35 flanking regions. These size analogues were then pulsed on T2 cells, and the reactivity (IFN-{gamma} release) of three different CTL clones was assessed. The three clones (A42, B10-98, and B10-106) utilized different Vß-chains and had different cytokine release patterns (Fig. 1GoA) and stringency of subtype restriction (Fig. 2Go). These clones demonstrated, however, quantitatively similar expression of CD3, TCR, CD54, CD56, Fas, and Fas ligand as determined by FACS analysis (data not shown) and, like the rest of the CTL clones analyzed in this study, their pattern of cytokine release reflected the TC1 type (43). As previously noted, the decamer MART-126–35 was more efficiently recognized than MART27–36 when pulsed in equimolar concentrations on T2 cells (Fig. 1GoB) (2). Furthermore, binding affinity to HLA-A*0201 correlated with recognition of MART-126–35 and MART-127–36 (125I-ß2m-HLA/125I-ß2m-HLA + 125I-ß2m x 100 = 67 and 14%, respectively). A similar correlation was observed for other size variants. The best binder among the four 12-mer sequences was MART-124–35, while the following three overlapping 12 mers demonstrated lower affinity (125I-ß2m-HLA/125I-ß2m-HLA + 125I-ß2m x 100 = 38, 11, 5, and 4, respectively). Similarly, among the 11 amino acid sequences, the highest binding was observed for MART-125–35 (125I-ß2m-HLA/125I-ß2m-HLA + 125I-ß2m x 100 = 22, 11, and 3 for MART-125–35, MART-126–36, and MART-127–37, respectively). Among 8-mer sequences, MART-128–35, which encompasses both anchor residues (P2 and P9) of the immunodominant nonamer MART-127–35, had good reconstitution values and was recognized (125I-HLA/125I-HLA + 125I-ß2m x 100 = 61). In contrast, MART-127–34 and MART-129–36, lacking either the Ala at P2 or Val at P9, did not bind to HLA-A*0201 (125I-HLA/125I-HLA + 125I-ß2m x 100 = 3 and 13, respectively) and were not recognized. Seven residue analogues did not bind to the HLA-A*0201. In summary, binding and recognition of MART-1/Melan A-immunodominant region: 1) requires the presence of both Ala28 and Val35; and 2) has higher tolerance for sequence extensions at the N terminus than at the C terminus of MART-127–35. MART-127–35 does not have the canonical Leu at P2, but has a C terminus highly compatible with the HLA-A*0201 binding motif, suggesting P9 as the dominant anchor residue for this peptide. It is possible that C-terminal variability could more deeply affect binding to HLA-A*0201.



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  		FIGURE 1. A, Dose-response plot of cytokine release (pg/ml/24 h) by three MART-127–35-specific CD8+ CTL clones stimulated with T2 cells exogenously pulsed with different concentrations of MART-127–35 peptide. Effector to stimulator ratio of 1:1. B, Cytokine release profile (IFN-{gamma}) of the same three clones stimulated with T2 cells exogenously pulsed with various size analogues of MART-127–35 peptide. The peptide sequence analyzed is underlined. In brackets is the percentage of heavy chain reconstitution rate (125I-HLA/125I-HLA + 125I-ß2m x 100).

 


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  		FIGURE 2. Cytokine release profile of MART-127–35-specific CD8+ CTL clones derived from the same patient (with the exception of A42). Data are presented as pg/ml/24 h. The CTL clones analyzed are, from top to bottom, B1-10, B1-30, B10-38, B10-45, B10-98, B10-106, B10-143, B10-176, B10-177, B10-182, A42, and gp100 TIL (control CTL for gp100–209). Effector cells were stimulated at a 1:1 ratio with EBV-B cells expressing different HLA-A*02 alleles and exogenously pulsed with equimolar (1 µM) concentrations of MART-127–35 (black bars) or gp100–209-2 M (white bars) peptide. Three different cytokine patterns were analyzed: GM-CSF (A), IFN-{gamma} (B), and TNF-{alpha} (C).

 
Subtype restriction of the anti-MART-1/Melan A (AAGIGILTV) sequence

Given the uniqueness of MART-127–35 as epitopic determinant for MART-1/Melan A immunodominance in the context of HLA-A*0201, we analyzed the stringency of its association with other HLA-A*0201 variants. The CTL clones derived from patient 4 were analyzed for cross-reactivity with EBV-B lines expressing various HLA-A*02 subtypes and exposed to exogenous MART-127–35. In particular, the following subtypes were selected: HLA-A*0204 (97 R -> M), A*0206 (9 F -> Y), and A*0226 (152 V -> E), representing single-residue variants from A*0201 and A*0205, differing from A*0201 by four residues and representing the second-most common HLA-A*02 allele in the Caucasian (44) and melanoma (4) population. The CTL clones exhibited different stringency of cross-reactivity, which was partially dependent upon the cytokine analyzed (Fig. 2Go, A–C). Clones A42 and B10-143 exhibited the highest level of specificity for the HLA-A*0201/MART-127–35 combination. Cross-reactivity with HLA-A*0204 was noted in these two clones when GM-CSF release was analyzed, suggesting that the absence of detectable IFN-{gamma} release was not an absolute indication of lack of interaction between TCR and HLA/peptide complex, but rather the result of declining avidity of the TCR upon gradual modification of the HLA/peptide conformation.

The majority of the CTL clones recognized the combination MART-127–35/HLA-A*0204, and only B10-48 revealed substantial cross-reactivity against HLA-A*0205 and A*0206/MART-127–35 complexes, establishing firmly that among the HLA-A*02 subtypes analyzed, HLA-A*0204 is functionally the closest allele to HLA-A*0201. HLA-A*0226 exhibited no cross-reactivity with any of the HLA-A*0201-restricted CTL clones. Binding of MART-127–35 to HLA-A*0204 and A*0205 has been already demonstrated (9). Therefore, an attempt was made to induce T cell reactivity against this epitope by pulsing DC with MART-127–35 in patients 15, 19, and 20 (all of them were A*0205). All such attempts were unsuccessful. HLA-A*0204 individuals were not tested because we could not identify A*0204 among 200 HLA-A2 melanoma patients or 500 normal HLA-A2 Caucasian subjects, suggesting that this allele is exceedingly rare in these populations (45). HLA-A*0226 did not allow cross-reactive recognition of MART-127–35 or other canonical HLA-A*0201 epitopes such as gp100–209 or Flu M158–66 by HLA-A*0201-restricted CTL. The functional difference between HLA-A*0226 and A*0204 is striking considering that both alleles vary from HLA-A*0201 by only one residue. It is possible, however, that the R -> M substitution at position 97 of HLA*0204 has only minor structural effects on the HLA/peptide complex and does not affect binding (9). On the other hand, V -> E substitution in position 152 of the HLA-A*0226 allele (Fig. 1Go) may directly affect the ability of this allele to bind canonical HLA-A*0201 epitopes due to the multiple interactions of position 152 with pockets C, D, and E (46). The ability of HLA-A*0226 to bind well-characterized, high affinity ligands of HLA-A*0201 was therefore tested (Table IGoX). None of the four peptides tested could stabilize the HLA-A*0226/ß2m complex. Endogenous processing and presentation of MART-1/Melan A by HLA-A*0226-expressing autologous DC was not capable of inducing MART-1/Melan A-specific CTL (Table IIGo). Similarly, DC infected with rVV-gp100 could not elicit gp100 CTL. Exogenous loading of DC with 1 µM of MART-127–35 or gp100–209-2 M (Tables X and XI) or Flu M158–66 (data not shown) failed to elicit a CTL response from postvaccination PBMC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The identification of MAA (47) has led to a number of clinical immunization trials. Although the identification of the first MAA occurred in association with HLA-A1 (48) due to the prevalence of HLA-A*0201 in the melanoma population (3, 4) a significant number of MAA have been subsequently identified in association with this allele. These MAA include MART-1/Melan A (1, 49), gp100/Pmel 17 (50), tyrosinase (51), MAGE-3 (52), and N-acetylglucosaminyltransferase V (53). Among the HLA-A*0201-associated MAA, MART-1/Melan A plays a dominant role: MART-1/Melan A is recognized by the majority of TIL from HLA-A*0201 melanoma patients (2); in vitro stimulation of PBMC with HLA-A*0201-matched melanoma targets predominantly induces anti-MART-1/Melan A reactivity (54); indirect evidence suggests that MART-1/Melan A reactivity is more readily induced than reactivity against other MAA (55, 56, 57). Furthermore, MART-1/Melan A is expressed by the majority of metastatic melanoma lesions (58), making it an appealing candidate for antitumor vaccines.

As candidate epitopes for some of these MAA were identified, trials were conducted at the National Cancer Institute in which patients were immunized with a s.c. injection of peptide dissolved in IFA. Monitoring of patients’ PBMC revealed a powerful enhancement of MAA recognition in postvaccination CTL cultures (6, 7). Thus, peptide vaccination is very effective in stimulating HLA class I-restricted, MAA-specific T cell reactivity in vivo. Ignorance of the immunogenicity of MAA in association with other HLA alleles has, however, restricted their usage as immunogens to a subset of patients bearing the HLA allele associated with their identification. Because of this restriction, a whole protein vaccine that could be administered regardless of HLA phenotype or knowledge of epitopic determinant has been theorized.

Few lines of evidence suggest that MAA can yield T cell epitopes in association with HLA alleles different from those that originally led to their identification. So far, there is no evidence that MART-1/Melan A can be recognized in association with HLA alleles other than A*0201. In this study, we explored the suitability of MART-1/Melan A, a small m.w. protein, to function as an immunogen in association with other HLA-A class I alleles. It was postulated that an autologous system based on endogenous processing and presentation of whole Ag could allow equal opportunities to putative HLA/epitope combinations to induce T cell reactivity (18). The statistical power of the system is supported by the efficient induction of HLA-A*0201-restricted anti-MART-1 reactivity, suggesting that the inability to induce antitumor reactivity in association with HLA alleles other than HLA-A*0201 was not related to technical failure. It has been shown that the subdominant OVA55–62 can function as an immunogen in the context of Kb only when the dominant sequence of OVA257–264 is removed from the antigenic molecule (59). Similarly, the inability to identify non-HLA-A*0201-restricted antitumor responses in HLA-A*0201 patients could, admittedly, be secondary to the dominance of MART-127–35. Subdominant CTL specificities could have been hidden by the predominant expansion of MART-127–35-reactive, HLA-A*0201-restricted CTL. Such a possibility, however, appears unlikely considering the failure to identify non-HLA*A-0201-restricted MART-1/Melan A-specific clones from early CTL cultures in HLA-A*0201-expressing patients and the inability to induce tumor-specific cultures in non-HLA-A*0201-expressing patients.

The stringency of the requirements for MART-1 immunodominance appears extreme: only one peptide of intermediate affinity for HLA-A*0201 (55) shows specific activation of bulk and clonal CTL. This epitope (MART-127–35), and possibly its N-terminal extension, are therefore uniquely responsible for the prevalence of the observed anti-MART-1/Melan A responses. Thus, the immunodominance of this small MAA does not appear to be related to molecular size or broadness of epitope repertoire but rather to a unique, yet unexplained, MHC/peptide interaction with the potential of the mature human TCR repertoire. Previous work has shown that, within the domain of HLA-A*0201 binding motif, single residue substitutions at different positions of the MART-127–35 peptide are relatively permissive to CTL recognition, with the exception of position 5 (60). Conservation of the glycine at position 5 and a search for compatible sequences in available protein databases led to the identification of multiple-potential MART-127–35 mimicry analogues occurring in a variety of common human pathogens that could be recognized by MART-127–35-specific CTL (60). These findings underlie the hypothesis that T cells might encounter a variety of analogue peptide sequences in vivo and that epitope mimicry may play a role in modulating the CTL response to MART-127–35. It is possible that the serendipitous similarity of MART-127–35 to other common epitopes might determine its unique role as an MAA in the context of HLA-A*0201.

MART-1 immunodominance is also highly restricted by the HLA polymorphism. Not only were we unable to generate CTL reactivity in association with HLA class I alleles different from HLA-A*02, but we also could not raise MART-1/Melan A reactivity in association with alleles belonging to the A2-like supertype shown to bind canonical HLA-A*0201 peptides (8) including MART-127–35 (9). It is important to emphasize that the uniqueness of the HLA-A*0201/MART-127–35 combination cannot be generalized, since other Ags are capable of stimulating CTL reactivity in association with different HLA-A*02 alleles (39).

The conclusion suggested by this study is that the suitability of a particular Ag/HLA combination as an immunogen cannot be assumed based on HLA binding motif nor structural relatedness among HLA molecules. Evidence of immunogenicity should be sought for each MAA/allele combination at an empirical level by identifying naturally occurring epitopes in relevant CTL populations. In particular, small m.w. proteins cannot be assumed to be comparable immunogens across the HLA polymorphism. Whole Ag vaccines, although conceptually appealing, may not, in practical terms, offer any additional advantage over peptide-based vaccination strategies.


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  		Table IX. Half-life of dissociation of ß2m (minutes) from HLA-A*0201 or HLA-A*0226 (152 V -> E) heterotrimer complexes containing different peptides

 

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  		Table X. Melanoma patient HLA-A*0226 (152 V -> E): MART-1 reactivitya

 

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  		Table XI. Melanoma patient HLA-A*0226 (152 V -> E): gp100–209-2M reactivitya

 

   Footnotes
 
1 Address correspondence and reprint requests to Dr. Francesco M. Marincola, Surgery Branch, National Cancer Institute, Building 10, room 2B56, Bethesda MD, 20892. E-mail address: Back

2 Abbreviations used in this paper: MAA, melanoma-associated Ags; CM, culture medium; DC, dendritic cells; MART-1, melanoma Ag recognized by T cells; rFP, recombinant fowl pox virus; rVV, recombinant vaccinia virus; TIL, tumor-infiltrating lymphocytes; gp100, glycoprotein 100; GM-CSF, granulocyte/macrophage-CSF; ß2m, ß2-microglobulin; 125I-ß2m, 125I-labeled ß2m. Back

Received for publication December 16, 1997. Accepted for publication March 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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