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The Journal of Immunology, 2001, 167: 5852-5861.
Copyright © 2001 by The American Association of Immunologists

A New Generation of Melan-A/MART-1 Peptides That Fulfill Both Increased Immunogenicity and High Resistance to Biodegradation: Implication for Molecular Anti-Melanoma Immunotherapy1

Jean-Sébastien Blanchet*, Danila Valmori{dagger}, Isabelle Dufau*, Maha Ayyoub{dagger}, Christophe Nguyen*, Philippe Guillaume{dagger}, Bernard Monsarrat*, Jean-Charles Cerottini{dagger}, Pedro Romero{dagger} and Jean Edouard Gairin2,*

* Laboratoire d’ImmunoPharmacologie Structurale, Institut de Pharmacologie et Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse, France; and {dagger} Ludwig Institute for Cancer Research, Division of Clinical Onco-Immunology, University Hospital and Lausanne Branch, Lausanne, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intense efforts of research are made for developing antitumor vaccines that stimulate T cell-mediated immunity. Tumor cells specifically express at their surfaces antigenic peptides presented by MHC class I and recognized by CTL. Tumor antigenic peptides hold promise for the development of novel cancer immunotherapies. However, peptide-based vaccines face two major limitations: the weak immunogenicity of tumor Ags and their low metabolic stability in biological fluids. These two hurdles, for which separate solutions exist, must, however, be solved simultaneously for developing improved vaccines. Unfortunately, attempts made to combine increased immunogenicity and stability of tumor Ags have failed until now. Here we report the successful design of synthetic derivatives of the human tumor Ag Melan-A/MART-1 that combine for the first time both higher immunogenicity and high peptidase resistance. A series of 36 nonnatural peptide derivatives was rationally designed on the basis of knowledge of the mechanism of degradation of Melan-A peptides in human serum and synthesized. Eight of them were efficiently protected against proteolysis and retained the antigenic properties of the parental peptide. Three of the eight analogs were twice as potent as the parental peptide in stimulating in vitro Melan-specific CTL responses in PBMC from normal donors. We isolated these CTL by tetramer-guided cell sorting and expanded them in vitro. The resulting CTL efficiently lysed tumor cells expressing Melan-A Ag. These Melan-A/MART-1 Ag derivatives should be considered as a new generation of potential immunogens in the development of molecular anti-melanoma vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The molecular characterization of tumor Ags that can be recognized by CTL has opened new opportunities for the development of Ag-specific cancer vaccines (1, 2). In the case of melanoma, a candidate Ag is Melan-A/MART-1 (Melan-A hereafter), a melanocyte lineage-specific protein expressed in ~75–100% of primary and metastatic melanomas depending on their clinical stage (3). Melan-A-specific CTL have been frequently isolated from PBMC of both HLA-A*0201 healthy donors and melanoma patients and from tumor-infiltrating lymphocytes of melanoma patients (4, 5). These CTL have been shown to primarily recognize peptide Melan-A26(27)–35 ((E)AAGIGILTV) (4) in an HLA-A*0201-restricted fashion (6).

Because of the high frequency of Melan-A circulating precursors (5, 7) and evidence of strong immune responses to this Ag in melanoma patients (8), Melan-A represents an attractive candidate for generic immunotherapy of HLA-A*0201+ melanoma patients. A superagonist variant of the nonameric Melan-A27–35 peptide has been shown to elicit an enhanced anti-melanoma CD8+ CTL response (9). We have recently undertaken clinical trials of peptide vaccination using the decameric analog Melan-A26–35A27L (ELAGIGILTV). In addition to exhibiting improved HLA-A*0201 binding properties (higher affinity and more stable HLA-A*0201/peptide complexes), Melan-A26–35A27L displays more potent antigenicity and immunogenicity than the natural Melan-A peptides (10, 11). Moreover, the large majority of CTL raised either in vitro or in vivo against Melan-A26–35A27L are fully cross-reactive with the Melan-A parental peptide sequences and able to specifically lyse Melan-A-expressing tumor cells (10, 11, 12).

When compared with other vaccination strategies (13), the use of antigenic peptides derived from tumor Ags (tumor Ag-derived peptides) as immunogens offers a number of advantages, including low cost and facility of administration of the vaccine, high specificity of elicited immune responses, and low toxicity (14, 15). However, the use of tumor Ag-derived peptides for cancer immunotherapy faces two major limitations: the weak antigenicity and immunogenicity of tumor Ags (16) and their high susceptibility to proteolytic degradation by proteases (17, 18, 19, 20). Weak immunogenicity can partially be ascribed to a certain level of immune tolerance to self-derived sequences (including suboptimal MHC binding and/or T cell recognition). Limiting the extent of peptide degradation by, for example, delivering the peptide incorporated in a water/oil emulsion often significantly increases immunogenicity (21). A more effective approach for avoiding rapid peptide degradation by proteases implies the design of protease-resistant peptide analogs. Approaches based on structural modifications to inhibit proteolytic degradation of bioactive peptides exist (22, 23) and have been applied to MHC class I-restricted (20, 24, 25) or class II-restricted (26) antigenic peptides. Unfortunately, the structural modifications introduced in the antigenic peptide sequence most often result in a dramatic reduction or even in a complete loss of peptide binding to MHC and/or T cell recognition (24, 27, 28, 29). This difficulty may be partly overcome by a detailed knowledge of the degradation pathway of the antigenic peptide in human serum that allows the rational design of minimally modified, peptidase-resistant, and still biologically active nonnatural analogs (20, 24). It is clear that synthetic derivatives of tumor Ags combining both high protection against peptidases and higher immunogenicity represent candidates of choice for cancer immunotherapy. However, none has been successfully designed and used until now.

The aim of the present study was to design such derivatives of the Melan-A tumor Ag. For that, we merged complementary approaches based on manipulation of the structural and pharmacological properties of MHC class I-restricted Ags and of the immune response against cancer. First we dissected the mechanism of Melan-A peptide degradation in human serum. Second, on the basis of these results, we designed a series of 36 nonnatural derivatives of the previously defined Melan-A26–35A27L analog. Among them, eight were fully resistant to proteolysis by serum proteases and retained the antigenicity and immunogenicity of the parental peptide. When used to stimulate in vitro Melan-specific CTL responses in PBMC from normal donors, three of these sequences were more potent activators of tumor-reactive CTL than the parental peptide. These analogs represent a new generation of Melan-A/MART-1 Ag derivatives that now combine enhanced immunogenicity with proteolysis resistance. Therefore, they should be considered potential immunogens in the development of molecular anti-melanoma vaccines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

The tumor cell lines Me 290 and Me 275 were established from surgically excised melanoma metastasis from patients LAU203 and LAU50, respectively, and maintained in culture as previously described (10). T2, an HLA-A*0201+ human T/B cell hybrid peptide transporter-deficient cell line (30), was cultured in DMEM/10% FCS supplemented with 0.55 mM Arg, 0.24 mM Asn, and 1.5 mM Gln. The tyrosinase-specific CTL clone 156/34 was derived after in vitro stimulation of tumor-infiltrating lymphocytes from patient LAU156 with the natural tyrosinase368–376 peptide (YMDGTMSQV) (12). The polyclonal monospecific CTL line used for analog recognition experiments was induced in vitro after stimulation of PBMC from patient LAU203 (10).

Peptide synthesis

Peptides were synthesized by the solid phase method using F-moc chemistry and the N,N'-diisopropylcarbodiimide/N-hydroxyaza-benzotriazole coupling procedure. Peptide with {beta} amino acids, d amino acids, {alpha}-methylated amino acids ({alpha}Meaa),3 or N-methylated amino acids (NMeaa) were synthesized by incorporation of the corresponding modified amino acids commercially available (Bachem, Budendorf, Switzerland (NMeaa); Fluka, Buchs, Switzerland ({beta} amino acids); and Acros Organic, Noisy-Le-Grand, France ({alpha}Meaa)). Peptides with {alpha}Meaa were obtained as racemics, because {alpha}Meaa were purchased in a racemic form. Whenever possible, stereoisomers were isolated by reverse phase HPLC (RP-HPLC) and named p1 and p2. The reduced bond {Psi}(CH2-NH) was formed by the reductive alkylation of a free amino group with an F-moc-protected preformed amino aldehyde (31). N-terminal hydroxypeptides were synthesized following a previously described procedure (32). The retro-inverso bond {Psi}(NH-CO) was obtained by replacement of two sequential amino acids with an (R,S)-2-substituted malonate derivative and a gem-diaminoalkyl residue (for example, the gem-diaminoalkyl residue corresponding to glutamic acid side chain and 2-substituted malonic acid corresponding to leucine side chain (2(R,S)-isobutylmalonic acid) were used for the synthesis of [{Psi}1–2(NH-CO)]-Melan-A26–35 A27L) (33, 34). A mixture of two diastereoisomers of the modified peptide was obtained. The two isomeric forms were not separated. Peptides were purified (purity, >=98%) by RP-HPLC on a C8 column (Aquapore Brownlee, PerkinElmer, Norwalk, CT). The identities of the purified peptides were confirmed by electrospray ionization-mass spectrometry (ESI-MS). Peptide stock solutions were made (10-3 M in 100% DMSO) and stored at -20°C.

Degradation of Melan-A peptides in human serum

Peptides were added to preheated (10 min at 37°C before the assay) human serum to a final concentration of ~0.5 x 10-4 M and incubated at 37°C. For the analysis of peptide persistence in serum with Melan-A-specific CTL recognition assay, aliquots (100 µl) were removed at different times and put in liquid nitrogen to stop the enzymatic reaction. 51Cr-labeled T2 cells were then pulsed with serial dilutions of each degradation sample at 4°C, and chromium release experiments were performed as previously described (18).

For on-line RP-HPLC/ESI-MS analysis, aliquots of the degradation solution (100 µl) were removed at different times, and the enzymatic reaction was stopped by addition of 11 µl of trifluoroacetic acid (TFA). Precipitated serum proteins were pelleted by centrifugation at 15,000 rpm for 10 min at 4°C. The supernatants were frozen and kept at -20°C until analysis. We checked that 1) the Melan-A peptides did not precipitate and were stable in the presence of 10% TFA, and 2) DMSO did not affect serum protease activities at the concentration used. The HPLC profile of serum alone precipitated by 10% TFA was recorded to detect nonprecipitated peptides present in the serum. Melan-A peptides and their degradation products were separated, and their sequences were determined and quantified by on-line RP-HPLC/ESI-MS using a C18 ultrasphere ODS column (Beckman Coulter, Palo Alto, CA), a Waters 600 MS chromatograph (Waters, Milford, MA), and a TSQ-700 Finnigan-MAT mass spectrometer (Thermo Finnigan, San Jose, CA) as previously described (19, 24). Quantitative determination of peptides and their degradation products were obtained operating in the selected ion monitoring mode.

HLA-A*0201 binding

Peptide binding to HLA-A*0201 was assessed in a functional competition assay based on inhibition of recognition of the antigenic peptide tyrosinase368–376 (YMDGTMSQV) by the HLA-A*0201-restricted CTL clone 156/34. Various concentrations of competitor peptides (50 µl) were incubated with 51Cr-labeled T2 cells (50 µl; 1000 cells/well) for 15 min at room temperature. A suboptimal dose (1 nM) of the antigenic peptide tyrosinase368–376 (50 µl) was then added together with specific CTL (5000 cells/well; 50 µl). Chromium release was measured after a 4-h incubation at 37°C. The concentration of each competitor peptide required to achieve 50% inhibition of target cell lysis was then determined (IC50).

Peptide recognition by Melan-A specific CTL

Ag recognition was assessed using chromium release assays. Target cells (T2 cells) were labeled with 51Cr for 1 h at 37°C and washed three times. 51Cr-labeled target cells (1000 cells/50 µl) were then added to various concentrations of antigenic peptide (50 µl) in V-bottom 96-well plates for 15 min before addition of effector cells. A polyclonal CTL line specific for Melan-A26–35A27L was used as effector cells and added (5000 cells/100 µl) at a defined E:T cell ratio. Chromium release was measured in 100 µl of supernatant harvested after 4 h of incubation at 37°C. The percentage of specific lysis was calculated as follows: % specific lysis = [(experimental release - spontaneous release)/(total release - spontaneous release)] x 100. The concentration of each peptide required to achieve 50% maximal lysis of target (EC50) was then determined.

Generation of Melan-A specific CTL

PBMC from HLA-A*0201+ healthy donors were isolated by centrifugation in Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). CD8+ lymphocytes were isolated using a miniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany). The resulting population routinely contained >75% CD8+ T cells and was used as the responder cell population. Purified CD8+ T cells were plated at 1 x 106 cells/well together with 2 x 106 stimulator cells/well in 24-well plate in a total volume of 2 ml of Iscove’s medium supplemented with 10% human serum, Asn, Arg, and Gln (complete medium) in the presence of IL-7 (10 ng/ml; R&D Systems, Oxon, U.K.), IL-2 (10 U/ml; Glaxo Wellcome, Geneva, Switzerland), and stimulating peptide (1 µM). Stimulator cells were prepared as follows. Cells (2 x 106) derived from the CD8- population after miniMACS CD8+ lymphocyte isolation were irradiated (3000 rad) and adjusted to the appropriate volume before addition to the CD8+-enriched responder cell population. On day 7 cells were restimulated with peptide-pulsed T2 cells. T2 cells were incubated for 2 h at 37°C in serum-free medium (X-VIVO 10; BioWhittaker, Walkersville, MD) with the appropriate stimulating peptide (1 µM) and human {beta}2-microglobulin (3 µg/ml; Sigma-Aldrich, St. Louis, MO). Peptide-pulsed T2 cells were washed, irradiated (10,000 rad), adjusted to the appropriate volume of complete medium supplemented with IL-7 (10 ng/ml) and IL-2 (10 U/ml), and added to the lymphocyte culture (2 x 105 cells/well). Subsequent restimulations were performed weekly with peptide-pulsed T2 cells. CTL activity was first tested at the end of the first restimulation using an ELISPOT assay for IFN-{gamma} production by peptide-reactive CTL.

Flow cytometric analysis and Melan-A tetramer-guided cell sorting of specific CD8+ CTL

The specificity of CTL recognition was monitored by flow cytometric analysis after Melan-A26–35A27L HLA-A*0201 tetramer staining (5, 8). Cells were stained with tetramers (200 ng/sample) in 20 µl of PBS/2% FCS for 20 min at room temperature, then 20 µl of anti-CD8-FITC mAb (BD Biosciences, Basel, Switzerland) was added, and cells were incubated for an additional 30 min at 4°C. Cells were washed once in the same buffer and analyzed on a FACScan (BD Biosciences, San Jose, CA) flow cytometer. Data analysis was performed using CellQuest software (BD Biosciences). The CD8+tetramer+ and CD8+tetramer- lymphocyte populations were sorted using a FACSVantage (BD Biosciences) cell sorter. After cell sorting the polyclonal monospecific CTL population was expanded by restimulation in a nonspecific fashion using PHA (1 µg/ml; Sigma-Aldrich), and IL-2 (150 U/ml).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Melan-A Ag-related peptides are rapidly degraded in human serum by amino- and dipeptidyl-carboxy-peptidases

The kinetics as well as the mechanism of Melan-A peptide proteolytic degradation were determined by incubation of synthetic peptides in human serum. We studied different peptides related to the Melan-A Ag: the nonamer Melan-A27–35 (AAGIGILTV), the decamer Melan-A26–35 (EAAGIGILTV), and the analog Melan-A26–35A27L (ELAGIGILTV). We first determined the persistence of the antigenic peptide over time in serum as monitored by specific CTL recognition using a chromium release assay. After different incubation times in serum, serial dilutions of the samples containing the synthetic peptide were pulsed onto T2 cells and incubated with Melan-A-specific CTL. Lysis of peptide-pulsed T2 cells represents the persistence of intact peptide in human serum over time. Clearly, Melan-A antigenic peptides incubated in serum very rapidly lost the ability to sensitize T2 target cells for lysis by specific CTL (Fig. 1GoA), suggesting that they were degraded by peptidases in human serum. Of note, the Melan-A nonamer AAGIGILTV seemed to be more sensitive than the two Melan-A decamers.



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  		FIGURE 1. Degradation of the Melan-A peptides in human serum. A, Melan-A peptides were incubated in human serum, and the persistence of the Ag was evaluated using a functional cytolytic assay. At each incubation time T (•, 0 min; {circ}, 30 min; {blacksquare}, 60 min; {square}, 120 min; {blacktriangleup}, 240 min; {triangleup}, 24 h), 51Cr-labeled T2 cells were pulsed with serial dilutions of each degradation sample and used as target cells in the chromium release assay. The CTL used was a polyclonal monospecific CTL line raised against Melan-A26–35A27L peptide. The E:T cell ratio used was 10:1. B, Degradation kinetics of Melan-A Ags analyzed by HPLC/ESI-MS. Melan-A peptides were incubated in human serum, and the presence of the peptide was addressed by direct detection using on-line HPLC/ESI-MS. Peptide quantification was achieved by analysis of the mass spectrometry data. For a given molecular species, the reconstructed ion current (RIC) intensity characteristic of the identified peptide or degradation product was determined. B, left panels, The percentage of peptide remaining was calculated for each sample with the amount of peptide detected at T0 as a reference and plotted against the time: (RIC intensity)T x 100)/(RIC intensity)T0). B, middle and right panels, Kinetics of appearance of the amino- and carboxyl-terminal degradation products of Melan-A Ags. The amount of each product is shown as the RIC intensity plotted against time.

 
The exact mechanism of antigenic peptide degradation in serum was addressed by on-line RP-HPLC/ESI-MS that allows unambiguous identification and precise quantification of the degradation products resulting from tumor Ag proteolysis (19, 24). As shown in Fig. 1GoB (left panels), the Melan-A27–35 nonamer was degraded very rapidly (t1/2, 5 min), in agreement with a previous study (20). The Melan-A decamers Melan-A26–35 (EAAGIGILTV) and Melan-A26–35A27L (ELAGIGILTV) were degraded less rapidly than the nonamer, thus confirming the initial observation, and displayed similar half-lives (t1/2, 45 and 40 min, respectively). It must be noted that Melan-A26–35A27L, at the lowest dilution tested, could sensitize T2 cells for lysis by Melan-A-specific CTL after a 24-h treatment in serum (Fig. 1GoA), although it appeared almost completely degraded after 2 h (Fig. 1GoB). This observation can be explained by a 2- to 3-log difference in the sensitivity of the assays (biological vs physicochemical) as we previously noted (24). It further suggests that few copies of Melan-A26–35A27L not detectable by MS remained in the medium and were still able to stimulate, even though very weakly, the Melan-A-specific CTL.

Identification of the amino- and carboxyl-terminal fragments allowed us to determine the mechanism of peptide degradation. The degradation process of the Melan-A peptides in human serum followed two main pathways involving amino- and dipeptidyl-carboxy-peptidase activities (Fig. 1GoB, middle and right panels, and Fig. 2Go), as previously observed for the MAGE-1.A1 tumor Ag (24). Degradation experiments using selective peptidase inhibitor bestatine (amino-peptidase inhibitor) or captopril (dipeptidyl-carboxy-peptidase inhibitor) confirmed this. In both cases peptide degradation was partially inhibited (data not shown). Thus, the first two peptide bonds to be sensitive to peptidases are Glu1-Leu2 (or Ala2) and Leu8-Thr9. On-line RP-HPLC/ESI-MS analysis also gave us an estimation of the quantity of a given degradation product present during the time course of degradation. As shown in Fig. 1GoB, the main amino-terminal degradation products of the Melan-A27–35 nonamer (AGIGILTV and GIGILTV) were more abundant than those of the Melan-A26–35 decamers. This observation suggests that the Melan-A27–35 nonamer is degraded more rapidly by amino-peptidases than the Melan-A decamers. This sensitivity could explain the very short half-life of the nonameric peptide.



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  		FIGURE 2. Proposed model for Melan-A26–35A27L degradation in human serum. Full and dotted arrows correspond to dipeptidyl-carboxy-peptidases and amino-peptidases, respectively.

 
Analysis of carboxyl-terminal degradation of the Melan-A peptides also revealed differences between the nonamer and the decamers. The first carboxyl-terminal product from Melan-A27–35 degradation (AAGIGIL) was detected very rapidly, peaked at 20 min, and was totally degraded within 60 min. In the case of the two decamers, the quantity of the first carboxyl-terminal degradation products (EAAGIGIL or ELAGIGIL) was maximal at 60 min. These products were then degraded, but were still present in a detectable quantity at 240 min. The scheme depicted in Fig. 2Go summarizes the degradation mechanism of Melan-A26–35A27L. The slower rate of degradation may reflect that glutamic acid at the amino terminus of the peptide is not optimal for amino-peptidase activity. As a consequence, these peptides were less susceptible to amino-terminal degradation and were more available for carboxy-peptidase activity. Altogether, the degradation data suggest that the nature of the amino-terminal residues of peptide has a direct or indirect effect on both amino- and carboxy-peptidase activities.

Designing synthetic antigenic peptides resistant to degradation by proteases

To identify Melan-A peptides resistant to proteolysis, we synthesized a series of peptide analogs with structural modifications at one or both peptidase-sensitive positions and tested their degradation properties. We used the Melan-A26–35A27L peptide as the starting sequence because of its enhanced immunogenicity (10). Protection against amino-peptidases was obtained by substitution of glutamic acid residue at position 1 (E1) by {beta}aa ({beta}E1, {beta}A1, or {beta}D1) (35), NMeaa (NMeE1), a pyro-glutamic acid (pE1), N-acetylated E, or an N-hydroxylated glycine (NOHG1) (32). Modification of leucine residue at position 2 (L2) was achieved by replacing it with methylated aa ({alpha}MeL2, NMeL2) or dL2. To prevent degradation by dipeptidyl-carboxy-peptidase we replaced L8 by {alpha}MeL8 or dL8 or T9 by NMeT9 or dT9. To avoid carboxy-peptidase degradation, we also synthesized carboxy-amidated (CO-NH2) peptide. Finally, peptide bond alterations, such as reduced bond {Psi}(CH2-NH) or retro-inverso bond {Psi}(NH-CO), were included to prevent amino- or carboxyl-terminal degradation. All the analogs are listed in Table IGo. As exemplified in Fig. 3Go for analogs [{beta}D1]-Melan-A and [{Psi}8–9(CH2-NH)]-Melan-A, the amino- and carboxyl-terminal structural modifications of the Melan-A Ag protected the peptidase-sensitive bond. However, one-site protection did not result in peptides with significantly improved stability, indicating that protection at both ends is needed to stabilize the peptide. Indeed, all the two-site protected analogs were rendered resistant to peptidase activities with t1/2 values varying from >10 to >>24 h, as exemplified in Fig. 3Go (analog [{beta}D1, {alpha}MeL8]-Melan-A) and summarized in Table IGo. Among the doubly protected analogs, we noted that those bearing either amidation or {alpha}-methylation at the C terminus displayed the shortest or longest half-lives, respectively.


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  		Table I. Sequences, HLA-A*0201 binding properties, Melan-A-specific CTL recognition, and stability in human serum of Melan-A peptides and nonnatural Melan-A derivatives

 


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  		FIGURE 3. Protease resistance of mono-protected and doubly protected nonnatural Melan-A analogs. Examples of stability of mono-protected ([{beta}D]LAGIGILTV: [{beta}D1]-Melan-A, left panel; ELAGIGIL-[{psi}(CH2-NH)]-TV: [{Psi}8–9(CH2-NH]-Melan-A, middle panel) and doubly protected ([{beta}D]-LAGIGI-[{alpha}MeL]-TV: [{beta}D1, {alpha}MeL8]-Melan-A, right panel) analogs in human serum are shown. Degradation was addressed using HPLC/ESI-MS detection as described for Melan-A peptides (see Fig. 1GoB). The amount of each peptide was determined after analysis of MS data and is presented as a percentage of the maximum amount of the parental peptide at time zero (T0). {blacksquare}, Amount of peptide remaining; • and {circ}, amounts of degradation products generated from amino- and dipeptidyl-carboxy-peptidase activities, respectively.

 
Nonnatural Melan-A derivatives are efficiently recognized by Melan-A-specific CTL

We and others have previously shown that structural modification of an antigenic peptide may considerably affect the efficiency of peptide-MHC binding and/or CTL recognition (24, 25, 27). We first determined the HLA-A*0201 binding properties of the 36 newly designed nonnatural analogs in a functional competition assay (10). As reported in Table IGo, a dramatic reduction in peptide-MHC binding was observed for about half the peptides. However, for the other half, the peptide affinity for HLA-A*0201 was either similar to that of the Melan-A26–35A27L peptide or only slightly reduced. Indeed, the structural alteration of the anchor residue L2 was often deleterious on MHC binding ([dL2]- or [NMeL2]-Melan-A with an IC50 >1000 nM, for example). As previously shown by others (27), peptide backbone alteration in reduced or retro-inverso analogs dramatically reduced the peptide affinity for MHC. Other modifications showed limited or no impact on peptide affinity for MHC even if they concerned anchor residue ([{beta}E1]-Melan-A IC50, 2 nM; [{alpha}MeL2]-Melan-A IC50, 6 nM). Finally, doubly protected Melan-A analogs such as [{beta}A1, {alpha}MeL8](p1), [NOHG1, {alpha}MeL8](p1), or [{alpha}MeL2, {alpha}MeL8] displayed HLA-A*0201 binding affinity very similar to if not identical with that of the parental Melan-A26–35A27L peptide.

Next, we tested the ability of the nonnatural Melan-A26–35A27L analogs to be recognized by specific CTL (exemplified in Fig. 4Go and summarized in Table IGo). As expected, CTL recognition of the nonnatural Melan-A analogs correlated with MHC binding for most of the analogs, with poor HLA-A2 binders being weakly recognized by CTL. However, strikingly, most of the structurally modified peptides were able to efficiently sensitize target cells to lysis by Melan-A-specific CTL, with EC50 values in the subnanomolar range. Interestingly, some doubly protected analogs displayed efficiencies comparable to or even higher than that of the parental Melan-A26–35A27L peptide ([{beta}A1, {alpha}MeL8]-, [{beta}D1, {alpha}MeL8]-, and [{beta}E1, {alpha}MeL8]-MelanA26–35 A27L, as presented in Fig. 4Go).



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  		FIGURE 4. Recognition of the nonnatural, doubly protected Melan-A analogs by Melan-A26–35A27L-specific CTL. Recognition of the doubly protected analogs [{beta}A1, {alpha}MeL8]-Melan-A, [{beta}E1, {alpha}MeL8]-Melan-A, [{beta}D1, {alpha}MeL8]-Melan-A, and [{beta}A1, V10-CONH2]-Melan-A (•, upper panels) by CTL was addressed in a chromium release assay using a polyclonal monospecific CTL line raised against Melan-A26–35A27L. The target cells were T2 cells pulsed with increasing concentrations of peptide, and the E:T cell ratio was 10:1. The Melan-A peptides Melan-A27–35, Melan-A26–35, Melan-A26–35A27L, and Melan-A26–35E26A were tested as controls ({circ}, lower panels).

 
Selected nonnatural Melan-A analogs efficiently stimulate the expansion of Melan-A-specific CTL precursors in the circulation

The eight doubly substituted derivatives displaying the highest efficiencies were selected for further studies. They are listed in Table IIGo. To assess their immunogenicity, we used them to stimulate CD8+-enriched cells isolated from PBMC. The Melan-A26–35A27L peptide was used as a positive control. For the second stimulation, irradiated T2 cells pulsed with the appropriate peptide were used. All cultures were tested by flow cytometry 7 days after the second stimulation (MC2) for the presence of CD8+A2/Melan-A tetramer+ cells. In cultures stimulated with the nonnatural Melan-A analogs, A2/Melan-A tetramer+ cells were detected in the CD8+ cell population, indicating that the A2/Melan-A tetramer was fully or partially cross-reactive with the analog-specific CTL, as shown in Fig. 5Go. Cross-reactivity of the A2/Melan-A tetramer allowed us to directly evaluate the efficacy of the nonnatural analogs to generate Melan-A-specific CTLs from PBMC of healthy donors. The percentage of Melan-A-specific cells obtained after stimulation with Melan-A26–35A27L differed from one donor to another, related to the frequency of Melan-A-reactive precursors in the initial CD8+ population (Table IIGo; HD224, 2.3%; HD410, 2.5%; HD220, 0.3%). Interestingly, three analogs were able to induce twice as many Melan-A-specific cells superior as the parental peptide Melan-A26–35A27L in all the three healthy donors: [{beta}A1, {alpha}MeL8]-, [{beta}E1, {alpha}MeL8]-, and [{beta}D, {alpha}MeL8]-Melan-A (Table IIGo and Fig. 5Go). Thus, these three analogs are more immunogenic in vitro than the parental peptide.


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  		Table II. In vitro immunogenicity of Melan-A26–35A27L and of nonnatural analogs

 


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  		FIGURE 5. Antigenic specificity of, and tumor recognition by, CTL induced with the nonnatural Melan-A analogs. After in vitro stimulation of PBMC from healthy donor HD224 with the Melan-A peptides, the tetramer+CD8+ lymphocyte population was sorted on a FACSVantage cell sorter (BD Biosciences). Cell sorting of the tetramer+CD8+ population and subsequent in vitro expansion of lymphocytes were realized for all stimulations with the different analogs under the same conditions. Cytolytic CTL activity was measured after in vitro expansion of the different CTL populations (see Materials and Methods). A, Dot plot representation of cytometry analysis of culture after in vitro stimulation with Melan-A26–35A27L and the doubly modified analogs. The gate defined for cell sorting of tetramer+CD8+ population is represented. B, Melan-A peptide recognition by the sorted tetramer+CD8+ CTL from the corresponding gate shown in A ({triangleup}, Melan-A27–35; {square}, Melan-A26–35; {circ}, Melan-A26–35A27L; •, Melan-A doubly modified analog). The chromium release assay was performed with T2 cells as target cells pulsed with increasing peptide concentrations and at an E:T cell ratio of 10:1. C, Tumor cell recognition by the sorted tetramer+CD8+ CTL from the corresponding gate shown in A. A cytolytic assay was performed with 51Cr-labeled cells (Mel275 and Mel290: HLA-A*0201+, Melan-A+; Na8: HLA-A*0201+, Melan-A-) with increasing E:T cell ratios. {blacksquare}, Mel275; •, Mel290; {circ}, Na8; +, Na8 and Melan-A peptide).

 
A2/Melan-A tetramer-guided isolation and functional characterization of in vitro stimulated nonnatural Melan-A analog-specific CTL

To further document the Ag specificity of CTL generated upon stimulation with the nonnatural Melan-A analogs, we sorted the CD8+A2/Melan-A tetramer+ cells. After in vitro expansion without specific stimulation, the antigenic specificity of the sorted cells was tested in a chromium release assay. As shown in Fig. 5Go, B and C, the sorted CTL specific for a nonnatural Melan-A analog were able to recognize and efficiently lyse not only T2 target cells presenting the parental Melan-A26–35A27L peptide, but also the natural Melan-A26–35 and Melan-A27–35 peptides. Importantly, tumor cell lines Me 290 and Me 275 that naturally express the Melan-A Ag on their surfaces were also recognized efficiently by nonnatural Melan-A-specific CTL. The Na8 cell line that does not express the Melan-A gene was used as a control. It was not susceptible or only weakly susceptible to lysis by the nonnatural Melan-A-specific CTL, indicating the absence of nonspecific lytic activity, but became efficiently lysed when pulsed with the Melan-A26–35 natural peptide. In conclusion, CTL induced with the nonnatural analogs are fully competent to recognize the parental Melan-A peptides and lyse tumor cells that naturally express the natural Melan-A Ag.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Improvement of immunogenicity of TDAP is critical for the development of efficient cancer vaccines. Introducing favorable anchor residue in the peptide sequence to increase peptide affinity for MHC is a first step to improved immunogenicity (36). Nevertheless, the use of such immunogenic peptide analogs as a vaccine may be restricted due to their high susceptibility to protease degradation. Thus, the next step was to introduce structural modifications to obtain proteolysis-resistant antigenic peptides. In the present study we first elucidated the degradation pathway of the Melan-A antigenic peptides. Then, according to these results we designed structurally modified nonnatural Melan-A analogs that were potent activators of tumor-reactive CTL.

Our results are in accord with previous findings that antigenic peptides can be rapidly degraded in human serum by proteases (17, 18, 20, 24). The analysis of antigenic peptide persistence in serum using a Melan-A-specific CTL recognition assay indicates that the Melan-A27–35 nonamer AAGIGILTV is degraded more rapidly than the two Melan-A decamers. This observation could be explained in part by the ability of the Melan-A26–35A27L-specific CTL used in the test to recognize shorter peptides that could be intermediate degradation products of the decamers. Indeed, the nonapeptides AAGIGILTV and LAGIGILTV resulting from removal of the first amino acid from Melan-A26–35 and Melan-A26–35A27L, respectively, are efficiently recognized by the Melan-A-specific CTL used in the degradation test (data not shown) (10). This limitation was overcome by the analytical on-line HPLC-ESI/MS approach (19). We confirmed the initial observation indicating that the Melan-A nonamer is degraded more rapidly than the two decamers. More importantly, we identified and quantified the degradation fragments leading to outlining the exact mechanism of Melan-A peptide degradation and identification of protease-sensitive bonds within the peptide sequence. This step is of critical importance for introducing minimal, but efficient, structural modifications in the tumor antigenic sequence. As we previously showed for other human or murine tumor antigenic peptides (19, 24), the Melan-A peptides are very sensitive to exopeptidases in serum. Thus, this mechanism of peptide degradation involving both amino- and dipeptidyl-carboxy-peptidases seems to be generic for antigenic peptide degradation in serum. In addition, because some doubly protected analogs were extremely stable in serum, we can deduce that the peptidic bonds left unprotected in the antigenic sequence are not affected by endopeptidase activity. This finding is in agreement with previous observations indicating that endopeptidases are not involved in the degradation of short peptides in serum (17, 37). Melan-A nonamer and decamers show marked quantitative and qualitative differences in their degradation properties. However, the differences in the half-life of the Melan-A peptides could not be explained solely by differences in peptide length, because the decamer Melan-A26–35 E26A (AAAGIGILTV) displayed a kinetic of degradation (t1/2, 15 min; data not shown) closer to that of Melan-A27–35 than that of the two Melan-A decamers. Thus, the nature of the first amino-terminal residues that greatly influence peptide susceptibility to amino-terminal degradation (37) seems to govern the kinetics of degradation of the Melan-A peptides. Knowledge of the exact mechanism of peptide degradation was indeed of critical importance for rationally and efficiently designing nonnatural Melan-A analogs resistant to protease degradation. To obtain fully protected peptides, the two protease-sensitive bonds (E1-L2 and L8-T9) of the Melan-A26–35A27L sequence needed to be modified. However, the doubly modified peptides did not show equal resistance to proteolysis. In particular, C-terminal amidated analogs showed the weakest resistance and were about twice as unstable as {alpha}-methylated ones. We may assume that this moderate protective effect is due to the capacity of a dipeptidyl-carboxy-peptidase, such as angiotensin-converting enzyme, to cleave C-terminal amidated substrates (38). Because studies have shown that peptide stability is similar in FCS and in the mouse (18, 37, 39), we may reasonably postulate that the doubly protected analogs resistant to human serum proteases would also be protected against proteolysis in vivo. However, in most antitumor vaccination protocols, the antigenic peptide is delivered either by direct s.c. injection or after pulsing onto dendritic cells (DCs). The biological significance of studying peptide degradation by serum proteases may then reasonably be questioned. Interestingly, both the serum proteases that degrade tumor Ags (Refs. 19 and 24 and this study), those expressed by human DCs (40) able to degrade synthetic class I peptides (41), and those expressed by T cells present in the skin and the afferent lymph (42), such as CD13 and CD26, display amino- and dipeptidyl-peptidase activities. We may thus assume that modified peptides that resist serum proteases probably resist peptidases expressed by DCs and remain stable after s.c. injection. Nevertheless, direct determination of the fate and stability of antigenic peptides in the interstitial space of the dermis and/or in the lymph would be informative.

Modifications of the peptide structure leading to resistance to proteolysis may alter both the affinity of peptide-MHC binding and the efficiency of CTL recognition. The impact on peptide-MHC binding and CTL recognition clearly differed among the structural modifications. As previously described, we show that peptide bond alteration is deleterious for peptide-MHC binding (20, 24, 27). The results presented here indicate that amino acid modifications such as {alpha}-methylation, N-hydroxylation, or {beta}-amino acids are among the most efficient to protect against proteolysis and yet cause minimal reduction of the peptide antigenicity. Interestingly, despite an overall decrease in MHC presentation, some of the doubly protected analogs sensitized target cells to lysis by the Melan-A-specific CTL with similar or even higher efficacy than the parental peptide. To our knowledge, this is the first time that such an observation has been reported. To further assess the immunogenicity of the nonnatural Melan-A peptides, we performed in vitro stimulation of PBMC from healthy donors with the modified peptide analogs. We used A2/Melan-A tetramer to quantify the number of Melan-A-specific T cells elicited after stimulation with the protected peptides. This analysis shows that stimulation with doubly substituted, fully protected analogs can give higher numbers of Melan-A-specific T cells compared with those obtained by stimulation with the parental peptide Melan-A26–35A27L. These results thus indicate that protection against proteolysis of antigenic peptides could significantly enhance in vitro immunogenicity. They further show the unique immunological properties of these tumor Ag derivatives.

Even though Melan-A26–35A27L is not expressed at the surface of Melan-A-expressing tumors, the CTL elicited by stimulation with Melan-A26–35A27L efficiently cross-recognize the endogenously expressed Melan-A sequences (10, 11). We show in the present study that CTL elicited by the nonnatural Melan-A analogs are also cross-reactive with the parental and endogenously expressed Melan-A peptides at the tumor cell surface. We thus validate and extend the tumor Ag cross-reactivity properties to the new analogs.

Antigenic peptide stability to protease degradation could have important implications to elicit an efficient immune response. In peptide-based cancer vaccines, after injection, the peptides have to be loaded onto professional APCs such as DCs. Under activation, DCs overexpress not only MHC, CD40, and CD80, but also CD13 and CD26, molecules that are amino- and dipeptidyl-peptidases, respectively (40, 41, 42). Induction of such ectopeptidases on the surface of activated DCs could increase the extracellular degradation process of exogenous antigenic peptide and reduce the peptide loading onto MHC molecules. If peptides are degraded before presentation by DCs, repeated administration and/or high doses of antigenic peptide are then required for immunization (43, 44). Thus, the effective dose of a degradable peptide cannot be exactly estimated from the dose injected. Yet the concentration of peptide used to elicit efficient antitumor-specific CTL is a critical parameter (45). Indeed, high-avidity CTL, which are highly efficient for antitumor response, were only induced in vitro and in vivo with DCs pulsed with low concentrations of peptide (46, 47, 48, 49). Therefore, the quality of a peptide vaccine is probably determined not only by the density, but also by the duration, of the presence of peptide at the injection site for loading onto activated DCs in vivo. Thus, peptide protection against protease degradation appears to be an attractive and effective way to control the concentration and local persistence of an antigenic peptide after injection. In this context, the use of peptides protected against degradation should then allow the optimization of immunization protocols for cancer vaccine. However, concerns may be raised about possible negative effects that injection of nondegradable peptides may have. In particular, it has been reported that due to persistence, diffusion of such peptides into the systemic circulation could induce peripheral immune tolerance (50). A recent study has also shown pharmacokinetic differences between a T cell-tolerizing and a T cell-activating peptide (51). Indeed, knowledge of the pharmacokinetics of peptides is a prerequisite before their possible use in peptide-based immunotherapy. Pharmacokinetic studies of the peptidase-resistant Melan-A peptides are thus currently underway in our laboratories. In addition, the demonstration of increased in vitro immunogenicity of the protected peptide should also be extended in vivo and validated. In particular, the correlation between high protease resistance and increased immunogenicity remains to be addressed in vivo. Immunization of transgenic HLA-A2/Kb mice with different kinds of adjuvant or several doses of peptide could allow an evaluation of the benefit of the increased resistance to proteolysis on immunogenicity of antigenic peptides. These experiments are currently being performed in our laboratories.

In conclusion, the results shown in the present study demonstrate that protease-resistant, nonnatural tumor Ag derivatives can be highly immunogenic and potent activators of melanoma-specific CTL. They may represent promising new tools for molecular anti-melanoma immunotherapy.


   Acknowledgments
 
We thank Immanuel Luescher for invaluable advice and support with the use of tetramers and Pascal Batard and Sophie Millot for technical assistance.


   Footnotes
 
1 This work was supported in part by grants from the Center National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer (Contract 5485), and the Conseil Régional Midi-Pyrénées (Contract 99001129). J.-S.B. is the recipient of a doctoral fellowship from the Association pour la Recherche sur le Cancer and a European Molecular Biology Organization short-term fellowship (ASF-9575). M.A. is a recipient of a postdoctoral fellowship from the European Community-Biomed program (EC-BMH4-CT98-3589). Back

2 Address correspondence and reprint requests to Dr. Jean Edouard Gairin, Institut de Pharmacologie et de Biologie Structurale, Unité Mixte de Recherche 5089, Centre National de la Recherche Scientifique, 205 route de Narbonne, 31400 Toulouse, France. E-mail address: gairin{at}ipbs.fr Back

3 Abbreviations used in this paper: {alpha}Meaa, {alpha}-methylated amino acid; ESI-MS, electrospray ionization-mass spectrometry; DC, dendritic cell; NMeaa, N-methylated amino acid; NOHG, N-hydroxylated glycine; RP-HPLC, reverse phase HPLC; TFA, trifluoroacetic acid; RIC, reconstructed ion current. Back

Received for publication July 20, 2001. Accepted for publication September 18, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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