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The Journal of Immunology, 2006, 176: 1796-1805.
Copyright © 2006 by The American Association of Immunologists

Identification of Cross-Reactive Peptides Using Combinatorial Libraries Circumvents Tolerance against Her-2/neu-Immunodominant Epitope1

Joseph Lustgarten2,*, Ana L. Dominguez* and Clemencia Pinilla2,{dagger}

* Sidney Kimmel Cancer Center and {dagger} Torrey Pines Institute for Molecular Studies, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The majority of the currently defined tumor-associated Ags are often overexpressed products of normal cellular genes. Therefore, tolerance deletes high-affinity T cells directed against the TAAs, leaving only a low-affinity repertoire. We have demonstrated previously that the T cell repertoire against the immunodominant p773–782 A2.1-Her-2/neu-restricted peptide has low affinity in A2xneu mice (Her-2/neu mice crossed with A2.1/Kb mice), compared with A2xFVB mice (A2.1/Kb crossed with FVB-wild-type mice). Immunizations with this peptide have a minor impact in preventing tumor growth in A2xneu mice. Therefore, attempts to expand these responses may be of little clinical value. We hypothesized that if not all possible cross-reactive peptides (CPs) are naturally processed and presented, the possibility exists that T cells against these CPs persist in the repertoire and can be used to induce antitumor responses with higher avidity against native epitopes present on the tumor cells. We have used the positional scanning synthetic peptide combinatorial library methodology to screen the p773–782 T cell clone. The screening data identified potential amino acids that can be substituted in the primary sequences of the p773–782 peptide. The designed CPs induce CTL responses of higher affinity in A2xneu mice compared with the native p773–783 peptide. These CTLs recognize A2+-Her-2/neu+ tumors with high efficiency. Moreover, multiple immunizations with CPs significantly prolonged the survival of tumor-bearing A2xneu mice. These results have demonstrated that it was possible to circumvent tolerance with the identification of CPs and that these peptides could be of significant clinical value.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell immunity is a critical component of the immune response to a growing tumor. Although the identification of tumor-associated Ags (TAAs)3 encoding mutated cellular genes serves as targets for T cell immunity, the majority of the currently defined TAAs are often overexpressed products of normal cellular genes. Therefore, in practice, these overexpressed proteins pose a significant challenge to the design of effective T cell immunotherapies due to the consideration of self-tolerance. Her-2/neu (1, 2) is an important tumor Ag that can be used to target a wide range of tumors from different origins (3, 4). The Her-2/neu is a transmembrane glycoprotein with tyrosine kinase activity, the structure of which is similar to the epidermal growth factor receptor (5). The Her-2/neu protein is a component of a four-member family of closely related growth factor receptors, including EGFR or Her-1, Her-3, and Her-4. The Her-family receptors play a role in the process of growth signal transduction across the cell membrane. Consequently, overexpression of this protein contributes to uncontrolled growth signal transduction and, therefore, cellular transformation (6). Overexpression of Her-2/neu is associated with metastatic disease, poor prognosis, and low survival (7, 8).

By crossing Her-2/neu transgenic mice (9, 10) with A2.1/Kb transgenic mice (11) (A2xneu mice) and examining the CD8-T cell responses against the immunodominant p773–782 A2.1/Her-2neu-restricted epitope (12), we demonstrated that A2xneu mice are tolerant and only contain a low-avidity repertoire against Her-2/neu Ags (13). This raises the following question: What is the useful contribution to the immune defense of these low-avidity T cells? The significance of understanding the mechanism responsible for the persistence of low-avidity T cells could be 2-fold: 1) the potential to target such cells against self-tumor Ags for tumor destruction: we have demonstrated that multiple immunizations with A2.1-Her-2neu-restricted peptides (13) or Her-2/neu Ags (14, 15) in the presence of costimulatory molecules, such as anti-OX40 and/or anti4-1BB mAbs, could delay the tumor growth in A2xneu mice; and 2) it is possible that T cells recognize self-Ags with low avidity and may be able to recognize other Ags with high avidity. Complete elimination of all T cells reactive against self-Ags would severely restrict the diversity of the immune repertoire. Although TCRs are specific for particular epitopes, it is also clear that such TCRs can recognize a variety of related ligands (16, 17), and even peptides, without apparent homology to the original antigenic peptide (18, 19). This represents an optimal repertoire that is capable of recognizing a maximal diversity, while being functionally tolerant to self-Ags. Considering that the interaction of the TCR with the peptide-MHC ligand is highly flexible and that the same TCR can recognize many different epitopes (20, 21), several laboratories have demonstrated that amino acid alterations in T cell epitopes could enhance stimulation of T cell populations for the nominal epitope (22, 23). The alteration of tumor-specific epitopes has become an attractive strategy for modifying immune responses and enhancing Ag-specific immunotherapies (24, 25). The finding that a single alteration in the epitope can significantly enhance the antitumor effect raises the question of whether multiple alterations will have additive effects resulting in a more potent protection. An important issue becomes which strategy should be used to identify the specific amino acid in the epitope that must be altered and with which amino acid should be substituted. The screening of T cells with a positional scanning synthetic peptide combinatorial library (PS-SCL) allows for the identification of high-affinity ligands and provides information about the most effective residues at each position of the T cell epitope recognized by a T cell clone (26, 27, 28). In this regard, Borras et al. (29) concluded that after screening T cell clones with the PS-SCL, two types of T cell ligands with comparable or higher affinities than the native peptides can be identified: 1) peptides that do not necessarily correspond to sequences in described proteins and 2) peptides that are fragments of natural proteins. We hypothesized that T cells potentially directed against cross-reactive epitopes could have escaped tolerance induction and thereby be available for the induction of an antitumor response against the native p773–782 epitope presented by tumor cells. Using the PS-SCL strategy, we screened a decamer library with the p773–782 T cell clone, and derived cross-reactive peptides (CPs) having multiple amino acid substitutions of the native p773–782 peptide were recognized by the p773-specific CTLs. Immunization of A2xneu mice with the CP induced CTL responses of relatively higher affinity, and the CTLs were able to recognize A2+-Her-2/neu+ tumors. Moreover, multiple immunizations with CP eradicated tumors in A2xneu mice. These results have demonstrated that replacement of amino acids in the primary position of an immunodominant peptide could induce a CTL response while maintaining the T cell specificity against the native peptide presented by tumor cells. Together, our data strongly suggest that we could circumvent tolerance against immunodominant self-tumor Ags with the identification of CP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The neu transgenic mice (line 202) were commercially obtained from The Jackson Laboratory and maintained homozygously. The FVB mice were purchased from Harlan Laboratories. The HLA-A2.1/Kb transgenic mice were kindly provided by Dr. L. Sherman (Scripps Research Institute, La Jolla, CA). The neu and FVB mice were mated with the HLA-A2.1/Kb mice to generate A2xneu and A2xFVB mice.

Cell lines

The A2.1/neu p773-restricted CTLs were generated by immunizing A2.1/Kb mice with the p773–782 peptide as described previously (12). The N202.A2 cell line was established from a spontaneous tumor (13). N202.1A cells were kindly provided by Dr. P. L. Lollini (University of Bologna, Bologna, Italy). The T2-A2.1/Kb cell line was also provided by Dr. L. Sherman. Anti-OX40 (OX86) mAb was obtained from the European Cell Culture Collection, and the anti-4-1BB (3H3) was obtained from Dr. R. Mittler (Emory University, Atlanta, GA). All cell lines were maintained in complete RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 5 x 10–5 M 2-ME, and 50 µg/ml gentamicin.

Libraries and peptide synthesis

The L-amino acid decapeptide PS-SCL arranged in a positional scanning format was prepared at the Torrey Pines Institute for Molecular Studies (San Diego, CA) as described previously using the simultaneous multipeptide synthesis method (30). The decapeptide PS-SCL consists of 200 mixtures in the OX9 format, where O represents one of the 20 natural L-amino acids in a defined position and X represents a mixture of all of the natural amino acids, with the exception of cysteine, at each of the remaining positions. All mixtures of this library are N-terminal free and C-terminal amide. Each mixture in the library is composed of 3.2 x 1011 peptides, and the total number of decapeptides in the library consists of 6 x 1012 different decamer peptides in approximate equimolar concentrations. Assuming an average of Mr 1150,000 and a concentration of 100 mg/ml of the mixture, the concentration of each individual peptide is 2.7 x 10–16 M. Individual peptides were synthesized by the simultaneous multipeptide synthesis method (31). The purity and identity of each peptide were characterized using an electrospray mass spectrometer interfaced with a liquid chromatography system.

Recognition of the peptide library

To analyze the decamer library, we used the p773-CTL clone. This specific line was generated several years ago after analyzing different HLA-A2.1/Her-2/neu peptides in HLA-A2.1/Kb transgenic mice (12). The p773-CTL line is a representative of nontolerant animals and was used to analyze the library. The recognition of the decamer library by the p773-CTL was assayed by INF-{gamma} release assay. The T2-A2.1/Kb cells (104 cells/well) were pulsed with the peptide library and incubated with the p773-CTLs at a 3:1 E:T ratio for 20 h. Supernatants were collected and evaluated for the production of INF-{gamma}. Mouse IFN-{gamma} and OptEIA ELISA kit sets (BD Pharmingen) were used to measure the amount of cytokine according to the manufacturer’s instruction.

In vitro binding of CPs to A2.1/Kb

The efficiency with which each selected CP bound A2.1/Kb was determined in a competitive binding assay (12). Each of the selected peptides (see Table II) was incubated (1 µg) with 51Cr-labeled T2-A2.1/Kb cells in the presence of A2.1/neu-restricted p369 native peptide (0.1 µg) that has a high binding activity to the A2.1/Kb molecule (12). Target cells were next incubated with the p369 specific-CTL (from A2xFVB mice) to assay for the recognition of the pulsed target cells. The binding of the test peptide to the target cells could be detected by the competitive inhibition of the binding of the p369 peptide, as evidenced by a decrease in the ability of the p369-CTL to lyse the target cells.


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  		Table II. Immunogenicity and cross-reactivity of the CPsa

 
Generation of dendritic cells (DCs) and immunization of A2xneu and A2xFVB mice

For DC isolation, bone marrow cells were depleted of lymphocytes (with magnetic beads conjugated with anti-CD4, anti-CD8, and anti-B220; Dynal Biotech). The remaining cells were cultured in complete RPMI 1640 medium containing 3% GM-CSF (supernatant from J558L cells transfected with murine GM-CSF gene; obtained from Dr. R. Steinman, Rockefeller University, New York, NY). To mature the DCs, 100 U/ml TNF-{alpha} were added on day 7, and DCs were collected on day 9. Maturation of pulsed DCs was confirmed by evaluating the expression of B7-1, B7-2, MHC class I, and MHC class II. Mature DCs were pulsed with the peptides at 10 µg/ml for 3 h at 37°C.

Analysis of peptide-restricted CTL responses

Ten days after immunization with DCs, animals were killed and spleens were removed. For stimulation of cultures, T cells (106 cells/well) were incubated with autologous irradiated (3000 rad) LPS spleen blasts (2 x 105 cells/well) that were pulsed with the indicated peptides in 24-well plates. After 5 days, CTLs were assayed for lytic activity. The N202.A2, N202, and T2-A2.1/Kb cells pulsed with the peptides were incubated with 150 µCi of 51Cr sodium chromate for 1 h at 37°C. Cells were washed three times and resuspended in complete RPMI 1640 medium. For the cytotoxic assay, 51Cr-labeled target cells (104) were incubated with varying concentrations of effector cells in a final volume of 200 µl in U-bottom 96-well microtiter plates. Supernatants were recovered after 6 h of incubation.

Evaluation of antitumor responses in A2xneu mice

A2xneu mice were implanted s.c. with 106 N202.A2 cells on day 0. On day 7, animals were randomly divided into groups of five and immunized one to five times s.c. with 106 DCs pulsed with the CP or p773–782 peptides. Anti-OX40 and anti-4-1BB mAbs (100 µg/injection of each Ab) were injected 2 days after DC immunization. DC vaccinations were performed opposite the site where the tumors were implanted. Tumor growth was monitored every 5 days, and growth rates were determined by caliper measurements in two diameters. Tumor volume was expressed as follows: (minor diameter)2 x major diameter/2. Statistical analysis was determined by Student’s t test. Five animals were included per group.

ELISPOT analysis

For ELISPOT analysis, a mouse INF-{gamma} ELISPOT kit was purchased from BD Pharmingen, and the manufacturer’s protocol was followed. Briefly, splenic cells from primed animals were isolated by negative selection with magnetic beads (Miltenyi Biotec). Enriched CD8+ T cells were plated at different concentrations (1 x 105, 3 x 105, and 1 x 106) and incubated with 5 x 104-irradiated LPS blast cells from A2xFVB mice pulsed with 10 µg/ml of the respective peptides and cultured in flat-bottom 96-well nitrocellulose plates that had been precoated with anti-INF-{gamma} mAb. After a 20-h incubation at 37°C, plates were washed with washing buffer, and the wells were incubated with detection Ab (biotin anti-INF-{gamma} mAb) for 2 h at room temperature. Plates were washed, and avidin-HRP was added to the wells and incubated for 1 h at room temperature. Plates were washed, and spots were developed by adding substrate solution. To enumerate the spots, an ImmunoSpot Analyzer was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Recognition of decapeptide library by p773-CTLs

We have shown previously the identification of an A2.1/neu-restricted decamer peptide (VMAGVGSPYV) with the use of the HLA-A2.1/Kb transgenic mice (12). Based on this peptide, we evaluated the T cell responses in A2xneu mice, and our results demonstrated that these mice only contained a T cell repertoire with low-avidity repertoire to neu Ags (13). These results indicated that A2xneu mice were tolerant to neu Ags (13, 14, 15). We hypothesized that if CPs that mimic the p773–782 epitope were identified, it would be possible to circumvent or bypass self-tolerance. The p773-CTL clone was used to test the recognition of a decamer PS-SCL by secretion of INF-{gamma}. As seen in Fig. 1, the defined amino acids in the most active mixtures corresponded to the amino acid of the native peptide in 7 of the 10 positions (as shown in the open bars). Furthermore, the PS-SCL-based biometrical analysis (32), which allows the systematic comparison of the PS-SCL screening with protein databases, was used to assess the ranking of the native peptide. A matrix representing the values shown in Fig. 1 was used to calculate the predicted stimulatory of millions of decapeptides present in all the proteins of the tumor Ag-specific and human protein databases. The native peptide ranked 3rd and 148th, respectively, on these protein databases.


Figure 1
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  		FIGURE 1. Recognition of the nonapeptide library by the p773-CTL clone. Recognition was assayed in a 20-h INF-{gamma} release assay. T2-A2.1/Kb cells were incubated in the presence of the libraries for 1 h at 37°C, and effector cells were added with an E:T ratio of 3:1. Supernatants were harvested after a 20-h incubation at 37°C, and an ELISA was performed. The OD (OD 492) is shown in the y-axis. The bars represent the average, and the error bars represent the SD of duplicates in the same experiment (similar results were found in a different experiment). The open bars correspond to the amino acid in the native peptide (VMAGVGSPYV), and the asterisks show the amino acids selected for the synthesis of the potential CPs.

 
Additionally, a number of mixtures with defined amino acids different from sequence of the native epitope at some positions, and these changes are capable of inducing a strong stimulation. The p773-CTL also recognizes the following: in position 1, F; in position 3, H; in position 4, N; in position 6, P and A; in position 7, I, L, and M; in position 9, H; and in position 10, L and M. It seems to be that for this T cell clone in position 8, P is a critical amino acid because no other mixture with a different amino acid was recognized. Based on these results, we designed 32 individual peptides (Table I) corresponding to the combinations of the defined amino acids of selected active mixtures, which are marked with asterisks in Fig. 1. It is important to note that the predicted stimulatory score for all of these potential CPs is higher than the score of the first ranking peptides in protein databases derived from the PS-SCL biometrical analysis.


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  		Table I. Selection and analysis of potential p773-derived CPs

 
Analysis of the potential p773-CPs

Each peptide was assayed in a chromium release for recognition against the p773-CTL. As shown in Table I, the selected CP demonstrated different degrees of recognition by the CTLs, and those that showed >20% killing were selected for further evaluation. Also, the efficiency of each of the peptides to bind A2.1 molecules was determined in a competitive binding assay. As shown in Table I, the selected CP had different capacities to bind A2.1 molecules, as indicated by inhibition of the binding of the p369 peptide. All peptides demonstrated binding capacity, relative to the Kd binding of the p369 peptide. Based on these results, 11 peptides were selected: CP1, CP2, CP3, CP9, CP10, CP11, CP14, CP20, CP22, CP23, and CP31. Together, these results indicate that substitutions can be made to the native peptides and can still be recognized by the p773-CTLs.

Priming of animals with CPs

The CPs were evaluated to determine their ability to induce an immune response. We compared the immune responses of A2xneu (tolerant) and A2xFVB (nontolerant) mice. Both A2xneu and A2xFVB mice were immunized with DCs pulsed with each of the 11 selected CPs. Table II shows a summary of the accumulative responses of each of the individual peptides. Interestingly, we obtained a diverse immune response and, based on these results, classified the responses into four groups according to the immunogenicity of the peptide and cross-reactivity of the CTLs (Table II). Immunogenicity indicates that these peptides induced a good CTL response directly against the peptides they were primed with; cross-reactivity indicates the ability of the CP-specific CTL to recognize the native p773 peptide. We identified the following: 1) CP1, CP14, and CP23 peptides are highly immunogenic and highly cross-reactive with p773; 2) CP2 and CP3 peptides are immunogenic, but the CTLs elicited show little or no cross-reactivity with p773; 3) CP10, CP20, and CP22 peptides are intermediate immunogenic and are not cross-reactive with p773; and 4) CP9, CP11, and CP31 peptides are not immunogenic. It is important to observe that the cytolytic activity of the CTLs derived from the immunogenic CP was very similar between the CTLs from A2xneu and A2xFVB mice. More importantly, the cross-reactivity is also very similar between A2xneu and A2xFVBmice. In contrast, as shown previously (Ref.13 ; see also Table II), there was a drastic difference between the immune responses from A2xneu and A2xFVB after immunization with the native p773 peptide. We performed peptide dose-curve assays with the CP1-, CP14-, and CP23-CTLs, and our data demonstrate that the CP-CTLs generated on both A2xneu and A2xFVB mice have a similar affinity for the CP (data not shown).

Affinity of the CP-CTLs against the p773–782 peptide

We next compared the recognition of the p773 native peptide in a dose-response assay by the CP1-, CP14-, and CP23-CTLs and p773 CTLs from A2xneu and A2xFVB mice. Our results indicate that the CP1-, CP14-, and CP23-CTLs from A2xFVB or A2xFVB mice could recognize the p773 peptide (Fig. 2). Interestingly, the CP-derived CTLs from A2xneu or A2xFVB mice recognize the p773 peptide with lower efficiency, compared with the p773-CTLs from A2xFVB mice. However, the CP-derived CTLs were much more efficient in recognizing the native p773 peptide than p773-CTLs from A2xneu mice. Based on the pattern of recognition of the CP-derived CTLs against the native peptide, these results suggest that the identified CP induced a CTL response that is of intermediate avidity against the p773 peptide.


Figure 2
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  		FIGURE 2. Recognition of p773 peptide by p773- and cp-CTLs. The p773- and cp-CTLs from A2xFVB (A) and A2xneu (B) mice were compared for the recognition of the p773 native peptide. T2-A2.1/Kb cells were pulsed with a decreasing concentration of p773 peptide, and CTLs were analyzed for cytotoxic activity in a 6-h 51Cr release assay at an E:T ratio of 10:1. The HIV-POL CTL was used as a control for specificity.

 
Additionally, we evaluated the cytolytic capacity of the CP1-, CP14-, and CP23-CTL populations from A2xneu and A2xFVB mice to recognize and lyse the A2.1+/Her-2neu+, N202.A2 cell line. The recognition of the N202.A2 cells by the p773-CTLs from A2xFVB mice was stronger than the CP-CTLs from A2xneu or A2xFVB. As demonstrated with the T2-A2.1/Kb-p773-pulsed cells, the CP-CTLs were more effective in recognizing the N202.A2 cells than p773-CTLs from A2xneu mice (Fig. 3). The CP-derived-CTLs did not recognize the A2.1–/Her-neu+ N202.1A cells, indicating that the recognition of the N202.A2 was specific. These data indicate that the CTLs induced by the CP are capable of recognizing the native p773-immunodominant peptide presented by the tumor cells.


Figure 3
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  		FIGURE 3. Recognition of N202.A2 tumor cells by cp-CTLs. The cp-CTLs derived from A2xFVB (left column) and A2xneu (right column) mice were assayed for specific killing activity of 51Cr-labeled N202.A2 (A2.1+/Her-neu+) and N202.1A (A2.1–/Her-neu+) cells. The HIV-POL CTL was used as a control for specificity.

 
Antitumor effect of the CPs

Having demonstrated that CP-CTLs recognized the native p773 peptide presented by tumor cells, we tested the antitumor effect of CP1, CP14, and CP23 peptides. A2xneu mice were implanted with tumors. One week later, animals were immunized three times with DCs pulsed with CP1, CP14, or CP23. As shown in Fig. 4, immunizations with the native p773 peptide inhibited ~15% of the tumor growth, whereas immunizations with the CP1, CP14, or CP23 peptides induced a ~40–50% tumor growth inhibition. We have shown previously (13, 14, 15) that when immunizing with the native peptide to enhance the antitumor responses in A2xneu mice it is important to note the following: 1) immunizations are conducted in the presence of costimulatory molecules such as anti-OX40 or anti-4-1BB mAbs; 2) immunizations with multiple Ags induce a stronger antitumor response increasing the number of tumor-specific effector cells, resulting in a stronger protective immunity; and 3) continuous immunizations further enhance the antitumor immune responses. To evaluate whether the antitumor responses could be enhanced with CPs, animals were immunized with a mixture of the three CP plus anti-OX40 and anti-4-1BB mAbs. For these experiments, we compared animals that were immunized with a mixture of CP1, CP14, and CP23 three, four, or five times (days 7, 17, 27, 37, and 47) in the presence of anti-OX40 and anti-4-1BB mAbs. The A2xneu mice were implanted with the N202.A2 cells on day 0; on day 7, they started treatment. As a control, mice were immunized five times with DCs pulsed with HLA-A2.1/HIV-POL peptide plus anti-OX40/anti-4-1BB mAb. As shown in Fig. 5, the median survival time was significantly delayed in CP-immunized mice (p < 0.001), compared with control, HIV-POL-, or p773-immunized animals. Control and HIV-POL-immunized A2xneu mice survived for 42 days, p773-immunized animals survived for 51 days, and after three, four, or five CP immunizations, animals survived for 69, 91, and 112 days, respectively. After five immunizations with CP1 plus anti-OX40 and anti-4-1BB mAb, animals survived for 63 days (data not shown). The advantage of injecting the peptides at the same time is that they induced a diverse immune response allowing for better disease coverage. Thus, the observation that immunizations with CPs significantly prolonged the survival of A2xneu mice, offers a window of opportunity for exploring CP vaccination as a novel strategy for developing more effective vaccines against self-tumor Ags.


Figure 4
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  		FIGURE 4. CP immunization induces an antitumor response. A2xneu mice were inoculated s.c. on day 0 with 106 N202.A2 cells. Animals were immunized three times (on days 7, 17, and 27) with s.c. injections of 106 DCs pulsed with the CP1, CP14, and CP23 peptides.

 

Figure 5
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  		FIGURE 5. Continuous immunizations with a mixture of CP induce tumor rejection in A2xneu mice. Animals were immunized three times (on days 7, 17, and 27), four times (on days 7, 17, 27, and 37), or five times (on days 7, 17, 27, 37, and 47) with s.c. injections of 106 DCs pulsed with a mixture of CP1, CP14, and CP23 peptides plus anti-OX40 and anti-41BB (100 µg per injection). As a control, a group of animals were immunized five times with 106 DCs with A2.1-HIV-POL peptide plus anti-OX40 and anti-41BB (100 µg per injection).

 
Immunization with CPs enhances the peptide-specific CD8 T cell frequency, compared with p773 native peptide

Our results indicate that immunization with CPs induces stronger antitumor immune responses. We have shown that CP-CTLs are of higher affinity, compared with p773-CTLs. We wanted to determine whether this improvement in the antitumor response would also correlate with an increase in peptide-specific CTL frequency. A2xneu and A2xFVB mice were immunized with CP1, CP14, CP23, and p773 in the presence or absence of anti-OX40 and anti-4-1BB mAb, and the frequency of peptide-specific CTLs were evaluated by ELISPOT assay. As shown in Fig. 6, A2xneu mice immunized with CPs showed a 4- to 5-fold increase of the peptide-specific CD8 T cells, compared with p773 peptide-immunized mice. A2xFVB mice immunized with CP or p773 peptides showed similar frequencies. Mice treated with anti-OX40 or anti-4-1BB mAb showed a 3- to 5-fold increase in the frequency of peptide-specific CD8 T cells. These results support the notion that the presence of anti-OX40 and anti-4-1BB mAb enhanced the antitumor responses by increasing the frequency of the peptide-specific CTLs. Additionally, these results suggest that the enhanced antitumor immune response with CP peptides in A2xneu mice is not only due to the fact that CP-CD8 T cells are of higher affinity but also to the fact that the frequency of CP-CD8 T cells is higher, compared with the frequency of p773-CD8 T cells.


Figure 6
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  		FIGURE 6. The CP induced a higher frequency of CD8 T cells than p773 peptide in A2xneu mice. A2xneu and A2xFVB mice were immunized with p773, CP1, CP14, and CP23 peptides in the absence or presence of anti-OX40 and anti-4-1BB. Anti-OX40 and anti-4-1BB mAbs were injected 2 days after immunization (100 µg). As a control, mice were immunized with DCs that were not pulsed (No peptide). Mice were killed 10 days after immunization, and CD8+ T cells were enriched from the spleen. ELISPOT analysis was performed as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is suggested that the majority of tumor-reactive T cells against an immunodominant epitope for a self-Ag are tolerized, resulting in the deletion of high-affinity T cells as we have demonstrated with the p773 peptide in the A2xneu mice (13). We hypothesized that, if not all possible CPs are naturally processed and presented, the possibility exists that T cells against these peptides persist in the repertoire, which can be used as targets to induce a stronger antitumor response against the immunodominant epitopes present on the tumor cells. Consequently, it is possible that this subset of CPs may not have induced tolerance and yet represents immunogens that could elicit effective immune responses against the tumor targets. The results presented in these studies support our hypothesis. We have identified CPs having amino acid substitutions in the primary position of an immunodominant peptide could induce a CTL response while maintaining the T cell specificity against the native peptide. The data presented here show that the identified CPs are capable of inducing a T cell response able to recognize the native p773–782 peptide presented on tumor cells. Furthermore, our results suggest that with the CP it was possible to circumvent tolerance. Critical for identifying these CPs was the use of the PS-SCL strategy (33). Considering that almost all possible combinations L-amino acid decapeptides are present in the peptide library screened allows the identification of T cell ligands with sequences unrelated to the native peptide (33). Additionally, with PS-SCL methodology, we are able to optimize the recognition of new T cell ligands by a given TCR instead of selecting the optimal CP by testing individual substitutions in the amino acids of the native peptide (34, 35). These data confirm the importance of using PS-SCLs in identifying possible CPs recognized by T cells.

The screening results of the p773-CTLs with the decapeptide library revealed that, for 7 of the 10 positions, the amino acids of the native sequence correspond to the defined amino acids of the active mixtures. Similar results were found for Melan-A (28, 36), tyrosinase (37) and SSX-2 (34) after screening specific CTLs with nonapeptide or decapeptide libraries. It is important to note that the p773-CTLs used in these studies were originated from nontolerant mice, and as such, they are of relatively high affinity. We have recently cloned p773-CTLs from A2xneu mice (low affinity). It would be of interest to screen these CTLs with the decapeptide library. These experiments will help us to determine whether the same potential amino acids identified with the high-affinity T cells will also be identified by the low-affinity T cells. If we identify that the low-affinity and high-affinity T cells recognize the same alternative amino acids, this will indicate that the T cell repertoire for these immunodominant epitopes for either tolerant or nontolerant hosts behave similarly and that the identification of CPs could be based on specific changes within the sequence of the peptide. In contrast, we may find that low-affinity T cells recognize different alternative amino acids than the high-affinity T cells and that these CPs could still induce CTLs capable of recognizing the native p773 peptide with higher affinity. These experiments could suggest that the cross-reactivity is based on the specificity and degree of plasticity of each TCR.

Based on the accumulative data presented with the CP, we hypothesized that CP1, CP14, and CP23 circumvent tolerance based on the antigenic mimicry ability of CP1-, CP14-, and CP23-CTLs that were able to cross-react and recognize the p773 native peptide. The support for this hypothesis is based on several points. First, a BLAST (basic local alignment search tool) search against CP1, CP14, and CP23 peptide sequences revealed no matches against known proteins. Analysis of the T cell responses in A2xneu and A2xFVB mice indicate that the immune responses against the CPs in these animals were identical. Therefore, because these peptides were not self-Ags, we could predict that tolerance mechanisms have not deleted CD8 T cells directed against the CPs. Second, CP-CTL repertoires were of lower avidity toward the native p773 peptide, compared with p773-CTLs from A2xFVB mice but of higher avidity, compared with p773-CTLs from A2xneu mice. Based on this, we could make the assumption that the CP-specific CD8 T cells were of intermediate avidity against the p773 epitope. As such, the threshold of avidities of the CP-T cell repertoires was sufficiently low against the p773, and these cells were not eliminated by the p773-tolerance mechanism. Third, it is known that TCRs have a remarkable degree of plasticity and are able to recognize more than two distinct peptides that share little sequence homology (38, 39). Therefore, CP-CTLs could recognize both the CP and the native p773 peptide. These results are in agreement with previous studies demonstrating that the TCR has degenerate specificity and can bind and recognize unrelated MHC-peptide complexes (16, 17, 18, 19, 38, 39).

It is important to note that a few substitutions were made to the native p773 and still the CP1-, CP14-, and CP23-CTLs could recognize the native peptide presented on A2.1+ Her-2/neu+ tumors. It is also important to note that these substitutions made in the CPs were conservative changes. We do not yet understand the differences between the selected peptides in stimulating an immune response and CTL cross-reactivity. All peptides have been shown to bind HLA-A2.1 molecules with a similar degree. There are some CPs that have stronger binding to HLA-A2.1 molecules than CP14 or CP23, and these are not immunogenic or cross-reactive. Based on the data obtained, we cannot correlate immunogenicity with cross-reactivity. One of our future goals is to learn what makes some CPs induce CTLs that are cross-reactive against the native p773 peptide, whereas other peptides are immunogenic but the CTLs do not cross-react with the native p773 peptide. For example, we show that CP1 is immunogenic and induces CTLs that cross-react with the p773 peptide. In contrast, CP2 or CP3 has identical sequences to CP1, except for a single substitution (CP2 has a Y in position 9 instead of an H, and CP3 has an S in position 7 instead of an I). The CP2 and CP3 peptides are immunogenic, but the CTLs do not recognize the native p773 peptide. Further studies are required to better understand CTL cross-reactivity and elucidate whether there are defined rules governing T cell cross-reactivity allowing the design of CPs for different tumor Ags.

Attempts have been made to improve the immunogenicity of the peptide by modifying the amino acid sequence to enhance the binding capacity of the peptide to MHC class I molecules (40, 41, 42, 43). In this regard, several groups have demonstrated the ability to increase the immunogenicity of HLA-A2.1/Her-2/neu-restricted epitopes by modifying the sequence or the peptides. Vertuani et al. (44) demonstrated that by changing position 2 (I for V) and position 9 (L for V) in the p369 epitope, the resultant Her-2.369-V2V9 peptide had a greater HLA-A2 stability and was more immunogenic than the wild-type peptide after immunizing HLA-A2.1/Kb transgenic mice. Importantly, the Her-2.369-V2V9-CTL still recognizes the native Her-2.369 epitope. Castilleja et al. (23) also demonstrated that, by modifying the Her-2.369 epitope in the central region of the peptide (position 5, S for G or A), the derived peptides enhance the production of INF-{gamma} and cytotoxic activity of Her-2.369-specific CTLs from donors that react against the Her-2.369 epitope. Although these studies demonstrated that peptides can be modified and still react against the native epitope, there is no evaluation in a tolerant host as to whether these mutated peptides overcome or circumvent tolerance and whether these peptides induce an antitumor immune response capable of controlling the tumor growth. Other heteroclitic peptides have been examined for other TAAs. For example, substituting a threonine with a methionine at position 210 of the gp:209–217 peptide increases the affinity of the peptide 5-fold. However, vaccination of patients with this peptide shows no objective clinical response (45). This raises the question of whether vaccination with heteroclitic peptides of high affinity for a self-Ag best suits the induction of an antitumor response against a self-tumor Ag. We believe that high-affinity heteroclitic peptides are not the best choice for tumor vaccines because the CTL repertoire against these heteroclitic peptides will probably also be of high avidity, even against the native immunodominant peptide. As such, tolerance might have eliminated the CTL repertoire against the heteroclitic peptides. Therefore, immunizations with the high-affinity heteroclitic peptide might not be of clinical value for developing cancer vaccines, as demonstrated with vaccinations using the gp:209–217 peptide. In contrast, the CPs that we have identified induce CTLs that are of intermediate avidity against the native p773 peptide and may have escaped tolerance against immunodominant epitopes, allowing the identification of CPs of intermediate affinity that could be a better choice for developing vaccines against self-tumor Ags.

Previous studies have demonstrated that using of the PS-SCL makes it possible to identify peptides able to stimulate CD4+ or CD8+ T cells as well as induce immune responses cross-reactive to the native peptides (46, 47, 48). However, none of these studies have evaluated the antitumor effect of these peptides. The data presented here clearly indicate that the CPs are more effective in inducing an immune response than the p773 peptide in A2xneu mice, resulting in a stronger antitumor response. This is mainly for two reasons: 1) CP-CTLs are of higher affinity than p773-CTLs from A2xneu mice; and 2) CP immunizations induce a higher frequency of peptide-specific CTLs, compared with p773 immunization. Even after these characteristics were observed, it was necessary to use multiple immunizations in the presence of costimulatory molecules (anti-OX40 and anti-4-1BB mAbs) to obtain a stronger antitumor response. We do not yet fully understand the significance of applying multiple immunizations. This could be interpreted as follows: 1) because CP immunization induced a CTL response of intermediate affinity against the p773 native peptide, it might be necessary to stimulate and sustain the antitumor response overtime; 2) it might be necessary to constantly restimulate an immune response because the interaction of the CP-specific T cells and tumor cells might not be sufficient to maintain active T cells as might happen with high-affinity T cells; and 3) it could be possible that the stimulated CP-CTLs, which are not of high affinity, are further tolerized or deleted by the tumor. Not much is known about the in vivo interaction of intermediate or low-affinity CTLs and the tumors. Further analysis of how these CTLs interact and behave with tumors will help us to optimize immune responses against self-tumor Ags. Overall, our data show for the first time that with the identification of CPs it is possible to circumvent tolerance, and these peptides are capable of inducing an immune response that significantly prolonged the survival of A2xneu tumor-bearing mice. These studies offer a window of opportunity for exploring and optimizing CP vaccination as a novel strategy to develop more effective vaccines against self-tumor Ags.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (CA 78579) and the University of California Breast Cancer Research Program (9WB-0100 to J.L.). Back

2 Address correspondence and reprint requests to Dr. Joseph Lustgarten, Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121. E-mail address: jlustgarten{at}skcc.org; and Dr. Clemencia Pinilla, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. E-mail address:cpinilla{at}tpims.org Back

3 Abbreviations used in this paper: TAA, tumor-associated Ags; PS-SCL, positional scanning synthetic peptide combinatorial library; CP, cross-reactive peptide; DC, dendritic cell. Back

Received for publication June 21, 2005. Accepted for publication November 8, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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