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The Journal of Immunology, 2005, 174: 7104-7110.
Copyright © 2005 by The American Association of Immunologists

Differential Immunogenicity of Two Peptides Isolated by High Molecular Weight-Melanoma-Associated Antigen-Specific Monoclonal Antibodies with Different Affinities1

Wei Luo, Jeff Chi-feng Hsu, Chun-Yen Tsao, Eric Ko, Xinhui Wang and Soldano Ferrone2

Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peptide mimics isolated from phage display peptide libraries by panning with self-tumor-associated Ag (TAA)-specific mAbs are being evaluated as immunogens to implement active specific immunotherapy. Although TAA-specific mAb are commonly used to isolate peptide mimics, no information is available regarding the Ab characteristics required to isolate immunogenic TAA peptide mimics. To address this question, we have used mAb 763.74 and mAb GH786, which recognize the same or spatially close antigenic determinant(s) of the human high m.w.-melanoma-associated Ag (HMW-MAA), although with different affinity. mAb 763.74 affinity is higher than that of mAb GH786. Panning of phage display peptide libraries with mAb 763.74 and mAb GH786 resulted in the isolation of peptides P763.74 and PGH786, respectively. When compared for their ability to induce HMW-MAA-specific immune responses in BALB/c mice, HMW-MAA-specific Ab titers were significantly higher in mice immunized with P763.74 than in those immunized with PGH786. The HMW-MAA-specific Ab titers were markedly increased by a booster with HMW-MAA-bearing melanoma cells, an effect that was significantly higher in mice primed with P763.74 than in those primed with PGH786. Lastly, P763.74, but not PGH786, induced a delayed-type hypersensitivity response to HMW-MAA-bearing melanoma cells. These findings suggest that affinity for TAA is a variable to take into account when selecting mAb to isolate peptide mimics from a phage display peptide library.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The identification and molecular characterization of human tumor-associated Ags (TAA) 3 have provided well-defined moieties to implement active specific immunotherapy of malignant diseases (1, 2). At variance with their murine counterparts, most human TAA are shared, nonmutated self-Ags that are weakly or nonimmunogenic in patients with malignant diseases (3, 4). Therefore, one of the challenges facing tumor immunologists is the development of strategies to induce a strong immune response to these self-TAAs, because in animal model systems an association has been found between the magnitude of the elicited TAA-specific immune responses and therapeutic efficacy of active specific immunotherapy (5, 6).

Among the strategies used to overcome unresponsiveness to a self-TAA, one relies on the use of TAA mimics as immunogens (7). The validity of this strategy has been demonstrated by the ability of anti-idiotypic (anti-id) mAb, which mimic TAA, to induce TAA-specific immune responses in patients with malignant diseases when the nominal Ag is not immunogenic (8, 9, 10, 11, 12, 13). In a phase I clinical trial, we have demonstrated that administration of the mouse anti-id mAb MK2–23, which mimics the determinant defined by the mouse anti-high m.w.-melanoma-associated Ag (HMW-MAA) mAb 763.74, induced HMW-MAA-specific Abs in ~60% of the immunized melanoma patients (10). The survival of patients who had developed anti-HMW-MAA Abs was significantly longer than that of patients who had not developed this immune response. These clinical findings have prompted us to continue to optimize the immunization strategy with HMW-MAA mimics. In the present study we have investigated the potential of HMW-MAA peptide mimics as an alternative immunogen to anti-id mAb, because peptide mimics have several advantages over anti-id Abs. The relative ease of modifying peptide mimics facilitates the application of strategies to enhance their ability to induce nominal TAA-specific immune responses. Furthermore, peptide mimics can be easily synthesized and standardized, thus expediting the steps required to obtain approval from regulatory agencies for their use in clinical trials.

In the present study we have chosen to pan phage display peptide libraries with anti-HMW-MAA mAb to isolate peptide mimics. This strategy has already been successfully used to isolate peptide mimics of human TAA, including the Lewis Y (LeY) and sialyl Lewis X Ags as well as the oncogenic protein Her-2/neu (14, 15, 16). To date, no information is available about the characteristics of Abs that are required to isolate TAA peptide mimics that are effective in inducing immune responses to the nominal TAA. Therefore, in the present study we have investigated the relationship between the immunogenic properties of HMW-MAA peptide mimics and the affinity of the HMW-MAA-specific mAb used to isolate these peptides from phage display peptide libraries. To address this question, we have used the HMW-MAA-specific mAb 763.74 and anti-anti-id mAb GH786. These mAb recognize the same or spatially close antigenic determinant(s) of HMW-MAA, because mAb 763.74 and mAb GH786 cross-block the binding of each other to HMW-MAA-bearing melanoma cells. However, mAb 763.74 exhibits a markedly higher affinity for HMW-MAA than mAb GH786 (17). Our previous studies have shown that HMW-MAA-binding Abs, elicited by anti-id mAb MK2–23 in mice, rabbits, and melanoma patients, exhibit only a low level of reactivity with melanoma cells (10, 18, 19). This finding does not reflect the poor immunogenicity of mAb MK2–23, because the immunized animals or patients developed high titer anti-anti-id Abs reacting with idiotopes in the Ag-combining site of mAb MK2–23. Because a positive association has been demonstrated between the development of HMW-MAA-binding Abs and clinical outcome (10), we have also investigated in the present study whether HMW-MAA-specific immune responses elicited by the isolated peptide mimics could be enhanced by a booster with HMW-MAA-bearing melanoma cells. This strategy is based on the knowledge that B cell clones undergo somatic hypermutations during the course of an immune response. It is our working hypothesis that after peptide mimic immunization, a booster with HMW-MAA-bearing melanoma cells may selectively expand the mutated B cell clones that secrete Abs with enhanced affinities for HMW-MAA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Phage display peptide libraries

Phage display peptide libraries X15 and LX-8 (XCX8CX), which display random linear 15-mer and random disulfide-constrained 12-mer peptides, respectively, were constructed as previously described (20, 21) and were provided by Dr. J. K. Scott (Simon Fraser University, Burnaby, Canada).

Cell lines

Human HMW-MAA-bearing Colo38 melanoma cells, HMW-MAA-negative M14 clone 5 melanoma cells (provided by Dr. J. B. McCarthy, University of Minnesota, Minneapolis, MN), and HMW-MAA-negative LG2 B-lymphoid cells were maintained at 37°C in a 5% CO2 atmosphere in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (BioWhittaker) and 2 mM L-glutamine (Invitrogen Life Technologies). The M14 clone 5/HMW-MAA cells, which express HMW-MAA after transfection of M14 clone 5 cells with a pcDNA3.1+ plasmid containing the HMW-MAA cDNA, were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and 0.4 mg/ml G418 (Promega).

Antibodies

Mouse anti-HMW-MAA mAb 763.74, generated with human melanoma cells, and mouse anti-HMW-MAA anti-anti-id mAb GH786, generated with anti-id mAb MK2–23, were developed and characterized as previously described (17, 22). Anti-HLA class I Ag mAb TP25.99 was developed as previously described (23). Affinity-purified rabbit anti-mouse IgG Abs and HRP-conjugated goat anti-mouse Ig Abs were purchased from Jackson ImmunoResearch Laboratories. R-PE-labeled F(ab')2 of goat anti-mouse Ig Abs were purchased from BD Pharmingen.

Synthetic peptides

Peptides P763.74 (QCTGPNVATNCR), PGH786 (NQLPQYMGPAPAYMR), and MB1194–208 (CDNVADLHEKYSGSTP; a control peptide derived from the human proteasome subunit MB1) were purchased from the Molecular Genetics Instrumentation Facility, University of Georgia (Athens, GA). A cysteine residue was added to the N terminus of each synthetic peptide for conjugation to keyhole limpet hemocyanin (KLH) (24), using the coupling agent m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce).

Animals

Eight-week-old female BALB/c mice were purchased from Taconic Farms.

Isolation of peptides from phage display peptide libraries with anti-HMW-MAA mAb

Panning of amplified X15 and LX-8 peptide libraries with mAb was performed as previously described (25). Screening of randomly selected phage clones that react with mAb 763.74 or mAb GH786 was performed by the immunoscreening assay, as previously described (21). DNA sequences encoding the peptide inserts from isolated phage clones were determined by the dideoxynucleotide chain termination method as previously described (26), using the M13 primer (5'-AGTAGCAGAAGCCTGAAGA-3').

Binding assays

The binding assays to assess the reactivity of mAb with peptides and of sera with peptides and cells were performed as previously described (23, 27). The Ab titer was defined as the highest serum dilution that resulted in 50% of maximal binding of serum to peptides or cells. The inhibition assay to determine the ability of synthetic peptides to inhibit binding of mAb to HMW-MAA was performed by incubating increasing amounts of synthetic peptides with biotinylated anti-HMW-MAA Abs and then measuring the reactivity of the mixture with HMW-MAA-expressing human melanoma cells, as previously described (27). Results are expressed as percent inhibition by peptides (inhibitor) of mAb binding to HMW-MAA-bearing Colo38 melanoma cells, calculated using the formula: % inhibition = ((OD450 in the absence of inhibitor – OD450 in the presence of inhibitor)/OD450 in the absence of inhibitor) x 100.

Immunization of mice

BALB/c mice (eight per group) were immunized s.c. at 3-wk intervals with peptides that had been conjugated to KLH. Fifty micrograms of each KLH-conjugated peptide was mixed 1/1 (v/v) with CFA for priming (day 0) and with IFA for subsequent injections (days 21, 42, 63, 84, and 105). Mice were boosted with an s.c. injection of HMW-MAA-bearing Colo38 melanoma cells (5 x 105 cells/mouse) on day 132. HMW-MAA-negative LG2 human B lymphoid cells (5 x 105 cells/mouse) served as a specificity control. Mice were bled on day 0, and 1 wk after each injection.

Flow cytometric analysis

Flow cytometric analysis of melanoma cells stained with sera from immunized mice was performed as previously described (28). Briefly, 5 x 105 cells were incubated for 1 h on ice with 100 µl of mouse immune sera (1/60 dilution). Cells were then washed twice with 0.5% BSA in PBS (BSA-PBS) and incubated for an additional 30 min on ice with R-PE-labeled F(ab')2 of goat anti-mouse Ig Abs (1/100 dilution). After two washes, cells were fixed in 2% paraformaldehyde and analyzed with a FACScan flow cytometer (BD Biosciences). Results were analyzed with CellQuest software (BD Biosciences) and are expressed as fluorescence intensity units.

Immunochemical assays

Cells labeled with 125I (Na125I; Amersham Biosciences) by the lactoperoxidase method (29) were solubilized in 1% Triton X-100-containing lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin, pH 7.4). Indirect immunoprecipitation, SDS-PAGE, and autoradiography were performed following the methodology previously described (30).

Delayed-type hypersensitivity (DTH) reaction

Irradiated (20K rad) cultured Colo38 human melanoma cells (5 x 105 cells/mouse) were injected into the right hind footpads of mice that had received six immunizations with HMW-MAA peptide mimics on days 0, 21, 42, 63, 84, and 105. The thickness of each footpad was measured at the indicated times as previously described (18). The amount of swelling induced by the injected HMW-MAA-bearing cells was calculated by subtracting the thickness of the footpad measured at 0 h from that measured at the indicated time point (24, 48, or 72 h) after injection of cells. Mice immunized with peptide MB1194–208 and mice injected with HMW-MAA-negative LG2 cells (5 x 105 cells/mouse) served as specificity controls. Results are expressed as the mean swelling ± SD.

Statistical analysis

The statistical significance of differences in the experimental results was analyzed using two-tailed, unpaired, Student’s t test. A difference was considered statistically significant at p ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Isolation of peptides from phage display peptide libraries LX-8 and X15 by panning with anti-HMW-MAA mAb 763.74 and mAb GH786

Panning of LX-8 and X15 libraries with mAb 763.74 resulted in the isolation of eight phage clones from the LX-8 library, but no clones from the X15 library. All of these clones reacted specifically with mAb 763.74. Sequencing of DNA encoding the peptides displayed by these clones identified the peptide sequence QCTGPNVATNCR (P763.74) in six of eight clones, and QCTGPNFATNCR and TCNGPSVYMNCL in each of the remaining two clones. P763.74 displayed the highest binding reactivity with mAb 763.74 and was chosen for subsequent experiments (data not shown). Panning of LX-8 and X15 libraries with mAb GH786 resulted in the isolation of four and six phage clones from the LX-8 and X15 libraries, respectively. Sequencing of DNA encoding the peptides displayed by these clones identified the peptide sequence TCRLPFQNVACH in two clones and SCLLPFQNIFCS in the other two clones isolated from the LX-8 library. The sequence NQLPQYMGPAPAYMR (PGH786) was identified in all six clones isolated from the X15 library. PGH786 displayed the highest binding reactivity to mAb GH786 and was chosen for subsequent experiments (data not shown).

Reactivity of synthetic peptides P763.74 and PGH786 with mAb 763.74 and mAb GH786

Peptides P763.74 and PGH786 were compared for their specific binding reactivity to their corresponding panning mAb and for their ability to inhibit the binding of these mAb to HMW-MAA. Each peptide reacted with its corresponding panning mAb in a dose-dependent manner (Fig. 1, A and B). Each peptide also specifically inhibited the binding of its corresponding panning mAb to HMW-MAA-bearing melanoma cells in a dose-dependent manner, but did not cross-inhibit the binding of the other mAb to HMW-MAA-bearing melanoma cells (Fig. 1, C and D). These results indicate that peptides P763.74 and PGH786 react specifically with the Ag-combining sites of mAb 763.74 and mAb GH786, respectively.



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  		FIGURE 1. Reactivity of peptides P763.74 and PGH786 with anti-HMW-MAA mAb 763.74 and mAb GH786. A and B, Increasing amounts of peptide P763.74 (•; A) or PGH786 (•; B) were coated on 96-well flexible plastic plates and incubated with biotinylated forms of their corresponding panning mAb (100 µg/ml biotinylated mAb 763.74 or mAb GH786, respectively). After the additional incubation with HRP-conjugated streptavidin, the reaction was developed using 3,3',5,5'-tetramethylbenzidine substrate. Results are presented as OD measured at 450 nm. Peptide MB1194–208 was used as a control ({circ}; A and B). C and D, Increasing amounts of peptide P763.74 ({blacktriangleup}) or PGH786 ({circ}) were incubated with 0.25 µg/ml biotinylated mAb 763.74 (C) or mAb GH786 (D), and then incubated with HMW-MAA-bearing Colo38 melanoma cells (2 x 105 cells/well). After the additional incubation with HRP-conjugated streptavidin, the reaction was developed using 3,3',5,5'-tetramethylbenzidine substrate. OD was measured at 450 nm. Results are expressed as the percent inhibition of the binding of mAb to HMW-MAA-bearing Colo38 melanoma cells.

 
Immunogenicity of HMW-MAA peptide mimics P763.74 and PGH786 in BALB/c mice

Mice immunized with peptide mimic P763.74 or PGH786 were tested for the development of Abs reacting with the immunizing peptide and with HMW-MAA-bearing melanoma cells in binding assays. The kinetics of the development of Ab populations are shown in Fig. 2. Analysis of sera harvested from mice immunized with peptide P763.74 or PGH786 demonstrated increasing titers of peptide-reactive Abs. The anti-PGH786 Ab titer obtained from PGH786-immunized mice was significantly (p < 0.05) higher than the anti-P763.74 Ab titer obtained from P763.74-immunized mice (Fig. 2A). Reactivity with HMW-MAA-bearing Colo38 melanoma cells, but not with HMW-MAA-negative LG2 B lymphoid cells, was detected in sera from six of the eight mice immunized with peptide P763.74 and in four of the eight mice immunized with peptide PGH786. In contrast to the peptide-reactive Ab titers, the mean titers of HMW-MAA-bearing melanoma cell-reacting Abs were significantly (p < 0.05) higher in sera obtained from P763.74-immunized mice than in sera obtained from PGH786-immunized mice (Fig. 2C). The selective reactivity of these sera with HMW-MAA-bearing melanoma cells was confirmed by their reactivity with M14 clone 5/HMW-MAA cells that express HMW-MAA (Fig. 3), but not with the parental M14 clone 5 cells or empty plasmid-transfected M14 clone 5 cells. The latter cells do not express HMW-MAA. Sera from mice immunized with the control MB1194–208 peptide displayed strong reactivity with MB1194–208 peptide, but no reactivity with HMW-MAA-bearing Colo38 cells. These sera also did not stain M14 clone 5/HMW-MAA cells (Fig. 3). Despite their reactivity with HMW-MAA-bearing melanoma cells in binding assays, sera from P763.74- or PGH786-immunized mice were not able to immunoprecipitate any components from radiolabeled HMW-MAA-bearing Colo38 melanoma cell lysates (data not shown).



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  		FIGURE 2. Development of Abs with selective reactivity with HMW-MAA-bearing melanoma cells in BALB/c mice immunized with peptide P763.74 or PGH786. Mice were immunized with KLH-conjugated peptide P763.74 ({blacksquare}), PGH786 ({cjs2108}), or control peptide MB1194–208 ({square}; 50 µg of each peptide/injection/mouse) on days 0, 21, 42, 63, 84, and 105 and with HMW-MAA-bearing Colo38 melanoma cells (5 x 105 cells/injection/mouse) on day 132. Sera were harvested on day 0, and 7 days after each immunization. A, Two-fold dilutions of sera were incubated with plate-bound P763.74 or PGH786. After the additional incubation with HRP-conjugated goat anti-mouse IgG Abs, the reaction was developed using 3,3',5,5'-tetramethylbenzidine substrate. OD was measured at 450 nm. Results are expressed as the mean ± SD of the highest dilution of sera from eight mice giving 50% of the maximal binding to the immunizing peptide. Peptide MB1194–208 was used as a control. *, p < 0.05. B, Sera harvested from mice immunized with control peptide MB1194–208 react with MB1194–208, but not with P763.74 or PGH786. C, Two-fold dilutions of sera (100 µl/well) were incubated with human HMW-MAA-bearing Colo38 melanoma cells (1 x 105/well). After the additional incubation with HRP-conjugated goat anti-mouse IgG Abs, the reaction was developed using 3,3',5,5'-tetramethylbenzidine substrate. OD was measured at 450 nm. Sera from mice immunized with irrelevant peptide MB1194–208 were used as controls. Results are expressed as the mean ± SD of the highest dilution of sera giving 50% of the maximal binding to HMW-MAA-bearing melanoma cells. *, p < 0.05. D, Sera from mice immunized with P763.74, PGH786, or the control peptide MB1194–208 did not react with non-HMW-MAA-bearing LG2 human lymphoid cells. Anti-HLA class I Ag mAb TP25.99 was used as a positive control and reacted with LG2 cells (data not shown).

 


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  		FIGURE 3. Flow cytometric analysis of HMW-MAA-bearing M14 clone 5/HMW-MAA melanoma cells stained with Abs elicited by peptides P763.74 and PGH786 in BALB/c mice. M14 clone 5/HMW-MAA transfectants and parental non-HMW-MAA-bearing M14 clone 5 cells were incubated on ice with 100 µl of sera (1/60 dilution) from mice that were immunized with peptide P763.74, PGH786, or control peptide MB1194–208 only (A) or with sera from mice immunized with P763.74, PGH786, or control peptide MB1194–208 and boosted with Colo38 melanoma cells (B). After washing, cells were incubated on ice with R-PE-labeled F(ab')2 of goat anti-mouse Ig Abs. Cells were then washed, fixed in 2% paraformaldehyde, and analyzed with a FACScan flow cytometer. Results are expressed as fluorescence intensity (empty histogram). Preimmune sera were used as controls (gray histogram). M14 clone 5/HMW-MAA and M14 clone 5 melanoma cells incubated with sera from mice that had been immunized with P763.74 or PGH786 and boosted with HMW-MAA-negative LG2 cells (C) or incubated with anti-HMW-MAA mAb 763.74 (10 µg/ml; D) were used as controls.

 
Induction of a DTH response to HMW-MAA-bearing melanoma cells in BALB/c mice immunized with HMW-MAA peptide mimic P763.74 or PGH786

Injection of HMW-MAA-bearing Colo38 melanoma cells into the right hind footpad of mice immunized with either peptide P763.74 or PGH786 induced enhanced swelling compared with that of mice immunized with the same peptide but injected with LG2 human B-lymphoid cells, which do not express HMW-MAA. The increase in swelling was statistically significant (p < 0.05) in peptide P763.74-immunized mice, but not in PGH786-immunized mice compared at 24 h after cell injection (Fig. 4), and the swelling persisted for 48 h (data not shown). The DTH response to melanoma cells induced by HMW-MAA peptide mimics was specific, because no swelling was observed in Colo38 cell-injected mice that had been immunized with the control MB1194–208 peptide.



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  		FIGURE 4. DTH reaction to HMW-MAA-bearing Colo38 melanoma cells in BALB/c mice immunized with peptide P763.74 or PGH786. BALB/c mice (eight per group) were immunized with KLH-conjugated HMW-MAA peptide mimics (P763.74 or PGH786; 50 µg of each peptide/injection/mouse) on days 0, 21, 42, 63, 84, and 105. On day 132, irradiated HMW-MAA-bearing Colo38 melanoma cells (5 x 105 cells/injection/mouse; {cjs2108}) and HMW-MAA-negative LG2 lymphoid cells (5 x 105 cells/injection/mouse; {square}) were injected into the right and left hind footpads, respectively. Swelling was determined by subtracting footpad thickness at 0 h from that measured 24 h after the injection of cells. Results are expressed as the mean swelling ± SD. BALB/c mice immunized with control peptide MB1194–208 by the same schedule were also injected at the same time point with either Colo38 or LG2 cells and were used as controls. *, p < 0.05.

 
Enhancement of immune responses elicited with HMW-MAA peptide mimics by a booster with HMW-MAA-bearing melanoma cells

To investigate whether HMW-MAA-specific reactivity induced by peptides P763.74 and PGH786 can be augmented by a booster with the original Ag, mice immunized with these peptide mimics were boosted s.c. with HMW-MAA-bearing Colo38 melanoma cells (5 x 105 cells/mouse) on day 132. HMW-MAA-bearing melanoma cells were used for this purpose due to the practical difficulties in isolating or producing sufficient quantities of purified HMW-MAA. The booster with Colo38 melanoma cells resulted in a markedly enhanced reactivity of the sera with HMW-MAA-bearing melanoma cells; the mean Ab titer increased from 1/60 to 1/250 in P763.74-immunized mice and from 1/40 to 1/80 in PGH786-immunized mice, with a significant (p < 0.05) difference between these two groups (Fig. 2). However, this booster did not increase the reactivity with HMW-MAA-bearing melanoma cells of sera from mice that had failed to develop HMW-MAA-binding Ab responses after immunization with peptide P763.74 or PGH786. The enhanced reactivity is specific to HMW-MAA-bearing melanoma cells, because the same sera did not react with HMW-MAA-negative LG2 B-lymphoid cells. Flow cytometric analysis also showed that these sera strongly stained M14 clone 5/HMW-MAA cells, but did not stain the parental M14 clone 5 cells (Fig. 3) or empty plasmid-transfected M14 clone 5 cells. This finding is in contrast to the lack of reactivity with M14 clone 5/HMW-MAA cells of sera from mice immunized with the control peptide MB1194–208 and boosted with HMW-MAA-bearing Colo38 melanoma cells (Fig. 3). The specificity of the Ab response against HMW-MAA in mice immunized with peptide P763.74 or PGH786 and boosted with HMW-MAA-bearing melanoma cells was further analyzed by immunochemical assays. SDS-PAGE analysis showed that sera from mice that had been immunized with peptide P763.74 and boosted with HMW-MAA-bearing Colo38 melanoma cells immunoprecipitated the two subunits of HMW-MAA from radiolabeled Colo38 cell lysate (Fig. 5). Somewhat surprisingly, sera from mice immunized with peptide PGH786 and boosted with Colo38 cells failed to immunoprecipitate any HMW-MAA components from radiolabeled Colo38 cell lysate. However, as expected, neither sera from mice immunized with the control MB1194–208 peptide and boosted with Colo38 cells nor sera from mice immunized with either P763.74 or PGH786 and boosted with HMW-MAA-negative LG2 cells were able to immunoprecipitate HMW-MAA from radiolabeled Colo38 cell lysate.



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  		FIGURE 5. SDS-PAGE analysis of Ags immunoprecipitated from Colo38 melanoma cell lysates by sera from mice immunized with peptide P763.74 or PGH786 and boosted with HMW-MAA-bearing Colo38 melanoma cells. 125I-labeled Colo38 melanoma cells were solubilized with 1% Triton X-100-containing lysis buffer. Ags were immunoprecipitated from the radiolabeled cell lysate with sera from mice sequentially immunized with peptide P763.74 and with HMW-MAA-bearing Colo38 melanoma cells (B) or with sera from mice sequentially immunized with peptide PGH786 and with HMW-MAA-bearing Colo38 melanoma cells (C). Anti-HMW-MAA mAb 763.74 (A), sera from mice sequentially immunized with irrelevant peptide MB1194–208 and with HMW-MAA-bearing Colo38 melanoma cells (D), sera from mice sequentially immunized with peptide P763.74 and with LG2 lymphoid cells (E), sera from mice sequentially immunized with peptide PGH786 and with LG2 lymphoid cells (F), and preimmune sera (G) were used as controls. Ags were eluted from the immunoadsorbent, separated by 8% SDS-PAGE, and processed for autoradiography using Hyperfilm (Amersham Biosciences).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Several lines of evidence indicate that peptides P763.74 and PGH786, isolated by panning phage display peptide libraries with mAb 763.74 and mAb GH786, respectively, are mimics of HMW-MAA. First, each peptide reacted in vitro with the Ag-combining site of the respective anti-HMW-MAA mAb used to carry out the panning process. Second, each peptide induced selective Ab responses (albeit at low titers) directed against HMW-MAA-bearing melanoma cells. Interestingly, the sequences of the two peptides displayed no homology with the published amino acid sequence of HMW-MAA core protein. These findings suggest that mimicry of the HMW-MAA determinant(s) by these two peptides reflects structural homology. In addition, although mAb 763.74 and mAb GH786 share ~90% amino acid sequence identity in their VH and VL regions (S. Ferrone, unpublished observations), peptides P763.74 and PGH786 reacted only with the binding sites of their corresponding panning mAb and did not cross-react with the other mAb.

The immune responses elicited by peptide mimics P763.74 and PGH786 resemble that elicited by anti-id mAb MK2–23, which also mimics the HMW-MAA antigenic determinant defined by mAb 763.74. First, a marked variability in the level of HMW-MAA-specific immunity has been found in mice immunized with peptide P763.74 or PGH786. This variability ranged from lack of detectable HMW-MAA-specific immune response to relatively strong immunity, although the immunized hosts were syngeneic mice obtained from one colony. This variability is also frequently found in mice that have been immunized with other TAA mimics (18, 31). The mechanism(s) underlying this observation has not been elucidated. Furthermore, consistent with our previous findings in mice immunized with anti-id mAb MK2–23 (18), these two peptides elicited two major Ab populations: one subset reacts with only the immunizing peptide, and the other reacts with the immunizing peptide and cross-reacts with HMW-MAA. The titer of Abs reacting with HMW-MAA-bearing melanoma cells induced by HMW-MAA peptide mimics is low, although that of Abs reacting with the immunizing peptides is high. The high titer of the latter Abs indicates that the immunization schedule used is effective in generating a robust humoral immune response and cannot account for the low titer of anti-HMW-MAA Abs.

The present study has also shown that HMW-MAA peptide mimics could induce a DTH reaction to HMW-MAA-bearing melanoma cells in BALB/c mice. This finding is consistent with the induction of a DTH reaction to HMW-MAA-bearing melanoma cells after immunization with anti-id mAb MK2–23 (18) and raises the possibility that a peptide mimic can elicit both humoral and cellular immune responses to the nominal TAA.

In the present study we have demonstrated that HMW-MAA-specific Ab titers induced by peptides P763.74 and PGH786 can be specifically and significantly enhanced by boosting with HMW-MAA-bearing melanoma cells. This prime-boost immunization strategy has previously been used with TAA mimics in several carbohydrate TAA systems (15, 32, 33). However, the results reported in the literature are conflicting. Chapman et al. (33) have reported that melanoma patients immunized with anti-id mAb BEC2, which mimics GD3 ganglioside, followed by GD3-L-KLH did not develop GD3-specific Ab responses. In contrast, Kieber-Emmons et al. (15) have shown that anti-LeY responses in mice primed with the minigene encoding LeY mimics is significantly enhanced by boosting with LeY. Our findings are consistent with those from the latter report and support the hypothesis that hypersomatic mutations in the variable regions of Abs elicited by a TAA mimic may enhance the association constant of the mutated Abs for the nominal TAA. Selective expansion of a B cell population(s) producing mutated Abs by a booster with the nominal TAA may eventually result in a marked increase in Abs that are highly reactive to TAA in the immunized host.

Several lines of evidence indicate that peptide P763.74 is more effective in inducing HMW-MAA-specific immune responses than peptide PGH786. First, although P763.74 induced a lower titer of peptide-specific Abs than PGH786, P763.74 induced a significantly higher titer of Abs reacting with HMW-MAA-bearing melanoma cells than PGH786. Second, the enhancement of anti-HMW-MAA Ab responses after a booster with HMW-MAA-bearing melanoma cells was much stronger in mice immunized with peptide P763.74 than in mice immunized with peptide PGH786. Third, P763.74, but not PGH786, was able to specifically induce a DTH reaction to HMW-MAA-bearing melanoma cells.

Characterization of the immune response elicited by peptide mimics in several TAA systems has shown that the level of Abs reacting with the nominal TAA is lower than that of the Abs reacting with the immunizing peptide mimics. Similar results have been reported for other types of TAA mimics, including xenogeneic Ags, which display homology in their amino acid sequence with human TAA (34, 35), and anti-id Abs, which bear the internal image of human TAA (19, 36). To overcome this limitation, investigators are exploring several approaches to augment the TAA-specific immune response elicited by TAA mimics. These approaches include 1) prime-boost immunization strategies, which we have used in this study; 2) administration of TAA mimics with adjuvants (37) or cytokines (38); and 3) enhancement of the ability of peptide mimics to mimic the nominal TAA, based on information derived from structural analysis of the interactions of TAA peptide mimics with the corresponding Abs (39, 40).

Our present study demonstrates that the affinity for nominal TAA of the Ab used to isolate TAA peptide mimics from phage display peptide libraries may play an important role in the identification of TAA mimics that can effectively induce TAA-specific immune responses. This finding may suggest a new strategy to enhance the immunogenicity of TAA mimics. However, caution must be exercised in the interpretation of these results, because only one peptide dose, one immunization schedule, and one Ag-Ab system were used. Nevertheless, the results we have obtained argue in favor of the possibility that an Ab with higher affinity to the nominal Ag may be more effective for isolating TAA peptide mimics with a strong ability to induce TAA-specific immune responses than one with lower affinity to the nominal Ag.


   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 U.S. Public Health Service Grants P01CA89480 and R01CA105500 awarded by the National Cancer Institute, Department of Health and Human Services, and by a grant from the Harry J. Lloyd Charitable Trust. Back

2 Address correspondence and reprint requests to Dr. Soldano Ferrone, Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. E-mail address: soldano.ferrone{at}roswellpark.org Back

3 Abbreviations used in this paper: TAA, tumor-associated Ag; anti-id, anti-idiotypic; DTH, delayed-type hypersensitivity; HMW-MAA, high m.w.-melanoma-associated Ag; KLH, keyhole limpet hemocyanin; LeY, Lewis Y Ag. Back

Received for publication December 29, 2004. Accepted for publication March 25, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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