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Identification of an Antigenic and Immunogenic Motif Expressed by Two 7-Mer Rituximab-Specific Cyclic Peptide Mimotopes: Implication for Peptide-Based Active Immunotherapy¹

Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Two 7-mer cyclic peptides—Rp15-C and Rp13-C—which bear the antigenic motif recognized by the anti-CD20 mAb rituximab, but have different motif-surrounding amino acids, show a comparable avidity for rituximab and inhibit the binding of rituximab to raft-associated CD20 and rituximab-induced membrane ceramide on human lymphoid Daudi cells. Their immunogenic profiles differed: Abs recognizing CD20 were induced in two and five of five BALB/c mice immunized with Rp15-C and Rp13-C, respectively. Analysis of immunogenic motif, performed by panning a 7-mer phage-display peptide library with purified anti-peptide IgGs, showed that the motif defined by anti-Rp15-C mostly included amino acids surrounding the rituximab-specific antigenic motif <aNPS>, whereas that defined by anti-Rp13-C was <NPS>. These data indicate that their motif-surrounding amino acids can markedly influence the specificity of Abs, even when elicited with a short 7-mer peptide. Because these anti-peptide Abs are of IgG isotype, their specificity is likely to reflect how peptides are processed at the T cell level and suggest that, within a short peptide, the motifs defined by T cells during the initial phase and upon their stimulation may be different. Our findings may account for the failure of most forms of peptide-based immunotherapy in cancer and autoimmune diseases in which anti-mimotope Abs are expected to play a relevant therapeutic effect. They also suggest strategies to implement the specificity of peptide-induced Abs against the target Ag.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The CD4⁺ T cell and the humoral immune response play an important role in active immunotherapy against tumor cells and can reject a tumor even more efficiently than CD8⁺ cells (1). Thus, the major challenge for active specific immunotherapy is the development of an effective method to elicit a therapeutic B cell immune response against Ags, which are successfully targeted in passive immunotherapy (PIT)³ with the corresponding mAb.

This approach has been hampered because purified Ag in sufficient amounts for vaccination protocols cannot be readily obtained and purification itself may result in denaturation. However, as observed with rCD4 (2, 3) or the CD20-derived 44-mer large peptide (4), even when purified Ags are available, they may expose hidden epitopes that are more immunogenic than those normally exposed on the native (membrane-associated) molecule. The immune response generated against the target cells by these epitopes is usually poorly effective or not effective at all.

One way of eliciting a more vigorous response, specifically directed against the nominal Ags, is to use small peptides that mimic the epitope recognized by the therapeutic mAb. Peptides are easily synthesized and can be readily manipulated to implement the specificity of the immune response. Furthermore, their small size means that they are likely to express only the functional epitope, i.e., the one that mimics the target Ag.

In this context, the phage-display peptide library (PDPL) has emerged as a powerful technique for the isolation of small peptides that are mimics of Ags successfully used as PIT targets. PDPL consists of phage particles, each expressing peptides differing in their sequence to build up a repertoire of at least 1 x 10⁹ different sequences. Screening of PDPL with the target Ag-specific mAb results in the isolation of clones expressing mAb-specific peptides. Their sequence can be deduced by using appropriate primers and phage clone-derived nucleic acids. If the peptides thus isolated are complementary to the V region of the mAb (i.e., able to inhibit the mAb binding to the target Ag), the alignment of their sequences will provide information on the epitope defined by the mAb (antigenic motif). This peptide is likely to induce an immune response against the target Ag in the immunized host and it will be referred to as mimotope.

Mimotopes have been successfully isolated and shown to elicit Abs against the nominal Ags and/or to trigger biological effects similar to those of the PIT in different clinical settings, namely tumors (5, 6, 7, 8), autoimmune diseases (8, 9), and infectious diseases (10, 11).

One major concern has been the degree of variability in the levels of the Abs against the nominal Ag detected (or that which is undetectable, in some cases) in the immunized host. When detected, their level and their relative avidity binding are always lower than those of anti-peptides Ab (7, 10, 12). These considerations, in conjunction with recent observations indicating that the effectiveness of peptide-based vaccine is associated with the magnitude of the immune response against the nominal Ag (13, 14), prompted us to define the mechanism(s) underlying mimotope failure to induce an effective response against the target Ag.

One possibility is that the amino acids of the mimotope, which are essential for mAb binding (antigenic motif), may be weakly or not immunogenic. In this case, the peptide will raise Abs recognizing amino acids (immunogenic motif) located either partly or completely outside the antigenic motif. These Abs will not bind the target Ag.

To test this hypothesis, we used a panel of 11 cysteine-constrained 7-mer rituximab-specific peptides, all expressing the rituximab-specific antigenic motif a/sNPS (matching the ¹⁷⁰ANPS¹⁷³ aa stretch of CD20), but with different motif-surrounding amino acids (8).

Having demonstrated that one of them (peptide Rp15-C) induced anti-CD20 Abs (8) and found that these Abs were detected in only one of the five immunized BALB/c mice (our unpublished observations), we selected (from the panel) peptide Rp13-C with motif-surrounding amino acids chemically different from those of Rp15-C and compared their antigenic and immunogenic profiles.

In this study, we show that, though antigenically comparable with respect to their reactivity with rituximab, Rp13-C elicited anti-CD20 Abs more consistently than Rp15-C. Screening of PDPL with purified mouse anti-peptide IgG and sequence analysis of the isolated phage-clone inserts indicate that the differences between them reflect the recognition by anti-peptide Abs of motif (immunogenic motif), the one defined by anti-Rp13-C Abs being almost identical with the antigenic motif, whereas that seen by anti-Rp15-C IgG is determined by amino acids surrounding it.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

Female BALB/c mice (8- to 12-wk old) were purchased from Charles River Laboratories.

Cells

The human B lymphoid CD20⁺ cell lines Raji and Daudi, and the T lymphoid cell line CEM (CD20^–), were grown in RPMI 1640 medium, supplemented with 10% FCS (complete medium; HyClone) and 5 mM L-glutamine.

Conventional reagents, mAb, and PDPL

Electrophoresis reagents were purchased from Bio-Rad. Unless otherwise specified, all chemicals were purchased from BDH Chemicals. The anti-CD20 rituximab (chimeric IgG1) and its isotype-matched TNF--specific mAb infliximab were purchased from IDEC Pharmaceutical and Centocor, respectively. The anti-HLA class I mAb TP25.99 was described previously (15).

The anti-HLA class I mAb HC-10-specific peptide Qp-1a (9) and peptide Rp15-C, mimic of the CD20 epitope recognized by rituximab (8), were previously characterized. The rituximab-specific phage-clone insert sequences, including that expressed by R15-C phage clone (template for the synthesis of Rp15-C), were previously identified (Table I) (8) by screening a PDPL, expressing cysteine-constrained 7-mer peptide (c7c PDPL), with rituximab. All inserts expressed the rituximab-specific antigenic motif <a/sNPS> and had different motif-surrounding amino acids.

F(ab')₂ were obtained by pepsin digestion of chimeric rituximab and infliximab as previously described (16). Their purity was assessed by SDS-PAGE. mAb concentration was determined in a bicinchoninic acid assay (Pierce). Purified mAb were coupled to biotin using the biotin-N-hydroxysuccinimide ester (Sigma-Aldrich), as previously described (17). The c7c PDPL was purchased from New England Biolabs. Its characteristic have been previously described in detail (9).

Synthesis of peptides

Cyclic peptides were synthesized at Sigma-Genosys and Primm "Peptide Synthesis Service." Their quality was assessed by analytical reverse-phase chromatography and mass spectral analysis. Their purity was >80%.

Keyhole limpet hemocyanin (KLH) conjugation of peptides and BALB/c mice immunization

The animal studies were reviewed and approved by the institutional review committee. Peptide was coupled to KLH by means of glutaraldehyde as previously described (8). BALB/c mice were primed with 1 µg of peptide-KLH (or KLH only) mixed with CFA, then boosted with the same amount of immunogen mixed with IFA on days 7, 14, 21, and 28. Sera were harvested on day 28 and then weekly until the eighth week. Sera drawn on day 35 displayed the highest binding titer with the corresponding immunogen and were used for the assay. Sera (38 µl) drawn on days 35, 42, 49, and 56 were pooled and used as the source for the purification of peptide-specific Ig.

Purification and specificity of anti-peptide IgG from mouse serum

Ig was purified by precipitation with caprylic acid, as described (18). Briefly, 150 µl of pooled mouse serum were diluted with 600 µl of acetate buffer (60 mM, pH 4.0) and the pH was adjusted to 4.5 with 1 M Tris. Then, caprylic acid (25 µl/ml of diluted sample) was added dropwise with vigorous mixing. After a 30-min stirring at room temperature, the insoluble material was centrifuged for 30 min at 10,000 x g. The supernatant was harvested, passed through a 0.8-µm filter (Albet) to remove insoluble particles, and extensively dialyzed against PBS for 24 h. Purity and concentration were assessed as described above. IgG recovery was 1.6 µg/µl. Anti-KLH IgG were removed from the preparation by four incubations, each for at least 2 h at 25°C (under continuous shaking), in wells of a 96-well polyvinylchloride microtiter plate previously coated with KLH by an overnight incubation with 100 µl/well of a PBS solution containing 100 µg/ml KLH. The specificity of purified anti-peptide IgG was assessed in ELISA, as previously described (9).

Immunofluorescence assays

Confocal microscopy. Confocal microscopy inhibition assay was performed by preincubating 50 µl of a PBS solution containing rituximab (2.5 µg/ml) with an equal volume of PBS solution containing 100 µg/ml peptide for 1 h at 4°C. Then, the mixture was added to human B lymphoid cells Raji (1 x 10⁶/tube) and incubation was prolonged for 10 min at 37°C. Cells were next washed once with 4 ml of PBS-BSA and fixed with paraformaldehyde (2% final dilution) for 15 min at 25°C. They were then washed and incubated with biotinylated CTB for 30 min on ice. After washing, cells were incubated with 50 µl of PBS containing an appropriate dilution of affinity purified FITC-xenoantibodies to human IgG (to detect rituximab) and PE-streptavidin (to detect CTB in the raft) for 30 min on ice. Following an additional washing, cells were mounted on glass coverslips with DABCO mounting medium (Sigma-Aldrich) and examined with a Nikon confocal microscope attached to a CCD camera (Nikon digital sight DS-U1), using a x60 Plan Apo VC objective. An argon laser at 488 nm was used to excite FITC and a helium-neon laser was filtered at 560 nm to excite PE. Cells stimulated with rituximab in the presence of a negative control peptide or in the absence of inhibitor were used as negative controls.

FACS analysis. Inhibition by peptide of rituximab-induced membrane ceramide increase of Daudi cells was conducted as previously described (19), with minor modifications. Briefly, 50 µl of PBS solution containing rituximab (10 µg/ml) were mixed with an equal volume of PBS containing 10-fold scalar concentrations (starting concentration 400 µg/ml) of peptide or plain PBS. After a 1-h incubation at 4°C, the mixture was added to human B lymphoid cell line Daudi (1 x 10⁶/tube) and incubation prolonged for 10 min at 37°C to stimulate cells. Next, cells were washed once with 4 ml of PBS-BSA, fixed with paraformaldehyde, and incubated with 50 µl of PBS containing rabbit IgG (50 µg/ml) for 30-min at 4°C to block FcR-binding sites. Ceramide expression was evaluated by sequential incubation (30 min at 4°C) of cells with an appropriate dilution of anti-ceramide IgM mAb and FITC-xenoantibodies to mouse IgM. Immunofluorescence was measured using a FACScan cytometer. The reactivity of anti-peptide serum with lymphoid cells and the inhibition by peptide or F(ab')₂ of mAb of anti-peptide serum or mAb binding to lymphoid cells were assessed as previously described (8).

Immunochemical assays

Immunochemical assays were performed as previously described (8). The relative avidity of rituximab for peptides Rp15-C and Rp13-C was assessed in a solid-phase ELISA by testing the binding of different concentrations of rituximab with saturated concentrations of the peptides, following procedures previously described (8, 20).

Affinity selection, immunoscreening, and sequence analysis

Biopanning of PDPL with purified anti-peptide mouse IgG was performed as described (8), the only difference being that anti-KLH mouse IgG were used to remove either anti-peptide IgG allotype/isotype-matched IgG or anti-KLH Ig-specific phage particles. ELISA screening of phage clones and nucleotide sequence analysis of Ab-specific phage-clone inserts was completed as previously reported (9).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Peptide selection

To evaluate whether the changing of antigenic motif-surrounding amino acids influences the ability to induce an anti-CD20 response in terms of consistency and relative binding avidity (percentage of stained cells and fluorescence intensity), we selected a peptide bearing the same Rp15-C antigenic motif <aNPS>, but with chemically different motif-surrounding amino acids (Table I). Insert sequences of phage-clone R3-C, R7-C, R8-C, R14-C, R16-C, R17-C, and R19-C were excluded in that, similarly to Rp15-C, they expressed aromatic amino acids such as W, Y, or F at the N-terminal side of the antigenic motif (Table I). Furthermore, like Rp15-C, R3-C, R7-C, R14-C, and R19-C phage-clone inserts also expressed proline at the N-terminal side of these aromatic amino acids. Of the remaining three phage clones R2-C, R10-C, and R13-C, the insert sequence of the last was the most suitable for consideration as the template for the synthesis of the corresponding peptide Rp13-C, in that it was the only one to bear the Rp15-C-antigenic motif <aNPS>, whereas sNPS was expressed by R2-C- and R10-C-phage inserts.

Rp13-C and Rp15-C display similar antigenic profile and mimic raft-associated (functional) CD20

To rule out the possibility that the outcome of the analysis of the immunogenic properties of Rp15-C and Rp13-C was the result of their different antigenic properties, they were compared by testing their relative avidity for rituximab, their ability to inhibit the binding of rituximab to raft-associated CD20 (21), and rituximab-triggered membrane-ceramide increase (19).

Fig. 1A shows that different concentrations of rituximab similarly reacted with Rp15-C and Rp13-C, though the first peptide displayed a slightly higher relative avidity for rituximab. The specificity of the reactivity was assessed by the lack of rituximab binding with the negative control peptide Qp-1a and of infliximab reactivity with all three peptides. Similar results were obtained by testing the ability of different concentrations of Rp15-C and Rp13-C to inhibit the binding of rituximab to CD20⁺ Raji cells in immunofluorescence assay. Fig. 1B shows that the two peptides displayed an overlapping profile in their ability to inhibit the rituximab-CD20 interaction. The inhibition was specific, because the negative control peptide Qp-1a did not affect the reactivity.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Peptides Rp15-C and Rp13-C display comparable reactivity for rituximab in a solid-phase ELISA (A) and inhibition fluorescence assay (B). A, Ninety-six-well polyvinyl-chloride microtiter plates were incubated with 50 µl of PBS solution containing saturating concentrations of KLH-cyclic peptide Rp15-C () and Rp13-C () (10 µg/ml) for 12 h at 4°C. After one washing and blockage of free protein-binding sites, 4-fold serial dilutions of rituximab (continuous line) or infliximab (negative control) (dotted line; starting concentration 2.5 µg/ml) were added to the plate and incubation was prolonged for 4 h at 25°C. Wells were then washed three times and mAb binding to the peptide was detected by sequential addition of an appropriate dilution of HRP-conjugated xenoantibodies to human IgG (Fc portion) and OPD-substrate solution. Color reaction was stopped with 100 µl of 2 N H2SO4. Absorbance was read at 492 nm. Background binding was determined by absorbance generated in wells with blocking solution alone. Bindings of rituximab and infliximab to KLH-Qp-1a (+) were included as negative controls. The peptide with the highest reactivity at the lowest concentration of rituximab has the greatest relative avidity for rituximab. B, Fifty microliters of a PBS solution containing rituximab at the highest dilution (2.5 µg/ml) staining 100% of cells were preincubated with an equal volume of PBS solution containing 2-fold serial dilutions of KLH-free peptide Rp15-C () and Rp13-C () (starting concentration 800 µg/ml). Following 2-h incubation at 4°C, the mixture was added to human B lymphoid Daudi cells (5 x 105/tube) and incubation was prolonged for 10 min at 4°C. Then, cells were washed and incubated with an appropriate dilution of a FITC-xenoantiserum to human IgG (Fc portion). Afterward, cells were washed once with 4 ml of PBS-BSA and fixed with paraformaldehyde (2% final dilution). Binding of rituximab in the presence of peptide Qp-1a (*) was included as negative control. Results are expressed as percentage inhibition of cell fluorescence staining as compared with the staining of cells in the absence of inhibitor.

View larger version (67K): [in this window] [in a new window]   FIGURE 2. Inhibition by peptide of rituximab binding to raft microdomain-associated CD20. Fifty microliters of a PBS solution containing rituximab (2.5 µg/ml) were preincubated with an equal volume of PBS solution containing 100 µg/ml peptide Rp15-C (A) and Rp13-C (B). Following 1-h incubation at 4°C, the mixture was added to human B lymphoid Raji cells (1 x 106/tube) and incubation was prolonged for 10 min at 37°C. Then, cells were washed once with 4 ml of PBS-BSA and fixed with paraformaldehyde (2% final dilution) for 15 min at 25°C. Afterward, cells were washed and incubated with biotinylated CTB for 30 min on ice. After an additional washing, cells were incubated with an appropriate dilution of affinity purified FITC-xenoantibodies to human IgG (to detect rituximab) and PE-streptavidin (to detect CTB in the raft) and incubation was prolonged for an additional 30 min. Cells were washed, mounted on glass coverslips with DABCO mounting medium (Sigma-Aldrich), and examined with a Nikon confocal microscope using a x60 Plan Apo VC objective. An argon laser at 488 nm was used to excite FITC and a helium-neon laser was filtered at 560 nm to excite PE. Stimulation of cells with rituximab in the presence of unrelated peptide Qp-1a (C) or in the absence of inhibitor (D) was included as control.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Inhibition by peptide of the rituximab-induced membrane ceramide increase on human B lymphoid Daudi cells. Fifty microliters of PBS solution containing rituximab (10 µg/ml) were mixed with an equal volume of PBS containing 10-fold scalar concentrations (starting concentration 400 µg/ml) of peptide Rp13-C (A) and Rp15-C (B). After 1-h incubation at 4°C, the mixture was added to human B lymphoid cell line Daudi (1 x 106/tube), and incubation was prolonged for 10 min at 37°C to stimulate cells. After that, cells were washed once with 4 ml of PBS-BSA, fixed with paraformaldehyde, and incubated with 50 µl of PBS containing rabbit IgG (50 µg/ml) for 30-min at 4°C to block FcR-binding sites. Ceramide expression was evaluated by sequential incubation of cells with an appropriate dilution of anti-ceramide IgM mAb and FITC-xenoantibodies to mouse IgM (FITC-anti-IgM). Immunofluorescence was measured with a FACScan cytometer. Ceramide expression induced by rituximab in the absence of inhibitor (thick continuous line) and background fluorescence profile of cells (FITC probe only) (shaded area) were included as controls.

Immunization of five BALB/c mice with Rp15-C elicited anti-CD20 Abs in serum from mouse 1 (33.23% of stained cells) and to a lower extent in that from mouse 2 (12.6% of stained cells) (Fig. 4, left panel), despite the high titer of anti-peptides Abs observed in all immunized mice (data not shown). In contrast, sera from all five Rp13-C-immunized mice reacted with CD20⁺ human B lymphoid Daudi cells (CD20⁺) (Fig. 4, right panel), though to a variable extent. Three sera (nos. 1B, 2B, and 3B) stained at least 40% of cells. The highest percentage of staining was obtained with serum no. 1B (50.45%), the lowest with serum no. 5B (28.43%). The specificity of the reactivity was indicated by the lack of reactivity of either anti-Rp13-C or anti-Rp15-C sera with CD20^– human T lymphoid CEM cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Occurrence of anti-CD20 Abs in sera from mice immunized with Rp15-C and Rp13-C, respectively. Fifty microliters of a 1/20 dilution of sera from mice immunized with Rp15-C (sera nos. 1–5) and Rp13-C (sera nos. 1B–5B) were incubated with rabbit IgG-treated CD20+ Raji cells (2 x 105 cells) for 30 min on ice. Then, cells were washed and bound Abs were detected with an appropriate dilution of FITC-xenoantibodies to mouse (or human, to detect rituximab) IgG (Fc portion). Cells were then washed, fixed in 2% paraformaldehyde, and analyzed with a FACScan cytometer. Results are expressed as fluorescence intensity (empty histograms). The percentage of stained cells is indicated in each panel. Binding of sera to CD20– CEM cells was included as specificity control. Binding of mAb rituximab (RIT) and anti-HLA class I mAb TP25.99 (TP) to Raji and CEM cells, respectively (bottom panels) were included as positive controls. The background fluorescence staining (FITC probe only) is indicated (shaded area).

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Specificity of the reactivity with CD20+ human B lymphoid cell line Daudi of anti-Rp13-C Abs elicited in BALB/c mice. A and B, Fifty microliters of a 1/20 dilution of anti-Rp13-C serum was preincubated with 50 µl of a 10-fold serial dilutions of peptide Rp13-C (A) and Rp15-C (B) (starting concentration 400 µg/ml) for 1 h at 4°C. Then, the mixture was added to rabbit IgG-treated Daudi cells (5 x 105) and incubation was prolonged for 30 min on ice. Afterward, cells were washed and bound Abs were detected with an appropriate dilution of FITC-xenoantibodies to mouse IgG (Fc portion). Immunofluorescence was measured with a FACScan cytometer. Background fluorescence staining (FITC probe only) is indicated (shaded area). Binding of anti-Rp13-C in the presence of unrelated peptide Qp-1a was used as negative control. C, Fifty microliters of a 1/20 dilution of anti-Rp13-C serum were added to rabbit IgG-treated Daudi cells (2 x 105 cells), previously preincubated for 1 h on ice with different concentrations of F(ab')2 of mAb rituximab (Fab R). The assay was continued as described in A. The binding of anti-peptide sera to cells preincubated with F(ab')2 of mAb infliximab (Fab I) was used as negative control.

To determine whether the difference between Rp15-C and Rp13-C in eliciting anti-CD20 Ab reflects the recognition by anti-peptide Abs of motifs different in their degree of similarity from that recognized by rituximab, 38 µl of sera drawn on days 35, 42, 49, and 56 from BALB/c mouse immunized with Rp15-C and Rp13-C, respectively, which had developed anti-CD20 Abs, were pooled and IgG purified as described (see Materials and Methods). After the assessment of their purity (Fig. 6A) and an extensive adsorption on insolubilized KLH, IgG were tested in ELISA with the immunizing KLH peptide to ascertain whether their specificity was preserved. As shown in Fig. 6, B and C, IgG anti-Rp15-C and anti-Rp13-C cross-reacted with KLH-immunizing peptide. The reactivity was dose-dependent and specific, because neither serum reacted with negative control peptide KLH-Qp-1a (Fig. 6D), while rituximab (positive control) reacted with both peptides.

View larger version (45K): [in this window] [in a new window]   FIGURE 6. Purity (A) and specificity (B–D) of anti-peptide IgG purified from sera of BALB/c mice immunized with the rituximab-specific cyclic peptides Rp15-C and Rp13-C. A, Anti-peptide IgG were purified from sera of immunized BALB/c mice by precipitation with caprylic acid. Two micrograms of purified product were run on SDS-PAGE under reducing conditions and stained with Coomassie brilliant blue (Bio-Rad). MW, m.w. markers. B–D, Ninety-six-well polyvinyl-chloride microtiter plates were incubated with 50 µl of PBS solution containing KLH-Rp15-C (B), KLH-Rp13-C (C), and negative-control KLH peptide Qp-1a (D) (10 µg/ml) for 12 h at 4°C. After one washing and blockade of free protein-binding sites, 4-fold serial dilutions of anti-peptide IgG from sera of BALB/c mice immunized with Rp15-C () and Rp13-C () were added to the plates (starting concentration 2.5 µg/ml). Following 4-h incubation at 25°C and three washings, IgG-peptide interaction was detected with an appropriate dilution of HRP-xenoantibodies to mouse IgG (Fc portion) and OPD-substrate solution. Color reaction was stopped with 100 µl of 2 N H2SO4. Absorbance was read at 492 nm. Background binding was determined by absorbance generated in wells with blocking solution alone. Binding to the KLH peptide of rituximab (+) and infliximab (X), detected with peroxidase-xenoantibodies to human IgG, was included as positive and negative controls.

Phage clones were isolated by panning the c7c PDPLs with anti-Rp15-C and -Rp13-C purified IgG, respectively. At each round, phage particles binding to isotypic and allotypic determinants were removed by a preadsorption step on purified mouse anti-KLH IgG.

Immunoscreening of 30 randomly selected colonies from the panning of c7c PDPL either with anti-Rp15-C-IgG or with anti-Rp13-C-IgG showed that 20 (66%) and 22 (73.3%) clones specifically reacted with anti-Rp15-C IgG and anti-Rp13-C IgG, respectively (Table II). Nucleotide sequence of the selected anti-Rp15-C IgG-specific clones and the alignment of their insert sequences showed that only 20% of them expressed the antigenic motif <aNPS> recognized by rituximab (Table II, sequences underlined), the resulting motif <pyxnxxl> (immunogenic motif) being markedly different from the antigenic motif and located mostly at its NH₂-terminal side.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
To be effective in active immunotherapy, a peptide has to recognize the binding site of the therapeutic mAb used for the panning procedure and to induce Abs against the nominal Ag. In addition, these Abs should be consistently expressed at high levels in the immunized host (13, 14). Although the first two criteria can be easily verified, there is no way to select from a panel of peptides that which induces more consistently and at high titers Abs against nominal Ag (effective mimotope).

The 7-mer cyclic peptides Rp15-C and Rp13-C, bearing the antigenic motif <aNPS> recognized by rituximab (8) but with different motif-surrounding amino acids, meet the first two criteria. Even so, anti-CD20 Abs were induced more consistently in Rp13-C- than in Rp15-C-immunized animals.

The failure of Rp15-C to consistently elicit anti-CD20 Abs is not unique to the CD20 system and parallels similar findings in mice (7, 10), rabbits (6, 11), and humans (12) immunized with peptides that are mimics of other target cell-associated Ag systems in different clinical settings, namely infectious diseases (10, 11) and cancer (6, 7, 12).

To overcome these limits, two strategies have been exploited to direct the immune response induced by mimotope more specifically against the nominal Ag, namely: 1) the synthesis of a peptide including phage-derived NH₂-terminal flanking amino acid sequence, on the assumption that a conformation reflecting that of phage particles expressing the peptide more closely will be reproduced (6); and 2) the injections of purified (10) or cell extract-containing nominal Ag (7) in the host, before (10) or after (7) peptide treatment. The latter approach has been successfully used in the melanoma system by Luo et al. (7). The level of anti-high m.w. melanoma-associated Ag (HMW-MAA) Abs induced by peptide P763.74 (mimics of the HMW-MAA) was markedly increased when peptide-treated mice were boosted with HMW-MAA-bearing cell lysate. Even so, clinical application of this strategy is questionable and hard to conceive because of ethical commitment.

The different immunogenicity of our two peptides in terms of their ability to induce anti-CD20 Abs is unlikely to be due to their antigenic properties, because three lines of evidence indicate that these are similar, if not identical with respect to their reactivity with rituximab. Indeed, Rp15-C and Rp13-C: 1) displayed a comparable avidity for rituximab; 2) competed with raft-associated CD20 for their binding to rituximab; and 3) inhibited membrane ceramide increase triggered by rituximab. In addition, they displayed similar immunogenic properties, in that Rp15-C and Rp13-C induced cross-reacting anti-peptide Abs and Rp15-C efficiently inhibited Rp13-C-induced anti-CD20 Abs.

As a possible explanation of the ineffectiveness of a mimotope, we sought to determine whether the immunogenic motif was different from the antigenic motif. These experiments were performed with anti-peptide IgG purified with caprylic acid from 150 µl of mouse sera and without affecting their specificity. These IgG were efficiently used to pan a PDPL to isolate the specific phage clones.

The alignment of insert sequences expressed by anti-Rp15-C-specific phage clones indicates that the amino acids PY, located at the NH₂-terminal side of the antigenic motif <aNPS>, are strongly immunogenic and can account for the specificity of the majority of Abs elicited with Rp15-C. In contrast, only 20% of anti-Rp15-C IgG-specific phage-clone insert sequences expressed <aNPS>. If this percentage reflects either the expression percentage of Abs with that specificity or the probability that these can be generated during a peptide immunization course, then only a small portion of anti-Rp15-C IgG recognizes the rituximab-specific antigenic motif <aNPS>, hence CD20. This would explain the low fluorescence intensity and/or percentage of cells stained by anti-peptide serum in two of five mice immunized with Rp15-C and the failure to detect anti-CD20 Abs in the remaining three mice.

The replacement of amino acids <PY> with <WA>, as in peptide Rp13-C, markedly changed the specificity of the corresponding anti-Rp13-C Abs which exclusively recognized <NPS>, a significant portion of the antigenic motif, and consequently were more specifically directed against the nominal Ag CD20. Even so, their relative binding avidity was always lower than that of rituximab, probably because of the lack of A (or S) before the immunogenic motif <NPS>.

The data presented here show that one explanation of the failure of most peptide-based vaccinations stems from differences in the amino acids involved in the formation of the antigenic and the immunogenic motif. They also suggest that appropriate amino acid substitutions can be used to enhance the efficacy of a mimotope, once the antigenic and immunogenic motifs are defined and found to be different.

Two possibilities can be envisaged to explain the influence of motif-surrounding amino acids on the specificity of anti-peptide Abs. First, because the CD20 peptide mimotope expressing the motif is conformational, it is likely that motif-surrounding amino acids may influence the spatial orientation of the antigenic motif-amino acids. This spatial orientation, which can be appropriate for the motif <aNPS> to be recognized by rituximab when expressed on peptide Rp15-C and Rp13-C, may not be as effective on Rp15-C as on Rp13-C to prime the immune system to produce anti-<ANPS> (anti-CD20) Abs. The second possibility is that motif-surrounding amino acids may influence the peptide cleavage site during processation (22).

Given the complexity of the immune response, the extension of this study to the specificity of Abs elicited with a larger number of rituximab-specific peptides, in relation to the host’s genetic background (MHC and Ig haplotypes) and the use of other carriers, should hopefully provide useful information to define whether criteria for appropriate amino acids substitutions (in the region surrounding the motif) can be established to optimize mimotopes for human use.

The present study has centered on the analysis of the fine specificities of peptide-induced Abs with the specific aim of implementing their effector functions (8). These, similar to those triggered by rituximab, are expected to mediate the most relevant therapeutic effect in an active immunotherapy setting (4, 23). Nevertheless, because the anti-peptide Abs are of IgG isotype and carrier-free peptides are not immunogenic (8), it is likely that an Ag-specific T cell-B cell cooperation takes place in this response (4, 7, 24). Whether the most relevant T cell help is provided by CD4⁺ T cells recognizing KLH-derived MHC class II-bound peptides, and/or by MHC class II-bound peptides alone, remains to be determined. Ongoing studies on peptide-related differences in T cell activation and specificity will be useful to assess whether T cells can also be expected to play a therapeutic role in the CD20 mimotope-based active immunotherapy.

Lastly, as the difference in the fine specificity between rituximab and a portion of Abs elicited with rituximab-specific peptides reflects differences in the specificity of the BCR of the corresponding Ab-producing B cells, in the context of a cognate B cell-T cell cooperation, it is tempting to speculate that amino acids recognized by T cells within a short peptide in the initial phase of the immune response can be different from those they recognized later when stimulated on the same peptide. These considerations suggest that even for a short peptide, functional editing of a target Ag-specific B and/or T cell repertoire is a likely mechanism for escaping immune surveillance (12, 25).

	Acknowledgment

 
We are grateful to Vito Iacovizzi for his excellent secretarial assistance.

	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 a grant from Associazione Italiana per la Ricerca sul Cancro (Milan, Italy).

² Address correspondence and reprint requests to Dr. Federico Perosa, Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Piazza G. Cesare 11, 70124-Bari, Italy. E-mail address: f.perosa{at}dimo.uniba.it

³ Abbreviations used in this paper: PIT, passive immunotherapy; PDPL, phage-display peptide library; CTB, cholera toxin subunit B; KLH, keyhole limpet hemocyanin; HMW-MAA, high m.w. melanoma-associated Ag.

Received for publication June 21, 2007. Accepted for publication September 20, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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