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Originally published In Press as doi:10.1074/jbc.M507562200 on October 14, 2005

J. Biol. Chem., Vol. 280, Issue 50, 41546-41552, December 16, 2005
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Structural Basis of Antigen Mimicry in a Clinically Relevant Melanoma Antigen System*

Chien-Chung Chang{ddagger}1, Francisco G. Hernandez-Guzman§, Wei Luo{ddagger}, Xinhui Wang{ddagger}, Soldano Ferrone{ddagger}2, and Debashis Ghosh§3

From the {ddagger}Department of Immunology and of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263 and the §Hauptman-Woodward Medical Research Institute, Inc., Buffalo, New York 14203

Received for publication, July 12, 2005 , and in revised form, October 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although mimics of human tumor antigens are effective immunogens to overcome host unresponsiveness to the nominal antigen, the structural basis of this mimicry remains poorly defined. Therefore, in this study we have characterized the structural basis of the human high molecular weight-melanoma-associated antigen (HMW-MAA) mimicry by the mouse anti-idiotypic (anti-id) monoclonal antibody (mAb) MK2-23. Using x-ray crystallography, we have characterized the three-dimensional structure of the anti-id mAb MK2-23 Fab' and shown that its heavy chain complementarity-determining region (CDR3) (H3) and its light chain CDR1 (L1) are closely associated. These moieties are the source of HMW-MAA mimicry, since they display partial amino acid sequence homology along with a similar structural fold with the HMW-MAA core protein. Furthermore, a 15-residue peptide comprising the H3 loop of anti-id mAb MK2-23 demonstrates HMW-MAA-like in vitro and in vivo reactivity. This peptide in conjunction with the structural data will facilitate the characterization of the effect of the degree of antigen mimicry on the induction of a self-antigen-specific immune response by a mimic.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antigen mimicry has been implicated in the pathogenesis of several pathophysiological conditions such as viral immune evasion (1-3) and autoimmunity (4-7). In these situations, the structurally similar foreign moieties either interfere with the normal biological functions mediated by their nominal counterparts or elicit unwanted immune responses against the host antigens. The consequences sometimes can be quite detrimental. Nevertheless, in the case of autoimmune responses mediated by molecular mimicry, an intriguing finding is the restriction of the damage to an organ or a tissue. This pattern is distinct from the general immune hyper-responsiveness caused by suppression of the regulatory arm of the immune system, such as cytotoxic T lymphocyte antigen-4 (CTLA-4) blockade (8, 9).

The potential ability of molecular mimicry to target a specific host antigen has provided the rationale for its use to elicit and/or enhance an immune response against human tumor antigens that are mostly non-mutated self-antigens and therefore poorly or non-immunogenic in patients (for review, see Refs. 10 and 11). Various types of tumor antigen mimics have been identified (for review, see Ref. 11). Among them the most extensively utilized is represented by antiidiotypic (anti-id)4 monoclonal antibodies (mAb), which have been developed in several human tumor antigen systems (for review, see Refs. 10 and 11). Anti-id mAb markedly differ in their immunogenicity as measured by their ability to elicit a humoral immune response to the corresponding self-tumor antigen. The cause of this variability is not known. The lack of this information reflects, at least in part, the limited knowledge about the structural basis of antigen mimicry by anti-id antibodies and about the relationship between the extent of antigen mimicry and ability of a mimic to overcome unresponsiveness to a self-tumor antigen. Since this knowledge may improve the design of tumor vaccines, in the present study we have investigated the structural basis of human high molecular weight-melanoma associated antigen (HMW-MAA) (12) mimicry by the mouse anti-id mAb MK2-23 (13). The latter anti-id mAb was derived from a mouse immunized with the HMW-MAA-specific idiotypic mAb 763.74 (13). This antigen system was selected for our studies, since anti-id mAb MK2-23 has been found to elicit HMW-MAA-specific antibodies in mice and rabbits (14, 15), both of which express orthologs of human HMW-MAA constitutively (16, 17). In addition, anti-id mAb MK2-23 was able to induce HMW-MAA-specific humoral responses in patients with melanoma (18, 19). This immune response appears to be clinically relevant, since it was associated with regression of metastases in a few patients (20) and with a statistically significant survival prolongation (18, 19). The elicited HMW-MAA-specific antibodies, or anti-anti-id antibodies, were also found to inhibit the binding of mAb 763.74 (21) to HMW-MAA in vitro (14, 15, 18), suggesting that structural rather than functional homology underlies the mAb 763.74 defined HMW-MAA epitope mimicry by the anti-id mAb MK2-23.

Using x-ray crystallography and immunological studies, we have found that the complementarity determining region 3 (CDR3) of the anti-id mAb MK2-23 heavy chain plays an important role in the three-dimensional structural basis of HMW-MAA mimicry by anti-id mAb MK2-23.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Purification of Anti-id mAb MK2-23 F(ab')2 Fragments—F(ab')2 fragments were generated by digesting with immobilized pepsin (Pierce) mouse mAb IgG1 MK2-23 (14), which was purified from ascites by protein A chromatography (Bio-Rad). High purity mAb MK2-23 F(ab')2 fragments were obtained by sequential protein A column and S-200 gel filtration chromatography. Peak S-200 fractions were pooled and concentrated to 7.9-10 mg/ml in low salt phosphate buffer (50 mM KH2PO4, 10 mM NaCl, pH 7.4). Purified F(ab')2 fragment preparations were preincubated at room temperature with 5 mM dithiothreitol at pH 5.5 for 1.5 h, yielding monomeric Fab' fragments.

Crystallization and Data Collection—Diffraction-quality crystals were obtained from 20% polyethylene glycol 6000, in 0.1 M HEPES buffer, pH 7.5, at a protein concentration of 7.9 mg/ml. Protein and precipitant solutions were mixed at the 3:2 ratio, and droplets were allowed to vapor diffuse against wells of precipitant solutions in hanging droplets. Fab' fragments were crystallized in the orthorhombic space group P212121 having unit-cell dimensions a = 75.05 Å, b = 76.89 Å, c = 82.18 Å, {alpha} = {beta} = {gamma} = 90° and one Fab' molecule in the asymmetric unit. Diffraction data to 2.50 Å resolution were collected with a crystal flash frozen in liquid nitrogen on a R-AXIS IV area detector equipped with a rotating anode x-ray source (93,266 measured intensities, 17,003 unique reflections, 99.9% complete, intensity/{sigma} (intensity) = 4.2 in the highest resolution shell, Rmerge = 0.067).

Structure Solution and Refinement of the Model of Anti-id mAb MK2-23 Fab' Fragments—The three-dimensional structure of the mAb MK2-23 Fab' fragments was determined by the molecular replacement method using a known Fab structure (Protein Data Bank (PDB) code 2RCS) as the search model and XPLOR routines (22). The experimentally determined sequences for the variable domains were built into the electron density, and the model was subjected to several rounds of refinement and rebuilding using the CNS package of software (23). The final crystallographic R factor for 441 residues and 66 water molecules (3444 total atoms) is 0.236 with an R-free value of 0.297 for 16,656 reflections between 33 and 2.50 Å resolution. TABLE ONE provides a summary of data collection and structure refinement results.


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  		TABLE ONE
Data collection and refinement summary

 
Purification and Biotinylation of mAb 763.74—mAb 763.74 was purified from ascitic fluid by sequential ammonium sulfate and caprylic acid precipitation (24). The purity and activity of mAb preparations were assessed by SDS-PAGE and by testing with the corresponding antigen in a binding assay, respectively. Biotinylation was performed using NHS-LC-biotin (succinimidyl-6-(biotinamido)hexanoate) (Pierce) according to the manufacturer's instructions.

Assessment of Peptide PMK2-23H3 Antigenicity—Synthetic peptides derived from anti-id mAb MK2-23CDR3H (PMK2-23H3, ARSNYV-GYHVRWYFD), anti-id mAb MK2-23CDR1L (PMK2-23L1, SVEYYGSSLMQ), and HMW-MAA (PHMW-MAA.D2.7, IRSG-DEVHYHVTAGPRW) were purchased from N. C. Wang, Hospital for Sick Children, Toronto, Ontario, Canada. A synthetic peptide derived from {beta}2-microglobulin (Pb2m, KNGERIEKVEHS), used as a negative control, was purchased from University of Georgia Molecular Genetics Instrumentation Facility (Athens, GA). Escalating concentrations of peptides as indicated were incubated with biotinylated mAb 763.74 (0.5 mg/ml) at 4 °C overnight in U-bottom 96-well plates. The mixture was then incubated with HMW-MAA-bearing melanoma cells Colo38 (105/well) for 1 h at 4 °C. After three washes with 1% bovine serum albumin in phosphate-buffered saline, an optimal amount of horseradish peroxidase-conjugated streptavidin was added. Reactions were then developed and visualized with the 3,3',5,5'-tetramethylbenzidine substrate system (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Reactions were reported as OD measured with an ELISA reader. Percent inhibition was calculated by the formula: 100% x (ODirrelevant peptide - ODtest peptide)/ODirrelevant peptide. Dissociation constant (Kd) was calculated as the molar concentration of the peptide required to cause a 50% inhibition of binding of biotinylated mAb 763.74 to HMW-MAA-bearing melanoma cells Colo38 (25).

Assessment of Peptide PMK2-23H3 Immunogenicity—Peptide PMK2-23H3 conjugated to the carrier protein keyhole limpet hemocyanin with the cross-linking agent m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce) was mixed with complete Freund adjuvant for priming (100 µg/injection) and with incomplete Freund adjuvant for boosting (50 µg/injection). Immunizations were given subcutaneously to 8-week-old female BALB/c mice (Taconic Farms, Germantown, NY) (five per group) on days 0, 21, and 42. Sera were harvested before immunization and on days 7 and 28. They were tested for reactivity with the immunizing peptide coated on 96-well plates (24) and with HMW-MAA-bearing melanoma cells in ELISA and fluorescence-activated cell sorting (FACS) analysis, as described previously (14, 26).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Amino Acid (aa) Sequence Homology and Structural Similarity of Anti-id mAb MK2-23 CDRs with HMW-MAA Core Protein—To investigate whether aa sequence homology plays a role in the HMW-MAA mimicry by anti-id mAb MK2-23, we compared the aa sequence of the anti-id mAb MK2-23 CDRs with that of the HMW-MAA core protein (27), since CDRs constitute the idiotope of an antibody. Only a partial homology was found between the CDR3H (H3) and the HMW-MAA core protein (Fig. 1 and data not shown). This region corresponds to residues 94-102 within anti-id mAb MK2-23 H3, displaying eight identical, although discontinuous, and one conservative matches with the region spanning from residues 1159 to 1174 in the HMW-MAA core protein. In addition, the region corresponding to residues 29-33 within anti-id mAb MK2-23 L1 shows four identical and one conservative matches with residues 1132-1136 of the HMW-MAA core protein (Fig. 1). Interestingly, the two homologous regions map, with only 21 aa apart, to the seventh of the 15 putative chondroitin sulfate proteoglycan (CSPG) repeat units of the domain 2 of HMW-MAA (designated HMW-MAA.D2.7, residues 1128-1216) (Fig. 1) (28). The HMW-MAA.D2.7 segment is predicted to adopt an all-{beta}-fold conformation, comprising eight {beta}-strands (28). This prediction is supported by the alignment of the putative HMW-MAA.D2.7 {beta}-strands to the six {beta}-strands of a N-cadherin fragment with a known three-dimensional structure (PDB code 1NCJ). Interestingly, the two HMW-MAA.D2.7 regions (residues 1132-1135 and residues 1159-1174), with which anti-id mAb MK2-23 L1 and H3 have homology, are located within the predicted first and fourth {beta}-strands, respectively, in the HMW-MAA segment (Fig. 1) (28). This observation suggests that the anti-id mAb MK2-23 L1 and H3 loops represent the moiety that mimics the HMW-MAA epitope.



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  		FIGURE 1.
Partial sequence homology and structural similarity between HMW-MAA seventh CSPG repeat (HMW-MAA.D2.7) and anti-id mAb MK2-23 L1 and H3 loops. The putative domain organization of HMW-MAA is illustrated according to Staub et al. (28). The numbering of anti-id mAb MK2-23 L1 and H3 follows the Kabat convention (29). There are 15 CSPG repeat units (filled trapezoid) located in domain 2 of HMW-MAA. The predicted {beta}-strand structures of HMW-MAA.D2.7 are shown in red letters. The homologous regions are boxed. The residues in anti-id mAb MK2-23 L1 and H3 that show 100% identity with the HMW-MAA residues are shown in blue. The alignment was performed with the ClustalW program (European Bioinformatics Institute). D, domain; TM, transmembrane; CY, cytoplasmic tail.

 



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  		FIGURE 2.
Crystal structure of an anti-id mAb MK2-23 Fab' fragment at 2. 5 Å resolution shown in ribbon diagram. A, the CDR loops of the light (CDR1L) (L1) (green) and heavy (CDR3H) (H3) (yellow) chains are shown as labeled. The boxed area is shown in stereo images (B), detailing the secondary and tertiary structures of anti-id mAb MK2-23 L1 and H3 and their final electron density maps. Hydrogen bonds are shown by dotted lines.

 
Overall Three-dimensional Structure of Anti-id mAb MK2-23 Fab' Fragments—To determine whether anti-id mAb MK2-23 L1 and H3 loops display a structural fold similar to that of the HMW-MAA.D2.7 segment, we crystallized and solved the structure of the anti-id mAb MK2-23 Fab' fragments. The overall structure of anti-id mAb MK2-23 Fab' fragment exhibits the typical immunoglobulin fold with 377 of 379 non-Gly and non-Pro residues in the allowed regions (Fig. 2A and TABLE ONE). The two residues that are in the disallowed region are Ala51 of the L chain and Ser172 of the H chain, both of which are located on turns with well defined electron densities. Our experimental electron densities agree well with the deduced aa sequences of anti-id mAb MK2-23 Fab' L and H chains, except for the following discrepancies. An Ala side chain was better accommodated than an Arg at residue 71 of the H chain. A few solvent-exposed side chains, such as Tyr100 and Arg100C of the H chain (on the H3 loop), did not show appreciable electron densities. It is likely that these side chains participate in antigen recognition and are dynamically disordered in absence of an antigen.



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  		FIGURE 3.
Three-dimensional alignment of five available anti-id mAb: 409.5.3 (PDB code 1AIF), 6A6 (PDB code 1PG7), E225 (PDB code 1CIC [PDB] ), E5.2 (PDB code 1DVF), and MK2-23 (this study), two anti-anti-id mAb: 131 (PDB code 2CK0) and GH1002 (PDB code 1GHF) and two randomly selected idiotypic mAb: Mopc21 (PDB code 1IGC), and R24 (PDB code 1R24 [PDB] ) by using least squares fitting of the conserved residues.

 
Secondary and Tertiary Conformations of Anti-id mAb MK2-23 L1 and H3—Next we examined the structural features of L1 and H3 loops of the anti-id mAb MK2-23. L1 consists of residues Arg24, Ala25, Ser26, Glu27, Ser27A, Val27B, Glu27C, Tyr27D, Tyr28, Gly29, Ser30, Ser31, Leu32, Met33, and Gln34 (29). According to current classifications (30), anti-id mAb MK2-23 L1 belongs to the Vk L1 canonical structure 5, containing four insertions. An interesting feature of this L1 loop is the formation of a pair of anti-parallel strands linked by three inter-strand hydrogen bonds (27BCO-HN32, 27DNH-OC30, and 27DCO-HN30) and a left-handed hairpin turn having Tyr28 and Gly29 backbones in the (+,+) helical conformational space of the Ramachandran plot. Whether the latter geometry is a common feature among all canonical structure 5 L1 loops or a unique characteristic of anti-id mAb MK2-23 L1 remains to be determined, since the three-dimensional structure of at least another canonical structure 5 L1 loop is not yet available at the time of preparation of this manuscript (30). Of note here is that the average temperature factor for L1 is about 45 Å2, which is significantly higher than the average temperature factor of the entire molecule (36 Å2).

The H3 loop, consisting of residues Ser95, Asn96, Tyr97, Val98, Gly99, Tyr100, His100A, Val100B, Arg100C, Trp100D, Tyr100E, Phe100F, Asp101, and Val102, contains six insertions (29). H3 is relatively longer than that of other mouse immunoglobulins; it is only shorter than that of mAb R19.9 (PDB code 1FAI) and mAb R45-45-11 (PDB code 1IKF) by 1 and 3 residues, respectively. In accordance with the structural patterns of the immunoglobulin H3 loop torso region (31), anti-id mAb MK2-23 H3 belongs to a class in which the torso region does not contain a {beta}-bulge, which is the most common class, but a regular {beta}-sheet hairpin structure (31). The salt bridge between Arg94 and Asp101 is absent, but like in most immunoglobulins, the combination of the length and sequence of the loop between Arg94 and Asp101 dictates to a large extent the specificity. The anti-parallel strands at the loop termini are held by hydrogen bonding (95CO-HN101, 96NH—OC100E, 96CO-HN100E, and 98NH-OC100C). At the tip of the loop, residues Tyr100 to Arg100C form a distorted type III helical turn with a 100CO-HN100C hydrogen bond. Similar to L1, H3 also displays high thermal motion (average B ~60 Å2); however, the main chain and most of the side chain electron densities, except those of Tyr97 and Arg100C, are well defined to allow an unequivocal tracing of the backbone. It is noteworthy that the L1 and H3 loops pack closely against each other through hydrophobic interactions (L1 Leu32 side chain against the H3 main chain, Val106 and Tyr27D side chains) and through the formation of one hydrogen bond (L1 Gln34 to H3 Trp100D CO).



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  		FIGURE 4.
Dose-dependent inhibition by anti-id mAb MK2-23H3-derived peptide PMK2-23H3 of binding of mAb 763.74 to HMW-MAA-bearing melanoma cells Colo38. Peptides PHMW.D2.7 (HMW-MAA-derived), PMK2-23H3 (anti-id mAb MK2-23H3-derived), and an irrelevant control peptide Pb2m ({beta}2-microglobulin-derived) at increasing concentrations were incubated with biotinylated mAb 763.74 (0.5 µg/ml) and assayed as described under "Experimental Procedures." Results of one of three representative experiments are shown.

 



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  		FIGURE 5.
Immunogenicity of peptide PMK2-23H3 in BALB/c mice. Sera harvested before immunization (circle) and on days 7 (square) and 28 (triangle) after immunization from BALB/c mice immunized with anti-id mAb-derived peptide PMK2-23H3 were tested for their reactivity in ELISA with the immunizing peptide (closed symbols, left panel of A) and with HMW-MAA-derived peptide PHMW.D2.7 (open symbols, right panel of A). Sera harvested before immunization (circles) and on day 28 (triangles) were tested in ELISA (left panel in B) with HMW-MAA-transfected M14#5 cells (closed symbols) and with their mock-transfected counterparts (open symbols, left panel in B). Sera harvested on day 28 were tested by FACS analysis (right panel in B, open profiles) with HMW-MAA-transfected M14#5 cells (M14#5/HMW) and with their mock-transfected counterparts (M14#5/neo). Preimmune sera (filled profile) were used as controls.

 
Comparison of L1 and H3 Loops of Anti-id mAb MK2-23 with Those of Other Anti-id mAb—To examine the structural variation of the anti-id mAb MK2-23 L1 and H3 loops as compared with other anti-id antibodies, we aligned the three-dimensional structure of these two loops with those of the four anti-id mAb structures available thus far: 409.5.3 (PDB code 1AIF), 6A6 (PDB code 1PG7), E225 (PDB code 1CIC [PDB] ), and E5.2 (PDB code 1DVF). Two anti-anti-id mAb, 131 (PDB code 2CK0) and GH1002 (PDB code 1GHF), and two idiotypic mAb, Mopc21 (PDB code 1IGC) and R24 (PDB code 1R24 [PDB] ), were used for comparison. Using least squares fitting, the loop termini, which correspond to the conserved residues 23 and 37 of the L chain and 91 and 106 of the H chain, superimpose well with root mean square deviations (r.m.s.d.) between 0.2 and 1 Å (Fig. 3). Between the termini, the loops, and especially H3, adopt varying conformations with the largest r.m.s.d. around 6.5 Å. The H3 loop of anti-id mAb MK2-23 is long and forms a pair of anti-parallel {beta}-strands linked by four hydrogen bonds (Figs. 2B and 3). Together with the L1 loop, the H3 loop of anti-id mAb MK2-23 projects a 41-residue-long surface, which is the most protruded among all the anti-id antibodies with a known three-dimensional structure.

Similarity in the in Vitro Reactivity of the Anti-id mAb MK2-23-derived Peptide PMK2-23H3 and of the HMW-MAA.D2.7-derived Peptide PHMW.D2.7—The peptide ARSNYVGYHVRWYFD (designated as PMK2-23H3), which was derived from the H3 loop of anti-id mAb MK2-23 encompassing the described HMW-MAA-homologous aa sequence, was synthesized and analyzed for reactivity with the HMW-MAA-specific idiotypic mAb 763.74. As shown in Fig. 4, peptide PMK2-23H3 inhibits the binding of mAb 763.74 to HMW-MAA-bearing melanoma cells to a similar extent as the HMW-MAA.D2.7-derived peptide (IRSGDEVHYHVTAGPRW, designated as PHMW.D2.7). The inhibition is specific, since the irrelevant peptide Pb2m had no detectable effect on the binding of mAb 763.74 to melanoma cells. The Kd values for peptides PMK2-23H3 and PHMW.D.2.7 are 871 and 900 nM, respectively. The peptide SVEYYGSSLMQ (designated as PMK2-23L1), which was derived from the L1 loop of anti-id mAb MK2-23 encompassing the described HMW-MAA-homologous aa sequence, was also synthesized. Because of its low solubility, peptide PMK2-23L1 could not be used alone or in combination with peptide PMK2-23H3 in peptide binding assays.

HMW-MAA-like Immunogenicity of the Anti-id mAb MK2-23-derived Peptide PMK2-23H3—Anti-id mAb MK2-23H3-derived peptide PMK2-23H3 elicited in BALB/c mice antibodies that reacted with the immunizing peptide, with the HMW-MAA-derived peptide PHMW.D2.7 and with anti-id mAb MK2-23, as measured in a peptide binding assay (Fig. 5A). The elicited antibody response is specific, since the sera from the mice immunized with the irrelevant peptide Pb2m displayed no detectable reactivity with peptides PMK2-23H3 and PHMW.D2.7. More importantly, peptide PMK2-23H3 elicited in BALB/c mice antibodies with selective reactivity with HMW-MAA-bearing cells. As shown in Fig. 5B, sera from peptide PMK2-23H3-immunized mice reacted with HMW-MAA-transfected M14 melanoma cells (M14.HMW-MAA) but did not react with the mock-transfected counterpart (M14.neo) both in ELISA and in FACS analysis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The crystal structure of the anti-id mAb MK2-23 Fab' fragments revealed distinctive features of the three-dimensional conformations of CDR loops L1 and H3, strongly suggesting that the two loops play important roles in the interaction with mAb 763.74 and in the mimicry of the HMW-MAA epitope defined by mAb 763.74. Interestingly, when compared with H3 loops of anti-id mAb 409.5.3 (PDB code 1AIF), 6A6 (PDB code 1PG7), E225 (PDB code 1CIC [PDB] ), and E5.2 (PDB code 1DVF) with known three-dimensional structures, anti-id mAb MK2-23 L1 and H3 appear to be more protruded, displaying large variations in conformations. Of note is the left-handed hairpin turn present in L1 and a pair of anti-parallel {beta}-strands with strong interstrand hydrogen-bonding present in H3. These findings are in agreement with the predicted structure of the corresponding HMW-MAA.D2.7 fragment, which may also adopt a similar {beta}-strand conformation (28).

Several lines of evidence indicate the involvement of the anti-id mAb MK2-23 H3 loop (and perhaps also of the L1 loop) in the HMW-MAA mimicry by anti-id mAb MK2-23. First, the two loops exhibit moderate aa sequence homology with the HMW-MAA core protein, and the homologous regions are located in the same CSPG repeat unit (aa 1128-1216) only 21 residues apart. Supported by secondary structure prediction, these two HMW-MAA regions may be spatially close to form the epitope recognized by the HMW-MAA-specific mAb 763.74. Second, the anti-id mAb MK2-23H3-derived peptide PMK2-23H3 inhibits the binding of mAb 763.74 to HMW-MAA-bearing cells to the same extent as the corresponding HMW-MAA-derived peptide PHMW.D2.7. Last, peptide PMK2-23H3 elicited HMW-MAA-specific antibodies in BALB/c mice which express the antigen AN2. The latter displays at least 80% homology with HMW-MAA. These results imply that the peptide PMK2-23H3 is able to break unresponsiveness to a self-tumor antigen. The role of the anti-id mAb MK2-23 L1 loop could not be tested in this study because of the low solubility of the synthesized peptide. Whether the L1 loop is part of the scaffold that helps form the HMW-MAA-mimicking moiety remains to be determined. Taken together, these results support the role of a 15-residue motif of the anti-id mAb MK2-23 H3 loop in the structural basis of HMW-MAA mimicry by the anti-id mAb MK2-23.

The role of the H3 loop of anti-id antibodies in antigen mimicry has been reported in other antigenic systems. Fields et al. (32) have shown that in a hen egg lysozyme system the anti-id mAb VH3 accounts for 77% of the interaction with the idiotypic antibody. Furthermore, Ban et al. (33) have demonstrated that in the feline infectious peritonitis virus E2 peplomer system the anti-id mAb VH constitutes 63% of the binding interface with the idiotypic antibody. Notably, in these systems the anti-id antibodies were found to represent the mirror images of their nominal antigens, because they are both structurally and functionally similar to the nominal antigen, i.e. they are able to elicit antigen-binding, anti-anti-id antibodies. Whether this structural organization of the idiotypic cascade is a general rule or is restricted to some antigenic system remains to be determined. From a practical viewpoint, the identification of a single peptide loop as an important structure-mimicking element allows us to investigate the relationship between the degree of antigen mimicry by a peptide mimic and its ability to overcome unresponsiveness to the nominal self-antigen. In the case of anti-id mAb MK2-23, the 15-residue H3 loop is a partial {beta}-strand and is self-stabilized by 4 interchain hydrogen bonds, suggesting that the anti-id mAb MK2-23 H3 loop is quite stable in solution. This feature will facilitate introduction of aa substitutions into the loop to modulate its structure and functional properties. It is tempting to speculate that subtle changes in the structural property of the H3 loop may change its ability to induce nominal antigen-specific immune response. This possibility is supported by the finding that the isoaspartyl form of a cytochrome c peptide but not its nominal (aspartyl) counterpart can break humoral tolerance to the self-cytochrome c (34).


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2AAB) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) DQ241816 [GenBank] and DQ241817 [GenBank] .

* This work was supported in part by United States Public Health Service Grants P01 CA89480 and R01 CA105500 awarded by the NCI/National Institutes of Health, DHHS, and by a grant from the Harry J. Lloyd Charitable Trust. 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. Back

1 Supported in part by a Susan G. Komen Breast Cancer Foundation predoctoral fellowship. Back

2 To whom correspondence may be addressed: Dept. of Immunology, Roswell Park Cancer Inst., Elm and Carlton Sts., Buffalo, NY 14263. Tel.: 716-845-8534; Fax: 716-8457613; E-mail: soldano.ferrone{at}roswellpark.org.

3 To whom correspondence may be addressed: Hauptman-Woodward Medical Research Inst., Inc., 700 Ellicott St., Buffalo, NY 14203. Tel.: 716-898-8617; Fax: 716-898-8660; E-mail: ghosh{at}hwi.buffalo.edu.

4 The abbreviations used are: anti-id, anti-idiotypic; CDR, complementarity-determining region; CSPG, chondroitin sulfate proteoglycan; FACS; fluorescence-activated cell sorting; H3, heavy chain CDR3; HMW-MAA, human high molecular weight-melanoma-associated antigen; L1, light chain CDR1; mAb, monoclonal antibody; PDB, Protein Data Bank; ELISA, enzyme-linked immunosorbent assay; aa, amino acid(s); r.m.s.d., root mean square deviation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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