Abstract
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.
Highlights
Antigen mimicry has been implicated in the pathogenesis of several pathophysiological conditions such as viral immune evasion [1,2,3] and autoimmunity (4 –7)
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 weightmelanoma associated antigen (HMW-MAA) [12] mimicry by the mouse anti-id monoclonal antibodies (mAb) MK2-23 [13]
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 threedimensional structural basis of HMW-MAA mimicry by anti-id mAb MK2-23
Summary
Purification of Anti-id mAb MK2-23 F(abЈ) Fragments—F(abЈ) 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(abЈ) fragments were obtained by sequential protein A column and S-200 gel filtration chromatography. 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. Structure Solution and Refinement of the Model of Anti-id mAb MK2-23 FabЈ Fragments—The three-dimensional structure of the mAb MK2-23 FabЈ 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]. Reactions were developed and visualized with the 3,3Ј,5,5Ј-tetramethylbenzidine substrate system (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD)
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