Antigenic and immunological mimicry of peptide mimotopes of Lewis carbohydrate antigens
Introduction
The production of specific antibodies against polysaccharides play a pivotal role in the defense against gram-negative and gram-positive bacteria, and have shown promise in vaccine approaches in the immunotherapy of cancer (Livingston et al., 1997; Livingston and Ragupathi, 1997; Ravindranath et al., 1997a, Ravindranath et al., 1997b). However, carbohydrate antigens are typically poorly immunogenic, very difficult to purify in large quantities, difficult to synthesize and usually induce mostly short-lived IgM type antibodies in a vaccinated host without long lasting immunity. Most carbohydrate antigens belong to the category of T cell-independent antigens that reflect their inability to stimulate MHC class II-dependent T cell help (Mond et al., 1995). Consequently, carbohydrates alone are not capable of induction of a sufficient anamnestic or secondary immune response and require extensive adjuvanticity. An alternative approach for augmentation of carbohydrate immunity is the use of peptide or polypeptide surrogate antigens.
The idea of using surrogate antigens as immunogens, requires that antigenic mimicry, accomplished using amino acids in place of sugars, induces a precisely reproduced immune specificity pattern for the nominal antigen (Olsson, 1987; Diakun and Matta, 1989; Hutchins et al., 1996; Tsuyuoka et al., 1996). Mimicking peptides represent a new and very promising tool to overcome T cell-independence and to increase the efficiency of the immune response to carbohydrates (Shikhman and Cunningham, 1997). Peptides with a high prevalence of tryptophan and tyrosine occur in peptides that mimic carbohydrates (Oldenburg et al., 1992; Scott et al., 1992; Hoess et al., 1993; Shikhman et al., 1994; Shikhman and Cunningham, 1994; Westerink et al., 1995; Valadon et al., 1996; Harris et al., 1997; Zhang et al., 1997b; Agadjanyan et al., 1997; Phalipon et al., 1997; Taki et al., 1997). A more precise understanding of the binding properties of carbohydrate-mimicking peptides is required to determine whether the occurrence of aromatic containing motifs is due to molecular mimicry or simply reflects an advantage provided by aromatic rings for interaction between proteins (Valadon et al., 1996).
Peptides with the aromatic motif Wā§¹YXY have been previously defined to mimic several carbohydrate subunits that include YPY as a mimic of mannose as identified from peptide phage screening with Con A (Oldenburg et al., 1992; Scott et al., 1992) WRY found to mimic a (1ā4)glucose as identified from analysis of protein that bind to a-amylase (Murai et al., 1985; Mirkov et al., 1995), WLY found to mimic Lewis Y (LeY) as identified from peptide phage screening with an anti-LeY antibody, B3 (Hoess et al., 1993) and YRY derived from an anti-idiotypic antibody found to mimic the major C polysaccharide a (2ā9) sialic acid (MCP) of Neisseria meningitidis (Westerink et al., 1995). This aromatic motif is also implicated as a mimic for a Cryptococcus neoformans epitope (Valadon et al., 1996).
The sequence similarities that define this motif suggest that antibodies to homologous peptides might cross-react with similar subunits expressed on what are otherwise dissimilar carbohydrate structures; if so the basis of cross-reactivity would be structural. Mapping peptide epitopes containing the Wā§¹YXY motif with anti-carbohydrate antibodies could lend to defining fine specificities that might go undetected by screening carbohydrate antigens alone. Molecular modeling suggests that the neolactoseries LeY and sialyl-lex (sLeX) tetrasaccharide structure is similar to the core structure of MCP, providing a structural basis for potential cross-reactivity of antibodies (Agadjanyan et al., 1997). Consequently, evaluating responses to neolactoseries antigens would further define recognition specificities for antibodies directed to these important carbohydrate forms.
Here, we delineate specificity patterns associated with the antigenic and immunological mimicry of peptide mimotopes that induce antibodies to Lewis (Le) antigens. The blood group-related neolactoseries carbohydrate structures Lewis X (LeX), sLeX, Lewis a (Lea), sialyl-Lea (sLea) and LeY are examples of terminal carbohydrate structures related to tumor prognosis (Miyake et al., 1992; Dabelsteen, 1996). These antigens (Fig. 1) constitute carbohydrate moieties on some tumor associated gangliosides, the human carcinoembryonic antigen family, the human pancreatic MUC-1 antigen and identified on glycoproteins and glycolipids on carcinomas of the skin, stomach, pancreas, lung, colon, breast and prostate. SLeX and sLea, are implicated as immunogenic antigens in human melanoma as well (Ravindranath et al., 1997a). Histo-blood group related antigens are observed only at the secretory borders on normal tissues (Zhang et al., 1997a). This location appears to be inaccessible to the immune system, inducing neither tolerance nor autoimmune responses. Consequently, some Le antigens, notably LeY, are excellent targets for passive immunotherapy or a vaccine approach in the treatment of cancer.
Contrasting the antigenic and immunological properties of peptide mimotopes forms provides information about how amino acid differences lend to specific carbohydrate mimicry. We observe that the immunological presentation of Wā§¹YXY containing peptides influences the specificity pattern for histo-blood group synthetic carbohydrate probes. We also observe that the peptide mimotopes elicit polyclonal sera that specifically bind to human tumor cells, but not to normal tissues, and can mediate complement dependent cytotoxicity (CDC) of representative human breast cell lines, albeit at low titers.
Section snippets
Preparation of peptide immunogens
Several peptides were synthesized, repeating putative centralized motifs, suggested to mimic carbohydrate forms and correspond to GGIYYPYDIYYPYDIYYPYD (K61105), GGIYWRYDIYWRYDIYWRYD (K61106), GGIYYRYDIYYRYDIYYRYD (K61107), GGGAPWLYGAPWLYGAPWLY (K61223), GGAPWLYGGAPWLYAPWLY (K61108). Other peptides were synthesized as variants that include GGAGRWVFSAPGVRSIL (K6111), GGGWPYLRFSPWVSPLG (K61110), GGARVSFWRYSSFAPTY (K61109). Peptides were synthesized with the addition of a tripeptide YGG spacer and
Antigenic mimicry of peptide motifs
To determine the extent of antigenic mimicry of the Wā§¹YXY putative motifs in shown in Table 1, a variety of peptides were synthesized, some repeating the respective putative centralized motifs. MAP forms were synthesized for detection of reactivity patterns with the anti-LeY monoclonal antibodies BR55-2 and 15.6A and the anti-sialylated LeX reactive antibody FH-6 (Fig. 2a). These antibodies are very specific for their respective antigens. It is hypothesized that MAP forms may represent
Discussion
Immunization with simple synthetic Lewis antigen-conjugates, as observed with simple LeY-conjugates, only results in sera and MAbs, reactive with the immunizing antigen (Kitamura et al., 1994). In contrast, immunization with LeY expressing cell lines or multivalent LeY forms, yield MAbs that react with both synthetic carbohydrate forms and native carbohydrate configurations (Vlasova et al., 1994). One difference between these antigen sources is the distribution of LeY epitopes on the carrier ā
Acknowledgements
This work was supported by the USAMRMC (DAMD17-94-J-4310) Breast Cancer Program. Computer equipment support from the Cancer Center of the University of Pennsylvania is also gratefully acknowledged. We also thank Charlotte Read Kensil of Aquilia Pharmaceuticals (Worcester, MA) for supplying the QS-21.
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