Human heterophile antibodies that agglutinate animal erythrocytes are known to detect the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). This monosaccharide cannot by itself fill the binding site (paratope) of an antibody and can also be modified and presented in various linkages, on diverse underlying glycans. Thus, we hypothesized that the human anti-Neu5Gc antibody response is diverse and polyclonal. Here we use a novel set of natural and chemoenzymatically-synthesized glycans to show that normal humans have an abundant and diverse spectrum of such anti-Neu5Gc antibodies, directed against a variety of Neu5Gc-containing epitopes. High sensitivity and specificity assays were achieved by using N-acetylneuraminic acid (Neu5Ac)-containing probes (differing from Neu5Gc by one less oxygen atom) as optimal background controls. The commonest anti-Neu5Gc antibodies are of the IgG class. Moreover, the range of reactivity and Ig classes of antibodies varies greatly amongst normal humans, with some individuals having remarkably large amounts, even surpassing levels of some well-known natural blood group and xenoreactive antibodies. We purified these anti-Neu5Gc antibodies from individual human sera using a newly developed affinity method and show that they bind to wild-type but not Neu5Gc-deficient mouse tissues. Moreover, they bind back to human carcinomas that have accumulated Neu5Gc in vivo. As dietary Neu5Gc is primarily found in red meat and milk products, we suggest that this ongoing antigen-antibody reaction may generate chronic inflammation, possibly contributing to the high frequency of diet-related carcinomas and other diseases in humans.
Recombinant glycosylated biotherapeutic agents are usually produced in non-human mammalian cell lines, which can synthesize and/or metabolically incorporate the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). This contamination was previously known but ignored, as normal humans were thought to not react against Neu5Gc. However, recent findings indicate that humans have variable spectra of sometimes high levels of circulating anti-Neu5Gc antibodies. We studied two monoclonal antibodies in clinical use (Cetuximab and Panitumumab), and show covalently-bound Neu5Gc on Cetuximab. Anti-Neu5Gc antibodies from normal humans interact with Cetuximab in a Neu5Gc-specific manner and generate immune complexes in vitro. Mice with a human-like defect in Neu5Gc synthesis generate anti-Neu5Gc antibodies upon injection with Cetuximab. Circulating anti-Neu5Gc antibodies enhance Cetuximab clearance. These findings have potential relevance to half-life, efficacy and immune reactions in patients given such drugs. Finally, we show a method to reduce the Neu5Gc content of cultured cell lines and their secreted glycoproteins.
Human carcinomas can metabolically incorporate and present the dietary non-human sialic acid Neu5Gc, which differs from the human sialic acid N-acetylneuraminic acid (Neu5Ac) by one oxygen atom. Tumor-associated Neu5Gc can interact with low levels of circulating anti-Neu5Gc antibodies, thereby facilitating tumor progression via chronic inflammation in a human-like Neu5Gc-deficient mouse model. Here we show that human anti-Neu5Gc antibodies can be affinity-purified in substantial amounts from clinically-approved intravenous IgG (IVIG) and used at higher concentrations to suppress growth of the same Neu5Gc-expressing tumors. Hypothesizing that this polyclonal spectrum of human anti-Neu5Gc antibodies also includes potential cancer biomarkers, we then characterize them in cancer and non-cancer patients’ sera, using a novel sialoglycan-microarray presenting multiple Neu5Gc-glycans and control Neu5Ac-glycans. Antibodies against Neu5Gcα2–6GalNAcα1-O-Ser/Thr (GcSTn) were found to be more prominent in patients with carcinomas than with other diseases. This unusual epitope arises from dietary Neu5Gc incorporation into the carcinoma marker Sialyl-Tn, and is the first example of such a novel mechanism for biomarker generation. Finally, human serum or purified antibodies rich in anti-GcSTn-reactivity kill GcSTn-expressing human tumors via complement-dependent-cytotoxicity or antibody-dependent-cellular-cytotoxicity. Such xeno-autoantibodies and xenoautoantigens have potential for novel diagnostics, prognostics and therapeutics in human carcinomas.
Patients with cancer have circulating heterophile antibodies that agglutinate animal red cells via recognition of the mammalian cell surface sialic acid N-glycolylneuraminic acid (Neu5Gc), which was long considered an oncofetal antigen in humans. However, humans are genetically deficient in Neu5Gc production and instead metabolically accumulate Neu5Gc from dietary sources, particularly red meats and milk products. Moreover, mice with a humanlike defect showed no alternate pathway for Neu5Gc synthesis and even normal humans express anti-Neu5Gc antibodies. We show here that human tumors accumulate Neu5Gc that is covalently attached to multiple classes of glycans. The paradox of human tumor Neu5Gc accumulation in the face of circulating anti-Neu5Gc antibodies was hypothesized to be due to facilitation of tumor progression by the resulting low-grade chronic inflammation. Indeed, murine tumors expressing human-like levels of Neu5Gc show accelerated growth in syngeneic mice with a human-like Neu5Gc deficiency, coincident with the induction of anti-Neu5Gc antibodies and increased infiltration of inflammatory cells. Transfer of polyclonal monospecific syngeneic mouse anti-Neu5Gc serum also enhanced growth of transplanted syngeneic tumors bearing human-like levels of Neu5Gc, with tumors showing evidence for antibody deposition, enhanced angiogenesis and chronic inflammation. These effects were suppressed by a cyclooxygenase-2 inhibitor, a drug type known to reduce human carcinoma risk. Finally, affinity-purified human anti-Neu5Gc antibodies also accelerate growth of Neu5Gc-containing tumors in Neu5Gc-deficient mice. Taken together, the data suggest that the human propensity to develop diet-related carcinomas is contributed to by local chronic inflammation, resulting from interaction of metabolicallyaccumulated dietary Neu5Gc with circulating anti-Neu5Gc antibodies.antibodies ͉ COX-2 ͉ Neu5Gc ͉ red meat ͉ sialic acid
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