Human xeno-autoantibodies against a non-human sialic acid serve as novel serum biomarkers and immunotherapeutics in cancer

Abstract

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 1 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 noncancer 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 xeno-autoantigens have potential for novel diagnostics, prognostics, and therapeutics in human carcinomas.

Figure 1. Anti-Neu5Gc IgG antibodies can be affinity-purified from IVIG

(A) Diagram of Neu5Ac and Neu5Gc. (B) Anti-Neu5Gc antibodies were affinity-purified from IVIG over sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, fractions collected and analyzed by ELISA against Neu5Gcα-PAA (mean±SD of triplicates; representative of multiple independent experiments). (C) Eluted fractions were pooled, concentrated, filtered from free glycans, and analyzed by ELISA against multiple Neu5Gc-glycans (mean±SD; representative of two independent experiments).

Figure 2. Affinity-purified anti-Neu5Gc IgG antibodies can specifically kill tumors expressing cell-surface Neu5Gc in vivo

(A) qPCR reveals down regulation of CMAH gene expression in MC38 cells with siRNA to CMAH (MC38si) compared to the wild-type MC38 cells (mean±SD of triplicates; Two tailed unpaired t-test P<0.0001). (B) FACS analysis using a polyclonal chicken anti-Neu5Gc antibody confirms reduced expression of Neu5Gc on the cell surface (representative of two independent experiments). (C) Cmah^−/− mice were injected subcutaneously with MC38 (right flank) and MC38si (left flank). Affinity-purified anti-Neu5Gc IgG can specifically kill tumors expressing Neu5Gc on the cell surface (MC38) (n=6) compared to the control-treated mice (n=5), but no significant effect is observed when the Neu5Gc expression on the cell surface is diminished (MC38si), as determined by daily measurements of tumor volumes (left panels; mean±SEM; Two-way ANOVA P<0.001) or terminal tumor weights on day 16 (right panels; mean±SEM; Two-tailed t-test * p=0.036), representative of 3 independent experiments. Direct comparison of the effects of anti-Neu5Gc IgG on these tumors in the same mice revealed attenuated MC38 tumors compared to the control MC38si tumors (P=0.0392, Two-way-ANOVA).

Figure 3. Validation of sialoglycan-microarray slides for detection of anti-Neu5Gc antibodies

Various glycan-pairs (glycans #1–40 as detailed in Table 1) with terminal Neu5Gc or Neu5Ac were spotted on Epoxy-coated slides, then developed using (A) affinity-purified chicken anti-Neu5Gc IgY (1:10,000) (32) detected by Cy3-anti-chicken IgY (0.5 µg/ml); or, (B) human anti-Neu5Gc Ig positive human serum (1:100; S34 (5) detected by Cy3-anti-human IgG (1.5 µg/ml). Data were analyzed with an Excel pivot table, are representative of more than three independent experiments and show mean±SD of 4 replicate spots (of the glycans printed at 125 µM).

Figure 4. Anti-GcSTn is a classifier for cancer cases/controls and is suggested to be a human-specific and tumor-associated carcinoma biomarker

Probabilities of being a cancer case were calculated using logistic regression where predictors were the two parameters, α and β, which summarized the anti-Neu5Gc antibody response to glycan 6 (Neu5Gc-sialyl-Tn; GcSTn) against the pan antibody level of 20 Neu5Ac glycans. (A) ROC curve for training data, used to select glycan 6, that had 67 non-metastatic breast cancer cases and 25 controls. (B) ROC curve for the first validation data set, which had 74 new non-metastatic breast cancer cases and 25 new controls. (C) ROC curve for a second validation data set, which had 99 cases of other cancer types and 55 controls. The biochemical and genetic rationale for the generation of the novel human carcinoma biomarker is schematically presented. (D) Somatic Cosmc mutations generate incomplete O-linked glycosylation, resulting in tumor-associated expression of the sialylated-Tn antigen in many carcinomas (left panel). Incorporation of dietary-Neu5Gc by such carcinomas generates Neu5Gc-sialyl Tn, detected by the humoral adaptive immune system as foreign, thus generating antibodies against it. Such xeno-auto-antibodies specific for Neu5Gc-sialyl Tn, are hypothesized to be novel biomarkers for early screening of carcinomas and/or potential immunotherapeutic tools (right panel).

Figure 5. CDC and ADCC of cells expressing cell surface GcSTn

Jurkat cells were chased-out of pre-existing media-derived Neu5Gc then fed with 3 mM Neu5Ac or Neu5Gc. (A) FACS analysis using a polyclonal chicken anti-Neu5Gc antibody (highly specific to all Neu5Gc but not Neu5Ac glycans (32)) confirms feeding with Neu5Gc. A mouse monoclonal antibody specific for sialyl-Tn (STn) demonstrates cell-surface expression of this structure with either terminal Neu5Ac (STn; on Neu5Ac fed cells) or Neu5Gc (GcSTn; on Neu5Gc fed cells). (B) Human serum S34 (high in anti-GcSTn reactivity; Fig. 3B) can promote complement dependent cytotoxicity (CDC) of cells fed with Neu5Gc but not with Neu5Ac (left panel). Similarly, affinity-purified human anti-Neu5Gc IgG (high in anti-GcSTn reactivity; Fig. 1C) can promote CDC of Jurkat cells in a Neu5Gc-dependent manner (right panel; two independent experiments each; mean±SD; Two-way ANOVA, * P<0.01, ** P<0.05). (C) Human serum S34 (10%) can promote Neu5Gc-specific antibody-dependent cellular-mediated cytotoxicity (ADCC) with increasing effector:target ratios (target cells, T, Jurkat cell fed with Neu5Ac/Neu5Gc; effector cells, E, PBMCs in RPMI), in contrast to human serum S30 (10%) (5). However, when human serum S30 (10%) was supplemented with 10 µg/ml of the purified anti-Neu5Gc IgG it could promote ADCC similar to human serum S34 (representative of two independent experiments; two-way ANOVA * P<0.05).