Cancer Relevance of Circulating Antibodies Against LINE-1 Antigens in Humans

Abstract

Long interspersed nuclear element-1 (LINE-1 or L1), the most abundant family of autonomous retrotransposons occupying over 17% of human DNA, is epigenetically silenced in normal tissues by the mechanisms involving p53 but is frequently derepressed in cancer, suggesting that L1-encoded proteins may act as tumor-associated antigens recognized by the immune system. In this study, we established an immunoassay to detect circulating autoantibodies against L1 proteins in human blood. Using this assay in >2,800 individuals with or without cancer, we observed significantly higher IgG titers against L1-encoded ORF1p and ORF2p in patients with lung, pancreatic, ovarian, esophageal, and liver cancers than in healthy individuals. Remarkably, elevated levels of anti-ORF1p-reactive IgG were observed in patients with cancer with disease stages 1 and 2, indicating that the immune response to L1 antigens can occur in the early phases of carcinogenesis. We concluded that the antibody response against L1 antigens could contribute to the diagnosis and determination of immunoreactivity of tumors among cancer types that frequently escape early detection.

Significance: The discovery of autoantibodies against antigens encoded by L1 retrotransposons in patients with five poorly curable cancer types has potential implications for the detection of an ongoing carcinogenic process and tumor immunoreactivity.

FIGURE 1

Approach and proof of concept. A, Hypotheses: proteins encoded by L1 are expected to be recognized as tumor-associated antigens and become targets for antibody response. This response is stimulated by the cGAS-STING-mediated induction of inflammation. Circulating Abs to L1 antigens can be used as cancer biomarkers if detected by anti-ORF1p and anti-ORF2p immunoassays. Comparison of antibody levels in healthy individuals (N = 137) and patients with cancer (N = 331) in the assays for anti-ORF1p (B), anti-ORF2p (C), and p53 (D). The significance of differences is assessed by Mann–Whitney U test. E, Spearman correlation between anti-ORF1p IgG titers and anti-p53 IgG signals in serum samples of patients with cancer (N = 331). All P value less than 0.05 is considered significant.

FIGURE 2

Relevance of antibodies detected by immunoassay to L1 ORF1p. The results of immunofluorescent staining (A) and immunoblot analysis (B) of HeLa cells transduced with Tet-inducible L1/Gluc reporter construct with (+DOX) and without (−DOX) L1 induction by doxycycline (Supplementary Fig. S1). “Rabbit αORF1p Ab” – polyclonal anti-ORF1p rabbit antiserum. The other panels represent staining with 50,000-fold diluted serum samples from patients with ovarian cancer with 10⁷–10⁸ anti-ORF1p IgG titers in anti-ORF1p immunoassay. “M” stands for the protein size marker added to the sample along with HeLa cells lysate without DOX L1 induction. The asterisks on the bottom panel correspond to ORF1p standard (40-kDa) band.

FIGURE 3

Anti-ORF1p IgG response in serum samples from patients with five cancer types. A, Selection of cancers for analysis in anti-ORF1p immunoassay. Sector sizes are proportional to the incidence of these cancers in the United States in 2021 (https://seer.cancer.gov/statfacts/). Color code indicates a 5-year survival. “S” stands for the existence of approved cancer screening protocols. B, Detection of anti-ORF1p IgG titers in serum samples of patients with the indicated cancer types of all stages. Sample size: ovary (N = 979), esophagus (N = 377), lung (N = 907), pancreas (N = 124), liver (N = 217) cancers and healthy (N = 352). C, The same as panel B; only samples from patients with cancer stages 1 and 2 are shown. Sample size: ovary (N = 193), esophagus (N = 79), lung (N = 90), pancreas (N = 40), and liver (N = 67) cancers and healthy (N = 352). The gray areas in B and C mark samples below the anti-ORF1p IgG titer threshold of 450. Statistics were calculated by Dunn multiple comparison test with adjusted P value for anti-ORF1p IgG titers. All P value less than 0.05 is considered significant. D, Anti-ORF1p immunoassay threshold determination. E, Spearman r correlation analysis of anti-ORF1p IgG titers and age among healthy individuals (20–89 years of age) or patients with cancer (18–95 years of age).

FIGURE 4

Schematic description of a hypothetical relationship between circulating IgG titers to LINE-1 antigens and cancer susceptibility to immunotherapy by immune checkpoint inhibitors. Presence of low titers of anti-LINE-1 IgG in healthy subjects can be explained by immune response that occurred to precancerous lesions associated with derepression of LINE-1 that were aborted due to effective T-cell response. These events generate memory B cells and the presence of low titers of anti-LINE-1 antibodies even in a cancer-free organism. Development of cancer that passivate T-cell response due to the engagement of immune checkpoint factors PD-L1 and CTLA-4 becomes a growing source of LINE-1 antigens. If this cancer expresses LINE-1 and retains the ability to be recognized by the immune system (e.g., the ability to present antigens), it will then induce elevation of anti-LINE1 IgG levels, which will signal about principal immunoreactivity of the tumor. *This response is stimulated by the cGAS-STING–mediated induction of inflammation.