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US-12618839-B2 - Antibodies for detecting Epstein Barr virus-positive gastric cancer

US12618839B2US 12618839 B2US12618839 B2US 12618839B2US-12618839-B2

Abstract

Provided herein are methods, compositions, kits, and systems for detecting Epstein Barr virus infection (EBV) in gastric cancer (GC) patients. In particular, provided herein are methods, composition, kits, and systems for diagnosing and treating EBV-positive gastric cancer (EBV + GC) in a biological sample of an individual based on the presence and level of antibodies against particular Epstein Barr virus proteins. EBV + GC is a distinct subtype of gastric cancer and is associated with unique molecular profiles. Also provided herein are methods for providing more personalized therapy for each of these distinct cancer subtypes and methods for determining an Epstein Barr virus-positive gastric cancer antibody signature, and kits comprising components and protocols for performing the methods of this disclosure.

Inventors

  • Joshua Labaer
  • Lusheng Song
  • Ji Qiu
  • Yunro Chung

Assignees

  • ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY

Dates

Publication Date
20260505
Application Date
20201023

Claims (20)

  1. 1 . A method for identifying a subject having increased risk of developing Epstein-Barr Virus-positive gastric cancer (EBV + GC), the method comprising: (a) reacting a biological sample obtained from the subject with a reagent composition that comprises components for detecting in the biological sample the presence of one or more IgG antibodies selected from anti-BORF2, anti-LF2, anti-BDLF2, anti-BXLF1, anti-BRLF1, anti-BaRF1, anti-BGLF5, anti-BOLF1, anti-BRRF1, anti-BALF2, anti-BLLF3, and anti-BSLF2; wherein the components comprise a panel of EBV antigens that bind to the anti-BORF2, anti-LF2, anti-BDLF2, anti-BXLF1, anti-BRLF1, anti-BaRF1, anti-BGLF5, anti-BOLF1, anti-BRRF1, anti-BALF2, anti-BLLF3, and anti-BSLF2 antibodies; (b) contacting the biological sample and reagent composition with an anti-IgG antibody comprising a label; and (c) detecting the presence of the one or more IgG antibodies in the sample, wherein increased seroreactivity of the one or more IgG antibodies relative to the seroreactivity of the one or more IgG antibodies in a control sample obtained from a subject that does not have EBV + GC is indicative of increased risk of EBV + GC.
  2. 2 . The method of claim 1 , wherein the detected antibodies comprise anti-BALF2, anti-BORF2, and anti-BRRF1.
  3. 3 . The method of claim 1 , wherein the biological sample is one or more of a whole blood sample, a serum sample, and a plasma sample.
  4. 4 . The method of claim 1 , wherein the EBV antigens are encoded by SEQ ID NOs: 42, 138, 76, 26, 60, 4, 34, 20, 148, 14, 30, 8, and 36.
  5. 5 . The method of claim 1 , wherein the detection step is carried out using an ELISA assay or a Western Blot assay.
  6. 6 . The method of claim 1 , further comprising administering a vaccine-based gastric cancer treatment to the subject if identified as having an increased risk of EBV + GC gastric cancer.
  7. 7 . A method to detect EBV + GC in a subject at risk of having EBV + GC, the method comprising: (a) contacting a biological sample obtained from the subject with a set of reagents, wherein the set of reagents binds to at least three biomarkers in the biological sample, wherein the biomarkers are IgG antibodies selected from the group consisting of anti-BORF2, anti-LF2, anti-BDLF2, anti-BXLF1, anti-BRLF1, anti-BaRF1, anti-BGLF5, anti-BOLF1, anti-BRRF1, anti-BALF2, anti-BLLF3, and anti-BSLF2; wherein the set of reagents comprises a panel of EBV antigens that bind to the anti-BORF2, anti-LF2, anti-BDLF2, anti-BXLF1, anti-BRLF1, anti-BaRF1, anti-BGLF5, anti-BOLF1, anti-BRRF1, anti-BALF2, anti-BLLF3, and anti-BSLF2 antibodies; (b) measuring the level of the at least three biomarkers in the biological sample; and (c) detecting that the level of the at least three biomarkers is increased in the biological sample relative to a level of the at least three biomarkers in a control sample from a subject without EBV + GC, thereby detecting the presence of EBV + GC in the subject.
  8. 8 . The method according to claim 7 , wherein the at least three biomarkers comprise anti-BALF2, anti-BORF2, and anti-BRRF1.
  9. 9 . The method of claim 7 , further comprising (d) administering an EBV+ gastric cancer therapy to the subject, wherein the EBV + GC therapy is selected from the group consisting of chemotherapy, hormonal therapy, radiotherapy, immunotherapy, and surgical removal of stomach tissue.
  10. 10 . The method of claim 7 , wherein the biological sample is one or more of a whole blood sample, a serum sample, and a plasma sample.
  11. 11 . The method of claim 7 , wherein the detection step is carried out using an immunoassay.
  12. 12 . A method of determining an Epstein Barr Virus (EBV) gastric cancer antibody signature comprising IgG antibodies, contained in a biological sample from an individual, that bind to immobilized EBV antigens, the method comprising: (a) contacting the sample to a panel of immobilized EBV antigens under conditions that promote formation of antigen-antibody complexes, wherein the immobilized EBV antigens comprise one or more of BORF2, LF2, BDLF2, BXLF1, BRLF1, BaRF1, BGLF5, BOLF1, BRRF1, BALF2, BLLF3, and BSLF2; and (b) identifying complexes formed by immobilized EBV antigens and antibody in the sample, to determine an EBV antibody signature.
  13. 13 . The method of claim 12 , wherein the antibody signature is expressed as a level of antibody binding to each immobilized antigen.
  14. 14 . The method of claim 12 , further comprising comparing an antibody signature from one individual to the antibody signature from another individual.
  15. 15 . The method of claim 12 , wherein one individual has a disease process, and one individual is a healthy individual and the method allows comparison of the antibody signature in the healthy individual and the individual with a disease.
  16. 16 . The method of claim 15 , wherein the disease process comprises EBV + GC.
  17. 17 . The method of claim 12 , wherein the immobilized EBV antigens are encoded by SEQ ID NOs: 42, 138, 76, 26, 60, 4, 34, 20, 148, 14, 30, 8, and 36.
  18. 18 . A kit for performing the method of claim 1 , the kit comprising a panel of EBV antigens comprising at least three of BORF2, LF2, BDLF2, BXLF1, BRLF1, BaRF1, BGLF5, BOLF1, BRRF1, BALF2, BLLF3, and BSLF2.
  19. 19 . The kit of claim 18 , wherein the EBV antigens are encoded by SEQ ID NOs: 42, 138, 76, 26, 60, 4, 34, 20, 148, 14, 30, 8, and 36.
  20. 20 . The method of claim 7 , wherein the EBV antigens are encoded by SEQ ID NOs: 42, 138, 76, 26, 60, 4, 34, 20, 148, 14, 30, 8, and 36.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national stage application filed under 35 U.S.C. § 371 of International Application No. PCT/US2020/057010, filed Oct. 23, 2020, which claims priority to U.S. Provisional Application No. 62/925,584, filed Oct. 24, 2019, which is hereby incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under R01 CA199948 and U01 CA214201 awarded by the National Institutes of Health. The government has certain rights in the invention. In accordance with 37 C.F.R. § 1.821(c), the present specification makes reference to a Sequence Listing submitted electronically in the form of an ASCII text file (named “112624.01327_ST25.txt”, 448,936 bytes in size, created on Nov. 6, 2025). The sequence listing is electronically submitted via Patent Center with the application and is incorporated herein by reference in its entirety, with the intention that, upon publication (including issuance), this incorporated Sequence Listing will be inserted in the published document immediately before the claims. BACKGROUND Gastric cancer (GC) is a major public health problem. GC represents the third leading cause of cancer mortality in the world, with approximately 1,000,000 new diagnoses and over 783,000 deaths in 2018 (equal to 1 in every 12 deaths globally). Besides gastric cancer induced by Helicobacter pylori (H. pylori) infection, around 10% of gastric cancer present the evidence of Epstein Barr Virus (EBV) involvement. As a ubiquitous virus that infects over 90% of adults, the association of EBV infection and gastric cancer is still unclear. Like other cancers associated with EBV (e.g., nasopharyngeal carcinoma (NPC), Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and non-Hodgkin lymphoma (NHL)), EBV-associated gastric cancer (EBV+ GC) is conventionally detected by in situ hybridization of EBV-encoded small RNA (EBER), a specific marker for EBV presence. EBV+ GC is a distinct subtype of gastric cancer classified by The Cancer Genome Atlas (TCGA) with overall lower mortality, occurs more frequently in male than female, is non-cardiac gastric carcinoma type with 90% prevalence of lymphoepithelioma-like gastric carcinoma, and displays significant intra- or peritumoral immune cell infiltration compared to EBV− GC. Current knowledge of EBV+ GC has been focused on epigenetic and genetic aberrance. EBV+ GC displays unique molecular characteristics, recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, PD-L1 and PD-L2. Consideration of its unique molecular characterization and distinct gastric cancer subtype, distinguishing EBV+ GC from EBV− GC would benefit a following targeted therapy and precision medicine. Previously, anti-VCAp18 IgG/IgA, anti-EBNA1 IgG/IgA, and anti-Early protein (EA) IgG/IgA, which were used to detect EBV induced cancers, NPC, BL, and HL, as serological biomarkers, had been applied for EBV+ GC detection. However, their presence in EBV+ GC has been controversial. Some groups reported elevated anti-VCA IgA and anti-EA IgG in EBV+ GC as compared to EBV− GC or healthy controls. Other groups have studied these antibodies but did not find a significant association between them and EBV+ GC. Other than anti-EBV antibodies, Chung et al. had reported the present of circulating EBER in serum for limited EBV+ GC cases (n=5) but not in controls (n=197). However, to date, none of these proposed markers have demonstrated enough discriminative power to be used for EBV+ GC diagnosis. Accordingly, there remains a need in the art for improved reagents and methods for detecting EBV-associated gastric cancer, assessing risk of developing EBV+ gastric cancer, and identifying subjects in need of treatment for this is unique subtype of gastric cancer, meaning reagents and methods that are more reliable and have sufficient discriminatory power for risk stratification and for early detection in asymptomatic individuals. SUMMARY In a first aspect, provided herein is a method for identifying a subject having increased risk of developing EBV-positive gastric cancer (EBV+ GC). The method can comprise or consist essentially of (a) reacting a biological sample obtained from a subject with a reagent composition that comprises components for detecting in the biological sample the presence of one or more antibodies selected from anti-BORF2, anti-LF2, anti-BDLF2, anti-BXLF1, anti-BRLF1, anti-BaRF1, anti-BGLF5, anti-BRRF1, anti-BALF2, anti-BLLF3, and anti-BSLF2; and (b) detecting the presence of the antibodies in the sample, wherein increased seroreactivity relative to a control for one or more of the antibodies is indicative of at least a four-fold increased risk of EBV+ GC. The detected antibodies can comprise anti-BALF2, anti-BORF2, and anti-BRRF1. The biological sample can be one or more of a whole blood sample, a serum sample, and a plasma sample. The method can detect EBV+ GC