Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies

Abstract

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease.

Targeting Epstein-Barr virus–transformed B lymphoblastoid cells using antibodies with T-cell receptor–like specificities

“…[39][40][41] Using E1, we have similarly reported the direct detection of a high number of HLA-A2/EBNA1 complexes on nasopharyngeal carcinoma biopsies and EBV-positive cell lines. 13 These findings supported our current observation that EBNA1 could indeed be presented endogenously by BLCLs. Notably, because EBNA1 is expressed in both lytic and latent EBV infections and is present across all EBV malignancies, E1 might potentially be applied to latency I-and latency II-associated diseases.…”

“…L1 displayed the best binding avidity (lowest K D value) among the 3 antibodies, consistent with our earlier SPR data. 13 These results indicate that our TCR-like mAbs bind specifically to their respective pMHC complexes, akin to that of a TCR.…”

“…13 Briefly, splenocytes from immunized mice were magnetically enriched with the specific biotinylated pMHC complexes before fusion, resulting in an enhanced percentage of hybridomas secreting target-specific antibodies. 13 To demonstrate the specificities of these TCR-like mAbs for their pMHC, we used a pMHC ELISA with HLA-A*0201 monomers substituted with the relevant peptides. Figure 1A).…”

“…These TCR-like mAbs were shown to detect endogenous targets found on EBVpositive cell lines, splenic lesions of EBV-infected humanized mice, as well as nasopharyngeal carcinoma biopsies, underscoring their ability to recognize these EBV epitopes in EBV-associated malignancies. 13 Despite the ability to recognize endogenous EBV antigens, the therapeutic potential of these antibodies in targeting EBV-associated tumors has not been directly assessed. B lymphoblastoid cell lines (BLCLs) are the in vitro counterparts of B cells found in EBV-PTLD, with both displaying the characteristic latency III expression pattern.…”

“…As antibodies, TCR-like mAbs also exhibit better stabilities and higher affinities than TCRs. 9 Over the years, increasing interest in TCR-like mAbs has led to a rapid expansion of the repertoire of TCRlike mAbs targeting both viral [10][11][12][13] and tumor-associated antigens. [14][15][16][17][18][19] These studies have demonstrated the feasibility of TCR-like mAbs to target cells expressing neoantigen, independent of the immunoregulatory microenvironment that might inhibit T-cell function.…”