Background Tumor-specific mutated proteins can create immunogenic non-self, mutation-containing ‘neoepitopes’ that are attractive targets for adoptive T-cell therapies. To avoid the complexity of defining patient-specific, private neoepitopes, there has been major interest in targeting common shared mutations in driver genes using off-the-shelf T-cell receptors (TCRs) engineered into autologous lymphocytes. However, identifying the precise naturally processed neoepitopes to pursue is a complex and challenging process. One method to definitively demonstrate whether an epitope is presented at the cell surface is to elute peptides bound to a specific major histocompatibility complex (MHC) allele and analyze them by mass spectrometry (MS). These MS data can then be prospectively applied to isolate TCRs specific to the neoepitope.
Methods We created mono-allelic cell lines expressing one class I HLA allele and one common mutated oncogene in order to eliminate HLA deconvolution requirements and increase the signal of recovered peptides. MHC-bound peptides on the surface of these cell lines were immunoprecipitated, purified, and analyzed using liquid chromatography-tandem mass spectrometry, producing a list of mutation-containing minimal epitopes. To validate the immunogenicity of these neoepitopes, HLA-transgenic mice were vaccinated using the minimal peptides identified by MS in order to generate neoepitope-reactive TCRs. Specificity of these candidate TCRs was confirmed by peptide titration and recognition of transduced targets.
Results We identified precise neoepitopes derived from mutated isoforms of KRAS, EGFR, BRAF, and PIK3CA presented by HLA-A*03:01 and/or HLA-A*11:01 across multiple biological replicates. From our MS data, we were able to successfully isolate murine TCRs that specifically recognize four HLA-A*11:01 restricted neoepitopes (KRAS G13D, PIK3CA E545K, EGFR L858R and BRAF V600E) and three HLA-A*03:01 restricted neoepitopes (KRAS G12V, EGFR L858R and BRAF V600E).
Conclusions Our data show that an MS approach can be used to demonstrate which shared oncogene-derived neoepitopes are processed and presented by common HLA alleles, and those MS data can rapidly be used to develop TCRs against these common tumor-specific antigens. Although further characterization of these neoepitope-specific murine TCRs is required, ultimately, they have the potential to be used clinically for adoptive cell therapy.
Tumor-specific mutations found across a wide variety of human cancers are becoming increasingly appreciated in the clinical setting. As investigators implement new highly effective immunotherapies, they have discovered that tumor-specific ‘neoantigens’ drive the immune responses underlying these therapies. One form of effective immunotherapy is adoptive cell therapy (ACT), wherein tumor-reactive T-cells, first procured from tumor infiltrating lymphocytes (TIL), are expanded in vitro and given to a properly prepared recipient. This has been reported to cause objective responses in over half of treated patients with metastatic melanoma with a quarter achieving complete and durable rejection of their cancer. More recently, similar successes have been reported in individual patients with common epithelial cancers. Extensive studies on the antigens recognized by effective TIL have again revealed that mutated neoantigens are playing a key role in these clinical outcomes.
Identifying mutation-reactive TIL from a patient is laborious and time-consuming, and sometimes does not generate robust T-cell responses. In one clinical study, most patients had detectable neoantigen reactive TIL, yet only 1.6% of tumor-associated mutations generated a T-cell response. Additionally, this strategy does not identify all reactivities in a patient, and unfortunately certain cancer histologies require additional measures to sufficiently enrich T-cell populations for effective neoantigen reactivity. Given the complexity and sometimes inefficient results of established TIL screening assays, there has been a push to identify T-cell responses against the most common shared activating oncogenic mutations in human cancers, which potentially could treat multiple patients with off-the-shelf therapies. Targeting these antigens has the additional advantage of avoiding antigen loss as a means of immune evasion, as these are essential to the malignancy.
Multiple neoepitope-reactive T-cell receptors (TCRs) have been successfully identified that recognize different mutated oncogenes by traditional TIL screening methods but this remains a hit-or-miss process. Several different strategies exist to determine whether a particular major histocompatibility complex (MHC)/mutation combination can create a neoepitope before T-cell isolation occurs. Epitope-prediction algorithms can be used to quickly predict the binding of mutated peptides to a diverse array of HLA alleles. One disadvantage to this approach is that these algorithms only predict binding of the peptide, not processing and presentation of the neoepitope. Additionally, the accuracy of the algorithms depend on how much data is available for a specific HLA restriction and which amino acids reside in the anchor residues. Mass spectrometry (MS) has emerged as a powerful tool that can assess the processing and presentation of precisely defined minimal epitopes at the cell surface. Recently, several neoepitopes have been identified by MS, showing the feasibility of this approach. Analyzing engineered monoallelic cell lines expressing a single HLA allele eliminates the need for in silico HLA-deconvolution, which is a considerable limitation and impediment in classic immunopeptidomic experiments.
Once a neoepitope and HLA molecule are known, neoepitope reactive TCRs need to be identified. Either a patient’s peripheral blood lymphocytes (PBL) can be stimulated, or HLA transgenic mice can be vaccinated before sorting using the appropriate minimal determinant tetramer in order to isolate these TCRs. Unmodified murine TCRs are highly active when inserted into human PBL, and these receptors have the theoretical advantage of not pairing with endogenous human TCR alpha and beta chains. TCR ‘mis-pairing’ can potentially generate hazardous new reactivities and often negatively affects expression at the cell surface. Multiple clinical protocols have demonstrated that human PBL modified with murine TCRs can mediate objective tumor regressions.
Here, we present an MS survey of 10 commonly occurring tumor-specific mutations presented by two high-frequency alleles in the US population: HLA-A*03:01 and HLA-A*11:01. We isolated naturally processed neoepitopes using MS and assessed their immunogenicity by identifying neoepitope reactive TCRs in HLA-transgenic mice. Using our pipeline, we identified four HLA-A*11:01 restricted neoepitopes and five HLA-A*03:01 restricted neoepitopes naturally processed and detected by MS. We were able to isolate TCRs reactive to all four HLA-A*11:01 neoepitopes and three HLA-A*03:01 neoepitopes. These off-the-shelf TCRs can potentially be used to conveniently and quickly engineer autologous peripheral blood lymphocytes for ACT clinical trials.
The 721.221 (American Type Culture Collection, RRID:CVCL_6263) and K562 (gifted from Paul Robbins, RRID:CVCL_0004) cell lines were confirmed to be HLA class-I negative by flow cytometric analysis (BD FACSCanto II) using pan-class-I antibody fluorescein isothiocyanate (FITC)-labeled W6/32 (BioLegend, RRID:AB_314873).
Confirmation of neoantigen reactivity of isolated TCRs: One week after transduction, transduced T-cells (5×10 4) were co-cultured with the appropriate target cells (5×10 4) for 24 hours. Target cell lines were first prepared by cognate HLA transduction. These HLA-monoallelic cell lines were transduced an additional time with a mutation-containing construct, as described in the supplemental materials and methods (online supplemental table 1) or pulsed with serially diluted peptides. For peptide titrations, both the mutant and matched wild-type peptides were used. The supernatants were harvested for human interferon gamma (IFN-γ) ELISA following the manufacturer’s protocol (DuoSet, R&D Systems). Experiments were performed in triplicate.
Other details and additional experimental procedures are provided in the online supplemental materials and methods.
The 721.221, a B-lymphoblastoid line, and K562, an erythroleukemia line, were selected for initial testing due to the lack of endogenous class I HLA expression. Both cell lines were retrovirally transduced with either HLA-A*03:01 or HLA-A*11:01, sorted, and evaluated to verify that both lines could maintain high levels of MHC expression in culture (online supplemental figure 1A). To test whether these cell lines could function as antigen presenting cells, KRAS G12V 7–16 and KRAS G12V 8–16 peptides were pulsed onto 721.221/A*11:01 and K562/A*11:01 cell lines as a positive control for a known neoepitope/HLA pair. Both lines pulsed with peptide were recognized by their respective murine TCR (online supplemental figure 1B,C). The 721.221 and K562/A*11:01 cell lines were subsequently transduced with the KRAS G12V minigene to assess endogenous processing and presentation of mutated epitopes from a retroviral minigene construct (online supplemental table 1). Both cell lines were recognized by the murine HLA-A*11:01 restricted anti-KRAS G12V TCR (online supplemental figure 1D). We repeated these preliminary experiments using our HLA-A*11:01 restricted KRAS G12D TCR, demonstrating that both cell lines are capable of processing and presenting multiple HLA-A*11-restricted neoepitopes (online supplemental figure 1E,F). Due to reports of differential epitope processing by the constitutive (standard) and immunoproteasome, we tested proteasomal subunit expression in each line. 721.221 expresses the immunoproteasome and constitutive proteasome while K562 expresses only the constitutive proteasome (online supplemental figure 1G). From these data we elected to move forward testing both cell lines to evaluate how differences in the proteasome affect processing and presentation of epitopes.
We developed an MS pipeline that could reliably and reproducibly identify neoepitopes presented on our cell lines (figure 1). We compared multiple reagents, conditions, procedures and software to optimize the recovery and identification of known HLA-A*11:01 restricted KRAS G12V and G12D epitopes (online supplemental figure 2A). KRAS G12V 8–16 (VVGAVGVGK), KRAS G12V 7–16 (VVVGAVGVGK), and KRAS G12D 7–16 (VVVGADGVGK) peptides were identified in all samples tested (online supplemental table 2 and figure 2B), consistent with the previously reported data for this epitope.
Immunoprecipitation and mass spectrometry strategy for isolating and identifying peptides. Schematic of the strategy used for isolating and identifying MHC class I peptides. (1) The 721.221 and K562 cells were stably transduced with either HLA-A*03:01 or HLA-A*11:01. Transduced cells were sorted based on their class I MHC expression, selecting for the cells expressing the most via the pan-class-I antibody W6/32. Sorted mono-allelic cell lines were stab
