The Mutation-Associated Neoantigen Functional Expansion of Specific T Cells (MANAFEST) Assay: A Sensitive Platform for Monitoring Antitumor Immunity

Abstract

Mutation-associated neoantigens (MANA) are a target of antitumor T-cell immunity. Sensitive, simple, and standardized assays are needed to assess the repertoire of functional MANA-specific T cells in oncology. Assays analyzing in vitro cytokine production such as ELISpot and intracellular cytokine staining have been useful but have limited sensitivity in assessing tumor-specific T-cell responses and do not analyze antigen-specific T-cell repertoires. The FEST (Functional Expansion of Specific T cells) assay described herein integrates T-cell receptor sequencing of short-term, peptide-stimulated cultures with a bioinformatic platform to identify antigen-specific clonotypic amplifications. This assay can be adapted for all types of antigens, including MANAs via tumor exome-guided prediction of MANAs. Following in vitro identification by the MANAFEST assay, the MANA-specific CDR3 sequence can be used as a molecular barcode to detect and monitor the dynamics of these clonotypes in blood, tumor, and normal tissue of patients receiving immunotherapy. MANAFEST is compatible with high-throughput routine clinical and lab practices. Cancer Immunol Res; 6(8); 888-99. ©2018 AACR.

Figure 1. IFNγ ELISpot underestimates the breadth of the antigen-specific T-cell response

T cells from healthy donor JH014 were stimulated with one of 13 known MHC class I-restricted epitopes (Supplementary Table S1) and cultured for 10 days. IFNγ ELISpot was performed in duplicate wells on an aliquot of cultured T cells (left) and TCR Vβ CDR3 sequencing was performed on the remaining T cells, T cells cultured without peptide, and uncultured T cells (right). ELISpot data are shown as the mean number of spot forming cells (SFC) per 10⁶ cells with background subtracted for 3 tested epitopes. Accompanying significant expansions (Fisher’s exact test with Benjamini-Hochberg correction for FDR, <0.05) of the 5 clonotypes with the highest abundance post-culture are also shown in response to these 3 epitopes, 4NP (A, green), 3A (B, blue), and EBV 1 (C, red). Each symbol represents a unique CDR3 clonotype. The full list of significantly expanded clones is shown in Supplementary Table S2. ELISpot background is the mean number of SFC detected without peptide stimulation in the ELISpot plus two standard deviations. TCR sequencing data are shown as the number of templates of each clone (abundance) that was detected in the relevant condition. TNTC, too numerous to count.

Figure 2. Validation of expanded clonotypes specific for 4NP

4NP pMHC⁺ T cells from donor JH014 were sorted by FACS prior to culture (A, left) or after a 10-day stimulation with the EBV EBNA 4NP epitope (B, left). TCR Vβ CDR3 sequencing was performed on the pMHC⁺ population and the Vβ gene segment usage was evaluated (A and B, right). The full list of clonotypes and their representation within the pMHC⁺ population are shown in Supplementary Table S3. Clonotypes identified in the pentamer-sorted population were compared with those found in the same peptide-stimulated 10-day culture. Outgrowth of these clones was detected in 5 separate experiments.

Figure 3. Validation of expanded clonotypes specific for 3A

3A pMHC⁺ T cells from donor JH014 were sorted by FACS prior to culture (A, left) or after a 10-day stimulation with the EBV EBNA 3A epitope (B, left). TCR Vβ CDR3 sequencing was performed on the pMHC⁺ population and the Vβ gene segment usage was evaluated (A and B, right). The full list of clonotypes and their representation within the pMHC⁺ population are shown in Supplementary Table S4. Clonotypes identified in the pentamer-sorted population were compared with those found in the same peptide-stimulated 10-day culture.

Figure 4. FEST assay sensitivity

Titrating numbers of T cells from healthy donors JH014 (red) and JH016 (blue) were stimulated with the A11-restricted 4NP and the A2-restricted flu M peptide epitopes (Supplementary Table S1), respectively, for 10 days. Clonotypes significantly expanded relative to the “no peptide” control were identified in each condition. (A) The number of unique clonotypes that were expanded, as well as (B) the total number of templates corresponding to these clonotypes, are shown for each titrating cell number. (C) The correlation between clonality and the percent of productive templates that were expanded after the 10-day culture is shown. Additionally, T cells from donor JH014 were stimulated for 10 days with titrating concentrations of the 4NP peptide epitope. Data are reported as (D) the number of unique expanded clonotypes detected at each concentration or (E) the number of templates detected for each of the four pMHC⁺-matched clonotypes (abundance).

Figure 5. The MANAFEST assay identifies multiple recognized MANAs and provides TCR barcodes to enable tracking of the antitumor immune response in tissue and peripheral blood

Recognition of candidate MANAs was evaluated by the MANAFEST assay in PBMC obtained at the time of surgical resection from JH124, a patient with NSCLC being treated with neoadjuvant anti-PD-1. (A) A heatmap generated by the FEST analysis platform shows all MANA/clone pairs to which significant antigen-specific expansion was detected, with expansions to MANA #7 outlined in black. (B) T-cell clonotypes specific for MANA #7 as determined by the FEST analysis platform are shown as the number of sequencing templates (cells) detected after culture (abundance). (C) The frequency (%) of each of these clonotypes among all templates detected via TCRseq is also shown in post-treatment fresh-frozen and FFPE tissue and in peripheral blood obtained >1 year after surgical resection. The parameters used for this analysis are shown in Supplementary Data 2. The FDR for the CASSLTGGYTGELFF clonotype was 5.63 × 10^–22 and the FDR for the CASNKLGYQPQHF and CASSLLENQPQHF clonotypes was 0.001. These 3 clonotypes were also identified as being specific for MANA #7 in an assay of T cells obtained from this patient 44 days after surgical resection (16).