Combined TCR Repertoire Profiles and Blood Cell Phenotypes Predict Melanoma Patient Response to Personalized Neoantigen Therapy plus Anti-PD-1

Abstract

T cells use highly diverse receptors (TCRs) to identify tumor cells presenting neoantigens arising from genetic mutations and establish anti-tumor activity. Immunotherapy harnessing neoantigen-specific T cells to target tumors has emerged as a promising clinical approach. To assess whether a comprehensive peripheral mononuclear blood cell analysis predicts responses to a personalized neoantigen cancer vaccine combined with anti-PD-1 therapy, we characterize the TCR repertoires and T and B cell frequencies in 21 patients with metastatic melanoma who received this regimen. TCR-α/β-chain sequencing reveals that prolonged progression-free survival (PFS) is strongly associated with increased clonal baseline TCR repertoires and longitudinal repertoire stability. Furthermore, the frequencies of antigen-experienced T and B cells in the peripheral blood correlate with repertoire characteristics. Analysis of these baseline immune features enables prediction of PFS following treatment. This method offers a pragmatic clinical approach to assess patients' immune state and to direct therapeutic decision making.

Trial registration: ClinicalTrials.gov NCT02897765.

Keywords: NEO-PV-01; T cell clonality; TCR repertoire; anti-PD-1; cancer vaccines; flow cytometry; immunotherapy; melanoma; predictive biomarkers.

Conflict of interest statement

A.P., J.S., M.E.B., R.B., K.N.B., A.W., R.G.W., J.P., Z.S.K., M.S.R., and L.S. are/were all employees and/or equity holders at BioNTech US (formerly Neon Therapeutics, Inc.). P.A.O.: research funding paid to the institution: BMS, Merck, AstraZeneca, Celldex, CytomX, Glaxo Smith Kline, ARMO Biosciences; Neon Therapeutics, Consultant: Array, BMS, Merck, Genentech, Pfizer, Novartis, Neon Therapeutics, CytomX, Celldex. S.H.-L.: consultant to Amgen, BMS, Genmab, Xencor; Research support from BMS, Merck and Vaccinex. R.B.G.: Board of Directors, Alkermes plc and Infinity Pharmaceuticals and Scientific Advisory Board, Leap Therapeutics; stockholder and employee of BioNTech US (formerly Neon Therapeutics, Inc.).

© 2020 The Author(s).

Figures

Graphical abstract

Figure 1

Patients with Lack of Progression at 9 Months Have a Higher Peripheral TCR Repertoire Clonality prior to and throughout Study Treatment (A) Treatment schedule outline of the single-arm clinical trial of nivolumab plus personalized neoantigen vaccine (NEO-PV-01). Timings are indicated for nivolumab (blue arrow), personalized vaccine (NEO-PV-01, green arrow), and leukaphereses (orange dots). Leukaphereses from three time points (pre-treatment, pre-vaccine, and post-vaccine) are used for CD3+ T cell isolation for TCR-α/β sequencing and PBMCs for immunophenotyping by flow cytometry. (B) The proportion of clones belonging to each clone size category, averaged across patients (or HDs), and time points. An inset focusing on larger bins is provided (right). The legend defines frequency-based categories. (C) The (log–) fraction of clones belonging to the large (left) or hyperexpanded (right), for patients with and without PFS-9 at each time point or HDs. Boxplots indicate 25%, 50%, and 75% percentiles, and whiskers extend to the smallest/largest value within 1.5 times the interquartile range. p values are derived from a two-tailed Student’s t test. (D) The skewedness of the TCR-β repertoire frequency distribution measured by the Gini coefficient, DE50, and normalized Shannon’s entropy of each HD and patient across time points. The black line indicates median. p values are derived from a two-tailed Student’s t test. See also Figure S1 and Table S1.

Figure 2

Peripheral TCR Repertoires Are More Stable over Time in Patients Who Do Not Progress at 9 Months (A) Jensen-Shannon Divergence (JSD) of TCR-β CDR3 sequences accounting for the top 20% of the repertoire frequency between pre-V (left) or post-V (right) and baseline (pre-T) of patients with and without PFS-9. Low JSD values represent repertoire stability as indicated by the arrow (left). Black line indicates median. p values are derived from a two-tailed Student’s t test. (B) Schematic of a 3-way repertoire comparison. Segments in the Venn diagram represent the cumulative frequencies of the TCR-β CDR3 sequences detected in the intersecting time points in that segment. (C) Comparison of the cumulative frequencies of TCR-β CDR3 sequences between patients with and without PFS-9, at each time point. High cumulative frequency of G represents repertoire stability is as indicated by the arrow (left). Black line indicates median. p values are derived from a two-tailed Student’s t test. Venn diagram on the bottom left illustrate repertoire stability of patients with and without PFS-9. (D) The number of unique TCR-β CDR3 AA sequences detected at all three time points in patients with and without PFS-9. The black line indicates median. Black line indicates median. p value derived from a two-tailed Student’s t test. See also Figure S2.

Figure 3

Peripheral TCR Repertoires Stability and Diversity Are Correlated and Positively Correlate with Effector/Memory Phenotypes (A) The Gini coefficient of each patient versus the cumulative frequency of the G segment from each time points. Pearson's correlation coefficient (R) and the associated p value are indicated. (B) The percentage of positive effector-memory CD8+, memory CD4+ T cell, class-switched memory B cell, naive CD8+, naive CD4+ T cell, and naive B cell populations versus the cumulative frequency of the G segment (the persistent TCR-β clones). Pearson's correlation coefficient (R) and the associated p value are indicated. (C) The ratio of class-switched memory B cells to naive B cells versus the ratio of CD8+ effector-memory T cells to naive CD8+ T cells. Pearson's correlation coefficient (R) and the associated p value are indicated. See also Figure S3, Figure S4, and Table S2.

Figure 4

PCA of Baseline Peripheral TCR-β Repertoire Features and Immunophenotyping Separates Patients by PFS Status at 9 Months (A) First 2 components from a PCA of the aggregate peripheral measurements from the TCR-β repertoire and immunophenotyping. (B) The contributions (loadings) of the measured features to PC1. Color indicates source of data. (C) Kaplan-Meier curves for PFS of patients with PC1 >0 (teal) versus patients with PC1