Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.thermofisher.com For Research Use Only. Not for use in diagnostic procedures. The content provided herein may relate to products that have not been officially released and is subject to change without notice. G Lowman 1 , T Looney 2 , E Linch 1 , L Miller 1 , D Topacio-Hall 1 , A Pankov 2 , J Zheng 2 , R Hartberg 3 , H Almåsbak 3 , T E Stav-Noraas 3 , A Kullmann 3 , F Hyland 2 , M Andersen 1 (1) Thermo Fisher Scientific, Carlsbad, CA, USA (2) Thermo Fisher Scientific, South San Francisco, CA, USA (3) Thermo Fisher Scientific, Oslo, Norway Insights into the tumor microenvironment and therapeutic T cell manufacture revealed by long amplicon immune repertoire sequencing ABSTRACT TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to cancer immunotherapy 1-4 . Here we describe a multiplex PCR-based TCRβ sequencing assay (Ion AmpliSeq TM Immune Repertoire Assay Plus – TCRβ) that leverages Ion AmpliSeq library construction chemistry and the long read capability of the Ion S5 530 TM chip to provide coverage of all three CDR domains of the human TCRβ chain. We demonstrate use of the assay to evaluate tumor-infiltrating T cell repertoire features and monitor manufacture of therapeutic T cells. CONCLUSIONS These results demonstrate: (1) The accuracy and versatility of immune repertoire sequencing using the Ion AmpliSeq TM Immune Repertoire Assay Plus – TCRβ, (2) The benefit of combining targeted gene expression and repertoire profiling for studies of the tumor microenvironment, (3) The utility of repertoire sequencing covering all CDR regions in monitoring the manufacture of therapeutic T cells. REFERENCES 1. Robins, H. S. et al. Blood 114:19 (2009) 2. Carlson, C. S. et al. Nat. Commun. 4, 2680 (2013) 3. Li, B. et al. Nature Genetics 48, 725–732 (2016) 4. Sheikh, N. et al. Cancer Res. 76:13 (2016) 5. Sandberg et al. Leukemia 21:2 (2007) 6. Liu, X. et al. PLOS One 11:3 (2016) 7. Thompson, J.R. et al. Nucleic Acids Res. 30:9 (2002) 8. Qiu, X. et al. App. and Env. Microbiology 67:2 (2001) 9. Wang, G. et al. App. and Env. Microbiology 63:12 (1997) 0.2 0.4 0.6 0.8 1.0 T cell evenness 1 = Most even sizes 0 = Least even sizes 19 NSCLC biopsies 3 4 5 1,000 10,000 100,000 1,000 10,000 100,000 Number of Input T cells Clones Detected Sequencing of Counted T cells Repressive Tumor Microenvironment Permissive Tumor Microenvironment Inhibits T cell responses to tumor Permits T cell expansion and anti-tumor activity High T cell evenness Low T cell evenness 0.6 0.7 0.8 0.9 1.0 T cell clone evenness during in vitro expansion via anti-CD3/CD28 beads Clone Evenness Donor 1 Donor 2 0.00001 0.0001 0.001 0.01 0.1 1 0.00001 0.0001 0.001 0.01 0.1 1 57 copies 566 copies 5,655 copies 56,552 copies Observed Plasmid Frequency 30 plasmid spike-in Detection of reference rearrangement spike-ins Plasmid Input (pg) Figure 1. Ion AmpliSeq Immune Repertoire Assay Plus – TCRβ Multiplex AmpliSeq primers target the framework region 1 (FR1) and constant (C) regions of the TCRβ producing a ~330bp amplicon which covers the entire variable gene and the CDR3 region. The assay utilizes RNA input from blood leukocytes, fresh-frozen tissue, or sorted T cells and has a flexible input range between 10ng and 1μg. INTRODUCTION To evaluate assay accuracy we sequenced libraries derived from 30 well-studied T cell lymphoma rearrangements 5,6 , then compared our results with those reported by another commercially available immune repertoire sequencing technology. We next used the assay to profile tumor infiltrating T cell repertoires for a cohort of 19 individuals with non-small cell lung cancer. We correlate repertoire features with gene expression profiling data. We then harnessed the long read capability of the assay to profile T cells at various stages of the therapeutic T cell manufacturing process. ASSAY ACCURACY Long read TCRβ sequencing of 30 reference T cell lymphoma rearrangements cloned into plasmids in a background of PBL yielded strong linearity in detection of clonal frequencies in reference spike-in experiments. We further demonstrate the quantitative nature of the assay by studying populations of counted T cells. N1 N2 FR1 FR2 Diversity(D) Joining (J) Constant Variable gene (V) CDR3 FR3 ~330bp Amplicon CDR1 CDR2 Figure 2. Detection of reference rearrangements Libraries were prepared using pools of 30 known lymphoma rearrangements at known input concentrations (calculated to equal ~50,000 to ~5 copies of RNA) in a background of 100ng of leukocyte RNA. We observe strong linearity across five orders of magnitude of control input concentration. Figure 3. Detection of counted T cells Libraries were prepared using RNA extracted from counted populations of T cells (1,000 – 100,000). The detected clone frequency is linear and in agreement with known T cell input. Importantly, there is not evidence of a large false positive rate for clone detection. To test the reproducibility of the assay between library replicates, we constructed 16 libraries from the peripheral blood leukocyte sample. Correlations between variable gene usage and Top 50 clone detection frequency yield minimum correlation values of r=0.97 for variable gene usage and r=0.96 for Top 50 clone detection frequency. Tumor biopsy revealed 589 unique TCR. • Oligoclonal repertoire with a small number of dominating clones; • Shannon diversity: 6.78 PBL revealed 45305 unique TCR. • Diverse, polyclonal repertoire with few highly expanded T cells • Shannon diversity: 13.95 RESULTS The Ion AmpliSeq Immune Repertoire Assay Plus TCRβ kit was applied to study the overlap between between circulating and tumor-infiltrating T cells. 100ng of total RNA derived from PBL and tumor biopsy from an individual with Stage 1B squamous cell carcinoma of lung was used as template. Sample Types: • Blood • Fresh Frozen Tissue • Sorted T cells Sequencing of matched TIL and peripheral blood exhibited differing repertoire features between samples. The tumor biopsy showed an oligoclonal repertoire with a small number of dominating clones and a Shannon diversity value of 6.78. The peripheral blood samples exhibited a diverse, polyclonal repertoire with few highly expanded T cells and a Shannon diversity value of 13.95. By correlating the repertoires, we observe 219 clones that are shared between samples. There are 370 clones that are unique to the tumor sample, with a subset of these clones which are highly expanded. This highly expanded set of clones unique to the tumor could potentially point to T cells responding to tumor-specific antigen. To further study the ability of immune repertoire sequencing to probe the tumor microenvironment (TME), we sought to correlate repertoire features with gene expression profiles derived from the Oncomine TM Immune Response Research Assay (OIRRA). In a repressive TME the T cell repertoire may exhibit high evenness (with few expanded clones) due to inhibition of T cell response. In a permissive TME the repertoire may exhibit low evenness (with T cell expansion) due to T cell response to the tumor. We sought to correlate these metrics with several immune response gene expression categories. Clone overlap for PBL and Tumor Log10 frequency in PBL Log10 frequency in tumor -8 0 -2 -4 -6 -8 -6 -4 -2 0 45086 clones unique to PBL 219 shared clones 370 clones unique to tumor Figure 6. Correlation of T cell evenness and gene expression profile (A) Schematic illustrating T cell response in repressive/permissive tumor microenvironments. (B-C) Libraries were prepared from 19 NSCLC biopsies using the Ion AmpliSeq TM Immune Repertoire Assay Plus – TCRβ and the Oncomine TM Immune Response Research Assay (OIRRA). The majority of this cohort exhibited high T cell evenness – suggesting repressive tumor microenvironments. Using a largest principle component analysis of each gene category within the OIRRA panel, we see correlation between evenness values and markers for myeloid response. (A) (B) Proliferation Drug_target Myeloid_marker,stem_cell Correlation -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Correlation with Oncomine ™ Immune Response Research Assay Gene categories (C) Figure 7. T cell evenness measurement during therapeutic T cell manufacturing process. T cell clone evenness is tracked for two separate donors from Day 0 (PBMC pre- & post-isolation), Day 3 (pre- and post-bead removal), and after Day 10 in culture. Importantly, T cell evenness increases over time, showing that there is no bias for expansion in particular T cell populations in culture using CTS TM Dynabeads TM anti- CD3/CD28 beads and CTS TM OpTimizer TM serum-free media. PBMC-derived T cells were subjected to in vitro expansion via CTS TM Dynabeads TM anti-CD3/CD28 beads and CTS TM OpTimizer TM serum-free media as part of a therapeutic T cell research study. T cell expansion was measured using T cell clone evenness output by Ion Reporter TM analysis of data generated by the Ion AmpliSeq Immune Repertoire Assay Plus – TCRβ panel. There is a consistent increase in evenness with cell culture time, suggesting that anti-CD3/CD28 beads and CTS™ OpTmizer™ media promote polyclonal (unbiased) T cell expansion. Table 2. Steps in the manufacture of therapeutic T cells. Explanation of the role of T cell repertoire sequencing in the steps involved in carrying T cells from isolation, through expansion, transduction, and introduction Figure 5. Overlap of clones identified in matched TIL and PBL samples Plot showing clones identified and unique to peripheral blood (45,086 - along x-axis), unique to TIL (370 - along y-axis), and found in both samples (219 - middle of plot). Figure 4. Correlation of variable gene usage and Top 50 clone frequency across 16 libraries. Plot showing correlation between 16 replicate libraries for (A) variable gene usage and (B) Top 50 clone detection frequency.