Neoantigen-specific CD8 T cell responses in the peripheral blood following PD-L1 blockade might predict therapy outcome in metastatic urothelial carcinoma

CD8+ T cell reactivity towards tumor mutation-derived neoantigens is widely believed to facilitate the antitumor immunity induced by immune checkpoint blockade (ICB). Here we show that broadening in the number of neoantigen-reactive CD8+ T cell (NART) populations between pre-treatment to 3-weeks post-treatment distinguishes patients with controlled disease compared to patients with progressive disease in metastatic urothelial carcinoma (mUC) treated with PD-L1-blockade. The longitudinal analysis of peripheral CD8+ T cell recognition of patient-specific neopeptide libraries consisting of DNA barcode-labelled pMHC multimers in a cohort of 24 patients from the clinical trial NCT02108652 also shows that peripheral NARTs derived from patients with disease control are characterised by a PD1+ Ki67+ effector phenotype and increased CD39 levels compared to bystander bulk- and virus-antigen reactive CD8+ T cells. The study provides insights into NART characteristics following ICB and suggests that early-stage NART expansion and activation are associated with response to ICB in patients with mUC.

Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All WES and RNAseq data is available upon application at dbGaP at https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001743.v1.p1. GRCh38 reference genome is available at https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.39. The source data presented in figures are provided as Source Data file. All other relevant data are available from the authors upon request.
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The number of patients was chosen based on availability of patient material and sufficient sequencing quality. HLA-matching healthy donor samples were included concurrently to as large extent as possible, 1-3 donors per screening.
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All patients were screened once with the patient-specific pMHC library. Detected T cell responses were evaluated by tetramer-pMHC staining to the extent possible (included in Supplementary Information), but not all detected T cell responses could be evaluated due to limited PBMC sample availability. Of 65 evaluated pMHC tetramers, 50 responses were verified, 9 borderline (combined 90%) and 6 could not be validated with this methodology. Randomization This is a correlative study to assess T cell reactivity in relation to therapeutic outcome. Randomization is not relevant to our study. All patients were screened with patient-specific neopeptideMHC library.

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The relevant institutional ethics committees approved the study and informed written consent was obtained in accordance with the Declaration of Helsinki. The Institution Review Board of Memorial Sloan Kettering Cancer Center approved a biospecimen protocol permitting tissue and blood collection, sequencing, and correlative studies for patients on this trial. All patients provided written informed consent to this protocol. All patients provided written informed consent to both the IMvigor 210 trial and an Institution Review Boardapproved biospecimen protocol permitting tissue and blood collection, sequencing, and correlative studies. We have included documentation to support this claim. Of note, all clinical and genomic data are publicly available for download by authorized investigators on dbGaP at http://view.ncbi.nlm.nih.gov/dbgap-controlled. HD samples were collected by approval of the local Scientific Ethics Committee for the Capital Region of Denmark, with donor written informed consent obtained according to the Declaration of Helsinki. HD blood samples were obtained from the blood bank at Rigshospitalet, Copenhagen, Denmark. All samples were obtained anonymously.
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Patients had mUC and were treated with atezolizumab 1200mg intravenously (IV) every 21 days at Memorial Sloan Kettering Cancer Center as part of the IMVigor210 trial (Rosenberg et al. 2016). Cross sectional imaging was performed every 9 weeks for the first 12 months following cycle 1 day 1 followed by every 12 weeks thereafter.

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Patient formalin-fixed paraffin-embedded (FFPE) tumor and PBMC samples were obtained and prepared as previously described (Snyder et al. 2017). Blood samples were drawn from patients prior to IV infusion on the day of treatment, pre-treatment and during treatment, and PBMCs were isolated and cryopreserved at -150°C in Human Serum Albumin (HSA)/10% DMSO until analysis.
Healthy donor blood samples were obtained from the blood bank at Rigshospitalet, Copenhagen, Denmark. All samples were obtained anonymously. PBMCs from HDs were obtained from whole blood by density centrifugation on Lymphoprep in Leucosep tubes and cryopreserved at -150°C in FCS + 10% DMSO.

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Cells were sorted on a BD FACSMelody, BD FACSAriaFusion or acquired on a BD LSR Fortessa.

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FlowJo software was used to gate and sort the population of interest Cell population abundance For every T cell population sorted the sorted cell fraction represented 0.5-25% of the total population.
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