Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy

Cristina Puig-Saus 1 2 3 4, Barbara Sennino 5, Songming Peng 5, Clifford L Wang 5, Zheng Pan 5, Benjamin Yuen 5, Bhamini Purandare 5, Duo An 5, Boi B Quach 5, Diana Nguyen 5, Huiming Xia 6, Sameeha Jilani 7, Kevin Shao 5, Claire McHugh 5, John Greer 5, Phillip Peabody 5, Saparya Nayak 5, Jonathan Hoover 5, Sara Said 5, Kyle Jacoby 5, Olivier Dalmas 5, Susan P Foy 5, Andrew Conroy 5, Michael C Yi 5, Christine Shieh 5, William Lu 5, Katharine Heeringa 5, Yan Ma 5, Shahab Chizari 5, Melissa J Pilling 5, Marc Ting 5, Ramya Tunuguntla 5, Salemiz Sandoval 5, Robert Moot 5, Theresa Hunter 5, Sidi Zhao 6, Justin D Saco 7, Ivan Perez-Garcilazo 7, Egmidio Medina 7, Agustin Vega-Crespo 7, Ignacio Baselga-Carretero 7, Gabriel Abril-Rodriguez 7 8, Grace Cherry 7, Deborah J Wong 7, Jasreet Hundal 6, Bartosz Chmielowski 7 9, Daniel E Speiser 10, Michael T Bethune 5, Xiaoyan R Bao 5, Alena Gros 11, Obi L Griffith 6, Malachi Griffith 6, James R Heath 12, Alex Franzusoff 5, Stefanie J Mandl # 5, Antoni Ribas # 13 14 15 16
Source: Nature
Publication Date: (2023)
Issue: 8: 2023
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

One day after thaw, or immediately if used fresh, CD4+ and CD8+ T cells were isolated by positive selection using the CliniMACs Prodigy (Miltenyi Biotec) system or using magnetic LS columns and CD4 and CD8 microbeads (Miltenyi Biotec). Cells were then reseeded in culture medium at a density of 1.46 × 106 cells
per ml plus 12.5 ng ml-1 IL-7 (Miltenyi) plus 12.5 ng ml-1 IL-15 (Miltenyi) plus 1:17.5 ratio of TransACT T cell activation reagent (Miltenyi Biotec) by volume. Then, 2–3 days after activation, 80 µl of T cells at a concentration of 5–10 million cells per µl resuspended in P3 buffer (Lonza) were electroporated with 30 µg of neoTCR plasmid and 6.67 µl of pre-complexed RNPs as prepared above using Lonza X-unit in 100 µl cuvettes and program EO-115. Cells were expanded in culture medium supplemented with 12.5 ng ml-1 IL-7 plus 12.5 ng ml-1 IL-15 in 24-well G-rex (Wilson Wolf). Supplemented medium was exchanged every 2–3 days.


Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.