Recent ground-breaking results from a randomized phase III clinical trial showed tumor-infiltrating lymphocyte (TIL)-therapy to be superior to ipilimumab in anti-PD-1 refractory advanced melanoma patients, paving the way for TIL-therapy to become a standard treatment (Ref 1). Responses to TIL-therapy are thought to be primarily mediated by expanded CD8+ TILs (CD8+ REP-TILs) via secretion of cytotoxic molecules, including granzymes (Gzms) of which five distinct types (A, B, H, K, and M) have been identified in humans. To better understand the role of Granzymes in TIL-therapy, we characterized the granzyme profile of CD8+ REP-TILs during tumor cell recognition.
Matched CD8+ REP-TILs and autologous tumor cell line (TCL) pairs were generated using fresh metastatic melanoma samples. TIL/TCL pairs with known high tumor-reactivity were selected and employed in in vitro co-culture systems reproducing TIL: tumor recognition. Gzm mRNA upregulation was assessed via bulk mRNA (n=10) and single-cell RNA sequencing (n=6) of expanded TILs, +8 hours post co-culture. Gzm protein secretion was quantified by ELISA and flow cytometry post co-culture (+2, +8, and +24 hours, n=11).
Bulk and single-cell RNAseq data analysis demonstrated distinct upregulation of only GzmB by CD8+ REP-TILs post TCL recognition. Protein levels were similarly dominated by GzmB, representing 61%, 73%, and 48% of total secreted Gzm at +2, +8 and +24 hours, respectively. In addition, GzmB showed by far the greatest post TCL recognition fold induction, up to 23.8-fold, whereas other Gzms were below 6.0-fold. Overall, GzmA was secreted at high levels, although the response was largely unspecific and only minimally induced by TCL recognition (up to 4.9-fold increase). All remaining granzymes were secreted and induced at minimal levels.
GzmB dominates the tumor-specific granzyme-related response of CD8+ REP-TILs, suggesting GzmB as the primary effector molecule in patients treated with TIL-therapy. GzmB secretion is therefore a potential candidate for a TIL-therapy potency biomarker. The potential of less inducible Gzms, such as GzmA, requires further clarification. Ref 1: Haanen et al. 2022 ESMO Congress (LBA3, Presidential Symposium I).
The authors.
Lundbeck Foundation (grants R286-2018-991, R307-2018-3636), The Danish Cancer Society (R309-A17896), Herlev and Gentofte Research Council (Clinician-Scientist grant to Marco Donia).
I. Svane: Financial Interests, Personal, Advisory Board: BMS, Pierre Fabre, Novartis; Financial Interests, Personal, Invited Speaker: MSD, Pierre Fabre, Novartis, Roche, BMS, MSD; Financial Interests, Personal, Stocks/Shares, Cofounder and Founder warrents: IO Biotech; Financial Interests, Institutional, Research Grant: Adaptimmune, Enara Bio, Lytix Biopharma, TILT Biotherapeutics; Financial Interests, Institutional, Funding: Evaxion; Non-Financial Interests, Personal, Principal Investigator: BMS, Roche, TILT Biotherapeutics, Lytix Biopharma, Novartis. M. Donia: Financial Interests, Personal, Invited Speaker, Teaching: Roche, Novartis; Non-Financial Interests, Personal, Other, Sub-investigator of clinical trial with connected translational research: Bristol Myers Squibb; Non-Financial Interests, Personal, Proprietary Information: Genentech, Bristol Myers Squibb; Financial Interests, Personal, Other, Advisor: Achilles Therapeutics; Other, Institutional, Other, Chairman of the Melanoma and Non-melanoma Skin Cancer Scientific Committee: Danish Medicines Council. All other authors have declared no conflicts of interest.