PMID- 34615703 OWN - NLM STAT- MEDLINE DCOM- 20220111 LR - 20220111 IS - 2051-1426 (Electronic) IS - 2051-1426 (Linking) VI - 9 IP - 10 DP - 2021 Oct TI - Immunostimulatory effects of targeted thorium-227 conjugates as single agent and in combination with anti-PD-L1 therapy. LID - 10.1136/jitc-2021-002387 [doi] LID - e002387 AB - BACKGROUND: Targeted thorium-227 conjugates (TTCs) are an emerging class of targeted alpha therapies (TATs). Their unique mode of action (MoA) is the induction of difficult-to-repair clustered DNA double-strand breaks. However, thus far, their effects on the immune system are largely unknown. Here, we investigated the immunostimulatory effects of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC) in vitro and in vivo in monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice. METHODS: The murine cell line MC38 was transfected with the human gene encoding for MSLN (hMSLN) to enable binding of the non-cross-reactive MSLN-TTC. The immunostimulatory effects of MSLN-TTC were studied in vitro on human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studied in vivo as monotherapy or in combination with anti-PD-L1 in MC38-hMSLN tumor-bearing immunocompetent C57BL/6 mice. Experiments were supported by RNA sequencing, flow cytometry, immunohistochemistry, mesoscale, and TaqMan PCR analyses to study the underlying immunostimulatory effects. In vivo depletion of CD8+ T cells and studies with Rag2/Il2Rg double knockout C57BL/6 mice were conducted to investigate the importance of immune cells to the efficacy of MSLN-TTC. RESULTS: MSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6, CCL20, CXCL10, and stimulator of interferon genes (STING)-related genes) in vitro as determined by RNASeq analysis. The results, including phospho-STING activation, were confirmed on the protein level. Danger-associated molecular pattern molecules were upregulated in parallel, leading to dendritic cell (DC) activation in vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, p<0.05) as a single agent in human MSLN-expressing MC38 tumor-bearing immunocompetent mice. Combining MSLN-TTC with anti-PD-L1 further enhanced the efficacy (T:C 0.08, p<0.001) as evidenced by the increased number of tumor-free surviving animals. MSLN-TTC monotherapy caused migration of CD103+ cDC1 DCs and infiltration of CD8+ T cells into tumors, which was enhanced on combination with anti-PD-L1. Intriguingly, CD8+ T-cell depletion decreased antitumor efficacy. CONCLUSIONS: These in vitro and in vivo data on MSLN-TTC demonstrate that the MoA of TTCs involves activation of the immune system. The findings are of relevance for other targeted radiotherapies and may guide clinical combination strategies. CI - (c) Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. FAU - Lejeune, Pascale AU - Lejeune P AD - Bayer AG, Berlin, Germany. FAU - Cruciani, Veronique AU - Cruciani V AD - Bayer AS, Oslo, Norway. FAU - Berg-Larsen, Axel AU - Berg-Larsen A AD - Bayer AS, Oslo, Norway. FAU - Schlicker, Andreas AU - Schlicker A AD - Bayer AG, Berlin, Germany. FAU - Mobergslien, Anne AU - Mobergslien A AD - Bayer AS, Oslo, Norway. FAU - Bartnitzky, Lisa AU - Bartnitzky L AD - Bayer AG, Berlin, Germany. FAU - Berndt, Sandra AU - Berndt S AD - Bayer AG, Berlin, Germany. FAU - Zitzmann-Kolbe, Sabine AU - Zitzmann-Kolbe S AD - Bayer AG, Berlin, Germany. FAU - Kamfenkel, Claudia AU - Kamfenkel C AD - Bayer AG, Berlin, Germany. FAU - Stargard, Stefan AU - Stargard S AD - Bayer AG, Berlin, Germany. FAU - Hammer, Stefanie AU - Hammer S AD - Bayer AG, Berlin, Germany. FAU - Jorgensen, Jennifer S AU - Jorgensen JS AD - Minerva Imaging ApS, Copenhagen, Denmark. FAU - Jackerott, Malene AU - Jackerott M AD - Minerva Imaging ApS, Copenhagen, Denmark. FAU - Nielsen, Carsten H AU - Nielsen CH AD - Minerva Imaging ApS, Copenhagen, Denmark. FAU - Schatz, Christoph A AU - Schatz CA AD - Bayer AG, Berlin, Germany. FAU - Hennekes, Hartwig AU - Hennekes H AD - Bayer AG, Berlin, Germany. FAU - Karlsson, Jenny AU - Karlsson J AD - Bayer AS, Oslo, Norway. FAU - Cuthbertson, Alan S AU - Cuthbertson AS AD - Bayer AS, Oslo, Norway. FAU - Mumberg, Dominik AU - Mumberg D AD - Bayer AG, Berlin, Germany. FAU - Hagemann, Urs B AU - Hagemann UB AD - Bayer AG, Berlin, Germany urs.hagemann@bayer.com. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - England TA - J Immunother Cancer JT - Journal for immunotherapy of cancer JID - 101620585 RN - 0 (B7-H1 Antigen) RN - 0 (Immunoconjugates) RN - 0 (Thorium-227) RN - 60YU5MIG9W (Thorium) SB - IM MH - Animals MH - B7-H1 Antigen/*antagonists & inhibitors MH - Gene Expression Profiling MH - Immunoconjugates/pharmacology/*therapeutic use MH - Immunotherapy MH - Mice MH - Thorium/pharmacology/*therapeutic use MH - Transfection MH - Xenograft Model Antitumor Assays PMC - PMC8496392 OTO - NOTNLM OT - gene expression profiling OT - immunotherapy OT - radiotherapy COIS- Competing interests: PL, VC, ABL, AS, AM, LB, SB, SZK, CK, SS, SH, CS, HH, JK, AC, DM, and UH are employees of Bayer AG or Bayer AS. AS, SB, SH, CS, HH, and DM are shareholders of Bayer AG. PL, SH, JK, AC, and UH hold patents on targeted thorium-227 conjugates. EDAT- 2021/10/08 06:00 MHDA- 2022/01/12 06:00 PMCR- 2021/10/06 CRDT- 2021/10/07 06:00 PHST- 2021/09/06 00:00 [accepted] PHST- 2021/10/07 06:00 [entrez] PHST- 2021/10/08 06:00 [pubmed] PHST- 2022/01/12 06:00 [medline] PHST- 2021/10/06 00:00 [pmc-release] AID - jitc-2021-002387 [pii] AID - 10.1136/jitc-2021-002387 [doi] PST - ppublish SO - J Immunother Cancer. 2021 Oct;9(10):e002387. doi: 10.1136/jitc-2021-002387.