Abstract 2995: Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma
Authors: Gibson Amber , Sukhumalchandra Pariya , Kerros Celine , Qiao Na , Philips Anne , Zhang Mao , Alkoutami Sami , Semelsberger Scott , Lu Yoshimi , Byers Lauren , Tian Ze , Shi Chunhua , Yan Jun , Zha Dongxing , Marinos Alejandro , Sergeeva Anne , He Hong , John Lisa St. , Molldrem Jeffrey J. , Alatrash Gheath 
Published: April 2023

Abstract
Background: Immunotherapy is the standard of care for many solid tumor malignancies. However, due to a paucity of known tumor antigens in non-small cell lung cancer (NSCLC) and osteosarcoma (OS), identification of novel immunogenic antigens is needed. Our group has experience with neutrophil serine proteases (NSP) derived from the azurophilic granules of polymorphonuclear leukocytes (PMN), specifically, cathepsin (CG), proteinase 3 (P3), and neutrophil elastase (NE). We have identified two HLA-A2 (HLA-A*0201)-restricted nonameric peptides: CG1 (FLLPTGAEA), derived from CG, and PR1 (VLQELNVTV), derived from NE and P3. We have previously shown that CG1 and PR1 are effective targets in hematological malignancies and that PR1 can be a target for some solid tumor malignancies. Here, we extend our findings to demonstrate that CG1 and PR1 can be immunotherapeutic targets for NSCLC and OS.
Methods: Healthy donor PMN were isolated from buffy coats using double Ficoll gradient centrifugation. To evaluate uptake, malignant cells were pulsed with PMN lysates, harvested, permeabilized, and stained with anti-CG, anti-NE or anti-P3. To evaluate cross-presentation, cells were cultured with PMN lysates, harvested and stained with CG1 TCR-like antibody, or PR1 TCR-like antibody (8F4). Flow cytometry was performed using the Fortessa X-20 Cell Analyzer. CG1- or PR1-specific cytotoxic T-lymphocyte (CTL) were expanded from healthy donor peripheral blood mononuclear cells. Standard cytotoxicity assays were used to evaluate the lysis of target cells co-cultured with CG1- or PR1-specific CTL.
Results: We have previously demonstrated that NE and P3 are taken up and cross-presented by NSCLC and become targets for PR1-CTL, thus in this study, we investigate if NSCLC cells take up CG, and whether OS cells take up NE and P3, resulting in enhanced cytotoxicity by peptide-specific CTLs. We first employed The Cancer Cell Line Encyclopedia to show the absence of NSP’s in NSCLC and OS cell lines. RT-PCR confirmed lack of endogenous CG mRNA expression in NSCLC and OS, and lack of NE and P3 in OS cell lines. Next, we analyzed antigen cross-presentation and showed that NSCLC cells take up and cross-present CG, leading a 13.3-fold increase in CG1-CTL killing of NSCLC cells pulsed with CG versus non-pulsed cells. In addition, OS cells take up and cross-present NE and P3, with a 13.1-fold increase in PR1-CTL killing of OS cells that were pulsed with NE and P3 vs. non-pulsed cells.
Conclusion: Our data define two novel HLA-A2 restricted peptides that can be targeted in NSCLC and OS. Specifically, CG1 and PR1 are presented on the surfaces of NSCLC and OS, increasing their susceptibility to killing by CG1 and PR1 specific CTL. Our work represents a novel link between innate and adaptive immune responses in solid tumors and identifies potential antigen targets for CTL, peptide vaccines, and TCR-like antibodies in the setting of NSCLC and OS.
Citation Format: Amber Gibson, Pariya Sukhumalchandra, Celine Kerros, Na Qiao, Anne Philips, Mao Zhang, Sami Alkoutami, Scott Semelsberger, Yoshimi Lu, Lauren Byers, Ze Tian, Chunhua Shi, Jun Yan, Dongxing Zha, Alejandro Marinos, Anne Sergeeva, Hong He, Lisa St. John, Jeffrey J. Molldrem, Gheath Alatrash. Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2995.