An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8+ T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell This information is current as Lines of September 29, 2021. Pedro O. Flores-Villanueva, Malathesha Ganachari, Heinner Guio, Jaime A. Mejia and Julio Granados J Immunol 2018; 200:2965-2977; Prepublished online 19 March 2018; Downloaded from doi: 10.4049/jimmunol.1701054 http://www.jimmunol.org/content/200/8/2965 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/03/17/jimmunol.170105 Material 4.DCSupplemental References This article cites 29 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/200/8/2965.full#ref-list-1 Why The JI? Submit online. by guest on September 29, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology An Isolated TCR ab Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8+ T Cells To Kill and Secrete IFN-g in Response to Lung Adenocarcinoma Cell Lines Pedro O. Flores-Villanueva,* Malathesha Ganachari,† Heinner Guio,‡ Jaime A. Mejia,x,1 and Julio Granados{ Lung cancer is a leading cause of cancer-related death among both men and women in the United States, where non–small cell lung cancer accounts for ∼85% of lung cancer. Lung adenocarcinoma (ADC) is the major histologic subtype. The presence of actionable mutations prompts the use of therapies designed to specifically address the deleterious effects of those cancer-driving mutations; these therapies have already shown promise in cases carrying those actionable mutations (∼30%). Innovative ther- + apeutic approaches are needed for the treatment of 70% of patients suffering from lung ADC. Adoptive transfer of CD8 T cells Downloaded from specific against cancer/testis (CT) Ags, whose protein expression is restricted to the gonads (testis and ovary) and cancerous cells, is an excellent alternative. In this study, we report the isolation of HLA-A*02:01/CT37 peptide–specific a and b TCR chains from a CD8+ T cell clone obtained from a patient suffering from lung ADC. We also report the development of an innovative CD3z construct. With those TCR chains and the engineered (modified) CD3z chain, we produced a construct that when transduced into CD8+ T cells is capable of redirecting transduced CD8+ T cell cytotoxic activity and IFN-g secretion against peptide-pulsed autologous cells and HLA-A*02:01–positive and CT37-expressing lung ADC cell lines. Our findings will launch the development of http://www.jimmunol.org/ innovative adoptive transfer immunotherapies for the treatment of lung ADC, targeting the most prevalent HLA molecules and CT37 peptides restricted by these molecules. The Journal of Immunology, 2018, 200: 2965–2977. he presence of actionable genetic mutations in epithelial Although various lung ADCs express a diverse number of CT growth factor receptor (EGFR) and rearrangements in Ags, there is a need to carefully select those whose expression is T anaplastic lymphoma kinase prompt the prescription of truly restricted to cancerous and testis/ovary cells for development specific tyrosine kinase inhibitors (1, 3). Precision treatment ap- of adoptive transfer therapies. Following that precept, we selected proaches are not available for mutations in the BRAF, HER2/neu, the CT37 Ag. Moreover, we have identified a CT37-Ag peptide and KRAS genes (3). Moreover, 70% of patients do not carry any restricted by the MHC HLA-A*02:01 class I molecule and were by guest on September 29, 2021 of these actionable mutations. Thus, we must develop innovative able to pull from peripheral blood of a patient suffering from lung therapeutic approaches to address the needs of those patients. An ADC CT37-peptide/HLA-A*02:01–restricted CD8+ T cells. We option for those cases is the use of Ab blockers of checkpoint isolated a CD8+ T cell clone from the polyclonal population of inhibitors (4, 5). Treatment with these Abs has shown that these CD8+ T cells, molecularly cloned its a and b TCR chains, de- tumors express immunogenic Ags (6). Some of the most immu- veloped constructs, and efficiently transduced heterologous CD8+ nogenic molecules (aberrantly) expressed in several tumors, in- cells with these constructs, redirecting them to recognize CT37- cluding lung adenocarcinoma (ADC), are cancer/testis (CT) Ags peptide/HLA-A*02:01 complexes on the cell surface of autolo- (7–11). Hence, adoptive transfer immunotherapies targeting CT gous CD3-depleted PBMCs, and on HLA-A*02:01–positive lung Ags may cover the needs of that important percentage of patients ADC cell lines HCC2935 (carrying a wild-type TP53 gene), and suffering from non–small cell lung cancer (NSCLC). the NCI-H1993 and H522 (carrying a disabling mutation in the *Genomic Medicine and Immunotherapy, INBIOMEDIC USA, International Con- The sequences presented in this article have been submitted to the Uniprot database sortium for the Study of Lung Cancer, The Woodlands, TX 77384; †Department (http://www.uniprot.org/uniprot/Q8N0W7) under accession number Q8N0W7. of Biology and Biochemistry, University of Houston, Houston, TX 77004; x Address correspondence and reprint requests to Dr. Pedro O. Flores-Villanueva or Dr. Julio ‡INBIOMEDIC, Lima 14, Peru; Strategic Research and Development in Oncology, { Granados, Genomic Medicine and Immunotherapy, INBIOMEDIC USA, International Houston Methodist Research Institute, Houston, TX 77030; and Divisio´n de Inmu- Consortium for the Study of Lung Cancer, 110 E. Whistlers Bend Circle, The Woodlands, nogene´tica, Departamento de Trasplantes, Instituto Nacional de Ciencias Me´dicas y TX 77384 (P.O.F.-V.) or Divisio´n de Inmunogene´tica, Departamento de Trasplantes, Insti- Nutricio´n Salvador Zubira´n, C.P. 14080 Mexico City, Mexico tuto Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubira´n, Avenida Vasco de 1Current address: Merck Research Laboratories, North Wales, PA. Quiroga No. 15, Colonia Belisario Domı´nguez Seccio´n XVI, Delegacio´n Tlalpan, C.P. 14080 Ciudad de Me´xico, Me´xico (J.G.). E-mail addresses: poflores-villanueva@post. ORCIDs: 0000-0002-6184-4616 (P.O.F.-V.); 0000-0002-0701-1891 (M.G.); 0000- harvard.edu (P.O.F.-V.) or [email protected] (J.G.) 0002-1315-6878 (J.A.M.). The online version of this article contains supplemental material. Received for publication July 21, 2017. Accepted for publication February 1, 2018. Abbreviations used in this article: ADC, adenocarcinoma; CT, cancer/testis; EGFR, This work was supported by the Houston Methodist Research Institute, the Instituto epithelial growth factor receptor; FW, forward; hr, human recombinant; IHC, immu- Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubira´n, and INBIOMEDIC nohistochemistry; NSCLC, non–small cell lung cancer; RT, reverse transcriptase; RV, private funds. reverse; siRNA, small interfering RNA; SOE, splicing by overlap extension. P.O.F.-V., J.A.M., and J.G. conceived, designed, and supervised the study; P.O.F.-V. This article is distributed under The American Association of Immunologists, Inc., and H.G. recruited patients; P.O.F.-V., M.G., and H.G. developed methodologies, Reuse Terms and Conditions for Author Choice articles. executed the experiments, and acquired data; and P.O.F.-V. and J.G. wrote the article. All authors contributed to the analysis and interpretation of data, the administrative, Ó technical, and material support, and the review of the manuscript. Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701054 2966 AN ADOPTIVE TRANSFER PLATFORM FOR LUNG ADENOCARCINOMA TP53 gene). In addition, we developed a modified CD3z chain IHC analysis of lung tissues + capable of enhancing both CD8 T cell–mediated Ag-specific For IHC, lung tissue samples from the 10 lung ADC patients that underwent cytotoxic (CTL) activity and secretion of IFN-g. Our a and b surgery were fixed in paraformaldehyde and embedded in paraffin. Testis TCR/CD3z construct could be used to redirect CD8+ CTL to lyse samples were provided by the Department of Pathology at the Methodist CT37-expressing lung ADC tumor cells in an MHC class I– Hospital. For IHC, we used heat-induced epitope retrieval in citrate buffer specific-peptide restricted fashion. (Thermo Fisher Scientific, Waltham, MA), rabbit anti-human CT37 poly- clonal Ab HPA011284 (Sigma-Aldrich, St. Louis, MI), and a Polymer Detection System (Lab Vision Products; Thermo Fisher Scientific, Kala- Materials and Methods mazoo, MI). We immunostained 5 mm thick sections. Negative controls Selection of the CT Ag were sections incubated with irrelevant normal rabbit IgG (KPL, Gai- thersburg, MD). We used 13 automation buffer (pH 7.5) in all wash steps. Using the CT database (http://www.cta.lncc.br) we selected those CT Ags SuperMount permanent aqueous mounting media (BioGenex, San Ramon, with protein expressed only in testis/ovary and cancer cells, according to CA) was used for mounting immunostained tissue sections. Mounted the Human Protein Atlas database (http://www.proteinatlas.org) and sections were assessed at 4003 total magnification and 0.8 numerical CT37’s GeneCard proteomics information (http://www.genecards.org).