An Isolated TCR βα 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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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). In aperture of the objective lenses. Image acquisition was performed using a addition, based on publicly available information, we selected those CT computerized analysis system comprising a BX41 microscope with a Ags reported to be expressed in NSCLC (7–11). An ideal CT Ag will have U-TVIX-2 and a U-CMAD3 tube and adapter attached for on-screen in silico–determined strong binding peptides (percentage rate ,0.1) re- viewing, a C3040 4.1 megapixel digital camera, and Magnafire-SP soft- stricted by the most frequent MHC class I molecules in several pop- ware (Olympus America, Central Valley, PA). ulations, including HLA-A*02:01, HLA-A*24:02, and at least one of the + following MHC class I molecules: HLA-A*03:01, HLA-A*11:01, HLA- Isolation of HLA-A*02:01/CT37 peptide–specific CD8 T cells

B*07:02, HLA-C*07:01 (allelefrequencies.net). Peptides and peptide Downloaded from Leukocytes were obtained from donor NSCLC_7 by leukapheresis. Half of specificities were determined using the NetMHCpan Server (http://www. ∼ 3 9 cbs.dtu.dk/services/NetMHCpan/). the bag ( 1 10 leukocytes per 100 ml) was used to isolate PBMCs by Ficoll-gradient centrifugation. Then, 3 3 108 CD8+ T cells were negatively Patients selected using the CD8+ T cell isolation Miltenyi Biotec kit, four large- capacity columns, and protocols furnished by Miltenyi Biotec. Next, A total of 43 patients were recruited, including 20 lung ADC patients, in 5 3 107 CD8+ T cells (the rest were frozen) were used to isolate peptide– 2010–2012. The rest were recruited in 2013. Patients provided informed Ag-specific cells by positive selection using biotinylated pentamers consent under protocols approved by the Institutional Review Board of the

(Proimmune), streptavidin microbeads, and one large-capacity column http://www.jimmunol.org/ Houston Methodist Research Institute in Houston (TX) and/or an Institu- (Miltenyi Biotec, Somerville, MA). We obtained 3 3 106 peptide-specific tional Review Board regulated by the Peruvian Ministry of Health. Selection CD8+ T cells. Then, 1 3 106 peptide-specific CD8+ T cells per well were criteria were patients suffering from lung ADC who had undergone surgery stimulated in vitro with 5 3 106 YLCSGSSYFV peptide-pulsed CD3+ and were under clinical observation; from this we had access to demographic cell–depleted autologous PMCs irradiated 3000 rad per well, in 24-well information, including age and gender, and clinical data such as tobacco Costar tissue-culture plates, and 2 ml final volume of media. We used smoking status, computerized tomography scanning information, histologi- Roswell Park Memorial Institute/RPMI 1640 medium (Life Technologies), cal classification, with available frozen tumor tissue suitable for CT37 supplemented with 10% heat-inactivated human AB serum, and 2 mM immunohistochemistry (IHC), mRNA, and genomic DNA extraction. We L-glutamine, 1 U/ml penicillin G, and 100 mg/ml streptomycin (Life selected nine HLA-A*02:01–positive patients, with their tumors express- Technologies). The CT37 peptide YLCSGSSYFV was used at 100 ng/ml ing CT37 mRNA. Four were freely willing to donate leukocytes for FACS final concentration. Human recombinant (hr) IL-2 (10 ng/ml; R&D Sys- analysis, and isolation and cloning of TCR a and b chains. tems, Minneapolis, MN) was added 72 h after peptide stimulation and the by guest on September 29, 2021 cultures left for 10 d (to let the cells rest). Then, 48 h after IL-2 stimulation HLA-A*02:01 genotyping and every 48 h thereafter, 100 ml of spent culture media was retrieved from We used the Micro SSP HLA-A*02 Allele Specific Trays (One L)and50ng each well and replaced by 100 ml of fresh complete media per well. After of genomic DNA, isolated from tissue or blood cell pellets, using DNeasy two cycles of stimulation the cells were harvested; live cells were obtained Blood and Tissue extraction kits (Qiagen, Valencia, CA). Available lung by Ficoll-gradient centrifugation and analyzed by FACS for quality con- 3 6 + ADC cell lines were also genotyped. trol. We harvested 4.5 10 peptide-specific CD8 T cells. Cultures were placed in a humidified tissue culture incubator, at 37˚C and 5% C02. The Real-time PCR assay highly purified YLCSGSSYFV and YYLCSGSSYF peptides were ob- tained from Creative Peptides (Shirley, NY). For the assessment of CT37 (FMR1NB/NY-SAR-35) expression, we used the 7500 Fast Real-Time PCR System and assay-on-demand primers for Cloning of HLA-A*02:01/CT37 peptide–specific CD8+ T cells

CT37 (ID Hs00896732_m1) and the housekeeping gene pyruvate dehy- + drogenase b (PDHB, ID Hs00168650) (Applied Biosystems, Carlsbad, HLA-A*02:01/CT37 peptide–specific CD8 T cells were cloned using the limiting dilution technique. We used a limiting dilution to dispense (ap- CA). To calculate the relative quantity of mRNA expression, we used the + 22DDcycle threshold method implemented in the software (Applied Bio- proximately) one HLA-A*02:01/CT37 peptide–specific CD8 T cell in systems). The data are presented as the fold change and/or log of fold 100 ml of media for every three wells, with each well already containing 2 3 5 change in gene expression normalized to the endogenous reference gene peptide YLCSGSSYFV-pulsed 5 10 CD3-depleted autologous PBMCs in PDHB. Total RNA was extracted from tumor tissues using RNeasy Fibrous 100 ml of media per well, in 96-well tissue culture plates from Costar. The Tissue Mini Kit RNA or from cells using Qiagen’s RNA isolation kit. final peptide concentration was 100 ng/ml. We added hrIL-2 (10 ng/ml; cDNA was obtained from 3 mg of total RNA using the High Capacity R&D Systems) 72 h after peptide stimulation. Cultures were placed in a cDNA Reverse Transcription Kit (Applied Biosystems). Approximately humidified tissue culture incubator for 10 d (to let the cells rest), at 37˚C and 100 ng of cDNA was used to determine the expression levels of these 5% C02. genes. The HCC2935, H1993, H1299, H522, H23, and H460 lung ADC Isolation and molecular cloning of CT37’s YLCSGSSYFV cell lines were tested as well. peptide–specific TCR a- and b-chain cDNAs EGFR AND KRAS cancer-driver mutation analysis Next, 1 3 106 CD8+ T cells specific to the YLCSGSSYFV peptide were Total RNA was reversed transcribed using an oligo(dT) primer and the Qiagen lysed using buffer RLT and total RNA extracted using Qiagen’s RNA Omniscript RT kit. The EGFR (exons 18–22) and KRAS (exons 2 and 3) were isolation kit. RNAs in RNase inhibitor (Promega, http://www.promega. PCR amplified using Platinum TAQ polymerase Supermix (Life Technologies, com) was added to the extracted total RNA. First-strand cDNA was pre- Carlsbad, CA) and primers: EGFR forward (FW) 59-TGAAGGCTGTC- pared from total RNA reverse-transcribed using the SMARTer RACE kit CAACGAATG-39 EGFR, reverse (RV) 59-AGGCGTTCTCCTTTCTCCAG-39, (TaKaRa Bio, Madison, WI). Primers specific for c-terminal Ca region or and KRAS FW 59-AGGCCTGCTGAAAATGACTG-39 and RV 59- Cb regions were used together with the SMARTScribe reverse transcrip- TGGTGAATATCTTCAAATGATTTAGT-39. The dye terminator cycle se- tase (RT) enzyme, capable of incorporating the SMARTer II-A oligonu- quencing method was used to detect gene mutations in a Beckman Coulter cleotide sequence in the 59 end of the first-strand cDNA during the RT GenomeLab GeXP Genetic Analysis System. All lung ADC cell lines were also process. This allows the amplification of TCR a-andb-chain cDNAs tested. without a priori knowledge of the V region sequences. RT primer The Journal of Immunology 2967 sequences were as follows: TCRa 59-GCT GGA CCT CAG CCG CAG CD3z/CD28/41BB/z-chain signaling domain PCR-added a Furin enzyme CGT CAT-39; TCRb159-TCA GAA ATC CTT TCT CTT GAC CCA-39; recognition site and a sequence encoding the P2A self-cleaving peptide. TCRb259-CTA GCC TCT GGA ATC CTT TCT CTT-39. We cloned a gel-purified PCR product in a TOPO XL PCR cloning vector The TCR a and b chains were amplified using the universal long 5‘ and proceeded to expansion and purification of vectors as explained above. PCR primer II-A and 39-specific reverse primers for Ca,orCb1, or Cb2 Construct 2 was transferred to the pBABEzeo amphotropic retroviral chains (three separate reactions), and the Platinum TAQ polymerase vector, cloned, and expanded in GigaSingles competent cells, and plasmids Supermix (Life Technologies). Then, a seminested PCR was performed containing the amplified sequences of interest were extracted from the using the universal short 59 PCR and each of the C-region PCR primers. bacteria using LPS-free Qiagen Plasmid Mini Kits, as explained above. The PCR products were then run in a 1.5% agarose/Tris–borate–EDTA The construct was sequenced using the primers described above for construct buffer gel and the DNA bands of interest gel purified using the QIAEX II 2, and primers: J_1: 59-AGG AGT AAG AGG AGC AGG CTC CTG-3, J_2: gel extraction kit (Qiagen), and cloned into the TOPO XL PCR cloning 59-AAC GGG GCA GAA AGA AAC TCC TGT-39,J_3:59-GGC CAC GTC vector. TOP10 competent cells were transfected with the vectors con- TCT TGT CCA AAA-39. We call this product construct 3. taining the PCR products by electroporation and cultured in Luria agar To engineer the CD3z and CD3/CD28/4-1BB/CD3z constructs we plates with 100 mg/ml ampicillin for the selection of clones. Four clones proceeded as follows: we first amplified the CD3z chain using primers: for each TCR chain were selected and expanded in Luria broth supple- CD3z FW: 59-ATG AAG TGG AAG GCG CTT TTC ACC-39 and CD3z mented with 100 mg/ml ampicillin. Plasmids containing the amplified RV1: 59-CGA CGA GGG GGC AGG GCC TGC ATG-39. To add the P2A sequences of interest were extracted from the bacteria using LPS-free peptide we used the following primers: FW P2A_1: 59-GGT GAC GTG Qiagen Plasmid Mini Kits and sequenced using M13 FW (–20) and GTT GTT AAT CCT GGT CCT ATG AAG TGG AAG GCG CTT TTC M13 RV primers. The primers used to engineer the constructs using ACC-39 (triplets coding GDVEENPGP peptide portion of P2A peptide in splicing by overlap extension (SOE) PCR are provided in the next section. black) or FW P2A_2: 59-GGC ACT AAT TTC TCG CTC CTC AAG CAA GGC GGT GAC GTG GTT GTT AAT CCT GGT CCT-39 (triplets TCR ab and CD3z construct engineering and sequencing coding ATNFSLLKQAGDVEENPGP peptide portion of P2A peptide in

black) and CD3z RV1: 59-CGA CGA GGG GGC AGG GCC TGC ATG- Downloaded from We engineered the constructs using the cloned TCR a- and b-chain cDNAs, 39, using the product of the first amplification in the second amplification. primers designed to add the T2A self-cleavage sequence preceded by a Furin enzyme cleavage-recognition site in between the 39 region of the The gel-purified final product was cloned in a TOPO XL vector and se- ab TCR a chain and the 59 region of the TCR b-chain, using the SOE PCR quenced. We then proceeded to remove the stop codon from the TCR a 9 method. construct with the following primers: FW1 TCR :5-ATG AAA TCC 9 b 9 The PCR products were run in a 1.5% agarose/Tris–borate–EDTA buffer TTG AGA GTT TTA-3 , RV1 TCR 1: 5 -GAA ATC CTT TCT CTT 9 gel; the DNA bands of interest were gel purified using the QIAEX II gel GAC CCA-3 and then added the overhang of the P2A-CD3 construct to the RV1 TCRb1 for the SOE PCR with the following primers: extraction kit (Qiagen) and cloned into TOPO XL PCR cloning vector. http://www.jimmunol.org/ FW1 TCRa:59-ATG AAA TCC TTG AGA GTT TTA-39 and RV1 TOP10-competent cells were transfected with the vectors and cultured in TCRb1_OVERHANG: 59-GAG GAG CGA GAA ATT AGT GCC GAA Luria agar plates with 100 mg/ml ampicillin for the selection of clones. ATC CTT TCT CTT GAC CCA-39. We then used the P2A-CD3 and the Four clones were expanded in Luria broth with 100 mg/ml ampicillin and TCRab P2A overhang AMPLICONS in an SOE PCR using the following sequenced. The plasmids amplified were extracted from the bacteria using primers: FW1 TCRa: 59-ATG AAA TCC TTG AGA GTT TTA-39 and LPS-free Qiagen Plasmid Mini Kits and sequenced using M13 FW (–20) CD3z RV1: 59-CGA CGA GGG GGC AGG GCC TGC ATG-39.We and M13 RV primers. The BamH1 restriction enzyme sequence in the 59 cloned the gel-purified product in a TOPO XL plasmid and PCR- region and Sal1 restriction enzyme sequence in the 39 region were PCR- sequenced the new construct. added to the construct for directional cloning in the pBABEzeo ampho- To engineer a CD3/CD28/4-1BB/z construct to be added to the TCRab tropic retroviral vector, which is derived from the Moloney Murine construct we used the following primers and amplifications: CD3z FW Leukemia Virus (Cell Biolabs, San Diego, CA). GigaSingles competent (extracellular and transmembrane domains): 59-ATG AAG TGG AAG by guest on September 29, 2021 cells (Thermo Fisher Scientific, http://www.fishersci.com) were transfected GCG CTT TTC ACC-39 and CD3z RV2: 59-CAG GAA CAA GGC AGT with the vectors containing the PCR constructs and cultured in Luria agar GAG AAT GAC-3’ or CD3z/CD28RV3: 59- CAG GAG CCT GCT CCT plates with 100 mg/ml ampicillin for selection of the clones. Five clones CTT ACT CCT CAG GAA CAA GGC AGT GAG AAT GAC -39 to add a were selected and expanded in Luria broth supplemented with 100 mg/ml CD28 signaling domain overhang to be used in an SOE PCR. The CD28 ampicillin. Plasmids containing the amplified sequences of interest were signaling domain was amplified using the following primers: CD28 FW1: extracted from the bacteria using LPS-free Qiagen Plasmid Mini Kits and 59-AGG AGT AAG AGG AGC AGG CTC CTG-39 and CD28 RV: 59- sequenced using the following primers: TCRa1: 59-ATG GCT TTG CAG GCG ATA GGC TGC GAA GTC GCG TGG-39, and then the primer CD3z AGC ACT CTG-39,TCRa2: 59-TCA GAA ATC CTT TCT CTT GAC-39, /CD28FW2: 59-GTC ATT CTC ACT GCC TTG TTC CTG AGG AGT Link1: 59-GCC ATG GTC AAG AGA AAG GAT-39,TCRb1_1: 59-ATG AAG AGG AGC AGG CTC CTG -39 to add a CD3z overhang. We linked CTG CTG CTT CTG CTG CTT CTG-39,TCRb1_2: 59-TCA GAA ATC the human CD3 z-chain extracellular and transmembrane domains and the CTT TCT CTT GAC CCA-39. Thus, we call this product construct 1. CD28 intracellular domain using SOE PCR and the following primers: After sequencing verification of the TCRab construct, we engineered CD3z FW: 59-ATG AAG TGG AAG GCG CTT TTC ACC-39 and CD28 two more constructs. We first amplified the complete CD3z chain from an RV: 59- GCG ATA GGC TGC GAA GTC GCG TGG-39. The 4-1BB oligo(dT) reverse-transcribed total RNA obtained from CD3-pulled cells, signaling domain was amplified using the following primers: 4-1BB FW: PCR-amplified the CD3z cDNA, and cloned the gel-purified PCR product 59-AAC GGG GCA GAA AGA AAC TCC TGT-39 and 4-1BB RV1: in a TOPO XL PCR cloning vector. We proceeded to the selection, ex- 59-CAG TTC ACA TCC TCC TTC TTC TTC TTT-39, and then an over- pansion, and sequencing verification of the clones as explained above. hang for the CD3z signaling domain was added using the primer: 4-1BB Then, we PCR-removed the stop codon in the 39 region of the TCR RV2: 59-CAG TTC ACA TCC TCC TTC TTC TTC TTT CTG CGC TCC b-chain in the TCRab construct and in between this region and the 59 TGCTGA ACT TCA CTC-39. The CD3z signaling domain was amplified region of the CD3z chain PCR-added a Furin enzyme recognition site and using the following primers: CD3z FW: 59-GAG TGA AGT TCA GCA a sequence encoding the P2A self-cleaving peptide. The construct 2 was GGA GCG CAG-39 and CD3z RV: 59-CGA CGA GGG GGC AGG GCC transferred to the pBABEzeo amphotropic retroviral vector, cloned and TGC ATG-39, and an overhang for 4-1BB added with primer: 4-1BB/CD3z expanded in GigaSingles competent cells, and plasmids containing the FW2:59- AAC GGG GCA GAA AGA AAC TCC TGT GAG TGA AGT amplified sequences of interest were extracted from the bacteria using TCA GCA GGA GCG CAG-39. To link the 4-1BB and CD3 z-chain sig- LPS-free Qiagen Plasmid Mini Kits, as explained above. The construct naling domains we used SOE PCR and the following primers: 4-1BB FW: was sequenced using the primers described above minus TCR b1_2 and 59-AAC GGG GCA GAA AGA AAC TCC TGT-39 and CD3z RV1: primers: TCRb1_3: 59-CTT CTC ACT AGG GGC GAT GTA-39, Link2: 59-CGA CGA GGG GGC AGG GCC TGC ATG-39. To produce the CD3 59-GGT GAA AAG CGC CTT CCA CTT-39, CD3_1: 59-ATG AAG TGG extracellular/transmembrane/CD28/4-1BB/ z construct we added the 4- AAG GCG CTT TTC ACC-3, CD3_2: 59-CGA CGA GGG GGC AGG 1BB overhang to the CD28 intracellular domain (first separate reaction): GCC TGC ATG-3. We call this product construct 2. After sequencing CD3z (extracellular and transmembrane domains) FW: 59-ATG AAG TGG verification of the CD3z chain, we PCR-engineered a third construct AAG GCG CTT TTC ACC-39 and RV JUNCTION: 59-ACA GGA GTT producing first a CD3z extracellular/transmembrane domain followed by a TCT TTC TGC CCC GTT GCG ATA GGC TGC GAA GTC GCG TGG- CD28/41BB/CD3z chain signaling domain, and cloned the PCR product in 39. To add the 4-1BB overhang to the CD3z intracellular domain (second a TOPO XL PCR cloning vector. We proceeded to selection, expansion, separate reaction), we used the primers: FW JUNCTION: 59-CCA CGC and sequencing verification of the clones as explained above. Then, we GAC TTC GCA GCC TAT CGC AAC GGG GCA GAA AGA AAC TCC PCR-removed the stop codon in the 39 region of the TCR b-chain in the TGT-39 and CD3z RV1: 59-CGA CGA GGG GGC AGG GCC TGC ATG- TCRab construct and in between this region and the 59 region of the 39. We then used an SOE PCR to join the amplicons of the first and second 2968 AN ADOPTIVE TRANSFER PLATFORM FOR LUNG ADENOCARCINOMA reaction using the following primers: CD3z FW: 59-ATG AAG TGG AAG proteins were eluted from the magnetic particles using the magnet and GCG CTT TTC ACC-39 and CD3z RV1: 59-CGA CGA GGG GGC AGG 30 ml SDS sample buffer (62.5 mM Tris-HCL, pH 6.8, 1% SDS, 10% GCC TGC ATG-39. We finally proceed to add overhangs (in black) for the glycerol). The eluted proteins were separated in 10% SDS-PAGE and P2A peptide using primers: FW P2A_1: 59-GGT GAC GTG GTT GTT transferred to a polyvinylidene difluoride membrane using the Bio-Rad AAT CCT GGT CCT ATG AAG TGG AAG GCG CTT TTC ACC-39 (for Trans-Blot system. Membranes were probed with 1:1000 HRP- the P2A portion GDVEENPGP in black) and then FW P2A_2: 59-GGC conjugated anti-biotin Ab ab1921 (Abcam, http://www.abcam.com), the ACT AAT TTC TCG CTC CTC AAG CAA GGC GGT GAC GTG GTT Immun-Star HRP kit, and chemiluminescence detected using a VersaDoc GTT AAT CCT GGT CCT-3 (for the P2A portion ATNFSLLKQAGD- Imaging system (Bio-Rad Laboratories, Hercules, CA). To determine the VEENPGP in black) with primer: CD3z RV1: 59-CGA CGA GGG GGC identity of CD3 chains, immunoprecipitated proteins obtained as indicated AGG GCC TGC ATG-39 in two sequential reactions. The gel-purified PCR above from cells transfected with construct 3 were blotted using rabbit product was cloned in a TOPO XL vector and the construct sequenced. We anti-CD3d EP4426, anti-CD3g 134684, anti-CD3ε 133628, and Armenian then proceeded to remove the stop codon from the TCRab construct with hamster anti-CD3z H136-968 Abs (Abcam), 1:2000 HRP-conjugated goat the following primers: FW1 TCRa:59-ATG AAA TCC TTG AGA GTT anti-rabbit IgG or 1:000 HPR-conjugated rabbit anti-Armenian hamster TTA-39 and RV1 TCRb1: 59-GAA ATC CTT TCT CTT GAC CCA-39, IgG (Abcam), and the Immun-Star HRP kit, and blots were revealed by and then added the overhang of the P2A-CD3/CD28/4-1BB/z construct to chemiluminescence using autoradiography Kodak BioMax Light film. the RV1 TCRb1 for the SOE PCR with the following primers: FW1 TCRa:59-ATG AAA TCC TTG AGA GTT TTA-39 and RV1 FACS analysis TCRb1_OVERHANG in black: 59-GAG GAG CGA GAA ATT AGT + CD8 T cells cultured as described above were harvested and washed twice GCC 9 GAA ATC CTT TCT CTT GAC CCA-3 . We then used the P2A- in cold incomplete RPMI 1640, then resuspended in 100 ml of cold in- z ab 21 CD3/CD28/4-1BB/ and the TCR P2A overhang AMPLICONS in an complete RPMI 1640 containing 10 mgml of human IgG (Sigma- a 9 ATG SOE PCR using the following primers: FW1 TCR :5- AAA TCC Aldrich), and incubated on ice for 15 min. Cells were washed once in 9 z 9 CGA 5 TTG AGA GTT TTA-3 and CD3 RV1: 5 - CGA GGG GGC AGG PBS+10% BSA (Sigma-Aldrich) and resuspended at 5 3 10 cells per 100 ml GCC TGC ATG-39. We cloned the gel-purified product in a TOPO XL Downloaded from of this buffer. FITC–anti-human CD8 mouse mAb HIT8a (BD Biosciences vector and sequenced the new construct. Pharmingen, bdbiosciences.com) and PE-labeled HLA-A*02:01/CT37 YLCSGSSYFV peptide pentamers or negative control PE-labeled HLA- Retroviral packing, purification, titer measuring, and A*02:01/NY-ESO-1 SLLMWITQV peptide pentamers (ProImmune) were + transfection of CD8 T cells added to the cells and incubated for 30 min on ice. Cells were then washed thoroughly and resuspended in cold PBS. Acquisition was done using the BD Amphotropic retroviral vector pBABEzeo containing constructs was LSR II instrument and software. We acquired 100 000 events. Data were transduced into PLAT-A cells (Cell Biolabs) with FUGENE 6 transfection analyzed using FlowJo 7.6.4 software. http://www.jimmunol.org/ reagent (Roche Applied Science) in Life Technologies Opti-MEM I reduced serum media. The transduced cells were grown in DMEM (Life Tech- [51Cr]-release cytotoxic assay nologies) containing Life Technologies’ 10% heat-inactivated FBS, 4 mM l-glutamine. The medium was replaced 16 h after transduction with fresh The ability of rested transduced CD8+ T cells (effector cells) to lyse HLA- medium containing 100 U/ml of penicillin and 100 mg/ml of streptomycin A*02:01 autologous CD3-depleted PBMCs pulsed with 100 mg/ml of (Life Technologies). After 24 h, virus particles in the media were collected CT37 YLCSGSSYFV peptide or control CT37 YYLCSGSSYF peptide, or by centrifugation overnight at 8000 rpm and 4˚C. The pellet was resus- the HLA-A*02:01-positive HCC2935, H1993, and H522, and HLA- pended in RPMI 1640 medium. Viral titer was determined using a A*02:01-negative H1299, H23, and H460 lung ADC cell lines (target QuickTiter retrovirus quantitation kit (Cell Biolabs). A total of 1 3 107 cells), was measured in [51Cr] release assays. Briefly, 1 3 106 target cells + 51 purified CD8 T cells from HLA-A*02:01 healthy donors of PBMCs were were labeled for 1 h at 37˚C with 200 mCi of Na2 CrO4 (Amersham, 3 3 activated with anti-CD3/CD28 Dynabeads at a ratio of three Dynabeads Arlington Heights, IL). Labeled target cells at 5 10 were incubated with by guest on September 29, 2021 per CD8+ T cell, in the presence of 5 3 107 CD3-depleted autologous effector cells at E:T ratios of 1:1, 4:1, and 16:1, for 4 h at 37˚C in 200 mlof PBMCs (irradiated 3000 rad). Activated CD8+ T cells were then trans- complete medium. Supernatants were harvested and counted using a 1470 duced with 5 3 1010 retrovirus particles using the ViraDucting Retrovirus wizard 30 automatic g counter (PerkinElmer, Gaithersburg, MD). Maxi- transduction kit and protocols provided (Cell Biolabs). Cultures were left mum and spontaneous [51Cr] release by each target was determined by for 10 d in six-well Costar tissue culture wells. After 72 h, the medium was incubating 5 3 103 labeled target cells in 2% SDS or medium, respec- replaced with fresh complete RPMI 1640 medium containing 10 ng/ml tively, for 4 h at 37˚C. Each point represented the average of triplicate hrIL-2 and 200 mg/ml Zeocin (InvivoGen, San Diego, CA). Then, spent wells, and percentage of specific lysis was calculated as follows: [(specific medium was replaced every 72 h with medium containing 200 mg/ml [51Cr] release 2 spontaneous [51Cr] release)/(maximum [51Cr] release 2 zeocin for the selection of stable cell clones. Live cells were then ob- spontaneous [51Cr] release)] 3 100. The highly purified and mass spec- tained by Ficoll-gradient centrifugation and restimulated in 24-well Costar trometry verified peptides YLCSGSSYFV and YYLCSGSSYF were ob- plates at 1 3 106 stable transduced cells per well with 5 3 106 CD3 cell– tained from Creative Peptides. Peptides were dissolved in DMSO and depleted autologous PBMCs irradiated 3000 rad and pulsed with 100 ng/ml diluted in incomplete RPMI 1640. We added the HLA-A2 mouse mAb of CT37 peptide YLCSGSSYF, in 2 ml of complete RPMI 1640 containing BB7.2 (Novus Biologicals) to the cultures to determine whether the lysis 100 U/ml of penicillin and 100 mg/ml of streptomycin. IL-2 (10 ng/ml) was activity was HLA-A*02:01 restricted at 1, 5, and 10 mg/ml. added 72 h later and 1 ml per well of spent media replaced every 72 h with fresh complete RPMI 1640 containing 100 U/ml of penicillin and 100 mg/ml IFN-g production and IFN-g ELISPOT assays of streptomycin. Cultures were left for 10 d. Similar cold experiments were set up simultaneously. Supernatants were harvested from E:T cocultures that were left for 4 h and tested for IFN-g Cell-surface protein isolation and Western blot analysis secretion using high-sensitivity ELISA kits (Thermo Fisher Scientific, Cell-surface proteins from stable retroviral transduced CD8+ T cells were http://www.thermofisher.com). ELISPOT assays were conducted using the g biotinylated and isolated for immunoprecipitation and Western blot anal- Human IFN- kit from Invitrogen (Thermo Fisher Scientific). Plates were ysis using the Thermo Fisher Scientific Pierce Cell Surface Protein Iso- conditioned according to protocols furnished by the manufacturer. In total, 8 3 104 transduced CD8+ T cells were stimulated with 3000 rad irradiated lation kit, anti-TCRb Ab clone 8A3 (Novus Biologicals, Littleton, CO) for 5 3 103 autologous CD3-depleted PBMCs pulsed with 100 mg/ml of CT37 the immunoprecipitation of the TCR complex, SDS-PAGE, transfer to membranes, and the Immun-Star HRP kit for detection of blots, respec- peptide YLCSGSSYFV or control CT37 peptide YYLCSGSSYF. Unsti- tively. Surface membrane proteins from 1 3 106 stable transduced cell mulated and PHA-stimulated cells were used as negative and positive m were biotinylated with EZ-Link sulfosuccinimidyl-2-(biotinamido) ethyl- controls, respectively. Cultures were placed in 100 l of complete RMPI 1, 3-dithioprpionate, a thiol-cleavage amine-reactive biotinylating reagent, 1640 per well in triplicate wells at 37˚C with 5% CO2 for 16 h. After decanting cells and medium from the wells, we proceeded according to the and then lysed with a mild detergent (M-PER; Thermo Fisher Scientific) in instructions provided. The plates were air dried, and the spots counted the presence of Halt Protease Inhibitor Cocktail. The biotinylated surface proteins were then isolated using Sera-Mag Neutravidin-Coated Magnetic using a stereomicroscope. Triplicate counts were averaged to give the final particles (GE Healthcare Bio-Sciences, Pittsburgh, PA) and resuspended in count. PBS with 0.01% Tween 20. The anti-TCRb Ab clone 8A3/Dynabeads- Gene expression silencing Protein G complex was added at 1:100 final concentration and the bio- tinylated proteins immunoprecipitated using the Immunoprecipitation Kit We used 30 pmol (final concentration) of each small interfering RNAs Dynabeads-Protein G. Biotinylated surface membrane TCR-complex (siRNAs) specifically designed to silence CD8A-chain or CT37, or negative The Journal of Immunology 2969 control siRNA (Thermo Fisher Scientific). This concentration of siRNAwas them, 12 expressed CT37 at the mRNA and protein level by IHC 6 used for 1 3 10 cells plated in six-well Costar tissue culture wells, using (35.29%). Thus, 21 out of 43 cases expressed CT37 mRNA and Lipofectamine RNAiMAX reagent, following protocols furnished with the were positive by IHC (∼48.83% of 43 cases) (data not shown). reagent (Thermo Fisher Scientific). To silence the CD8A chain, we tar- geted positions 1653 in exon 5 and position 1906 in exon 9. To silence From those 21, 5 expressed moderate levels (23.8%) and 16 low CT37, we targeted position 796 in exon 5. We used FACS analysis or levels (76.2%) of CT37 by IHC. However, we understand that a immunoprecipitation to assess the efficiency of silencing protocols. larger sample size is needed to address the issue more clearly. Statistical analysis Patient NSCLC_7, who was willing to donate leukocytes, had tumor cells expressing moderate levels of the CT37 Ag by IHC. The equality of more than two means or the equality of two means was CT37 expression was clearly observed in the cytoplasm and cell tested using the Kruskal–Wallis test or the Wilcoxon rank-sum (Mann– ∼ Whitney) test, respectively. Means, SDs, and results were calculated using membrane of malignant cells (Fig. 1A). In addition, 8% of their + Stata 11 software. circulating CD8 T cells were carrying a TCR specific for HLA- A*02:01/CT37 peptide YLCSGSSYFV, as determined by FACS Results analysis (Fig. 1B). This patient was a former smoker of more than We identified a CT Ag with peptide sequences restricted by one pack per d of tobacco cigarettes (20 cigarettes/pack), with a three of the most frequent MHC class I alleles across computerized tomography scan showing a solid solitary ground- populations of different ethnicities glass nodule of 2.8 cm located in the upper right pulmonary lobe of the lung. This patient had a stage 1 disease (T1bN0M0), with Three CT Ags, CT37, VCX3a, and XAGE1b, were selected as lung ADC carrying an EGFR driver mutation in exon 21 (L858R,

having protein expression restricted to testis/ovary and expressed in Downloaded from Table II). This patient remains cancer free 6 y after lobectomy (at NSCLC (http://www.proteinatlas.org, GeneCards, 7-11). CT37 the article submission date). Two more patients, NSCLC_5 and was the only one with strong binding peptides restricted by the most NSCLC_9, were also willing to donate leukocytes, had tumor cells frequent MHC class I molecules in several populations including expressing low levels of CT37 Ag by IHC analysis, and had ∼3 HLA-A*02:01, HLA-A*24:02,andHLA-B*07:02 (Table I, allele- and 4% circulating CD8+ T cells, respectively, but were within 5 y frequencies.net). Peptide specificities were determined using the of follow-up of surgery. NetMHCpan Server from the Technical University of Denmark Because weak IHC staining in healthy kidney and urinary http://www.jimmunol.org/ (http://www.cbs.dtu.dk/services/NetMHCpan/). We selected peptides bladder tissues is mentioned in the Human Protein Atlas using one with a percentage binding rate cut-off (percentage rate) ,0.1 to in- of two Abs, in this case using the rabbit anti-human CT37 poly- crease the likelihood of a peptide binding the selected MHC class I clonal IgG purified Ab HPA011284 (http://www.proteinatlas.org), molecules in vivo. We selected the HLA-A*02:01–restricted peptide we double-checked this in de novo processed frozen tissues from YLCSGSSYFV because it has a core amino acid sequence like that five randomly selected necropsy samples, five kidneys, and five of peptide YYLCSGSSYF. This later peptide YYLCSGSSYF has a urinary bladders. We observed only background staining like that percentage rate ,0.1 for the HLA-A*24:02, but not for HLA-A*02:01 in negative controls exposed to rabbit polyclonal isotype Ab (Table I). Thus, we decided to use those peptides for our studies be- control (Supplemental Fig. 1).

cause one is an excellent control for the other. by guest on September 29, 2021 We identified a patient carrying a high percentage of We molecularly cloned HLA-A*02:01/CT37 peptide–specific HLA-A*02:01/CT37 peptide–specific CD8+ T cells TCR a- and b-chains from a CD8+ T cell clone Patients recruited were of Hispanic ancestry. From the case review, Given that the patient NSCLC_7 was freely willing to donate 43 cases met the criteria and 9 were carriers of HLA-A*02:01 leukocytes, we decided to isolate their Ag-specific CD8+ T cells. alleles and expressed CT37 at the mRNA level (Table II). Four of After two in vitro stimulations we obtained a 98% pure population those nine cases expressed CT37 by IHC (∼44%) at the levels of polyclonal CD8+ T cells expressing a TCR restricted by HLA- indicated (Table II). In total, 34 were HLA-A*02:01 negative; of A*02:01 MHC class I/CT37 peptide YLCSGSSYFV complexes

Table I. CT37 Ag peptides selected according to in silico–determined HLA specificity and affinity

HLA Peptide CT37 Position Affinity (nM) Percentage Rank Binding Level Selection Criteria HLA-A*02:01 MLSIWILLFV 73 21.6 0.30 SB No HLA-A*02:01 FVCYYLSYYL 81 32.6 0.50 SB No HLA-A*02:01 YLCSGSSYFV 89 8.5 0.09 SB Yes HLA-A*02:01 MMQMFGLGAI 184 35.2 0.50 SB No HLA-A*02:01 MLMLSIWIL 71 7.0 0.07 SB Yes HLA-A*02:01 LMLSIWILL 72 21.9 0.30 SB No HLA-A*02:01 ALLNFFFPT 124 9.8 0.12 SB No HLA-A*02:01 ILLFVCYYL 78 14.0 0.17 SB No HLA-A*02:01 KQMMQMFGL 182 19.8 0.30 SB No HLA-A*02:01 SLFWRSEPA 206 22.7 0.30 SB No HLA-A*24:02 SYYLCSGSSYF 87 120.8 0.25 SB No HLA-A*24:02 YYLCSGSSYF 88 19.6 0.03 SB Yes HLA-A*24:02 YYLSYYLCSGSSYF 84 54.4 0.12 SB No HLA-B*07:02 DRPQPGWRESL 51 119.9 0.40 SB No HLA-B*07:02 RAMRVAHLEL 17 148.7 0.50 SB No HLA-B*07:02 RPQPGWRESL 52 7.1 0.01 SB Yes HLA-B*07:02 KPFGMLMLSI 67 131.0 0.40 SB No HLA-B*07:02 LPIYCRSLF 200 42.4 0.17 SB No The CT37 protein sequence with accession number Q8N0W7 was entered into the NetMHCpan server version 3.0 from the Technical University of Denmark (http://www. uniprot.org/uniprot/Q8N0W7). We inquired for peptide lengths of 8, 9, 10, 11, and 12 aa, setting the rank threshold for selection of strong binding peptides (SB) to 0.50. From the output, we selected those peptides with rank threshold ,0.10 (indicated in bold). 2970 AN ADOPTIVE TRANSFER PLATFORM FOR LUNG ADENOCARCINOMA

(Supplemental Fig. 2A). Using the limiting dilution method, we were able to isolate two cell clones from 300 HLA-A*02:01/CT37 YLCSGSSYFV peptide-specific polyclonal CD8+ T cells. One clone was expanded, reaching a final number of 1.6 3 106.We

CT37 IHC amplified, molecularly cloned, expanded, and sequenced the cDNA encoding the TCR a- and b-chains from that CD8+ T cell clone. All plasmids sequenced had the same TCR a or the same 2 TCR b-chains cDNA sequences and they were in frame. The TCR a- and TCR b-chains were 813 and 909 nucleotides long 6.11 Negative 5.93 Negative 6.10 Low (+) 5.97 Low (+) CT37 (Supplemental Fig. 2B), and their deduced V and J rearrangements RT-PCR Log were TRAV2S1_AJ24S1 and TRBV22S1_BJ2S1, respectively, indicating monoclonality (Supplemental Fig. 2C, 2D). 50% of neoplastic cells staining (high staining). . , We produced three constructs and successfully transferred them to and expressed them in allogeneic purified CD8+ T cells 111 E746-A750 E746-A750 The features of the constructs produced are depicted in Fig. 2A. A D D

A; G12D typical retroviral transduction efficiency of 60% is shown in the A; G12AD exon 21 L858R 6.0 Negative exon 21 L858R 6.27 Moderate (++) exon 21 L858R 5.88 Negative exon 19 in-frame exon 19 in-frame .

. Supplemental Fig. 2. In vitro expansion of stable transduced cells exon 2 codon 12 35G exon 2 codon 12 35G

+ Downloaded from or EGFR mutations) yielded 93% pure CD8 T cells (Supplemental Fig. 2E). Using FACS EGFR EGFR EGFR EGFR EGFR Deletion of Deletion of analysis we observed high expression of our TCRab constructs on Molecular Classification (KRAS KRAS KRAS transduced CD8+ T cells (Fig. 2B). The expression of TCRab con- structs was higher for constructs 2 and 3, carrying the CD3z chain or the engineered CD3z chain (Fig. 2B). We immunoprecipitated the TCRab complexes from transduced CD8+ T cells whose membrane

proteins were biotinylated, and observed that all TCRab constructs http://www.jimmunol.org/ coprecipitated with CD3d,CD3ε,CD3g,andCD3z chains, indicating that the engineering strategy used did not hamper the ability of these chains to polymerize in vivo (Fig. 2C). Western blot analysis with 3.2 T2aN0M0 1.6 T1aN0M0 specific Abs corroborated the identity of those CD3 chains (Fig. 2D). 5 and 1.5 T3N1M0 , 10–50% of neoplastic cells staining (moderate staining); 2.8, 1, and 0.8 T4N2M0 Tumor Size (cm) Stage TCR-transduced CD8+ T cell lysis autologous CD3 cell– 11 depleted PBMCs in an HLA-A*02:01/CT37 peptide YLCSGSSYFV with high functional avidity

To determine the functional avidity of the TCR cloned for HLA- by guest on September 29, 2021 A*02:01/CT37 peptide YLCSGSSYF complexes, we first calculated the peptide concentration needed to obtain 50% of maximal lysis nodes (EC50) using a serial dilution of the peptide YLCSGSSYFV (Fig. 3A, lymph nodes

compromised 51 portion 0.4 cm Scan Appearance left) and the [ Cr]-release cytotoxic assay as explained in Materials

in the same lobe with and Methods. We then proceeded to extrapolate the HLA-A*02:01– Computerized Tomography ipsilateral subcarinal lymph metastasis to ipsilateral hilar lobe; the larger invasive to the main carina with metastasis to visceral pleura was minimally specific peptide dissociation constant value (Koff), using existing plots + of EC50 values obtained from the interactions of human CD8 T cell clones specific for HLA-A*02:01/NY-ESO-1 SLLMWITQC peptide or the antimelanoma CD8+ T cell clone recognizing HLA-A*02:01/ 10% of neoplastic cells staining (low staining);

, MART-1

, Melan-A ELAGIGILTV peptide versus monomeric TCR/ 1 Histology HLA-A*02:01 peptide complexes Koff values obtained from disso- ciation kinetic measurements (12). Finding the Koff iskeytodeter- mining functional avidity because a lower Koff is correlated with

, no staining; higher functional avidity for HLA-A*02:01–specific peptide (higher 2 activation and calcium mobilization) (12). 1 pack/d Favoring ADC Three solid GGN in the same 1 pack/d ADC Solitary part-solid GGN; solid 1 pack/d ADC Solitary solid GGN 2 T1aN0M0 1 pack/d ADC Solitary solid GGN 2.8 T1bN0M0 1 pack/d1 pack/d ADC ADC Solitary solid GGN Solitary solid GGN 3 2.2 T1bN0M0 T1aN0M0 Not found 5.81 Negative 1 pack/d1 pack/d Favoring ADC ADC Presence of two solid nodules Solitary solid GGN 1.6 T1aN0M0 Not found 6.15 Low (+) 1 pack/d ADC Solitary solid GGN; the . , , , . , , . , To experimentally obtain EC50 values, we used a 16:1 E:T ratio of CD8+ T cells isolated from six HLA-A*02:01–positive healthy donors, which were transduced with construct 1, and their autol-

signs was used. ogous CD3-cells depleted PBMCs pulsed with indicated concen- 2

or trations of the CT37 YYLCSGSSYF peptide (Fig. 3A, left). The

1 mean values obtained from the six experiments were used to calculate the EC50. The EC50 was extrapolated using a plot of mean percentage specific lysis versus peptide concentration used (Fig. 3A, right). To find the EC50 we used y = 49.85. We converted nanograms per milliliter into moles per liter based on the m.w. of CT37 peptide YLCSGSSYF (m.w. = 1125.28). The extrapolated 212 EC50 value was 0.005 ng/ml (4.44 3 10 mol/l). The Koff values 21 21 extrapolated for this EC50 are ,1 3 10 s , placing our TCR Identifier Age (y) Gender Smoking NSCLC_2 69 Male Current NSCLC_1 64 Female Current NSCLC_7 58 Male Former NSCLC_8NSCLC_9 63 59 Female Female Former Former NSCLC_5NSCLC_6 70 68 Male Male Former Former NSCLC_4 61 Male Current NSCLC_3 56 Male Former

A semiquantitative scoring with among those elite clones exhibiting the lowest Koff values and Table II. Patients included: characteristics GGN, ground-glass nodule; KRAS, K-Ras protein 21 (p21)highest oncogene. functional avidity (12). The Journal of Immunology 2971

FIGURE 1. Clinical laboratory features con- sidered for selection of patient NSCLC_7. IHC of NSCLC_7 patient’s tumor sample showing CT37 Ag staining (A); FACS analysis of PBMCs obtained from patient NSCLC_7; upper section, HLA-A*02:01/CT37 YLCSGSSYFV peptide pentamer; lower section, the control HLA-A*02:01/NY-ESO-1 SLLMWITQV pep- tide pentamer (B). Procedures and reagents are Downloaded from explained in the Materials and Methods. http://www.jimmunol.org/

Given the fact that TCRs exhibiting such dissociation constants can Construct-transduced CD8+ T cells produced IFN-g when stably interact with HLA-specific peptide complexes in the absence of stimulated with autologous CD3 cell–depleted PBMCs in an by guest on September 29, 2021 CD8 complexes, we silenced the expression of the CD8A-chain in an HLA-A*02:01/CT37 peptide YLCSGSSYFV–restricted fashion + aliquot of our CD8 cell clone NSCLC_7 (from which we isolated Both the levels of IFN-g secretion and the frequency of IFN-g– our TCR). We used FACS analysis to detect any change in the per- secreting CD8+ T cells were significantly higher in CD8+ T cells centage of cells stained with HLA-A*02:01/CT37 YYLCSGSSYF transduced with constructs 2 or 3, as compared with CD8+ peptide complexes (Fig. 3B). We did not observe a significant change T cells transduced with construct 1, when exposed to autologous in the percentage of cells stained with HLA-A*02:01/CT37 cells pulsed with CT37 peptide YLCSGSSYFV (Fig. 4C, 4D), YYLCSGSSYF peptide complexes between CD8A silenced and + + with the highest observed for CD8 T cells transduced with control unsilenced NSCLC_7 CD8 T cell clone (Fig. 3B). Thus, we construct 3. We could not detect IFN-g secretion or frequency of concluded that our cloned TCR exhibit a high functional avidity for IFN-g–secreting CD8+ T cells higher than background in cultures HLA-A*02:01/CT37 YYLCSGSSYF peptide complexes. where PBMCs were pulsed with control CT37 YYLCSGSSYF Construct-transduced CD8+ T cells lysed autologous CD3 peptide (data not shown). Notably, we also observed in silico cell–depleted PBMCs in a highly specific HLA-A*02:01/CT37 that both the CT37 HLA-A*02:01 peptide YLCSGSSYFV and peptide YLCSGSSYFV–restricted fashion the CT37 HLA-A*24:02 peptide YYLCSGSSYF are predicted to be produced by the proteasome (Supplemental Fig. 4). Non- To determine whether expressed TCRab constructs were func- transduced CD8+ T cells did not secrete IFN-g when exposed to tional and highly restricted by the appropriate HLA/peptide target cells pulsed with appropriate peptide. HLA-A*02:01 re- complexes, we tested whether transduced CD8+ T cells were striction was evident in cultures where PBMCs were pulsed able to lyse HLA-A*02:01 autologous cells pulsed with CT37 with CT37 YLCSGSSYFV peptide but processed in the pres- peptide YLCSGSSYFV or control peptide using [51Cr] release ence of an anti–HLA-A02 Ab. The anti–HLA-A02 Ab blocked assays. We observed that CD8+ T cells transduced with all our IFN-g secretion in a dose-dependent manner (Kruskal–Wallis tests constructs were able to lyse autologous targets pulsed with CT37 for equality of IFN-g secretion levels in cultures containing 0, 1, 5, peptide YLCSGSSYFV, but not those pulsed with control CT37- or 10 mg/ml of anti–HLA-A02 Ab: p=0.002for construct 1, YYLCSGSSYF peptide (Fig. 4A). At E:T ratios of 4:1 and 16:1, p = 0.001 for construct 2, and p = 0.001 for construct 3; Fig. 4C). the lysis activities of CD8+ T cells transduced with constructs 2 and 3 were significantly higher than those of cells transduced with + construct 1, with the highest being that of the CD8+ T cells Construct-transduced CD8 T cell lysed lung ADC cell lines transduced with construct 3 (Fig. 4A). Blocking the HLA- expressing the CT37 Ag in an HLA-A*02:01–restricted fashion A*02:01 molecule with anti–HLA-A2 Ab abrogated the lysis We next examined whether CD8+ T cells transduced with our activity of CD8+ T cells transduced with construct 1, 2, and 3 in a constructs will exhibit the same functions when exposed to lung dose-dependent manner (Fig. 4B). ADC cell lines. We genotyped available (American Type Culture 2972 AN ADOPTIVE TRANSFER PLATFORM FOR LUNG ADENOCARCINOMA Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Constructs features (A), levels of TCRab-chain expression in CD8+ T cells transduced with the three different constructs (B), polymerization ability of TCRab and CD3 chains encoded in the three constructs and host-encoded CD3 chains (C), and identity of the polymerized CD3 chains (D). Depicted in (A) are the three constructs. The boxes represent the amino acid sequences of the chains indicated inside the boxes. T2A: self-cleavage 2A region of the Thosea asigna virus; P2A: self-cleavage 2A region of the porcine Teschovirus 1; CD3: in construct 3 represent the extracellular and transmembrane domains of the CD3z chain, followed by the signaling domains of CD28, 4-1BB, and CD3z chains. The lines represent amino acid se- quences of linkers: RAKR is the Furin consensus amino acid recognition site; SGSG is a hydrophilic tetrapeptide added to prevent steric hindrance. The arrows represent cleavage sites. In (B), FACS analysis results; transduced CD8+ T cells were stained with FITC–anti-CD8 Ab and PE-HLA-A*02:01/CT37 YLCSGSSYFV peptide complexes. A representative of three experiments is shown. Results using FITC-isotype Ab and PE-control pentamer complexes were negative (data not shown). In (C), biotinylated membrane proteins from CD8+ T cells were immunoprecipitated with an anti-TCRb Ab and molecular complexes separated by SDS-PAGE, transferred to a membrane, and blots detected by chemiluminescence. A representative of three experiments is shown. In (D), immunoprecipitated proteins obtained as indicated in (C) for construct 3, were blotted using rabbit anti-CD3d EP4426, anti-CD3g 134684, anti- CD3ε 133628 Abs, and Armenian hamster anti-CD3z H136-968 Abs (Abcam), HRP-conjugated goat anti-rabbit IgG or HPR-conjugated rabbit anti-Ar- menian hamster IgG, and blots were detected by chemiluminescence using autoradiography films (Kodak BioMax Light). A representative of two ex- periments is shown.

Collection) lung ADC cell lines for detection of those carrying the CD8+ T cells transduced with construct 3 significantly lysed HLA-A*02:01 allele. We first tested the HLA-A*02:01–positive HCC2935 and H1993 in a HLA-A*02:01–restricted fashion, as lines HCC2935 and H1299, and the HLA-A*02:01–negative line the anti–HLA-A2 Ab abrogated this lytic activity (Fig. 5Bi, ii). H1299 as control. HCC2935 was the only line carrying one of the Remarkably, CD8+ T cells transduced with any of our con- cancer-driver mutations tested. The line carries an allele with an structs were unable to lyse HLA-A*02:01–negative H1299 line EGFR deletion of 15 nucleotides in exon 19 (DE746-A750) and a (Fig. 5Biii). We also observed that CD8+ T cells transduced with wild-type EGFR allele. Regardless of the cancer-driver mutation construct 3 secreted the highest levels of IFN-g when cultured that these lines carried, we observed that all of them expressed in the presence of the HLA-A*0201–positive HCC2935 and CT37 mRNA and protein (Fig. 5Ai, ii). H1993 cell lines, but not when cultured in the presence of the The Journal of Immunology 2973 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. TCR-transduced CD8+ T cell lysis autologous CD3 cell–depleted PBMCs in a HLA-A*02:01/CT37 peptide YLCSGSSYFV with high functional avidity. (A) Results from [51Cr] release assays (left) at 16:1 E:T ratio. Dashed lines are the results from six experiments with cells isolated from six different HLA-A*02:01–positive donors. The solid line represents the mean values. Effector cells were purified CD8+ T cells transduced with construct 1. Target cells were CD3-depleted autologous PBMCs handled as in Materials and Methods and pulsed with indicated amounts of HLA-A*A02:01–restricted CT37 YLCSGSSYF peptide. [51Cr]-released cytotoxic assay proceeded for 4 h. Each experiment was done in triplicate. In (A), on the right we show the curve obtained with the mean values (in blue). We show the EC50 valueextrapolatedfromthiscurve.(B) Results from FACS analysis experiments conducted to determine whether silencing of the CD8A chain will affect HLA-A*0201/CT37 YLCSGSSYF peptide complex recognition by the NSCLC_7 CD8+ Tcellline. Left, The negative FACS analysis control; middle, graphic showing results of NSCLC_7 CD8+ T cell line transduced with control siRNA; right, results from NSCLC_7 cell line transduced with CT37-specific siRNA.

HLA-A*02:01–negative H1299 line. Notably, the supernatants H522 and HLA*02:01-negative H23 and H460 lung ADC cell tested were obtained from cultures that were left for the 4 h in- lines, and obtained similar results (Supplemental Fig. 3). cubation period and under conditions like those used to perform To ensure Ag specificity in CTL activity against HLA-A*02:01/ the [51Cr] release assay (Fig. 6A). Only background levels of IFN- CT37 peptide complexes, we silenced the expression of CT37 in g were observed when CD8+ T cells transduced with any of the CT37-expressing and HLA-A*02:01–positive cell lines HCC2935, three constructs were exposed to the H1299 line (Fig. 6A). In H1993, and H522, and tested the effect of this using CD8+ T cells concordance, CD8+ T cells transduced with construct 3 showed transduced with construct 1 in [51Cr] release assays (Fig. 7). Si- the highest frequency of IFN-g–secreting cells (Fig. 6B). Of note, lencing of CT37 expression abrogated CTL activity. we did not see significant differences in the frequency of IFN-g– secreting cells between CD8+ T cells transduced with construct 1 Discussion and those transduced with construct 2 when exposed to HLA- Our studies in patients suffering from lung ADC combined with our A*02:01–positive HCC2935 and H1993 cell lines (Fig. 6B). Only ex vivo and in vitro experiments led us to the identification and the background frequency of cells was observed when CD8+ isolation of a CT37 peptide-specific CD8+ T cell clone, and the T cells transduced with any of the three constructs were exposed molecular cloning of unique TCR a- and b-chains from this clone to the HLA-A*02:01–negative H1299 line (Fig. 6B). Non- through: 1) the ex vivo analysis of tissues using CT37 IHC; 2) the transduced CD8+ T cells did not lyse or secrete IFN-g when ex- in-silico identification of the CT37 YLCSGSSYFV peptide fol- posed to target lung ADC cells. We conducted these assays with lowed by the identification of a carrier of relatively high per- an additional set of CT37-expressing and HLA-A*02:01–positive centage of polyclonal CD8+ T cells restricted by HLA-A*02:01/ 2974 AN ADOPTIVE TRANSFER PLATFORM FOR LUNG ADENOCARCINOMA Downloaded from http://www.jimmunol.org/

FIGURE 4. Functional analysis of constructs in allogeneic CD8+ T cells. Retroviral transduction of effector (E) allogeneic CD8+ T cells was performed as explained in Materials and Methods. For [51Cr] release assay results presented in (A) and (B), six experiments, with each variable in triplicate, were performed. (A) After two cycles of 10 d stimulation in vitro, E cells were harvested, washed, counted, and plated to be tested at appropriate E:T ratios. 51 Target (T) cells were CD3-depleted autologous PBMCs (HLA-A*02:01–positive) labeled with 200 mCi of Na2 CrO4 for 1 h and washed. These cells were by guest on September 29, 2021 then 1) left untreated, 2) pulsed with 100 ng/ml of control CT37-YYLCSGSSYF peptide, or 3) pulsed with 100 ng/ml of CT37–YLCSGSSYFV peptide with high affinity for the HLA-A*02:01 class I allele. [51Cr]-released cytotoxic assay proceeded for 4 h. The p values from Mann–Whitney comparison of two means are presented. In (B), [51Cr]-released cytotoxic assay proceeded at a 16:1 E:T ratio. Target cells (as in A) were pulsed with 100 ng/ml of CT37- YLCSGSSYFV peptide. Cultures proceeded for 4 h as indicated above, without anti–HLA-A2 Ab or in the presence of this Ab at the concentrations indicated. The p values from Kruskal–Wallis comparison of more than two means are presented. (C) IFN-g–secretion assays proceeded as in (B), but the target cells were not labeled with [51Cr]. The supernatants were harvested and tested with a highly sensitive ELISA kit. The p values from Mann–Whitney comparison of two means resulting from cultures that proceeded without Ab are presented. (D) The results from ELISPOT assay to detect the frequency of Ag-specific IFN-g–secreting CD8+ T cells are presented. Target cells were pulsed with 100 ng/ml of CT37-YLCSGSSYFV peptide and cultures proceeded for 16 h at a 16:1 E:T ratio. The p values from Mann–Whitney comparison of two means are presented.

CT37 peptide complexes in peripheral blood; and 3) our in vitro frequent MHC class I molecules including: HLA-A*02:01, HLA- model using autologous CD3-depleted PBMCs. All TCR a- and A*24:02, and HLA-B*07:02 alleles (Table I, allelefrequencies. b-chains isolated from that cellular clone were TRAV2S1_AJ24S1 net). Moreover, the CT37 encoding gene is in the X chromosome and TRBV22S1_BJ2S1, respectively, supporting the fact that we (Xq27.3-q28 chromosome region; http://www.genecards.org), obtained a CD8+ T cell clone. This TCR a and b complex where most CT Ags are located (http://www.cta.Incc.br). Our exhibited high functional avidity for HLA-A*02:01/CT37 study also demonstrates that under appropriate parameters, in YLCSGSSYFV–peptide complexes. silico approaches are useful to identify peptides and MHC class I The isolation of those unique TCR a- and b-chains allowed us specificities suitable for development of adoptive transfer pre- to engineer constructs that when transferred to allogeneic CD8+ clinical platforms. Stringent criteria for selection of peptides T cells redirect them to express those TCR chains and lysis, with (a percentage rate ,0.1), may have increased the likelihood of high specificity and functional avidity, CD3-depleted autologous finding a peptide most likely binding in vivo the selected MHC PBMCs pulsed with the CT37 YLCSGSSYFV peptide, but not class I molecule. those PBMCs pulsed with control peptide. Moreover, TCR- The fact that patient NSCLC_7 had clinical features of a long- transfected CD8+ T cells lysed and secreted IFN-g when ex- term cancer-free survivor after appropriate clinical intervention, posed only to HLA-A*02:01–positive lung ADC cell lines and coincidentally had relatively large percentages of circulating expressing CT37 Ag, but not HLA-A mismatched or HLA- CD8+ T cells carrying TCRs recognizing HLA-A*02:01/CT37 A*02:01–positive lung ADC cell lines with a silenced CT37 Ag. Ag–peptide complexes may prompt the identification of similar Of note, IFN-g is a key stimulator of the proteasome and MHC cases for isolation of unique TCRs and for the development of class I molecule expression (13–15). timely immunotherapy interventions. As mentioned, CT37 was the only CT Ag among those selected Weak IHC staining in healthy kidney and urinary bladder tissues that had in silico strong binding peptides restricted by the most is mentioned in the Human Protein Atlas, using one of two Abs, in The Journal of Immunology 2975 Downloaded from http://www.jimmunol.org/

FIGURE 5. Features of lung ADC cell lines used (A) and cytotoxic capacity of CD8+ T cells transduced with the three different constructs (B). In (A), total RNA was isolated from cell lines and negative control (A549 cell line) as indicated, and RT-PCR results obtained. The mean of triplicates is shown (Ai). Protein lysates from cell lines indicted and negative control A549 were immunoprecipitated with rabbit polyclonal anti-human CT37 Ab HPA011284, proteins were separated by SDS-PAGE, transferred to a membrane, and CT37 blotted using rabbit polyclonal AP51690PU-N Ab and HRP-conjugated goat anti-rabbit IgG. Blots were detected by chemiluminescence using autoradiography films [Kodak BioMax Light; (Aii)]. In (B), retroviral transduction of effector (E) allogeneic CD8+ T cells was performed as explained in Materials and Methods. For results presented in (Bi)–(Biii), six [51Cr] release assays, with each variable in triplicate, were performed. After 2 cycles of 10 d stimulation in vitro, E cells were harvested, washed, counted, and plated to be tested at 16:1 E:T ratios. Target (T) lung ADC lines HLA-A*A-02:01–positive HCC2935 (Bi), and H1993 (Bii), and HLA-A*02:01–negative line H1299, were 51 51 labeled with 200 mCi of Na2 CrO4 for 1 h and washed. [ Cr]-released cytotoxic assay proceed for 4 h at a 16:1 E:T ratio. Cultures proceeded without or by guest on September 29, 2021 with an anti–HLA-A2 Ab to block the HLA-A*02:01 molecule, and at the indicated concentrations. The HCC2935 cell line carries wild type (wt) TP53 and KRAS genes, and an EGFR 2237_2254del10 (deletion E746-S752). The H1993 line carries a TP53 726C . G (C242W) substitution, and wt EGFR and KRAS genes. The H1299 carries a TP53 gene deletion, and wt EGFR and KRAS genes. Data on TP53 gene mutations was obtained from http://p53.free.fr/ Database/Cancer_cell_lines/NSCLC.html. We genotyped mutations in the EGFR and KRAS genes as explained in Materials and Methods. The p values from Mann–Whitney comparison of two means are presented. this case using the rabbit anti-human CT37 polyclonal IgG purified tissues mentioned in the Human Protein Atlas when using the Ab HPA011284 (http://www.proteinatlas.org). As mentioned HPA011284 polyclonal rabbit Ab. previously, we did not observe this in healthy tissue regions of de Our study agrees with the concept that some CT Ags are superb novo processed frozen tissues from five randomly selected cancer targets for the development of immune-cell–based immunother- cases, five kidneys, and five urinary bladders. We observed only apies (7–11). Indeed, protein expression of CT37 Ag is likely background staining in healthy tissue regions like that observed in restricted to testis/ovary and malignant cells, as determined by negative controls treated with a rabbit polyclonal isotype Ab (see proteomics (CT37 GeneCard), and by IHC (this study). In addi- the Supplemental Fig. 1). In addition, when testing lung ADC cell tion, lung ADC cells express CT37 [(8); this study]. However, as lines for CT37 protein expression we lysed these cells and these features are key to decreasing the chance of a patient suf- immunoprecipitated the protein lysate using the HPA011284 fering on-target off-tumor deleterious lysis of healthy tissue, we (binding CT37 epitopes in the region comprising aa 98–187) will conduct more extensive studies (with a larger sample of followed by Western blot analysis using a rabbit polyclonal normal tissues) to corroborate our findings. We think that the AP51690PU-N (binding CT37 epitopes in the region comprising production and validation of mAbs specific against CT37 will be aa 1–130) and observed a strong specific band of ∼29 kDa, and a necessary to conduct those studies. weaker band of ∼50 kDa. Because these Abs are targeted against Taken together, our IHC results, the ex vivo pulling of CD8+ highly specific CT37 amino acid sequences as we determined it T cells recognizing HLA-A*02:01/CT37 YLCSGSSYFV com- running a blast search for proteins carrying similar amino acid plexes from patient NSCLC_7, the isolation of the NSCLC_7 sequences (blast.ncbi.nlm.nih.gov/Blast.cgi), a possible explana- CD8+ T cell clone bearing a abTCR complex with high speci- tion is that the band of ∼50 kDa is a dimer of CT37 molecules. ficity, and high functional avidity for HLA-A*02:01/CT37 Arguing against this, CT37 is predicted to have a molecular mass YLCSGSSYFV complexes in vitro, we think that our cloned of 29.24 (web.expasy.org). Thus, those polyclonal Abs may harbor TCR a- and b-chains and constructs are suitable for preclinical an Ab recognizing a nonlinear (conformational) epitope exhibiting in vivo assessment of their antitumor activity, using animal mod- a nonspecific cross-reactivity. This feasible explanation may ex- els. If our adoptive transfer studies in animal models proves signifi- plain the weak IHC staining in healthy kidney and urinary bladder cant efficacy and we seek to move toward first-in-human studies, we 2976 AN ADOPTIVE TRANSFER PLATFORM FOR LUNG ADENOCARCINOMA Downloaded from

FIGURE 6. Retroviral transduction of effector (E) CD8+ T cells was performed as explained in Materials and Methods. After 2 cycles of 10 d stimulation http://www.jimmunol.org/ in vitro, transduced CD8+ T cells were harvested, washed, counted, and plated to be tested at a 16:1 E:T ratios to be tested against HLA-A*02:01–positive HCC2935 and H1993 cell lines, and HLA-A*02:01–negative H1299 cell line. For each experiment, six assays with each variable in triplicate, were performed. In (A), IFN-g secretion by transduced CD8+ T cells exposed to lung ADC cell lines. Cultures proceeded for 4 h, without or with an anti–HLA- A2 Ab to block the HLA-A*02:01 molecule, and at the indicated concentration. In (B), frequency of IFN-g–secreting transduced CD8+ T cells exposed to lung ADC cell lines. Cultures proceeded for 16 h. The p values from Mann–Whitney comparison of two means are presented. must thoroughly investigate CT37 expression in normal human tis- secretion capacity to CD8+ T cells exposed to HLA-A*02:01– sues to avoid potential hazards in targeting this Ag in human studies. positive lung ADC lines expressing CT37 Ag. This indicates that

We understand that species difference could conceal normal tissue TCR signaling should have profited by addition of the engineered by guest on September 29, 2021 toxicities that might occur in human patients. CD3z chain containing the costimulatory CD28 and 4-1BB We used the self-cleavage T2A polypeptide linkers in between fragments (17, 18). We also observed that those levels of IFN-g the TCR a- and b-chains to increase the chance that these two increased the expression of HLA-A*02:01 and of key immuno- chains will be dimerized among them. This may decrease the proteasome molecules (Supplemental Fig. 4). likelihood of creating new and potentially deleterious specificities To engineer constructs 2 and 3, we used a self-cleavage P2A by dimerization with native TCR a- and b-chains (16). Of the polypeptide linker in between the TCRab construct and the CD3z three constructs produced, construct 3 is the most promising be- chain to induce the expression of similar relative quantities of cause CD8+ T cells transduced with this construct exhibited the TCRab and CD3z chains (16), avoiding the ubiquitination and highest lytic, IFN-g secretion, and frequency of IFN-g–secreting destruction of the TCR a- and b-chains that do not find a CD3 cells. Indeed, the addition of the engineered CD3 chain containing chain available for polymerization (19). The molecular engineer- the CD28/4-1BB/CD3z signaling domains to the TCR a and b ing strategies used did not hamper the ability of those chains to construct (construct 3), conferred the highest lysis and IFN-g polymerize among them and with host cells encoded CD3d, CD3ε,

FIGURE 7. Cytotoxic capacity of CD8+ T cells transduced with the three different constructs against HLA-A*02:01–specific peptide under CT37 expression and CT37-expression silencing. Retroviral transduction of effector (E) CD8+ T cells was performed as explained in Materials and Methods. For results presented in (A)–(C), six [51Cr] release assays, with each variable in triplicate, were performed. After 2 cycles of 10 d stimulation in vitro, E cells were harvested, washed, counted, and plated to be tested at 16:1 E:T ratios. Target (T) lung ADC lines HLA-A*A-02:01–positive HCC2935, H1993, and H522 were used. CT37 expression was targeted using CT37-specific siRNA or control siRNA as explained in Materials and Methods. The cell lines were 51 51 labeled with 200 mCi of Na2 CrO4 for 1 h and washed. [ Cr]-released cytotoxic assay proceeded for 4 h. The p values from Mann–Whitney comparison of two means are presented. The Journal of Immunology 2977 and CD3g chains, nor did they affect the functions of the TCRab/ 5. Moon, E. K., C. J. Langer, and S. M. Albelda. 2017. The era of checkpoint + blockade in lung cancer: taking the brakes off the immune system. Ann. Am. CD3 complexes. Notably, CD8 T cells transduced with construct Thorac. Soc. 14: 1248–1260. 3 were able to express the highest lysis and IFN-g secretion ca- 6. Kamphorst, A. O., R. N. Pillai, S. Yang, T. H. Nasti, R. S. Akondy, A. Wieland, pacities as early as 4 h after exposure to lung ADC cell lines, and G. L. Sica, K. Yu, L. Koenig, N. T. Patel, et al. 2017. Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients. in a HLA-A*02:01–restricted fashion. We considered it likely that Proc. Natl. Acad. Sci. USA 114: 4993–4998. those HLA-A*02:01 molecules were loaded with the CT37 pep- 7. Lee, S. Y., Y. Obata, M. Yoshida, E. Stockert, B. Williamson, A. A. 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