{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T14:43:15Z","timestamp":1793371395495,"version":"4.5.0"},"reference-count":20,"publisher":"BMJ","issue":"7","license":[{"start":{"date-parts":[[2022,7,26]],"date-time":"2022-07-26T00:00:00Z","timestamp":1658793600000},"content-version":"unspecified","delay-in-days":25,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"Dunwoody-Edwards-Reese Ovarian Cancer Philanthropic Fund"},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["F32CA253968"],"award-info":[{"award-number":["F32CA253968"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["K07CA201013"],"award-info":[{"award-number":["K07CA201013"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P30CA016672"],"award-info":[{"award-number":["P30CA016672"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["TL1TR003169"],"award-info":[{"award-number":["TL1TR003169"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Miriam and Jim Mulva Research Fund"},{"name":"CPRIT Research Training Program","award":["RP170067"],"award-info":[{"award-number":["RP170067"]}]},{"DOI":"10.13039\/100005984","name":"Dr. Miriam and Sheldon G. Adelson Medical Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100005984","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["bmj.com"],"crossmark-restriction":true},"short-container-title":["J Immunother Cancer"],"accepted":{"date-parts":[[2022,6,9]]},"published-print":{"date-parts":[[2022,7]]},"abstract":"<jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>The correlation between elevated T-cell infiltration and improved survival of ovarian cancer (OvCa) patients suggests that endogenous tumor-infiltrating lymphocytes (TIL) possess some degree of antitumor activity that can be harnessed for OvCa immunotherapy. We previously optimized a protocol for ex vivo OvCa TIL expansion for adoptive cell therapy, which is now being tested in a clinical trial at our institution (<jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"clintrialgov\" xlink:href=\"NCT03610490\">NCT03610490<\/jats:ext-link>). Building on this success, we embarked on genetic modification of OvCa TIL to overcome key immunosuppressive factors present in the tumor microenvironment. Here, we present the preclinical optimization of CRISPR\/Cas9-mediated knockout of the TGF-\u03b2 receptor 2 (<jats:italic>TGFBR2<\/jats:italic>) in patient-derived OvCa TIL.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>OvCa TILs were generated from four patients\u2019 tumor samples obtained at surgical resection and subjected to CRISPR\/Cas9-mediated knockout of <jats:italic>TGFBR2<\/jats:italic> before undergoing a rapid expansion protocol. <jats:italic>TGFBR2<\/jats:italic>-directed gRNAs were comprehensively evaluated for their <jats:italic>TGFBR2<\/jats:italic> knockout efficiency and off-target activity. Furthermore, the impact of <jats:italic>TGFBR2<\/jats:italic> knockout on TIL expansion, function, and downstream signaling was assayed.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                     <jats:italic>TGFBR2<\/jats:italic> knockout efficiencies ranging from 59\u00b16% to 100%\u00b10% were achieved using 5 gRNAs tested in four independent OvCa TIL samples. <jats:italic>TGFBR2<\/jats:italic> knockout TIL were resistant to immunosuppressive TGF-\u03b2 signaling as evidenced by a lack of SMAD phosphorylation, a lack of global transcriptional changes in response to TGF-\u03b2 stimulation, equally strong secretion of proinflammatory cytokines in the presence and absence of TGF-\u03b2, and improved cytotoxicity in the presence of TGF-\u03b2. CRISPR-modification itself did not alter the <jats:italic>ex vivo<\/jats:italic> expansion efficiency, immunophenotype, nor the TCR clonal diversity of OvCa TIL. Importantly for clinical translation, comprehensive analysis of CRISPR off-target effects revealed no evidence of off-target activity for our top two <jats:italic>TGFBR2<\/jats:italic>-targeting gRNAs.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>CRISPR\/Cas9-mediated gene knockout is feasible and efficient in patient-derived OvCa TIL using clinically-scalable methods. We achieved efficient and specific <jats:italic>TGFBR2<\/jats:italic> knockout, yielding an expanded OvCa TIL product that was resistant to the immunosuppressive effects of TGF-\u03b2. This study lays the groundwork for clinical translation of CRISPR-modified TIL, providing opportunities for engineering more potent TIL therapies not only for OvCa treatment, but for the treatment of other solid cancers as well.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1136\/jitc-2021-003750","type":"journal-article","created":{"date-parts":[[2022,7,26]],"date-time":"2022-07-26T12:52:41Z","timestamp":1658839961000},"page":"e003750","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":49,"title":["CRISPR-mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-\u03b2 signaling"],"prefix":"10.1136","volume":"10","clinical-trial-number":[{"clinical-trial-number":"nct03610490","registry":"10.18810\/clinical-trials-gov"},{"clinical-trial-number":"nct03610490","registry":"10.18810\/clinical-trials-gov"},{"clinical-trial-number":"nct03449108","registry":"10.18810\/clinical-trials-gov"},{"clinical-trial-number":"nct01740557","registry":"10.18810\/clinical-trials-gov"},{"clinical-trial-number":"nct01955460","registry":"10.18810\/clinical-trials-gov"},{"clinical-trial-number":"nct01955460","registry":"10.18810\/clinical-trials-gov"}],"author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1608-4646","authenticated-orcid":false,"given":"Samantha M","family":"Fix","sequence":"first","affiliation":[{"name":"Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marie-Andr\u00e9e","family":"Forget","sequence":"additional","affiliation":[{"name":"Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5954-9557","authenticated-orcid":false,"given":"Donastas","family":"Sakellariou-Thompson","sequence":"additional","affiliation":[{"name":"Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunfei","family":"Wang","sequence":"additional","affiliation":[{"name":"Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4597-308X","authenticated-orcid":false,"given":"Tamara M","family":"Griffiths","sequence":"additional","affiliation":[{"name":"Biologics Development, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5499-044X","authenticated-orcid":false,"given":"Minjung","family":"Lee","sequence":"additional","affiliation":[{"name":"Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1317-9287","authenticated-orcid":false,"given":"Cara L","family":"Haymaker","sequence":"additional","affiliation":[{"name":"Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana Luc\u00eda","family":"Dominguez","sequence":"additional","affiliation":[{"name":"Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafet","family":"Basar","sequence":"additional","affiliation":[{"name":"Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher","family":"Reyes","sequence":"additional","affiliation":[{"name":"Cell Biology R&D, Thermo Fisher Scientific, Carlsbad, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanjay","family":"Kumar","sequence":"additional","affiliation":[{"name":"Cell Biology R&D, Thermo Fisher Scientific, Carlsbad, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2687-7463","authenticated-orcid":false,"given":"Larissa A","family":"Meyer","sequence":"additional","affiliation":[{"name":"Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, 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TGF-\u03b2 receptor enhances PSMA-Targeted human CAR T cell proliferation and augments prostate cancer eradication","volume":"26","author":"Kloss","year":"2018","journal-title":"Mol Ther"}],"container-title":["Journal for ImmunoTherapy of 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