Background Our ongoing clinical trial for the treatment of melanoma with TGF-β-resistant tumor-infiltrating lymphocytes (TIL) [TGF-β dominant negative receptor 2 (TGFβDNR2) transduced-TIL] has yielded long-term responses in checkpoint refractory patients (NCT01955460). Building on this success, we sought to extend the impact of TGF-β–resistant TIL therapy to additional cancers while optimizing a non-viral alternative to transduction with a TGFβDNR2. Ovarian cancer (OvCa), which is characterized by an abundance of TGF-β, a dysfunctional immune infiltrate, and a paucity of novel treatment options, is an ideal candidate for TGF-β–resistant TIL therapy. Here, we present an optimized and clinically-scalable method for CRISPR/Cas9-mediated deletion of the TGF-β receptor (TGFBR2) in OvCa TIL.
Methods OvCa TIL were generated from tumor fragments1 and subjected to CRISPR-mediated knockout of TGFBR2 before going through a rapid expansion protocol. Resistance of TGFBR2-knockout TIL to TGF-β signaling was evaluated via quantification of downstream SMAD-2/-3 phosphorylation, global transcriptional changes upon TGF-β exposure, and cytokine release following TCR stimulation in the presence of TGF-β. The impact of CRISPR modification on TIL expansion and TCR clonal diversity was evaluated. Finally, the risk of off-target CRISPR activity throughout the genome was evaluated using Target Enriched GUIDE-seq (TEG-seq)2 followed by next generation sequencing (NGS) validation of putative off-target sites.
Results Using five TGFBR2-directed guide RNAs (gRNAs), we achieved gene disruption efficiencies ranging from 48%–90%, which correlated inversely with the degree of SMAD phosphorylation after TGF-β exposure (r=-0.9440, p=0.0158, n=4 donors) (figure 1A-C). TGF-β exposure induced a strong transcriptional response in wild-type TIL but had little to no effect on TGFBR2-knockout TIL (figure 2). TGFBR2-knockout TIL functioned well in the presence of exogenous TGF-β as evidenced by equally strong secretion of pro-inflammatory cytokines in the presence and absence of TGF-β (figure 3). CRISPR-modification did not hamper the ex vivo expansion efficiency nor the TCR clonal diversity of expanded OvCa TIL (figure 4). Using TEG-seq, we identified ≤5 low-probability off-target sites for gRNA-#3 and gRNA-#4, each of which were attributed to background sequencing artifacts upon further validation by NGS of specific amplicons (figure 5).
Conclusions CRISPR/Cas9-mediated knockout of TGFBR2 is feasible and efficient in patient-derived OvCa TIL using clinically-scalable methods that yield little to no evidence of off-target activity. This study lays the groundwork for clinical translation of CRISPR-modified, TGF-β-resistant TIL for OvCa treatment, which will not only provide a novel immunotherapy for OvCa patients but also a platform for engineering more potent TIL therapies in general.
Sakellariou-Thompson D, Forget MA, Hinchcliff E, Celestino J, Hwu P, Jazaeri AA, et al. Potential clinical application of tumor-infiltrating lymphocyte therapy for ovarian epithelial cancer prior or post-resistance to chemotherapy. Cancer Immunology, Immunotherapy: CII 2019;68(11):1747–57.
Tang PZ, Ding B, Peng L, Mozhayskiy V, Potter J, Chesnut JD. TEG-seq: an ion torrent-adapted NGS workflow for in cellulo mapping of CRISPR specificity. Bio Techniques 2018;65(5):259–67.
Ethics Approval All procedures performed were in accordance with the 1975 Helsinki declaration. Ethical approval and tissue from surgical resections used to expand TIL were both obtained under protocols (PA16-0912 and LAB02-188) approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center. Written informed consent was obtained from all individual participants included in the study for their specimens and data to be used in research and for publication.
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