CRISPR-Edited Immune Effectors: The End of the Beginning
Authors: Feiyan Mo, Helen E. Heslop, Maksim Mamonkin
Journal: Molecular Therapy (2020)

Genome editing has emerged as a powerful tool to enhance the potency of effector cell therapies and potentially improve their safety.1Bailey S.R. Maus M.V. Gene editing for immune cell therapies.Nat. Biotechnol. 2019; 37: 1425-1434Crossref PubMed Scopus (107) Google Scholar Stadtmauer et al.2Stadtmauer E.A. Fraietta J.A. Davis M.M. Cohen A.D. Weber K.L. Lancaster E. Mangan P.A. Kulikovskaya I. Gupta M. Chen F. Tian L. et al.CRISPR-engineered T cells in patients with refractory cancer.Science. 2020; 367: eaba7365Crossref PubMed Scopus (584) Google Scholar at the University of Pennsylvania recently reported preliminary results of a first-in-man clinical study using CRISPR/Cas9-edited therapeutic T cells in patients with advanced malignancies. In this study, peripheral T cells were obtained from patients with multiple myeloma or sarcoma. To allow their engineered T cells to be more resistant to tumor-mediated immune suppression, the cells were then activated and treated with a mixture of CRISPR/Cas9 ribonucleoproteins (RNPs) to disrupt the PDCD1 gene that encodes inhibitory receptor PD-1 as well as TRAC and TRBC genes. TRAC and TRBC encode constant regions of endogenous TCR α and β chains, respectively. By eliminating endogenous T cell receptor (TCR) expression, the authors aimed to increase the expression of the transgenic NY-ESO-1/LAGE-1-specific TCR they subsequently incorporated by lentiviral transduction. The resulting NY-ESO-1 TCR-transgenic CRISPR triple-edited cells, or “NYCE cells,” were manufactured for four patients enrolled in the study, of whom three were infused. Even though the number of patients treated with NYCE T cells remains insufficient to draw definitive conclusions about the safety of their approach or the biological role of the mutations introduced, the study marks an important milestone in the development and clinical application of gene-edited effector cell therapy. Using recombinant Cas9 complexed with four distinct single guide RNAs (sgRNAs), the authors disrupted the TRAC, TRBC, and PDCD1 genes with efficiencies ranging from 15% (for the TRBC gene) to 45% (for the TRAC gene). The average lentiviral NY-ESO1 TCR transduction efficiency was 3.1%. Highlighting the inhibitory effect of endogenous TCR and/or PD-1 expression on the cytotoxicity of TCR-transgenic T cells, disruption of these genes modestly increased in vitro killing of tumor by NYCE T cells. The selected sgRNAs had minimal off-target activity, as evaluated using the iGUIDE method, and the identified non-specific sites were not mapped to known tumor suppressor genes or oncogenes in T cells. Of note, chromosomal translocations that arose from multiple simultaneous double-strand breaks in CRISPR-edited T cells were also detected in these patients. However, these genome alterations were unlikely to confer survival or proliferative advantage to the modified T cells, as the frequency of both NY-ESO-1 TCR-positive and -negative T cells carrying edited genes or translocations gradually decreased after infusion. Notably, PD-1-deficient T cells did not expand or persist better than T cells with unaltered PD-1 expression, consistent with previous findings that loss of PD-1 may reduce the ability of T cells to establish a long-lived memory population.3Odorizzi P.M. Pauken K.E. Paley M.A. Sharpe A. Wherry E.J. Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells.J. Exp. Med. 2015; 212: 1125-1137Crossref PubMed Scopus (268) Google Scholar Upon infusion, NYCE T cells expanded and tracked to tumor sites, stabilizing disease in two of three patients treated on the protocol. Although NYCE T cells remained detectable in circulation for up to 9 months after infusion, at least in one patient with myeloma, the authors documented downregulation of NY-ESO-1 and LAGE-1 antigens recognized by the transgenic TCR. Evidence of antigen escape, which has also been frequently observed in studies using CAR T cells for B cell malignancies,4Majzner R.G. Mackall C.L. Tumor antigen escape from CAR T-cell therapy.Cancer Discov. 2018; 8: 1219-1226Crossref PubMed Scopus (417) Google Scholar likely contributed to the limited antitumor activity of NYCE T cells and may warrant the use of T cells simultaneously targeting multiple tumor antigens. Despite the presence of pre-existing antibodies to the Cas9 protein from Streptococcus pyogenes,5Charlesworth C.T. Deshpande P.S. Dever D.P. Camarena J. Lemgart V.T. Cromer M.K. Vakulskas C.A. Collingwood M.A. Zhang L. Bode N.M. et al.Identification of preexisting adaptive immunity to Cas9 proteins in humans.Nat. Med. 2019; 25: 249-254Crossref PubMed Scopus (430) Google Scholar a component of the normal commensal flora in humans, no reactivation of Cas9-specific humoral immunity was observed after administration of NYCE T cells. The authors attributed this lack of response to the minimal presence of residual Cas9 protein in T cells at the time of cryopreservation and the transient immune suppression of patients induced by the lymphodepleting treatment and prior lymphotoxic therapies. While the authors did not measure activation of endogenous Cas9-specific T cells,6Wagner D.L. Amini L. Wendering D.J. Burkhardt L.M. Akyüz L. Reinke P. Volk H.D. Schmueck-Henneresse M. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population.Nat. Med. 2019; 25: 242-248Crossref PubMed Scopus (200) Google Scholar these responses, even if present, did not blunt the expansion of gene-edited T cells in vivo. Overall, these results indicate genome editing of therapeutic T cells ex vivo using CRISPR/Cas9-sgRNA RNP complexes results in minimal additional immunogenicity, largely due to a very transient persistence of electroporated Cas9 protein in rapidly dividing T cells prior to infusion. Numerous proof-of-concept reports have utilized genome-editing tools in T cells to minimize the risk of graft-versus-host disease,7Poirot L. Philip B. Schiffer-Mannioui C. Le Clerre D. Chion-Sotinel I. Derniame S. Potrel P. Bas C. Lemaire L. Galetto R. et al.Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies.Cancer Res. 2015; 75: 3853-3864Crossref PubMed Scopus (359) Google Scholar tumor immunosuppression,8Menger L. Sledzinska A. Bergerhoff K. Vargas F.A. Smith J. Poirot L. Pule M. Hererro J. Peggs K.S. Quezada S.A. TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors.Cancer Res. 2016; 76: 2087-2093Crossref PubMed Scopus (60) Google Scholar,9Ren J. Liu X. Fang C. Jiang S. June C.H. Zhao Y. Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition.Clin. Cancer Res. 2017; 23: 2255-2266Crossref PubMed Scopus (535) Google Scholar and T cell fratricide,10Gomes-Silva D. Srinivasan M. Sharma S. Lee C.M. Wagner D.L. Davis T.H. Rouce R.H. Bao G. Brenner M.K. Mamonkin M. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies.Blood. 2017; 130: 285-296Crossref PubMed Scopus (218) Google Scholar,11Gomes-Silva D. Atilla E. Atilla P.A. Mo F. Tashiro H. Srinivasan M. Lulla P. Rouce R.H. Cabral J.M.S. Ramos C.A. et al.CD7 CAR T Cells for the Therapy of Acute Myeloid Leukemia.Mol. Ther. 2019; 27: 272-280Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar among other applications. Further, commercial and academic entities have conducted several clinical studies using gene-edited T cells, including a landmark report in 2014 by Tebas et al.12Tebas P. Stein D. Tang W.W. Frank I. Wang S.Q. Lee G. Spratt S.K. Surosky R.T. Giedlin M.A. Nichol G. et al.Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.N. Engl. J. Med. 2014; 370: 901-910Crossref PubMed Scopus (1008) Google Scholar utilizing zinc-finger nucleases to create CCR5-edited T cells resistant to HIV infection. The latest study by Stadtmauer et al.2Stadtmauer E.A. Fraietta J.A. Davis M.M. Cohen A.D. Weber K.L. Lancaster E. Mangan P.A. Kulikovskaya I. Gupta M. Chen F. Tian L. et al.CRISPR-engineered T cells in patients with refractory cancer.Science. 2020; 367: eaba7365Crossref PubMed Scopus (584) Google Scholar is the first to report the manufacturing, safety, and clinical activity of CRISPR/Cas9-edited effector T cells. Recent technological advances could further improve the efficiency and precision of multiplexed genome editing and minimize its side effects. Examples include employing high-fidelity versions of Cas9 with reduced off-target activity,13Kleinstiver B.P. Pattanayak V. Prew M.S. Tsai S.Q. Nguyen N.T. Zheng Z. Joung J.K. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.Nature. 2016; 529: 490-495Crossref PubMed Scopus (1631) Google Scholar utilizing base editing in lieu of non-homologous end-joining (NHEJ) repair to minimize CRISPR-induced double-strand breaks and the resulting chromosomal translocations,14Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (2384) Google Scholar,15Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (1511) Google Scholar or knocking in desired constructs to replace endogenous genes via homology-directed recombination (HDR).16Eyquem J. Mansilla-Soto J. Giavridis T. van der Stegen S.J.C. Hamieh M. Cunanan K.M. Odak A. Gönen M. Sadelain M. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.Nature. 2017; 543: 113-117Crossref PubMed Scopus (942) Google Scholar With the first clinical testing of CRISPR/Cas9-edited therapeutic T cells, this pioneering study of NYCE T cells is perhaps “the end of the beginning” of the era of gene-edited immune effectors. F.M., H.E.H., and M.M. wrote the commentary. H.E.H. is a founder with equity in Allovir and Marker Therapeutics; has served on advisory boards for Gilead, Novartis, Tessa Therapeutics, Kiadis, and PACT Pharma; and receives research support from Tessa Therapeutics and Kuur Therapeutics. The National Cancer Institute (P50CA126752), the SU2C/AACR (604817) Meg Vosburg T cell Lymphoma Dream Team, and the Leukemia and Lymphoma Society supported the authors.