Non-viral precision T cell receptor replacement for personalized cell therapy

Creators: Foy, Susan P.¹; Jacoby, Kyle¹; Bota, Daniela A.²; Hunter, Theresa¹; Pan, Zheng¹; Stawiski, Eric¹; Ma, Yan¹; Lu, William¹; Peng, Songming¹; Wang, Clifford L.¹; Yuen, Benjamin¹; Dalmas, Olivier¹; Heeringa, Katharine¹; Sennino, Barbara¹; Conroy, Andy¹; Bethune, Michael T.¹; Mende, Ines¹; White, William¹; Kukreja, Monica¹; Gunturu, Swetha¹; Humphrey, Emily¹; Hussaini, Adeel¹; An, Duo¹; Litterman, Adam J.¹; Quach, Boi Bryant¹; Ng, Alphonsus H. C.³; Lu, Yue³; Smith, Chad¹; Campbell, Katie M.⁴; Anaya, Daniel¹; Skrdlant, Lindsey¹; Huang, Eva Yi-Hsuan¹; Mendoza, Ventura¹; Mathur, Jyoti¹; Dengler, Luke¹; Purandare, Bhamini¹; Moot, Robert¹; Yi, Michael C.¹; Funke, Roel¹; Sibley, Alison¹; Stallings-Schmitt, Todd¹; Oh, David Y.⁵; Chmielowski, Bartosz^{4, 6}; Abedi, Mehrdad⁷; Yuan, Yuan⁸; Sosman, Jeffrey A.⁹; Lee, Sylvia M.¹⁰; Schoenfeld, Adam J.^{11, 12}; Baltimore, David L.¹³; Heath, James R.³; Franzusoff, Alex¹; Ribas, Antoni^{4, 6}; Rao, Arati V.¹; Mandl, Stefanie J.¹

1. PACT Pharma, South San Francisco, CA, USA
2. University of California, Irvine
3. Institute for Systems Biology
4. University of California, Los Angeles
5. University of California, San Francisco
6. Jonsson Comprehensive Cancer Center at the University of California, Los Angeles, CA, USA
7. University of California, Davis
8. City Of Hope National Medical Center
9. Northwestern University
10. Fred Hutchinson Cancer Research Center
11. Cornell University
12. Memorial Sloan Kettering Cancer Center
13. California Institute of Technology

Abstract

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells^1,2,3. Here we developed a clinical-grade approach based on CRISPR–Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen–HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Copyright and License

© 2022, The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Acknowledgement

We thank the patients and their families and caregivers who participated in this study; and present and previous employees of PACT Pharma who contributed to this work, including the Upstream Operations, Process Development, Plasmid Manufacturing, Cell Manufacturing, Clinical Development, Clinical Operations, Quality Control, Quality Assurance and Program Management teams. The work was funded by PACT Pharma. J.R.H. and A.R. are funded by the Parker Institute for Cancer Immunotherapy. A.R. is funded by the National Institutes of Health (grant R35 CA197633). D.Y.O. is supported by NIH K08AI139375, a Young Investigator Award from the Prostate Cancer Foundation and a Damon Runyon Clinical Investigator Award (CI 110-21). K.M.C. is supported by a UCLA Tumor Immunology Training Grant (NIH T32CA009120), the Cancer Research Institute Irvington Postdoctoral Fellowship Program and a V Family Foundation Gil Nickel Melanoma Research Fellowship.

Data Availability

The following publicly available datasets were utilized: ExAc (3.1, https://gnomad.broadinstitute.org/downloads#exac-variants), dbSNP (v146, ftp://ftp.broadinstitute.org/bundle), GATK Resource Bundle (hg19/Grch37, ftp://ftp.broadinstitute.org/bundle), Human Proteome (Homo_sapiens.GRCh37.75.pep.all.fa, http://ensembl.org/), IMGT (TCR/HLA, 3.1.17, http://www.imgt.org/), RefSeq (1052019, ftp://hgdownload.cse.ucsc.edu/goldenPath), TCGA (v.1.0, https://portal.gdc.cancer.gov/) and Broad Institute (hg19, ftp://ftp.broadinstitute.org/bundle). The TCR sequences from the current study are provided in the supplementary files, and the genomics data are available following reasonable request from the European Genome-Phenome Archive repository.

Conflict of Interest

S.P.F., K.J., T.H., Z.P., E.S., Y.M., W.L., S.P., C.L.W., B.Y., O.D., K.H., B.S., A.C., M.T.B., I.M., W.W., M.K., S.G., E.H., A.H., D. An, A.J.L., B.B.Q., C.S., D. Anaya, L.S., E.Y.-H.H., V.M., J.M., L.D., B.P., R.M., M.C.Y., R.F., A.S., T.S.-S., A.F., A.V.R. and S.J.M. were employees of PACT Pharma during the conduct of this work. S.P., M.T.B., D.B., J.R.H. and A.R. are scientific co-founders of PACT Pharma. K.M.C. has received consulting fees from PACT Pharma and Tango Therapeutics and is a shareholder in Geneoscopy. D.Y.O. has received research support from Merck, PACT Pharma, the Parker Institute for Cancer Immunotherapy, Poseida Therapeutics, TCR2 Therapeutics, Roche/Genentech and Nutcracker Therapeutics. B.C. has advisory roles with the following companies: Iovance Biotherapeutics, IDEAYA Biosciences, Sanofi, OncoSec, Nektar, Genentech, Novartis and Instil Bio. D.Y.O. has also received research funding from the following companies: Iovance Biotherapeutics, Bristol-Myers Squibb, Macrogenics, Daiichi Sankyo, Merck, Karyopharm Therapeutics, Infinity Pharmaceuticals, Advenchen Laboratories, Idera, Neon Therapeutics, Xencor, Compugen, PACT Pharma, RAPT Therapeutics, Immunocore, Lilly, IDEAYA Biosciences, Tolero Pharmaceuticals, Ascentage Pharma, Novartis, Atreca, Replimune, Instil Bio, Trisalus and Kinnate. J.A.S. reports honorarium from Iovance, Apixogen, Jazz Pharm, and research with BMS, PACT Pharma and Corvus. A.J.S. reports consulting or advising roles to J&J, KSQ Therapeutics, BMS, Enara Bio, Perceptive Advisors, Heat Biologics and Iovance Biotherapeutics. Research funding: GSK (Inst), PACT pharma (Inst), Iovance Biotherapeutics (Inst), Achilles therapeutics (Inst), Merck (Inst), BMS (Inst), Harpoon Therapeutics (Inst). A.R. has received honoraria from consulting with Amgen, CStone, Merck, and Vedanta, is or has been a member of the scientific advisory board and holds stock in Advaxis, Appia, Apricity, Arcus, Compugen, CytomX, Highlight, ImaginAb, ImmPact, ImmuneSensor, Inspirna, Isoplexis, Kite-Gilead, Lutris, MapKure, Merus, PACT Pharma, Pluto, RAPT, Synthekine and Tango, has received research funding from Agilent and from Bristol-Myers Squibb through Stand Up to Cancer (SU2C), and patent royalties from Arsenal Bio. D.A.B., A.H.C.N., Y.L., M.A., Y.Y. and S.M.L. report no conflicts of interest for this work.

Supplemental Material

Supplementary Information: Supplementary Figs. 1–8, Supplementary Tables 1 and 3 and the clinical trial protocol.
Supplementary Table 2: Full-length TCR sequences.
Supplementary Table 4: List of flow cytometry reagents.

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