A Caltech Library Service

Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy

Stern, Lawrence A. and Gholamin, Sharareh and Moraga, Ignacio and Yang, Xin and Saravanakumar, Supraja and Cohen, Joseph R. and Starr, Renate and Aguilar, Brenda and Salvary, Vanessa and Hibbard, Jonathan C. and Kalbasi, Anusha and Shepphird, Jennifer K. and O’Hearn, James and Garcia, K. Christopher and Brown, Christine E. (2022) Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy. Proceedings of the National Academy of Sciences of the United States of America, 119 (33). Art. No. e2112006119. ISSN 0027-8424. doi:10.1073/pnas.2112006119.

[img] PDF - Published Version
Creative Commons Attribution.

[img] PDF - Supplemental Material
Creative Commons Attribution.


Use this Persistent URL to link to this item:


IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)–based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

Item Type:Article
Related URLs:
URLURL TypeDescription Information
Stern, Lawrence A.0000-0002-3369-7495
Gholamin, Sharareh0000-0001-7425-6074
Moraga, Ignacio0000-0001-9909-0701
Saravanakumar, Supraja0000-0002-0698-750X
Starr, Renate0000-0002-2200-6900
Kalbasi, Anusha0000-0001-8692-1457
Shepphird, Jennifer K.0000-0001-6963-0228
O’Hearn, James0000-0002-7287-2604
Garcia, K. Christopher0000-0001-9273-0278
Brown, Christine E.0000-0003-4915-8207
Additional Information:© 2022 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). Edited by Arthur Weiss, School of Medicine, University of California, San Francisco, CA; received June 30, 2021; accepted June 3, 2022. We thank Dr. Julie R. Ostberg for manuscript and figure preparation. L.A.S. was supported by the California Tobacco-Related Disease Research Grants Program Office of the University of California (Grant# 28FT-0072). Sponsored research support also received from Mustang Bio. S.G. and K.C.G. are supported by the Parker Institute for Cancer Immunotherapy. K.C.G. is supported by the National Institutes of Health (NIH-R01-AI51321). We acknowledge the Encyclopedia of DNA Elements (ENCODE) Consortium and the ENCODE Data Coordination Center for generating and sharing datasets used in SI Appendix, Fig. 5. L.A.S., S.G., and I.M. contributed equally to this work. K.C.G. and C.E.B. contributed equally to this work. Author contributions: L.A.S., I.M., J.R.C., K.C.G., and C.E.B. designed research; L.A.S.,S.G., X.Y., S.S., R.S., B.A., and V.S. performed research; L.A.S., J.H., A.K., and C.E.B.contributed new reagents/analytic tools; L.A.S., S.G., X.Y., S.S., R.S., B.A., J.H., J.O., andC.E.B. analyzed data; L.A.S., J.K.S., and C.E.B. wrote the paper; and J.O. reviewed and edited the paper. This article contains supporting information online at Data Availability. All study data are included in the article and/or SI Appendix. This article is a PNAS Direct Submission. Competing interest statement: Patents associated with CAR design, T cell manufacturing, and delivery have been licensed by Mustang Bio Inc. and ChimericTherapeutics, for which C.E.B. receives licensing and consulting payments. C4 and D7IL13-variant CARs are the subject of a patent filing for C.E.B., K.C.G, I.M, L.A.S. and X.Y.
Funding AgencyGrant Number
California Tobacco-Related Disease Research Program28FT-0072
Parker Institute for Cancer ImmunotherapyUNSPECIFIED
Subject Keywords:chimeric antigen receptors; CARs; T cells; IL13Rα2; glioblastoma
Issue or Number:33
Record Number:CaltechAUTHORS:20220809-494048000
Persistent URL:
Official Citation:Stern LA, Gholamin S, Moraga I, et al. Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy. Proc Natl Acad Sci U S A. 2022;119(33):e2112006119. doi:10.1073/pnas.2112006119
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:116187
Deposited By: George Porter
Deposited On:12 Aug 2022 20:05
Last Modified:12 Aug 2022 20:05

Repository Staff Only: item control page