Identification and characterisation of novel CAR-T cells to target IL13Rα2 positive human glioma in vitro and in vivo

Abstract

Background: Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB.

Methods: We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells.

Results: The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy.

Conclusion: Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.

Keywords

CAR-T cell therapy / genetically engineered T cells / IL-13Rα2 / immunotherapy / single-chain variable fragment

References

[1] Neelapu SS, Locke FL, Bartlett NL, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377:2531-2544.

[2] Davenport AJ, Cross RS, Watson KA, et al. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity. Proc Natl Acad Sci U S A. 2018;115:E2068-E2076.

[3] Schuster SJ, Svoboda J, Chong EA, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med. 2017;377:2545-2554.

[4] FDA: FDA approval brings first gene therapy to the United States. FDA; 2017.

[5] FDA: FDA approves First Cell-Based Gene Therapy For Adult Patientswith Relapsed or Refractory MCL. FDA; 2020.

[6] Lim WA, June CH. The principles of engineering immune cells to treat cancer. Cell. 2017;168:724-740.

[7] Ramos CA, Rouce R, Robertson CS, et al. In vivo fate and activity of second- versus third-generation CD19-specific CAR-T cells in B cell non-Hodgkin's lymphomas. Mol Ther. 2018;26:2727-2737.

[8] Ramos CA, Heslop HE, Brenner MK. CAR-T cell therapy for lymphoma. Annu Rev Med. 2016;67:165-183.

[9] Sadelain M. CD19 CAR T cells. Cell. 2017;171:1471.

[10] Curran KJ, Margossian SP, Kernan NA, et al. Toxicity and response after CD19-specific CAR T-cell therapy in pediatric/young adult relapsed/refractory B-ALL. Blood. 2019;134:2361-2368.

[11] Davila ML, Sadelain M. Biology and clinical application of CAR T cells for B cell malignancies. Int J Hematol. 2016;104:6-17.

[12] Joshi BH, Hogaboam C, Dover P, Husain SR, Puri RK. Role of interleukin-13 in cancer, pulmonary fibrosis, and other T(H)2-type diseases. Vitam Horm. 2006;74:479-504.

[13] Suzuki A, Leland P, Joshi BH, Puri RK. Targeting of IL-4 and IL-13 receptors for cancer therapy. Cytokine. 2015;75:79-88.

[14] Joshi BH, Plautz GE, Puri RK. Interleukin-13 receptor alpha chain: a novel tumor-associated transmembrane protein in primary explants of human malignant gliomas. Cancer Res. 2000;60:1168-1172.

[15] Knudson KM, Hwang S, McCann MS, Joshi BH, Husain SR, Puri RK. Recent advances in IL-13Ralpha2-directed cancer immunotherapy. Front Immunol. 2022;13:878365.

[16] Wykosky J, Gibo DM, Stanton C, Debinski W. Interleukin-13 receptor alpha 2, EphA2, and Fos-related antigen 1 as molecular denominators of high-grade astrocytomas and specific targets for combinatorial therapy. Clin Cancer Res. 2008;14:199-208.

[17] Zeng J, Zhang J, Yang YZ, et al. IL13RA2 is overexpressed in malignant gliomas and related to clinical outcome of patients. Am J Transl Res. 2020;12:4702-4714.

[18] Beard RE, Abate-Daga D, Rosati SF, et al. Gene expression profiling using nanostring digital RNA counting to identify potential target antigens for melanoma immunotherapy. Clin Cancer Res. 2013;19:4941-4950.

[19] Chiaramonte MG, Mentink-Kane M, Jacobson BA, et al. Regulation and function of the interleukin 13 receptor alpha 2 during a T helper cell type 2-dominant immune response. J Exp Med. 2003;197:687-701.

[20] Donaldson DD, Whitters MJ, Fitz LJ, et al. The murine IL-13 receptor alpha 2: molecular cloning, characterization, and comparison with murine IL-13 receptor alpha 1. J Immunol. 1998;161:2317-2324.

[21] Mentink-Kane MM, Wynn TA. Opposing roles for IL-13 and IL-13 receptor alpha 2 in health and disease. Immunol Rev. 2004;202:191-202.

[22] Jaen M, Martin-Regalado A, Bartolome RA, Robles J, Casal JI. Interleukin 13 receptor alpha 2 (IL13Ralpha2): expression, signaling pathways and therapeutic applications in cancer. Biochim Biophys Acta Rev Cancer. 2022;1877:188802.

[23] Gardner R, Wu D, Cherian S, et al. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy. Blood. 2016;127:2406-2410.

[24] Jacoby E, Nguyen SM, Fountaine TJ, et al. CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity. Nat Commun. 2016;7:12320.

[25] Yeku OO, Purdon TJ, Koneru M, Spriggs D, Brentjens RJ. Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment. Sci Rep. 2017;7:10541.

[26] Fichtner-Feigl S, Strober W, Kawakami K, Puri RK, Kitani A. IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med. 2006;12:99-106.

[27] Han J, Puri RK. Analysis of the cancer genome atlas (TCGA) database identifies an inverse relationship between interleukin-13 receptor alpha1 and alpha2 gene expression and poor prognosis and drug resistance in subjects with glioblastoma multiforme. J Neurooncol. 2018;136:463-474.

[28] Joshi BH, Kawakami K, Leland P, Puri RK. Heterogeneity in interleukin-13 receptor expression and subunit structure in squamous cell carcinoma of head and neck: differential sensitivity to chimeric fusion proteins comprised of interleukin-13 and a mutated form of Pseudomonas exotoxin. Clin Cancer Res. 2002;8:1948-1956.

[29] Joshi BH, Puri RK. Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli. Protein Expr Purif. 2005;39:189-198.

[30] Kioi M, Seetharam S, Puri RK. Targeting IL-13Ralpha2-positive cancer with a novel recombinant immunotoxin composed of a single-chain antibody and mutated Pseudomonas exotoxin. Mol Cancer Ther. 2008;7:1579-1587.

[31] Mitra A, Barua A, Huang L, Ganguly S, Feng Q, He B. From bench to bedside: the history and progress of CAR T cell therapy. Front Immunol. 2023;14:1188049.

[32] Roselli E, Boucher JC, Li G, et al. 4-1BB and optimized CD28 co-stimulation enhances function of human mono-specific and bi-specific third-generation CAR T cells. J Immunother Cancer. 2021;9:e003354.

[33] Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021;11:69.

[34] DeRenzo C, Krenciute G, Gottschalk S. The landscape of CAR T cells beyond acute lymphoblastic leukemia for pediatric solid tumors. Am Soc Clin Oncol Educ Book. 2018;38:830-837.

[35] Bradbury AR, Marks JD. Antibodies from phage antibody libraries. J Immunol Methods. 2004;290:29-49.

[36] Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991;222:581-597.

[37] Marks JD, Tristem M, Karpas A, Winter G. Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes. Eur J Immunol. 1991;21:985-991.

[38] Dunbar J, Deane CM. ANARCI: antigen receptor numbering and receptor classification. Bioinformatics. 2016;32:298-300.

[39] Leland P, Kumar D, Nimmagadda S, Bauer SR, Puri RK, Joshi BH. Characterization of chimeric antigen receptor modified T cells expressing scFv-IL-13Ralpha2 after radiolabeling with (89)Zirconium oxine for PET imaging. J Transl Med. 2023;21:367.

[40] Joshi BH, Leland P, Calvo A, Green JE, Puri RK. Human adrenomedullin up-regulates interleukin-13 receptor alpha2 chain in prostate cancer in vitro and in vivo: a novel approach to sensitize prostate cancer to anticancer therapy. Cancer Res. 2008;68:9311-9317.

[41] Barczak W, Suchorska W, Rubis B, Kulcenty K. Universal real-time PCR-based assay for lentiviral titration. Mol Biotechnol. 2015;57:195-200.

[42] Pituch KC, Miska J, Krenciute G, et al. Adoptive transfer of IL13Ralpha2-specific chimeric antigen receptor T cells creates a pro-inflammatory environment in glioblastoma. Mol Ther. 2018;26:986-995.

[43] Krenciute G, Krebs S, Torres D, et al. Characterization and functional analysis of scFv-based chimeric antigen receptors to redirect T cells to IL13Ralpha2-positive glioma. Mol Ther. 2016;24:354-363.

[44] Kreitman RJ. Immunotoxins for targeted cancer therapy. AAPS J. 2006;8:E532-551.

[45] Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ. Immunotoxin treatment of cancer. Annu Rev Med. 2007;58:221-237.

[46] Badger CC, Anasetti C, Davis J, Bernstein ID. Treatment of malignancy with unmodified antibody. Pathol Immunopathol Res. 1987;6:419-434.

[47] Khazaeli MB, Conry RM, LoBuglio AF. Human immune response to monoclonal antibodies. J Immunother Emphasis Tumor Immunol. 1994;15:42-52.

[48] Mountain A, Adair JR. Engineering antibodies for therapy. Biotechnol Genet Eng Rev. 1992;10:1-142.

[49] Wright A, Shin SU, Morrison SL. Genetically engineered antibodies: progress and prospects. Crit Rev Immunol. 1992;12:125-168.

[50] Kudo K, Imai C, Lorenzini P, et al. T lymphocytes expressing a CD16 signaling receptor exert antibody-dependent cancer cell killing. Cancer Res. 2014;74:93-103.

[51] Song DG, Ye Q, Carpenito C, et al. In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137 (4-1BB). Cancer Res. 2011;71:4617-4627.

[52] Kadayakkara DK, Damodaran K, Hitchens TK, Bulte JW, Ahrens ET. (19)F spin-lattice relaxation of perfluoropolyethers: dependence on temperature and magnetic field strength (7.0-14.1T). J Magn Reson. 2014;242:18-22.

[53] Brown CE, Badie B, Barish ME, et al. Bioactivity and safety of IL13Ralpha2-redirected chimeric antigen receptor CD8+ T cells in patients with recurrent glioblastoma. Clin Cancer Res. 2015;21:4062-4072.

[54] Brown CE, Starr R, Aguilar B, et al. Stem-like tumor-initiating cells isolated from IL13Ralpha2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells. Clin Cancer Res. 2012;18:2199-2209.

[55] Krebs S, Chow KK, Yi Z, et al. T cells redirected to interleukin-13Ralpha2 with interleukin-13 mutein-chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Ralpha1. Cytotherapy. 2014;16:1121-1131.

[56] Kahlon KS, Brown C, Cooper LJ, Raubitschek A, Forman SJ, Jensen MC. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res. 2004;64:9160-9166.

[57] Kawakami K, Taguchi J, Murata T, Puri RK. The interleukin-13 receptor alpha2 chain: an essential component for binding and internalization but not for interleukin-13-induced signal transduction through the STAT6 pathway. Blood. 2001;97:2673-2679.

[58] Kong S, Sengupta S, Tyler B, et al. Suppression of human glioma xenografts with second-generation IL13R-specific chimeric antigen receptor-modified T cells. Clin Cancer Res. 2012;18:5949-5960.

[59] Fujisawa T, Joshi BH, Puri RK. Histone modification enhances the effectiveness of IL-13 receptor targeted immunotoxin in murine models of human pancreatic cancer. J Transl Med. 2011;9:37.

[60] Goldman M, Lucke-Wold B, Martinez-Sosa M, et al. Steroid utility, immunotherapy, and brain tumor management: an update on conflicting therapies. Explor Target Antitumor Ther. 2022;3:659-675.

[61] Reddy R, Yan SC, Hasanpour Segherlou Z, et al. Oncolytic viral therapy: a review and promising future directions. J Neurosurg. 2024;140:319-327.

[62] Ostrand-Rosenberg S. Tolerance and immune suppression in the tumor microenvironment. Cell Immunol. 2016;299:23-29.

[63] Umansky V, Sevko A. Tumor microenvironment and myeloid-derived suppressor cells. Cancer Microenviron. 2013;6:169-177.

[64] Hall B, Nakashima H, Sun ZJ, et al. Targeting of interleukin-13 receptor alpha2 for treatment of head and neck squamous cell carcinoma induced by conditional deletion of TGF-beta and PTEN signaling. J Transl Med. 2013;11:45.

[65] Kioi M, Seetharam S, Puri RK. N-linked glycosylation of IL-13R alpha2 is essential for optimal IL-13 inhibitory activity. FASEB J. 2006;20:2378-2380.

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([0-9]+) The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.