Abstract
Cytokines that stimulate T cell proliferation, such as interleukin (IL)-15, have been explored as a means of boosting the antitumor activity of chimeric antigen receptor (CAR) T cells. However, constitutive cytokine signaling in T cells and activation of bystander cells may cause toxicity. IL-23 is a two-subunit cytokine known to promote proliferation of memory T cells and T helper type 17 cells. We found that, upon T cell antigen receptor (TCR) stimulation, T cells upregulated the IL-23 receptor and the IL-23α p19 subunit, but not the p40 subunit. We engineered expression of the p40 subunit in T cells (p40-Td cells) and obtained selective proliferative activity in activated T cells via autocrine IL-23 signaling. In comparison to CAR T cells, p40-Td CAR T cells showed improved antitumor capacity in vitro, with increased granzyme B and decreased PD-1 expression. In two xenograft and two syngeneic solid tumor mouse models, p40-Td CAR T cells showed superior efficacy in comparison to CAR T cells and attenuated side effects in comparison to CAR T cells expressing IL-18 or IL-15.
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Data availability
The RNA-seq datasets generated and analyzed during the current study are not publicly available (the genetic information from primary human T cells in this study was not consented to be published in the public domain) and will be available from the corresponding authors upon request.
References
Garber, K. Driving T-cell immunotherapy to solid tumors. Nat. Biotechnol. 36, 215–219 (2018).
D'Angelo, S. P. et al. Antitumor activity associated with prolonged persistence of adoptively transferred NY-ESO-1 c259T cells in synovial sarcoma. Cancer Discov. 8, 944–957 (2018).
Porter, D. L., Levine, B. L., Kalos, M., Bagg, A. & June, C. H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N. Engl. J. Med. 365, 725–733 (2011).
Maude, S. L., Teachey, D. T., Porter, D. L. & Grupp, S. A. CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood 125, 4017–4023 (2015).
Sanmamed, M. F. & Chen, L. A paradigm shift in cancer immunotherapy: from enhancement to normalization. Cell 175, 313–326 (2018).
Anderson, K. G., Stromnes, I. M. & Greenberg, P. D. Obstacles posed by the tumor microenvironment to T cell activity: a case for synergistic therapies. Cancer Cell 31, 311–325 (2017).
Kershaw, M. H., Westwood, J. A. & Darcy, P. K. Gene-engineered T cells for cancer therapy. Nat. Rev. Cancer 13, 525–541 (2013).
Yao, X. et al. Levels of peripheral CD4+FoxP3+ regulatory T cells are negatively associated with clinical response to adoptive immunotherapy of human cancer. Blood 119, 5688–5696 (2012).
Hurton, L. V. et al. Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc. Natl Acad. Sci. USA 113, E7788–E7797 (2016).
Hoyos, V. et al. Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 24, 1160–1170 (2010).
Waldmann, T. A. et al. Safety (toxicity), pharmacokinetics, immunogenicity, and impact on elements of the normal immune system of recombinant human IL-15 in rhesus macaques. Blood 117, 4787–4795 (2011).
Berger, C. et al. Safety and immunologic effects of IL-15 administration in nonhuman primates. Blood 114, 2417–2426 (2009).
Di Stasi, A. et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N. Engl. J. Med. 365, 1673–1683 (2011).
Sockolosky, J. T. et al. Selective targeting of engineered T cells using orthogonal IL-2 cytokine–receptor complexes. Science 359, 1037–1042 (2018).
Andersen, R. et al. Long-lasting complete responses in patients with metastatic melanoma after adoptive cell therapy with tumor-infiltrating lymphocytes and an attenuated IL2 regimen. Clin. Cancer Res. 22, 3734–3745 (2016).
Kagoya, Y. et al. A novel chimeric antigen receptor containing a JAK–STAT signaling domain mediates superior antitumor effects. Nat. Med. 24, 352–359 (2018).
Fraietta, J. A. et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat. Med. 24, 563–571 (2018).
Oppmann, B. et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13, 715–725 (2000).
Duvallet, E., Semerano, L., Assier, E., Falgarone, G. & Boissier, M. C. Interleukin-23: a key cytokine in inflammatory diseases. Ann. Med. 43, 503–511 (2011).
Ngiow, S. F., Teng, M. W. & Smyth, M. J. A balance of interleukin-12 and -23 in cancer. Trends Immunol. 34, 548–555 (2013).
Iwakura, Y. & Ishigame, H. The IL-23/IL-17 axis in inflammation. J. Clin. Invest. 116, 1218–1222 (2006).
Aggarwal, S., Ghilardi, N., Xie, M. H., de Sauvage, F. J. & Gurney, A. L. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J. Biol. Chem. 278, 1910–1914 (2003).
Guedan, S. et al. ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells. Blood 124, 1070–1080 (2014).
Xu, Y. et al. Glycolysis determines dichotomous regulation of T cell subsets in hypoxia. J. Clin. Invest. 126, 2678–2688 (2016).
Langrish, C. L. et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, 233–240 (2005).
Zhu, J., Yamane, H. & Paul, W. E. Differentiation of effector CD4 T cell populations. Annu. Rev. Immunol. 28, 445–489 (2010).
Muranski, P. & Restifo, N. P. Essentials of Th17 cell commitment and plasticity. Blood 121, 2402–2414 (2013).
Frankel, T. L. et al. Both CD4 and CD8 T cells mediate equally effective in vivo tumor treatment when engineered with a highly avid TCR targeting tyrosinase. J. Immunol. 184, 5988–5998 (2010).
Langowski, J. L. et al. IL-23 promotes tumour incidence and growth. Nature 442, 461–465 (2006).
Grivennikov, S. I. et al. Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth. Nature 491, 254–258 (2012).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).
Gargett, T. et al. GD2-specific CAR T cells undergo potent activation and deletion following antigen encounter but can be protected from activation-induced cell death by PD-1 blockade. Mol. Ther. 24, 1135–1149 (2016).
Philip, M. et al. Chromatin states define tumour-specific T cell dysfunction and reprogramming. Nature 545, 452–456 (2017).
Wherry, E. J. T cell exhaustion. Nat. Immunol. 12, 492–499 (2011).
Du, H. et al. Antitumor responses in the absence of toxicity in solid tumors by targeting B7-H3 via chimeric antigen receptor T cells. Cancer Cell 35, 221–237 (2019).
Lee, J. W., Komar, C. A., Bengsch, F., Graham, K. & Beatty, G. L. Genetically engineered mouse models of pancreatic cancer: the KPC model (LSL-Kras G12D/+;LSL-Trp53 R172H /+;Pdx-1-Cre), its variants, and their application in immuno-oncology drug discovery. Curr. Protoc. Pharmacol. 73, 14.39.11–14.39.20 (2016).
Mirlekar, B., Michaud, D., Searcy, R., Greene, K. & Pylayeva-Gupta, Y. IL35 hinders endogenous antitumor T-cell immunity and responsiveness to immunotherapy in pancreatic cancer. Cancer Immunol. Res. 6, 1014–1024 (2018).
Zeitouni, D., Pylayeva-Gupta, Y., Der, C. J. & Bryant, K. L. KRAS mutant pancreatic cancer: no lone path to an effective treatment. Cancers 8, E45 (2016).
Hu, B. et al. Augmentation of antitumor immunity by human and mouse CAR T cells secreting IL-18. Cell Rep. 20, 3025–3033 (2017).
Chmielewski, M. & Abken, H. CAR T cells releasing IL-18 convert to T-bethighFoxO1low effectors that exhibit augmented activity against advanced solid tumors. Cell Rep. 21, 3205–3219 (2017).
Avanzi, M. P. et al. Engineered tumor-targeted T cells mediate enhanced anti-tumor efficacy both directly and through activation of the endogenous immune system. Cell Rep. 23, 2130–2141 (2018).
Chen, Y. et al. Eradication of neuroblastoma by T cells redirected with an optimized GD2-specific chimeric antigen receptor and interleukin-15. Clin. Cancer Res. 25, 2915–2924 (2019).
Diaconu, I. et al. Inducible caspase-9 selectively modulates the toxicities of CD19-specific chimeric antigen receptor-modified T cells. Mol. Ther. 25, 580–592 (2017).
Tey, S. K., Dotti, G., Rooney, C. M., Heslop, H. E. & Brenner, M. K. Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation. Biol. Blood Marrow Transplant. 13, 913–924 (2007).
Teng, M. W. et al. Opposing roles for IL-23 and IL-12 in maintaining occult cancer in an equilibrium state. Cancer Res. 72, 3987–3996 (2012).
Zhang, L. et al. IL-23 selectively promotes the metastasis of colorectal carcinoma cells with impaired Socs3 expression via the STAT5 pathway. Carcinogenesis 35, 1330–1340 (2014).
Lo, C. H. et al. Antitumor and antimetastatic activity of IL-23. J. Immunol. 171, 600–607 (2003).
Overwijk, W. W. et al. Immunological and antitumor effects of IL-23 as a cancer vaccine adjuvant. J. Immunol. 176, 5213–5222 (2006).
Floss, D. M., Schroder, J., Franke, M. & Scheller, J. Insights into IL-23 biology: from structure to function. Cytokine Growth Factor Rev. 26, 569–578 (2015).
Hor, Y. T. et al. A role for RUNX3 in inflammation-induced expression of IL23A in gastric epithelial cells. Cell Rep. 8, 50–58 (2014).
Pulliam, S. R., Uzhachenko, R. V., Adunyah, S. E. & Shanker, A. Common γ chain cytokines in combinatorial immune strategies against cancer. Immunol. Lett. 169, 61–72 (2016).
Rochman, Y., Spolski, R. & Leonard, W. J. New insights into the regulation of T cells by γc family cytokines. Nat. Rev. Immunol. 9, 480–490 (2009).
Thurley, K., Gerecht, D., Friedmann, E. & Hofer, T. Three-dimensional gradients of cytokine signaling between T cells. PLoS Comput. Biol. 11, e1004206 (2015).
Oyler-Yaniv, A. et al. A tunable diffusion–consumption mechanism of cytokine propagation enables plasticity in cell-to-cell communication in the immune system. Immunity 46, 609–620 (2017).
Noman, M. Z. et al. The cooperative induction of hypoxia-inducible factor-1α and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis. J. Immunol. 182, 3510–3521 (2009).
Pawlus, M. R., Wang, L. & Hu, C. J. STAT3 and HIF1α cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells. Oncogene 33, 1670–1679 (2014).
Song, L. et al. Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma. J. Clin. Invest. 117, 2702–2712 (2007).
Tarek, N. et al. Unlicensed NK cells target neuroblastoma following anti-GD2 antibody treatment. J. Clin. Invest. 122, 3260–3270 (2012).
Caruana, I. et al. Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes. Nat. Med. 21, 524–529 (2015).
Louis, C. U. et al. Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood 118, 6050–6056 (2011).
Du, H. et al. Antitumor responses in the absence of toxicity in solid tumors by targeting B7-H3 via chimeric antigen receptor T cells. Cancer Cell 35, 221–237 (2018).
Vera, J. et al. T lymphocytes redirected against the κ light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells. Blood 108, 3890–3897 (2006).
Xu, Y. et al. Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15. Blood 123, 3750–3759 (2014).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Dauer, D. J. et al. Stat3 regulates genes common to both wound healing and cancer. Oncogene 24, 3397–3408 (2005).
Acknowledgements
This work was supported in part by the University Cancer Research Fund at the University of North Carolina at Chapel Hill (G.D.), R01-CA193140-03 from the National Cancer Institute (G.D.), 1R37-CA230786-01A1-01A1 from the National Institutes of Health (Y.P.-G.), W81XWH-19-1-0597 from the Department of Defense (Y.P.-G.) and W81XWH-18-1-0441 from the Department of Defense (H.D.). The UNC Small Animal Imaging Facility at the Biomedical Imaging Research Center, the Microscopy Services Laboratory and the Flow Cytometry Core Facilities are supported in part by an NCI Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center (P30-CA016086-40). The frozen tissues for RNA and protein extraction were obtained from the Tissue Procurement Facility at the UNC Lineberger Comprehensive Cancer Center. We thank C. Santos and the Animal Studies Core Facility for providing mice and surgery services for the orthotopic pancreatic cancer mouse models.
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X.M., G.D. and Y.X. designed the experiments, analyzed data and wrote the manuscript. X.M. and Y.X. performed the experiments. P.S., Y.C., H.D., C.S., N.P.K., D.M., S.A., B.S., Y.P.-G. and S.Z. provided critical help and discussion for experiments. C.S., B.V. and Y.X. performed analysis on RNA-seq data.
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G.D. and B.S. have sponsored research agreements with Bluebird Bio, Cell Medica and Bellicum Pharmaceutical. G.D. serves on the scientific advisory board of MolMed and Bellicum Pharmaceutical.
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Supplementary Fig. 1 Phenotypic analysis of p40-Td cells.
(a) Schematic representation of the retroviral vectors (upper panel) and representative flow cytometry plots illustrating the transduction efficiency of T cells measured by ΔNGFR expression (left panel) and qRT-PCR (right panel). Data shown as representative data and mean ± SD (n =4). (b) Cell counts of Ctrl cells and p40-Td cells cultured with IL-7 and IL-15. ΔNGFR+ T cells were sorted on day 5 post-transduction. Data shown as mean ± SD (n = 4). (c) Frequency of CD4+ and CD8+ T cells of Ctrl cells and p40-Td cells at day 12 post-transduction. Data shown as representative data and mean ± SD (n = 4). (d) Representative flow cytometry plots illustrating the expression of memory markers in Ctrl cells and p40-Td cells at day 12 post-transduction (n = 4). (e) Mean fluorescence intensity (MFI) of exhaustion and inhibitory receptors in Ctrl cells and p40-Td cells at day 12 post-transduction. Data shown as mean ± SD (n = 4).
Supplementary Fig. 2 p40-Td cells show enhanced proliferation and survival upon activation.
(a) Frequency of Ki67+ cells in CD4+ and CD8+ T cells of Ctrl cells and p40-Td cells stimulated with αCD3 and αCD28 Abs for 3 days. Data shown as individual value, mean ± SD (n = 5). p=0.0051 for CD4+T cells; p=0.0038 for CD8+ T cells determined by repeated measure 2-way ANOVA with Sidak post hoc test. (b) Expansion index of CellTrace Violet labeled Ctrl cells and p40-Td cells stimulated with αCD3 and αCD28 Abs for 5 days. Data shown as individual value, mean ± SD (n = 3). **p=0.0056 for CD4+; **p=0.0014 for CD8+ T cells determined by repeated measure 2-way ANOVA with Sidak post hoc test. (c) Representative flow plots (left panel) and summary (right panel) of Annexin V and 7AAD staining of Ctrl cells and p40-Td cells stimulated with αCD3 and αCD28 Abs for 5 days. Data shown as individual value, mean ± SD (n = 4). ****p<0.0001 determined by repeated measure 2-way ANOVA with Sidak post hoc test.
Supplementary Fig. 3 IL12 produced by p40-Td cells is insufficient to induce IL12 signaling.
Naïve CD4+ T cells were isolated from human peripheral blood mononuclear cells and activated with αCD3 and αCD28 Abs for 2 days with addition of (a) recombinant human IL12 in escalating concentration or (b) supernatant of activated Ctrl cells or p40-Td cells. Gene expression of IL12 target genes IFNG and TBX21 was measured by qRT-PCR. Data shown as individual values and mean ± SD (n = 3 for (a) and n = 4 for (b)). (c) Ctrl cells and p40-Td cells were activated with αCD3 and αCD28 Abs, and IFN-γ secretion was measured at 24 hrs post activation by ELISA. Data shown as individual values and mean ± SD (n = 4). (d) Ctrl cells and p40-Td cells were activated with αCD3 and αCD28 Abs for 2 days, and expression of TBX21 gene was measured by qPCR. Data shown as individual values and mean ± SD (n = 4).
Supplementary Fig. 4 Activation-inducible IL23R/IL23A gene expression and IL23 secretion.
Ex-TM cells (a-b) and p40-Td cells (c) were rested or stimulated with αCD3 and αCD28 Abs for 48 hrs and 5 days or stimulated for 48 hrs and then rested for additional 3 days (a-b) and 6 days (c). Gene expression of IL23R and IL23A was measured by qPCR at the indicated time points (a-b). IL23 protein in the supernatant was measured by ELISA (c). Data shown as individual values, mean ± SD (n = 3). (d) T cells transduced with the tyrosinase-specific TCR were rested (No stim) or stimulated with T2 cells pulsed with 1μM tyrosinase peptide for 24 hrs. Gene expression of IL23R and IL23A was measured by qPCR. Data shown as individual values, mean ± SD from 3 experiments using T cells obtained from a healthy donor.
Supplementary Fig. 5 p40 expression enhances the proliferation of CAR T cells targeting neuroblastoma.
(a) Schematic of the CAR design of the GD2-specific CAR (CAR) targeting neuroblastoma. (b) Representative flow plots (left panel) and summary (right panel) illustrating the co-expression of CAR and ΔNGFR in Ctrl CAR cells (CAR.Ctrl) and CAR cells expressing p40 (CAR.p40-Td). Data shown as individual values, mean ± SD (n = 4). (c) T cell counts of CAR.Ctrl cells or CAR.p40-Td cells activated by plate-bound anti- idiotype 1A7 Ab. Data shown as mean ± SD (n = 5). ****p<0.0001 determined by repeated measure 2-way ANOVA with Sidak post hoc test. (d) Frequency of apoptotic cells in CAR.Ctrl cells and CAR.p40-Td cells activated by the 1A7 Ab for 5 days using the Annexin V/7AAD staining. Data shown as individual values and mean ± SD (n = 4). ***p=0.0006 for live cells; ***p=0.0005 for late apoptotic cells determined by repeated measure 2-way ANOVA with Sidak post hoc test. (e) Detection of IL23 and IL2 in the supernatant of CAR.Ctrl cells and CAR.p40-Td cells post-stimulation with the 1A7 Ab. Data shown as individual values and mean ± SD (n = 3).
Supplementary Fig. 6 p40 expression enhances the anti-tumor activity of CAR T cells targeting neuroblastoma.
(a) Schematics of the repetitive coculture experiment. (b) CAR expression in T cells upon repetitive coculture. Data shown as individual value, mean ± SD (n = 3). (c) Tumor cell counts after multiple round coculture (R1, R2 and R3) of LAN-1 cells with CAR T cells cultured in IL2 instead of IL7 and IL15. Data shown as individual value, mean ± SD (n = 3). ***p=0.0007; ****p<0.0001 determined by repeated measure 2-way ANOVA with Sidak post hoc test.
Supplementary Fig. 7 p40 expression enhances the anti-tumor activity of CAR T cells encoding the 4-1BB endodomain.
(a) Schematics of CAR design of the GD2-specific CAR encoding the 4-1BB endodomain (GD2.CAR) and CAR expression in T cells. Data shown are representative of 10 independent experiments. (b-e) Counts of neuroblastoma tumor cells (LAN-1 (b-c) and CHLA-255 (d-e)) and CAR T cells after each round of repetitive coculture (R1, R2 and R3) with either control (Ctrl.) T cells, GD2.CAR T cells with 4-1BB endodomain (CAR.BB.Ctrl) or GD2.CAR T cells with 4-1BB endodomain coexpressing p40 (CAR.BB.p40-Td). Data shown as individual values and mean ± SD (n = 8 for LAN1, n =10 for CHLA). **p=0.008; ****p<0.0001 determined by repeated measure 2-way ANOVA with Sidak post hoc test. (f) Tumor bioluminescence (BLI) in mice engrafted with the neuroblastoma cell line CHA-225 and treated with 2 x 106 Ctrl cells, CAR.BB.Ctrl cells or CAR.BB.p40-Td cells (n = 3 mice/group).
Supplementary Fig. 8 p40 expression enhances the antitumor activity of B7-H3-specific CAR T cells.
(a) Schematics of the B7-H3-specific CAR design and CAR expression in T cells. Data shown are representative of 5 independent experiments. (b) Longer follow-up for the experiment described in Fig. 4c. Data shown are collected from 1 mouse experiment with 5 mice per group. (c) CAR expression in human T cells collected at the end of the in vivo experiment in the BXPC3 pancreatic tumor model. N.D.: not determined due to insufficient T cell events for analysis. Data shown as individual value and mean ± SD (n = 5 mice/group).
Supplementary Fig. 9 Immunohistochemistry staining determining infiltrating immune cells and tumor-associated fibroblasts in the tumor model in which KPC-mB7H3 tumor cells are orthotopically engrafted in immunocompetent mice.
(a) C57BL/6 mice were orthotopically engrafted with the KPC-mB7H3 pancreatic tumor cells. Tumors were collected at day 40 post-tumor engraftment and immunohistochemistry staining was performed using the indicated Abs. Section of tumor and adjacent normal tissue are shown. The scale bars indicate 100 μm. Data representative of 2 mice of 2 independent experiments.
Supplementary Fig. 10 Murine CAR T cells targeting murine B7-H3.
(a) Schematics of the B7-H3-specific CAR targeting murine B7-H3 and encoding murine CD28 and CD33ζ chain (mB7-H3.CAR) (upper panel). CAR expression in murine T cells co-transduced with an empty vector (EV) or a vector encoding murine p40 (mp40-Td). NT indicate control T cells non transduced. Data shown are representative of 3 independent experiments.
Supplementary Fig. 11 CAR T cells equipped with p40 show superior proliferative capacity upon stimulation and anti-tumor effects in vitro compared to CAR T cells engineered with IL18 or IL15.
(a) Schematics of IL18 expression vector and CAR.IL15-Td and expression of CAR molecule on CAR.Ctrl cells, CAR.p40-Td cells, CAR.IL18-Td cells and CAR.IL15-Td cells. Data shown are representative of 3 independent experiments. (b) Cell counts of CAR.Ctrl cells, CAR.p40-Td cells, CAR.IL18-Td cells and CAR.IL15-Td cells upon stimulation with the 1A7 Ab or cultured in media without addition of cytokines for the indicated days. Data shown as mean ± SD (n = 4). ****p<0.0001 determined by repeated measure 2-way ANOVA with Sidak post hoc test for cell count at day 5 post stimulation/culture. (c) Repetitive coculture assay described in Supplementary Fig. 6a with LAN-1 and CHLA-255 neuroblastoma tumor cells and GD2 specific CAR.Ctrl cells, CAR.p40-Td cells, CAR.IL18-Td cells and CAR.IL15-Td cells. Tumor cell numbers after each round of coculture (R1, R2 and R3) are shown. Data presented as individual values, mean ± SD (n = 3). **p=0.0028’; ***p=0.0003; **** p<0.0001 determined by repeated measure 2-way ANOVA with Sidak post hoc test.
Supplementary Fig. 12 CAR T cells equipped with p40 show superior anti-tumor effects in vivo as compared to CAR T cells engineered with IL18 or IL15.
NSG mice were systemically engrafted with the neuroblastoma cell line CHLA-255. Two weeks later, mice were treated with 2 x 106 CAR T cells. Data shown are collected from 2 independent experiments (n = 6 for Ctrl cells, n = 5 for CAR.Ctrl cells, n = 6 for CAR.p40-Td cells, n = 9 for CAR.IL18Td cells, n = 6 for CAR.IL15-Td cells). (a-b) Tumor progression indicated by tumor BLI signal was monitored by IVIS imaging. Mice euthanized unrelated to tumor growth are indicated by ”*”, and weight loss for each mice is shown in Supplementary Fig. 13. (c) Kaplan-Meier survival curve. *:p=0.0159; **p=0.0014; ***p=0.0004 determined by Log-rank (Mantel-Cox) test. Deaths due to tumor progression or weight loss were recorded.
Supplementary Fig. 13 CAR T cells equipped with p40 show superior safety profile as compared to CAR T cells engineered with IL18 or IL15.
(a) Weight loss of mice engrafted with CHLA-255 and treated with CAR T cells. * Indicates euthanasia due to excessive weight loss. Data shown are cumulative measurements from 2 independent experiments (n = 5 for CAR.Ctrl cells, n = 6 for CAR.p40-Td cells, n = 9 for CAR.IL18Td cells, n = 6 for CAR.IL15-Td cells).
Supplementary Fig. 14 OT-1-specific T cells co-expressing p40 promote superior antitumor effects as compared to OT-1 T cells co-expressing IL18 or IL15 in vivo.
(a) B16-OVA model described in Fig. 5e. Tumor bearing mice were treated with 2 x 106 OT-1 T cells modified to express murine p40 (OT1-mp40-Td), murine IL18 (OT1-mIL18-Td) or murine IL15 (OT1-mIL15-Td). Data shown as mean ± SD tumor volume post tumor engraftment from 2 independent experiments. Data shown as mean tumor volume ± SD (n = 7 for WT cells, OT1-EV cells, OT1-mIL18-Td cells and OT1-mIL15-Td cells, n = 6 for OT1-mp40-Td cells). EV indicates empty vector.
Supplementary Fig. 15 Engineered IL23 functions predominantly through an autocrine mode of action.
(a) ΔCD19-Td cells were rested or activated with αCD3 and αCD28 Abs for 7 days with or without 50 ng/mL recombinant human IL23 (rhIL23). Cell number were numerated by beads counting. Data shown as individual values and mean ± SD (n = 3). **p=0.0018 determined by repeated measure 2-way NAOVA with Sidak post hoc test. (b) Supernatants were collected from Ctrl cells or p40-Td cells incubated with murine splenocytes with or without recombinant human IL23 receptor (rhIL23R) protein. Murine IL17 released by the splenocytes in response to IL23 was measured by ELISA. Data are presented as individual values and mean ± SD (n = 4). p=0.0122 for Ctrl cells vs. p40-Td cells; p=0.0308 for p40-Td cells vs. p40-Td cells plus rhIL23R determined by repeated measure 1-way NAOVA with Sidak post hoc test.
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Ma, X., Shou, P., Smith, C. et al. Interleukin-23 engineering improves CAR T cell function in solid tumors. Nat Biotechnol 38, 448–459 (2020). https://doi.org/10.1038/s41587-019-0398-2
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DOI: https://doi.org/10.1038/s41587-019-0398-2
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