Abstract
In this prospective, interventional phase 1 study for individuals with advanced sarcoma, we infused autologous HER2-specific chimeric antigen receptor T cells (HER2 CAR T cells) after lymphodepletion with fludarabine (Flu) ± cyclophosphamide (Cy): 1 × 108 T cells per m2 after Flu (cohort A) or Flu/Cy (cohort B) and 1 × 108 CAR+ T cells per m2 after Flu/Cy (cohort C). The primary outcome was assessment of safety of one dose of HER2 CAR T cells after lymphodepletion. Determination of antitumor responses was the secondary outcome. Thirteen individuals were treated in 14 enrollments, and seven received multiple infusions. HER2 CAR T cells expanded after 19 of 21 infusions. Nine of 12 individuals in cohorts A and B developed grade 1–2 cytokine release syndrome. Two individuals in cohort C experienced dose-limiting toxicity with grade 3–4 cytokine release syndrome. Antitumor activity was observed with clinical benefit in 50% of individuals treated. The tumor samples analyzed showed spatial heterogeneity of immune cells and clustering by sarcoma type and by treatment response. Our results affirm HER2 as a CAR T cell target and demonstrate the safety of this therapeutic approach in sarcoma. ClinicalTrials.gov registration: NCT00902044.
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Data availability
Additional information may be requested from the corresponding authors if in alignment with the study consent and deidentifiable to protect research participant privacy. All other data supporting the findings of this study are available within the article and Supplementary Information. Source data are provided with this paper.
References
Isakoff, M. S., Bielack, S. S., Meltzer, P. & Gorlick, R. Osteosarcoma: current treatment and a collaborative pathway to success. J. Clin. Oncol. 33, 3029–3035 (2015).
Oh, D. Y. & Bang, Y. J. HER2-targeted therapies—a role beyond breast cancer. Nat. Rev. Clin. Oncol. 17, 33–48 (2020).
Ebb, D. et al. Phase II trial of trastuzumab in combination with cytotoxic chemotherapy for treatment of metastatic osteosarcoma with human epidermal growth factor receptor 2 overexpression: a report from the children’s oncology group. J. Clin. Oncol. 30, 2545–2551 (2012).
Scotlandi, K. et al. Prognostic and therapeutic relevance of HER2 expression in osteosarcoma and Ewing’s sarcoma. Eur. J. Cancer 41, 1349–1361 (2005).
Ahmed, N. et al. Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression. Mol. Ther. 17, 1779–1787 (2009).
Majzner, R. G. et al. Tuning the antigen density requirement for CAR T-cell activity. Cancer Discov. 10, 702–723 (2020).
Heitzeneder, S. et al. GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity. Cancer Cell 40, 53–69 (2022).
Ahmed, N. et al. Human epidermal growth factor receptor 2 (HER2)-specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma. J. Clin. Oncol. 33, 1688–1696 (2015).
Ahmed, N. et al. HER2-specific chimeric antigen receptor-modified virus-specific T cells for progressive glioblastoma: a phase 1 dose-escalation trial. JAMA Oncol. 3, 1094–1101 (2017).
Xu, X. J., Zhao, H. Z. & Tang, Y. M. Efficacy and safety of adoptive immunotherapy using anti-CD19 chimeric antigen receptor transduced T-cells: a systematic review of phase I clinical trials. Leuk. Lymphoma 54, 255–260 (2013).
Mueller, K. T. et al. Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia. Blood 130, 2317–2325 (2017).
Straathof, K. et al. Antitumor activity without on-target off-tumor toxicity of GD2-chimeric antigen receptor T cells in patients with neuroblastoma. Sci. Transl. Med. 12, eabd6169 (2020).
Majzner, R. G. et al. GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature 603, 934–941 (2022).
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).
Narayan, V. et al. PSMA-targeting TGFβ-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: a phase 1 trial. Nat. Med. 28, 724–734 (2022).
Srivastava, S. et al. Immunogenic chemotherapy enhances recruitment of CAR-T cells to lung tumors and improves antitumor efficacy when combined with checkpoint blockade. Cancer Cell 39, 193–208 (2021).
Del Bufalo, F. et al. GD2-CART01 for relapsed or refractory high-risk neuroblastoma. N. Engl. J. Med. 388, 1284–1295 (2023).
Shum, T. et al. Constitutive signaling from an engineered IL7 receptor promotes durable tumor elimination by tumor-redirected T cells. Cancer Discov. 7, 1238–1247 (2017).
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).
Morgan, R. A. et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol. Ther. 18, 843–851 (2010).
Schneeweiss, A. et al. Long-term efficacy analysis of the randomised, phase II TRYPHAENA cardiac safety study: evaluating pertuzumab and trastuzumab plus standard neoadjuvant anthracycline-containing and anthracycline-free chemotherapy regimens in patients with HER2-positive early breast cancer. Eur. J. Cancer 89, 27–35 (2018).
Feng, K. et al. Phase I study of chimeric antigen receptor modified T cells in treating HER2-positive advanced biliary tract cancers and pancreatic cancers. Protein Cell 9, 838–847 (2018).
Hegde, M. et al. Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma. Nat. Commun. 11, 3549 (2020).
Lee, D. W. et al. ASTCT consensus grading for cytokine release syndrome and neurologic toxicity associated with immune effector cells. Biol. Blood Marrow Transplant 25, 625–638 (2019).
Kempf-Bielack, B. et al. Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS). J. Clin. Oncol. 23, 559–568 (2005).
Pappo, A. S. & Dirksen, U. Rhabdomyosarcoma, Ewing sarcoma, and other round cell sarcomas. J. Clin. Oncol. 36, 168–179 (2018).
Robbins, P. F. et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J. Clin. Oncol. 29, 917–924 (2011).
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).
Terry, R. L. et al. Chimeric antigen receptor T cell therapy and the immunosuppressive tumor microenvironment in pediatric sarcoma. Cancers 13, 4704 (2021).
Modak, S., Kramer, K., Gultekin, S. H., Guo, H. F. & Cheung, N. K. Monoclonal antibody 8H9 targets a novel cell surface antigen expressed by a wide spectrum of human solid tumors. Cancer Res. 61, 4048–4054 (2001).
Gorlick, R. et al. Expression of HER2/erbB-2 correlates with survival in osteosarcoma. J. Clin. Oncol. 17, 2781–2788, (1999).
Parsons, D. W. et al. Actionable tumor alterations and treatment protocol enrollment of pediatric and young adult patients with refractory cancers in the National Cancer Institute-Children’s Oncology Group pediatric MATCH trial. J. Clin. Oncol. 40, 2224–2234 (2022).
O’Rourke, D. M. et al. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci. Transl. Med. 9, eaaa0984 (2017).
Davila, M. L. et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci. Transl. Med. 6, 224ra225 (2014).
Lee, D. W. et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 385, 517–528 (2015).
Portera, C. C. et al. Cardiac toxicity and efficacy of trastuzumab combined with pertuzumab in patients with [corrected] human epidermal growth factor receptor 2-positive metastatic breast cancer. Clin. Cancer Res. 14, 2710–2716 (2008).
Seidman, A. et al. Cardiac dysfunction in the trastuzumab clinical trials experience. J. Clin. Oncol. 20, 1215–1221 (2002).
Slamon, D. J. et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N. Engl. J. Med. 344, 783–792 (2001).
Stein, A. M. et al. Tisagenlecleucel model-based cellular kinetic analysis of chimeric antigen receptor-T cells. CPT Pharmacometrics Syst. Pharmacol. 8, 285–295 (2019).
Dudley, M. E. et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J. Clin. Oncol. 23, 2346–2357 (2005).
Heczey, A. et al. Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results. Nat. Med. 29, 1379–1388 (2023).
Lagmay, J. P. et al. Outcome of patients with recurrent osteosarcoma enrolled in seven phase II trials through Children’s Cancer Group, Pediatric Oncology Group, and Children’s Oncology Group: learning from the past to move forward. J. Clin. Oncol. 34, 3031–3038 (2016).
Smeland, S. et al. Survival and prognosis with osteosarcoma: outcomes in more than 2000 patients in the EURAMOS-1 (European and American Osteosarcoma Study) cohort. Eur. J. Cancer 109, 36–50 (2019).
Janeway, K. A. et al. Outcome for adolescent and young adult patients with osteosarcoma: a report from the Children’s Oncology Group. Cancer 118, 4597–4605 (2012).
Gardner, R. et al. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy. Blood 127, 2406–2410 (2016).
Grupp, S. A. et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N. Engl. J. Med. 368, 1509–1518 (2013).
Binnewies, M. et al. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat. Med. 24, 541–550 (2018).
Brohl, A. S. et al. Immuno-transcriptomic profiling of extracranial pediatric solid malignancies. Cell Rep. 37, 110047 (2021).
Das, R. K., Vernau, L., Grupp, S. A. & Barrett, D. M. Naive T-cell deficits at diagnosis and after chemotherapy impair cell therapy potential in pediatric cancers. Cancer Discov. 9, 492–499 (2019).
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).
Finney, O. C. et al. CD19 CAR T cell product and disease attributes predict leukemia remission durability. J. Clin. Invest. 129, 2123–2132 (2019).
Deng, Q. et al. Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas. Nat. Med. 26, 1878–1887 (2020).
Turtle, C. J. et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J. Clin. Invest. 126, 2123–2138 (2016).
Vitanza, N. A. et al. Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis. Nat. Med. 27, 1544–1552 (2021).
Hamann, D. et al. Phenotypic and functional separation of memory and effector human CD8+ T cells. J. Exp. Med. 186, 1407–1418 (1997).
Srivastava, S. & Riddell, S. R. Chimeric antigen receptor T cell therapy: challenges to bench-to-bedside efficacy. J. Immunol. 200, 459–468 (2018).
Lee, D. W. et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood 124, 188–195 (2014).
Eisenhauer, E. A. et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur. J. Cancer 45, 228–247 (2009).
Tung, J. W. et al. Modern flow cytometry: a practical approach. Clin. Lab. Med. 27, 453–468 (2007).
Corneau, A. et al. Mass cytometry: a robust platform for the comprehensive immunomonitoring of CAR-T-cell therapies. Br. J. Haematol. 194, 788–792 (2021).
Michelozzi, I. M. et al. High-dimensional functional phenotyping of preclinical human CAR T cells using mass cytometry. STAR Protoc. 3, 101174 (2022).
Kotecha, N., Krutzik, P. O. & Irish, J. M. Web-based analysis and publication of flow cytometry experiments. Curr. Protoc. Cytom. Chapter 10, Unit 10.17 (2010).
Jia, S. F., Worth, L. L. & Kleinerman, E. S. A nude mouse model of human osteosarcoma lung metastases for evaluating new therapeutic strategies. Clin. Exp. Metastasis 17, 501–506 (1999).
Liu, C. et al. Model-based cellular kinetic analysis of chimeric antigen receptor-T cells in humans. Clin. Pharmacol. Ther. 109, 716–727 (2021).
Vrisekoop, N. et al. Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool. Proc. Natl Acad. Sci. USA 105, 6115–6120 (2008).
Merritt, C. R. et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat. Biotechnol. 38, 586–599 (2020).
Feehley, T. et al. Healthy infants harbor intestinal bacteria that protect against food allergy. Nat. Med. 25, 448–453 (2019).
Acknowledgements
We thank all the participants and their families, the medical teams involved in caring for the participants and the Good Manufacturing Practice Facility staff who assisted in T cell product manufacturing.
The clinical trial was supported by Stand Up To Cancer St. Baldrick’s Pediatric Cancer Dream Team Translational Research Grant (SU2C-AACR-DT1113). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. This work was also supported by The V Foundation for Cancer Research, Triumph Over Kids Cancer Foundation, Cookies for Kids’ Cancer Foundation and Alex’s Lemonade Stand Pediatric Cancer Foundation. The Clinical Research Center at TCH and shared resources through Dan L. Duncan Cancer Center Support Grant P30CA125123 supported the trial conduct. M.H., S.K.J., K.S. and N.A. were supported by the NCI of the NIH under the Cancer Moonshot U54 project 1U54CA232568-01. M.H., S.N., S.K.J. and N.A. were supported by the NCI of the NIH under R01CA276684-01 and by The Faris Foundation. S.N. was supported by the NCI of the NIH under award number K12CA090433 and by the Curing Kids Cancer Foundation. C.D. was supported by the NCI of the NIH under award number K12CA090433. K.S. was supported by Cancer Prevention and Research Institute of Texas (CPRIT; RP160283), and A.Z.G. was supported by CPRIT RP160283, BCM Comprehensive Cancer Training Program. M.C. was supported by an NIH T32 training grant in Cell and Gene Therapy (2T32HL092332-16). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Manufacturing of some of the CAR T cell products was supported by CPRIT RP180785 Children’s Access to Regenerative Medicine in Texas. The CyTOF analysis was performed in the Flow Cytometry & Cellular Imaging Core Facility, which is supported, in part, by the NIH through MDACC’s Support Grant P30 CA016672, the NCI’s Research Specialist 1 R50 CA243707-01A1 and a Shared Instrumentation Award from the CPRIT (RP121010). Support was received from the National Gene Vector Biorepository at Indiana University (NHLBI contract 75N9019D00018). This project used the Hillman Cytometry Facility at the University of Pittsburgh Medical Center that is supported, in part, by award P30CA047904. Bioinformatics analyses were performed by Cancer Bioinformatics Services, supported, in part, by NCI through the UPMC Hillman Cancer Center CCSG award (P30CA047904).
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M.H., S.G. and N.A. developed and implemented the clinical trial, obtained funding, designed experimental methods and performed data analysis. M.H., S.N., C.D., S.K.J., K.S., M.W., K.A.J., M.C., D.M., C.R., H.Z., B.M., M.K., R.C., S.G.T., O.D., V.S.S., B.G., N.L., A.G., G.D., T.W., M.K.B., H.E.H., W.S.W., M.J.H., S.G. and N.A. were involved in study conception or trial implementation or data acquisition, statistical analysis and results interpretation. Z.N., P.R.M., R.R.B., A.M., A.S. and C.G. performed the laboratory experiments. A.Z.G. and A.H.S. modeled cellular kinetics. M.H., M.J.H. and R.B. designed and analyzed tumor profiling. M.H., S.N. and N.A. wrote the paper. All authors were involved in the critical review and editing of the paper.
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M.H., S.K.J., V.S.S., S.G. and N.A. are named inventors on patent applications in the field of CAR T cell therapy owned by BCM. C.D. has a patent and patent applications in the field of cell and gene therapy for cancer. K.A.J. is a consultant for Bayer, Illumina and Ipsen. B.G. owns QB Regulatory Consulting, LLC, which has consulting agreements with TESSA Therapeutics, Marker Therapeutics, LOKON Pharma, AlloVir and Proxima. N.L. is a consultant with Tessa Therapeutics. M.K.B. has equity in Allovir, Marker Therapeutics and Tessa Therapeutics, has served on advisory boards for Walking Fish Therapeutics, CellGenix, Marker Therapeutics, Tessa Therapeutics, Abintus, Allogene, Bellicum Pharmaceuticals, Bluebird Bio, Athenex, Memgen, Turnstone Biologics, Coya Therapeutics, TScan Therapeutics, Onkimmune, Poseida Therapeutics and Allovir and has received research support from Tessa Therapeutics. H.E.H. has equity in Allovir and Marker Therapeutics, has served on advisory boards for Tessa Therapeutics, Novartis, Gilead, GSK, Kiadis and Fresh Wind Biotechnologies and has received research support from Tessa Therapeutics and Kuur Therapeutics. S.G. has patent applications in the fields of natural killer cell and T cell and/or gene therapy for cancer. S.G. is also a consultant of Tessa Therapeutics, a Data and Safety Monitoring Board member of Immatics, and has received honoraria from Tidal, Catamaran Bio, Sanofi and Novartis within the last 2 years. N.A. received one-time royalties from Celgene and Cell Medica, consulted in the past for Adaptimmune and continues to consult for Equillium (pro bono) and The Children’s Cancer Hospital Egypt 57357. None of these relationships conflict with the published work. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Study-related cytopenia and pre-infusion serum cytokines.
(a) Absolute lymphocyte count (ALC) on day 0 fludarabine (Flu; n = 3 participants) and Flu and cyclophosphamide (Flu/Cy; n = 11 participants) lymphodepletion (Flu vs. Flu/Cy, ***p < 0.0001, two-tailed unpaired t-test). Error bars represent Mean with SD. (b) Trend in ALC in participants receiving Flu (n = 3) compared to Flu/Cy (n = 11) during the first 6 weeks after CAR T cell infusion. Data are shown as individual values for treated participants. Solid lines represent mean values overtime. D, day. Wk, week. (c) Absolute neutrophil count (ANC) nadir by lymphodepletion received (Flu vs. Flu/Cy, ***p < 0.0001, two-tailed unpaired t-test). (d) Time to recovery from severe neutropenia in Flu/Cy group (median time: 14 days, range: 7 to 28 days, **p = 0.009, two-tailed unpaired t-test) from CAR T cell infusion. Error bars represent Median with range. In panels (c) and (d) data are shown as individual values for Flu (n = 3) and Flu/Cy (n = 11) groups. (e) Heatmap of serum cytokine concentrations after lymphodepletion and prior to first CAR T cell infusion (day 0) in participants conditioned with fludarabine (Flu; n = 3) or Flu and cyclophosphamide (Flu/Cy; n = 11). UPN, unique participant number.
Extended Data Fig. 2 Nonlinear mixed-effects modeling of cellular kinetics.
(a) and (b) show model fit to individual cellular kinetic profiles. The population predicted vs. observed concentrations were randomly distributed across the line of unity, indicating model adequacy. UPN, unique participant number. HC, historical control. (c) Visual Predictive Check of the final cellular kinetic model. Lines represent the 5th, 50th, and 95th percentiles of the prediction-corrected observations. The blue bands represent 5th and 95th percentiles, and the pink band represents 50th percentiles of the prediction-corrected simulated data. (d) Final cellular kinetic model parameter estimates and the precision associated with their estimation.
Extended Data Fig. 3 CAR T cell kinetics and proinflammatory cytokines in peripheral blood.
(a) HER2 CAR T cell levels in peripheral blood measured using quantitative polymerase chain reaction (qPCR) after repeat CAR T cell infusions given with lymphodepletion. (b) Serum pro-inflammatory cytokine levels during the first week after the CAR T cell infusion, plotted in relation to CAR T cell copy numbers detected in peripheral blood. UPN, unique participant number.
Extended Data Fig. 4 Analysis of post-treatment tumor tissue.
(a) HER2-CAR transgene detection at tumor site(s) post-treatment. (b) Hematoxylin and eosin (H&E) staining of right lung biopsy tissue in participant 7 (previously participant 5) at 16 months off therapy showing benign perivascular lymphoid aggregate (yellow arrow) adjacent to muscularized vessel with intimal hyperplasia (black arrow) markedly obscuring the vessel (left panel; 200X) and area of organizing pneumonia (white arrow) obscuring airway and alveolar architecture of lung (right panel; 200X). Representative microscopic images shown; scale bar 100 µm.
Extended Data Fig. 5 Tumor HER2 expression.
(a) Tumor HER2 expression by immunohistochemistry (IHC) prior to study enrollment. (b) HER2 IHC of lung nodule from participant 4 resected at 5.9 months post first CAR T cell infusion. Left lung nodule from participant 12 resected at 6 weeks post-CAR T cells showing (c) viable tumor cells intermixed with osteoid and extensive angiolymphatic invasion on hematoxylin and eosin (H&E) staining, and no detectable HER2 (d) and vimentin (e) on IHC (independently validated by repeat testing). (f) HER2 expression in pre-treatment tumor sample confirmed by repeat IHC, done in parallel with post-treatment tumor tissue. Panels show representative microscopic images; scale bar 20 µm.
Extended Data Fig. 6 Spatial profiling of immune markers in tumor microenvironment (TME).
(a) Representative hematoxylin and eosin (H&E) staining showing viable tumor cells and immune infiltrates (TME-1) from different sarcoma histology evaluated; scale bar 200 μm. (b) Representative bubble plots from pre-treatment tumor samples showing heterogenous expression of immune cell markers (CD68 and CD11C shown). Blue denotes DNA staining. Squares represent region of interest (ROIs) selected on GeoMx® Digital Spatial Profiler. Bubbles represent the density of immune-related protein expression within the corresponding ROI. Expression of NK cell marker CD56 (c), and immune checkpoints PD-1 (d) and CTLA4 (e) in pre-treatment tumors from 9 participants grouped by sarcoma type. Individual data points represent a distinct ROI in a tumor sample. Box plots in (d) and (e) show min to max with horizontal line at the median. Protein expression shown as Signal-to-Noise Ratio (SNR). (f) Principal Component Analysis (PCA) showing immune-related protein expression in pre- and post-treatment samples by diagnosis and by best treatment response achieved. (g) Hierarchical clustering of immune cell markers in all ROIs from pre-treatment samples.** contrast was performed comparing complete response (CR) vs. progressive disease (PD). (h) Hierarchical clustering of immune cell markers in TME-1 by cytokine release syndrome (CRS) grade. Contrast was performed in (g) and (h) using two-sided t -tests with the Benjamini-Hochberg FDR (BH-FDR) adjustment for multiple comparisons. In UPN, unique patient number. OS, osteosarcoma. RMS, rhabdomyosarcoma. PNET, Primitive Neuroectodermal Tumor. CR, complete response. SD, stable disease. PD, progressive disease.
Extended Data Fig. 7 HER2 CAR T cell product characteristics and gating strategy for mass cytometry analysis.
(a) Transduction efficiency of HER2 CAR T cell products manufactured for all study participants (n = 14). Data shown as individual values. Horizontal lines represent the median. NT, nontransduced T cells. (b) 4-hour chromium release assay demonstrating HER2-specific cytotoxic function of infused CAR T cell products (n = 14 patients). LM7 and NCI-H1299 tumor cell lines expressed HER2. K562 and MDA-MB-468 tumor cell lines were used as negative controls. Data are shown as individual values for autologous CAR T cell products tested. Error bars represent the mean + /-SD. Correlation between duration of ex vivo expansion to the proportion of (c) CD3+CD45RA+ (r = −0.688, 95% CI −0.892 to −0.248, p = 0.0065; Pearson’s correlation) and (d) CD3+CD45RO+ (r = 0.648, 95% CI 0.179 to 0.877, p = 0.012; Pearson’s correlation) cells in the final CAR T cell products prior to cryopreservation. (e) Mass cytometry (CyTOF; Fluidigm) was performed on cryopreserved CAR T cell products and corresponding NT T cell samples for a subset of participants (n = 9). Pre-conjugated metal-tagged antibodies were purchased from Fluidigm. Calibration beads (Fluidigm) were added to all samples. Data were analyzed using Cytobank v9.1 (Beckman Coulter, IN). viSNE high-dimensionality reduction analysis was performed using all 36 metal-tagged antibody parameters. Gating was done to select for intact singlets and to exclude CD19+ and CD56+ cells. The resultant analysis on T cells with z-axis coloration for CD4+ (left upper panel) and CD8 (right upper panel), respectively, is shown for a representative CAR T cell product sample. Further analysis on T cell subsets was performed to examine co-expression of activation and exhaustion markers on CD8+PD-1+ T cells (lower panel). First, CD8+ T cells were gated on PD-1+ subsets. These were then analyzed for co-expression of TIM-3, LAG-3, and CD39, respectively.
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Clinical trial protocol.
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Supplementary Data 1
Statistical design and outcomes analysis plan for the phase 1 clinical trial reported in the paper.
Supplementary Data 2
CONSORT 2010 statement for the clinical trial reported in the paper.
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Hegde, M., Navai, S., DeRenzo, C. et al. Autologous HER2-specific CAR T cells after lymphodepletion for advanced sarcoma: a phase 1 trial. Nat Cancer (2024). https://doi.org/10.1038/s43018-024-00749-6
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DOI: https://doi.org/10.1038/s43018-024-00749-6