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Autologous HER2-specific CAR T cells after lymphodepletion for advanced sarcoma: a phase 1 trial

Nature Cancer (2024)Cite this article

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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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Fig. 1: HER2 CAR structure and CONSORT flow chart.
Fig. 2: Study schema and safety assessment.
Fig. 3: Homeostatic cytokines and HER2 CAR T cell kinetics.
Fig. 4: Histopathological findings, clinical responses and survival outcomes.
Fig. 5: Spatial profiling of immune markers in the TME.
Fig. 6: HER2 CAR T cell product characteristics.

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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.

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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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Author notes

  1. Christopher DeRenzo & Stephen Gottschalk

    Present address: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN, USA

Authors and Affiliations

  1. Texas Children’s Cancer and Hematology Center, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA

    Meenakshi Hegde, Shoba Navai, Christopher DeRenzo, Sujith K. Joseph, Khaled Sanber, Ahmed Z. Gad, Matthew Campbell, Dolores Mullikin, Zeid Nawas, Pretty R. Mathew, Raksha R. Bhat, Ankita Shree, Claudia Gerken, Mamta Kalra, Rikhia Chakraborty, Vita S. Salsman, Bambi Grilley, Adrian Gee, Malcolm K. Brenner, Helen E. Heslop, M. John Hicks, Stephen Gottschalk & Nabil Ahmed

  2. Center for Cell and Gene Therapy, Texas Children’s Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, TX, USA

    Meenakshi Hegde, Shoba Navai, Christopher DeRenzo, Sujith K. Joseph, Khaled Sanber, Ahmed Z. Gad, Matthew Campbell, Dolores Mullikin, Zeid Nawas, Catherine Robertson, Pretty R. Mathew, Huimin Zhang, Birju Mehta, Raksha R. Bhat, Ankita Shree, Claudia Gerken, Mamta Kalra, Sachin G. Thakkar, Olga Dakhova, Vita S. Salsman, Bambi Grilley, Natalia Lapteva, Adrian Gee, Malcolm K. Brenner, Helen E. Heslop, Stephen Gottschalk & Nabil Ahmed

  3. Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

    Meenakshi Hegde, Shoba Navai, Christopher DeRenzo, Sujith K. Joseph, Ahmed Z. Gad, Matthew Campbell, Dolores Mullikin, Zeid Nawas, Pretty R. Mathew, Raksha R. Bhat, Ankita Shree, Claudia Gerken, Mamta Kalra, Rikhia Chakraborty, Vita S. Salsman, Bambi Grilley, Adrian Gee, Malcolm K. Brenner, Helen E. Heslop, Stephen Gottschalk & Nabil Ahmed

  4. Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Meenakshi Hegde, Shoba Navai, Sujith K. Joseph, Mengfen Wu, Rikhia Chakraborty, Bambi Grilley, Tao Wang, Malcolm K. Brenner, Helen E. Heslop, Stephen Gottschalk & Nabil Ahmed

  5. Department of Medicine, Baylor College of Medicine, Houston, TX, USA

    Khaled Sanber, Malcolm K. Brenner & Helen E. Heslop

  6. Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX, USA

    Ahmed Z. Gad

  7. Department of Pediatrics, Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Katherine A. Janeway

  8. Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX, USA

    Angela Major, Natalia Lapteva, M. John Hicks & Nabil Ahmed

  9. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Gianpietro Dotti

  10. Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Riyue Bao

  11. UPMC Hillman Cancer Center, Pittsburgh, PA, USA

    Riyue Bao

  12. Department of Clinical Pharmacy, Ain Shams University, Cairo, Egypt

    Ahmed Hamed Salem

  13. Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt, Germany

    Winfried S. Wels

  14. German Cancer Consortium (DKTK), partner site Frankfurt/Mainz, Frankfurt, Germany

    Winfried S. Wels

  15. Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany

    Winfried S. Wels

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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.

Corresponding authors

Correspondence to Meenakshi Hegde or Nabil Ahmed.

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Competing interests

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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Nature Cancer thanks Richard Gorlick, Joseph Melenhorst and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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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.

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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.

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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.

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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.

Source data

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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Extended Data Table 1 Adverse events within the first 6 weeks post HER2 CAR T cell infusion
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Extended Data Table 2 CAR transduction efficiency in products and the cell dose infused
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Extended Data Table 3 HER2 grade and intensity in relation to treatment response
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Supplementary information

Supplementary Information

Clinical trial protocol.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–4.

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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