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

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 .

Extended Data Figure 1:. Study-related cytopenia and pre-infusion serum cytokines.

(a) Absolute lymphocyte count (ALC) on day 0 fludarabine (Flu; n=3 patients) and Flu and cyclophosphamide (Flu/Cy; n=11 patients) lymphodepletion (Flu vs. Flu/Cy, ***p<0.0001, two-tailed unpaired t-test). Error bars represent Mean with SD. (b) Trend in ALC in patients receiving Flu (n=3) compared to Flu/Cy (n=11) during the first 6 weeks after CART infusion. Data are shown as individual values for treated patients. 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 CART 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 patients conditioned with fludarabine (Flu; n=3) or Flu and cyclophosphamide (Flu/Cy; n=11).

Extended Data Figure 2:. Nonlinear mixed-effects modeling of cellular kinetics.

(a) and (b) show model fit to individual cellular kinetic profiles. UPN, unique patient number. HC, historical control. (c) Visual Predictive Check of the final cellular kinetic model. Lines represent the 5^th, 50^th and 95^th percentiles of the prediction-corrected observations. The blue bands represent 5^th and 95^th percentiles, and the pink band represents 50^th percentiles of the prediction-corrected simulated data. (d) Final cellular kinetic model parameter estimates.

Extended Data Figure 3:. CART kinetics and pro-inflammatory cytokines in peripheral blood.

(a) HER2-CART levels in peripheral blood measured using quantitative polymerase chain reaction (qPCR) after repeat CART infusions given with lymphodepletion. (b) Serum pro-inflammatory cytokine levels (right y-axis) during the first week after the CART infusion, plotted in relation to CART copy numbers (left y-axis) detected in peripheral blood. UPN, unique patient number. Pre, pre-infusion. Hr, hour. Wk, week.

Extended Data Figure 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 Patient 5/7 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 Figure 5:. Tumor HER2 expression.

(a) Tumor HER2 expression by immunohistochemistry (IHC) prior to study enrollment. (b) HER2 IHC of lung nodule from Patient 4 resected at 5.9 months post first CART infusion. Left lung nodule from Patient 12 resected at 6 weeks post-CART showing (c) viable tumor cells intermixed with osteoid and extensive angiolymphatic invasion on hematoxylin and eosin (H&E) staining, and no detectable HER2 (c) and vimentin (d) 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 Figure 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 patients 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 Figure 7:. HER2-CART product characteristics and gating strategy for mass cytometry analysis.

(a) Transduction efficiency of HER2-CART products manufactured for all study patients (n=14). Data shown as individual values. Horizontal lines represent the median. NT, non-transduced T-cells. (b) 4-hour chromium release assay demonstrating HER2-specific cytotoxic function of infused CART 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 CART 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 CART products prior to cryopreservation. (e) Mass cytometry (CyTOF; Fluidigm) was performed on cryopreserved CART products and corresponding NT T-cell samples for a subset of patients (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 CART 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.

Figure 1.. HER2-CAR structure and consort flow chart.

(a) Schema showing the components of the 2^nd-generation HER2-CAR and retroviral vector. (b) Consort flow chart summarizing the number of patients screened, enrolled, treated, and followed on the study. *One patient was reassessed for eligibility at recurrence and re-enrolled on the study.

Figure 2.. Study schema and safety assessment.

(a) Schematic representation of study screening, enrollment, treatment, and follow-up for safety and disease response assessment and other study evaluations. (b) Pro-inflammatory cytokines (IFN-γ, IL-6, IL-2, TNF-α, sIL-2R shown) measured from serum (n=8 patients) after the onset of fever following CART infusion (day +1 to +3) arranged by cytokine release syndrome (CRS) grade. Data shown as individual values. Dotted line represents the upper limit of the reference range derived from healthy donor serum in Cytokine 13 panel. UPN, Unique Patient Number. (c) Coronal view of chest computerized tomography (CT) of Patient 14 demonstrating near complete obstruction of the right middle lobe bronchus and mass effect on the bronchus intermedius and right lower lobe bronchus from the perihilar tumor post-treatment (day +4). (d) Longitudinal assessment of serum human anti-mouse antibody (HAMA) in treated patients (n=13). Dotted line represents the threshold for positive antibody activity.

Figure 3.. Homeostatic cytokines and HER2-CART cellular kinetics.

(a) Heatmaps demonstrating fold change in serum IL-15 and IL-7 levels during the first week compared to pre-CART infusion (day 0) baseline in treated patients (n=13). UPN, Unique Patient Number. (b) HER2-CART levels in the peripheral blood after the first infusion given with lymphodepletion, determined by quantitative polymerase chain reaction (qPCR) for HER2-CAR transgene detection. Flu, fludarabine. Flu/Cy, fludarabine and cyclophosphamide. Data are shown as individual values from independent samples analyzed overtime from total 14 patients. Solid lines represent the mean. (c) HER2-CART detected in peripheral blood following infusion of 1×10⁸ cells/m² after Flu in cohort A (n=3) and after Flu/Cy in cohort B (n=9), in comparison to the same dose of T-cells infused without lymphodepletion (LD; n=7) in a previous cohort of patients (historical controls). (d) Areas under the curve (AUC) were log-transformed to stabilize variances and analyzed using two-sided ANOVA with linear contrasts. Box plot (upper and lower quartiles with horizontal line at median) showing the difference in mean log₂ AUC for 6 weeks post-CART between patients in cohort A (n=3), and historical (no LD; n=7) controls (p=0.114), cohort B (n=9) and historical (no LD) controls (**p=0.002), cohort A and cohort B (p=0.264). Dots represent individual values, and diamond represents the mean. (e) Comparison of AUC between first and second treatment cycles in 5 patients receiving multiple cycles of LD and CART infusion (*p=0.029, paired two-tailed t-test).

Figure 4.. Histopathological findings, clinical responses, and survival outcomes.

Post-treatment hematoxylin and eosin (H&E)-stained biopsy of the left lung nodule in Patient 6 showing (a) dense central sclerosis with inflammatory infiltrates in the periphery (yellow arrows) and adjacent lung parenchyma (left panel) and subpleural nodule with sclerosis, inflammatory cells, and cholesterol clefts (right panel) without viable tumor identified. (b) Recurrent osteosarcoma lung metastasis in Patient 6 showing absence of HER2 expression (left panel) by immunohistochemistry (repeated independently with similar results) and positivity for vimentin (right panel). Vimentin staining was used as a control to account for the potential antigen loss during tissue processing. Panels (a) and (b) show representative microscopic images; scale bar 100 μm. (c) Swimmer plot showing disease status and treatment response for all study patients. *Denotes time point when Patient 5 is re-enrolled as a new patient (Patient 7). Kaplan-Meier estimate of (d) overall survival (OS; n=13) and (e) progression-free survival (PFS; n=14) for all study patients from the time of first HER2-CART infusion. For OS analysis, Patient 5 who was re-enrolled as Patient 7 at disease recurrence was counted according to the time from first study treatment only. UPN, unique patient number. SD, stable disease. PD, progressive disease. CR, complete response. LD, lymphodepletion.

Figure 5.. Spatial profiling of immune markers in tumor microenvironment (TME).

Total immune cell (CD45⁺) infiltrates (a), cytotoxic (CD8⁺) T-cells (b), and helper (CD4⁺) T-cells (c) in pre-treatment tumors from 9 patients grouped by sarcoma type. Protein expression shown as Signal-to-Noise Ratio (SNR). (d) Expression of B cell marker CD20 in pre-treatment samples by cytokine release syndrome (CRS) grade. Monocyte-macrophage marker CD68 by CRS grade (e) and by best response (f). In panels (a) to (f), individual data point represents a distinct region of interest (ROI) in a tumor sample (n=9 patients). UPN, unique patient number. OS, osteosarcoma. RMS, rhabdomyosarcoma. PNET, primitive neuroectodermal tumor. Gr, grade. CR, complete response. SD, stable disease. PD, progressive disease. Hierarchical clustering of immune cell markers by best treatment response in TME-1 (g) and TME-2 (h). **contrasts were performed by comparing CR vs. PD. (i) Hierarchical clustering of immune-related protein expression in TME-2 from pre- and post-treatment samples. **feature used for performing contrast comparison by two-sided t-tests with the Benjamini-Hochberg FDR (BH-FDR) adjustment for multiple comparisons.

Figure 6.. HER2-CART product characteristics.

(a) Heatmap shows the CART product (n=14) immunophenotype assessed prior to cryopreservation using flow cytometry. UPN, unique patient number. (b) CART product composition in CD8⁺ and CD4⁺ T-cell subsets shown by sarcoma type. OS, osteosarcoma. RMS, rhabdomyosarcoma. T_N/T_SCM, naïve/stem cell memory (CD45RO⁺CD62L⁺); T_CM, central memory (CD45RO⁺CD62L⁺ ); T_EM, effector memory (CD45RO⁺CCR7⁻CD62L⁻). Data are shown as individual values for autologous CART products (n=14). Error bars represent the median with 95% CI. two-tailed Mann-Whitney test, ns p >0.05. (c) Floating bars (min to max) showing CD8⁺ and CD4⁺ T-cell immunophenotype in CART products from patients achieving complete response (CR; n=3)) compared to others (n=11). SD, stable disease. PD, progressive disease. Lines represent the median. Two-tailed Mann-Whitney test, ns, p >0.05. (d) Proportion of CD8⁺CD45RA⁺CD27⁻ effector T-cells in CART products shown by best treatment response (*p=0.022, two-tailed Mann-Whitney test). Data are shown as individual values for each product tested (n=14). Lines represent median. (e) Single cell analysis of cryopreserved CART products (n=9) using mass cytometry (CyTOF) showing % of CD8⁺PD-1⁺ (*p=0.018, two-tailed unpaired t-test) and CD4⁺PD-1⁺ (**p=0.004, two-tailed unpaired t-test) T-cells from patients with osteosarcoma (n=4), in comparison to rhabdomyosarcoma (n=4) and primitive neuroectodermal tumor (not compared as n=1). Data are shown as individual values. Lines represent median. (f) Single-cell analysis (CyTOF) of cryopreserved CART products (n=9) showing the co-expression of immune checkpoint receptors in CD8⁺ T-cell subsets: % of CD8⁺PD-1⁺TIM-3⁺ (p=0.261, two-tailed Mann-Whitney test), % of CD8⁺PD-1⁺LAG-3⁺ (p=0.095, two-tailed Mann-Whitney test), and % of CD8⁺PD-1⁺CD39⁺ (p=0.166, two-tailed Mann-Whitney test) in patients with CR (n=3) vs. patients with SD/PD (n=6). Data are shown as individual values in box plots (min to max) with a horizontal line at the median.