Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma

Abstract

Refractory metastatic rhabdomyosarcoma is largely incurable. Here we analyze the response of a child with refractory bone marrow metastatic rhabdomyosarcoma to autologous HER2 CAR T cells. Three cycles of HER2 CAR T cells given after lymphodepleting chemotherapy induces remission which is consolidated with four more CAR T-cell infusions without lymphodepletion. Longitudinal immune-monitoring reveals remodeling of the T-cell receptor repertoire with immunodominant clones and serum autoantibodies reactive to oncogenic signaling pathway proteins. The disease relapses in the bone marrow at six months off-therapy. A second remission is achieved after one cycle of lymphodepletion and HER2 CAR T cells. Response consolidation with additional CAR T-cell infusions includes pembrolizumab to improve their efficacy. The patient described here is a participant in an ongoing phase I trial (NCT00902044; active, not recruiting), and is 20 months off T-cell infusions with no detectable disease at the time of this report.

Conflict of interest statement

M.H., S.K.J., T.T.B., and V.S.S. are named inventors on patent applications in the field of CAR T-cell therapy owned by Baylor College of Medicine (BCM). B.G. owns QBRegulatory Consultants, LLC (QBR) which provides clinical research regulatory and project management support to companies inclusive of Allovir, TESSA therapeutics, Marker Therapeutics Inc, and LOKON Pharma AB. H.E.H. reports ownership equity in Marker Therapeutics and Allovir, consulting with Tessa Therapeutics, Kiadis, Novartis, Gilead Biosciences, PACT Pharma, Marker Therapeutics, and Allovir, and research grants from Tessa Therapeutics and Cell Medica, outside the submitted work. M.K.B. reports ownership equity in Marker Therapeutics, Tessa Therapeutics, and Allovir, consulting with Tessa Therapeutics, Walking Fish Therapeutic, Memgen, TScan, Allogene, Marker Therapeutics, and Allovir, outside the submitted work. W.S.W. is named as an inventor on patents and patent applications in the field of cancer immunotherapy owned by Georg-Speyer-Haus. S.G. has patent applications in the fields of T-cell and/or gene therapy for cancer and a research collaboration with TESSA Therapeutics, is a DSMB member of Immatics, and on the scientific advisory board of Tidal. N.A. is named inventor on patents and patent applications owned by Baylor College of Medicine. 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 on medical education and research development. None of these relationships conflict with the published work. The remaining authors declare no competing financial interests.

Figures

Fig. 1. Clinical and pathological findings prior to enrollment, and the CAR T-cell infusion regimen.

a Histological examination showing hypocellular bone marrow (BM) containing alveolar-patterned rhabdomyosarcoma (RMS) cells on routine hematoxylin and eosin (H&E) staining and immunoreactivity to desmin and myogenin. b Positron emission tomography–computed tomography showing extensive BM involvement (upper panel) and a primary tumor in the right calf (lower panel). c Histological examination of the primary tumor showing RMS cells that are immunoreactive to desmin and myogenin. d HER2 immunoreactivity (grade 3, intensity score 3+) of the primary tumor and BM metastasis at baseline prior to study enrollment. Panels (a, c, d) show representative microscopic images; scale bar 100 µm. e Schematic outline of components of the HER2 CAR transgene introduced by retroviral vector transduction. TM transmembrane. f HER2 CAR expression in the autologous T-cell product released from the good manufacturing practice (GMP) laboratories for infusion. g Treatment regimen, including the induction and consolidation phases. Autologous HER2 CAR T-cell dose was 1 × 108 cells/m2 for each infusion. H&E hematoxylin and eosin stain. Cy/Flu cyclophosphamide and fludarabine.

Fig. 2. Measurement of serum cytokines and monitoring of HER2 CAR T cells after infusion.

a Analysis of serum cytokines after Cy/Flu administration and prior to T-cell infusion on day 0 showing the difference in IL-15 levels with (n = 3 infusion cycles, data presented as mean values ± standard deviation) and without (n = 2 infusions) lymphodepletion. b Kinetics of serum IL-15 levels prior to and after T-cell infusions given with cytoreducing chemotherapy (n = 3 infusion cycles). c Trends in the absolute lymphocyte count (ALC; shaded gray area) and levels of the HER2 CAR transgene detected by quantitative polymerase chain reaction (qPCR; solid black line) in the peripheral blood during the induction and consolidation phase leading to the initial complete response (CR1). d Detection of the HER2 CAR transgene in the peripheral blood and corresponding bone marrow levels at 6 weeks after infusions 2 and 5. e Analysis of pro-inflammatory cytokines (IL-6, GM-CSF, IFNγ, and TNFα) in the patient’s serum before and after CAR T-cell infusion given with (n = 3 infusion cycles) and without (n = 2 infusions) lymphodepletion. In panels (a, b, e), each dot in the graph represents an average of technical replicates from a biologically distinct serum sample. Inf CAR T-cell infusion, Cy/Flu cyclophosphamide and fludarabine.

Fig. 3. Clinical and pathological findings after autologous HER2 CAR T-cell infusions.

a Histological examination of the bone marrow 4 weeks after the salvage chemotherapy (ARST0921) and prior to initiating CAR T-cell infusions showing hypocellularity and presence of rhabdomyosarcoma (RMS) cells on hematoxylin and eosin (H&E) staining and immunoreactivity to desmin and myogenin, b complete disease response (CR1), evidenced by recovery of trilineage hematopoiesis and absence of immunoreactivity to desmin and myogenin after three HER2 CAR T-cell infusions. Panels (a, b) show representative microscopic images; scale bar 100 µm. c Representative image from positron emission tomography–computed tomography (PET-CT) with no evidence of FDG-avid disease in bone marrow or other sites 6 weeks after the third HER2 CAR T-cell infusion. d Detection of HER2 CAR-expressing T cells in the peripheral blood 7 days after the second infusion using flow cytometry. HER2 CAR was specifically recognized using HER2.Fc chimeric protein followed by a goat anti-human Fc conjugated with PE as a secondary antibody. SSC side scatter. e The proportion of CD3+ HER2 CAR-expressing T cells on day +7 after each infusion during the induction period. f Histograms showing the PD-1 and LAG3 surface expression in CAR-positive CD8+ (in blue) in comparison to CAR-negative CD8+ T cells (in black) at peak expansion (day +7) after each infusion during induction, and g the corresponding median fluorescence intensity (MFI) of PD-1 and LAG3 surface expression in CAR-positive and CAR-negative CD8+ T cells.

Fig. 4. Remodeling of TCRβ repertoire following HER2 CAR T-cell infusions.

a Longitudinal homeostatic space distribution of T-cell clones from the peripheral blood (PB) categorized as hyperexpanded/large (>1% frequency of productive rearrangements), medium (0.1–1% frequency), small (more than single event, but <0.1% frequency) and rare (single rearrangement events) before and 6 weeks after the first, second and third HER2 CAR T-cell infusions. The pre-infusion sample (Pre) was obtained at 4 weeks from the prior cyclophosphamide containing chemotherapy and serves as a chemotherapy only control. b Heat map representing Morisita’s overlap index of TCRβ CDR3 rearrangements between time-matched samples from the PB and bone marrow (BM) obtained 6 weeks after the second and third CAR T-cell infusions. The overlap index has values ranging from 0 to 1 representing low to high degree of overlap, respectively. c Amino acid (AA) length distribution of the TCRβ CDR3 in peripheral blood before and 6 weeks after the first, second and third HER2 CAR T-cell infusions. d TCRβV family genes and TCRβJ family genes in the CDR3 region of peripheral blood T cells before and 6 weeks after the first, second and third HER2 CAR T-cell infusions. Only TCRβV genes with >1% of cumulative productive frequencies are represented. Complete TCRβV family gene use is provided as Supplementary Table 4. Inf infusion, L left, R right.

Fig. 5. Longitudinal tracking of productive TCRβ CDR3 rearrangements.

a Fate of TCRβ CDR3 rearrangements which developed after the initiation of HER2 CAR T-cell infusions, from top 250 rearrangements (n = 127). b Heat map representing Morisita’s overlap index of TCRβ rearrangements between the infused CAR T-cell product and longitudinal samples from the peripheral blood demonstrating restructuring of the T-cell repertoire with each CAR T-cell infusion. The overlap index has values ranging from 0 to 1 depicting low to high degree of overlap, respectively. c Hyperexpanded (defined as having >1% frequency) TCRβ CDR3 rearrangements present in the peripheral blood prior to initiation of CAR T-cell infusions and their fate over the course of induction. d Longitudinal tracking of the productive TCRβ CDR3 rearrangements expanding in the peripheral blood analyzed 6 weeks after HER2 CAR T-cell infusions given during induction phase. Eight T-cell clones that were not detected pre-infusion or in the infused T-cell product were detected in the peripheral blood following CAR T-cell infusions (highlighted in blue). e Frequency distribution of top 10 TCRβ CDR3 rearrangements present in each peripheral blood (PB) and bone marrow (BM) sample 6 weeks after the T-cell infusion, with (induction) or without (consolidation) Cy/Flu lymphodepletion, in comparison to that of pre-infusion peripheral blood and the infused CAR T-cell product. Pre-infusion peripheral blood was obtained at study entry, 4 weeks after the prior cyclophosphamide containing chemotherapy. BM samples were unavailable for analysis after infusions 1 and 6. Pre pre-infusion, Inf infusion, Cy/Flu cyclophosphamide and fludarabine, L left, R right.

Fig. 6. Autoantibody responses identified in the patient’s serum before and after CAR T-cell infusions.

a Serum IgG levels obtained in the clinical laboratory prior to initiation of CAR T-cell infusion and 10 weeks after each infusion during the first induction period. b Indirect ELISA confirming serum antibody reactivity with recombinant FUT8, USP2, RAB7B, and GSK3A post HER2 CAR T-cell infusions during the first induction, in comparison to pre-infusion sample. Serum samples from each time point were tested in four dilutions as shown. c Waterfall plot depicting proteins with ≥2 fold change in autoantibody binding signal identified by ProtoArrayTM Human Protein Microarray analysis in the patient’s serum at tumor recurrence 6 months after stopping T-cell infusions (day 546) compared to the sample obtained during remission (day 425). Pre pre-infusion, Inf infusion.

Fig. 7. HER2 CAR T-cell infusions after bone marrow relapse and monitoring during the second remission.

a Surveillance bone marrow (BM) at 6 months after stopping HER2 CAR T-cell infusions showing hypocellularity and presence of HER2-expressing RMS cells (grade 2, intensity score 2+ by immunohistochemistry). b BM showing restoration of trilineage hematopoiesis and no morphological evidence of RMS (CR2) 6 weeks after one cycle of lymphodepletion and HER2 CAR T cells (1 × 108 cells/m2). Panels (a, b) show representative microscopic images; scale bar 100 µm. c Kinetics of serum IL-15 levels before and after HER2 CAR T-cell infusions given with Cy/Fly lymphodepletion (n = 3 infusion cycles). d HER2 CAR expression in the second autologous T-cell product manufactured and infused during consolidation of CR2. e Timeline of the initial treatment course, disease relapse, re-induction of CR2, and consolidation of the response shown in a schematic diagram. PD-1 antibody, pembrolizumab, was initiated 4 weeks after confirming the CR2 and continued every 3 weeks thereafter. f Analysis of pro-inflammatory cytokines (IFN-γ, TNFα, and GM-CSF) in the patient’s serum before and after CAR T-cell infusion given with (n = 3 infusion cycles) and without (n = 2 infusions) lymphodepletion. g Longitudinal monitoring of serum IL-6 and IL-4 levels during CR2, before and after adding PD-1-blocking antibody to the CAR T-cell infusion regimen, in comparison to CR1. Solid lines represent the mean values of sample duplicates tested. h Trends in the absolute lymphocyte count (ALC; shaded gray area) and levels of the HER2 CAR transgene detected by quantitative polymerase chain reaction (qPCR; solid black line) in the peripheral blood during the treatment phase of CR2 and the follow-up period. i Detection of HER2 CAR transgene in the matched BM and peripheral blood samples at 6 weeks after T-cell infusions during CR2. In panels (c, f), each dot in the graph represents an average of technical replicates from a biologically distinct serum sample. H&E hematoxylin and eosin stain, Cy/Flu cyclophosphamide and fludarabine.