Impact of prior therapies and subsequent transplantation on outcomes in adult patients with relapsed or refractory B-cell acute lymphoblastic leukemia treated with brexucabtagene autoleucel in ZUMA-3.

BACKGROUND: Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved in the USA for adults with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) and in the European Union for patients ≥26 years with R/R B-ALL. After 2 years of follow-up in ZUMA-3, the overall complete remission (CR) rate (CR+CR with incomplete hematological recovery (CRi)) was 73%, and the median overall survival (OS) was 25.4 months in 78 Phase 1 and 2 patients with R/R B-ALL who received the pivotal dose of brexu-cel. Outcomes by prior therapies and subsequent allogeneic stem cell transplantation (alloSCT) are reported. METHODS: Eligible adults had R/R B-ALL and received one infusion of brexu-cel (1×106 CAR T cells/kg) following conditioning chemotherapy. The primary endpoint was the CR/CRi rate per central review. Post hoc subgroup analyses were exploratory with descriptive statistics provided. RESULTS: Phase 1 and 2 patients (N=78) were included with median follow-up of 29.7 months (range, 20.7-58.3). High CR/CRi rates were observed across all prior therapy subgroups examined: 1 prior line of therapy (87%, n=15) and ≥2 prior lines (70%, n=63); prior blinatumomab (63%, n=38) and no prior blinatumomab (83%, n=40); prior inotuzumab (59%, n=17) and no prior inotuzumab (77%, n=61); and prior alloSCT (76%, n=29) and no prior alloSCT (71%, n=49). The frequency of Grade ≥3 cytokine release syndrome, neurological events, and treatment-related Grade 5 adverse events were largely similar among prior therapy subgroups.Median duration of remission (DOR) in responders with (n=14) and without (n=43) subsequent alloSCT was 44.2 (95% CI, 8.1 to not estimable (NE)) and 18.6 months (95% CI, 9.4 to NE); median OS was 47.0 months (95% CI, 10.2 to NE) and not reached (95% CI, 23.2 to NE), respectively. Median DOR and OS were not reached in responders without prior or subsequent alloSCT (n=22). CONCLUSIONS: In ZUMA-3, adults with R/R B-ALL benefited from brexu-cel, regardless of prior therapies and subsequent alloSCT status, though survival appeared better in patients without certain prior therapies and in earlier lines of therapy. Additional studies are needed to determine the impact prior therapies and subsequent alloSCT have on outcomes of patients who receive brexu-cel.

Three-year results from phase I of ZUMA-4: KTE-X19 in pediatric relapsed/refractory acute lymphoblastic leukemia.

Here we present the 3-year results of ZUMA-4, a phase I/II multicenter study evaluating the safety and efficacy of KTEX19, an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, in pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia. Phase I explored two dose levels and formulations. The primary endpoint was the incidence of dose-limiting toxicities. Thirty-one patients were enrolled; KTE-X19 was administered to 24 patients (median age 13.5 years, range 3-20; median follow-up 36.1 months). No dose-limiting toxicities were observed. All treated patients had grade ≥3 adverse events, commonly hypotension (50%) and anemia (42%). Grade 3 cytokine release syndrome rates were 33% in all treated patients, 75% in patients given the dose of 2×106 CAR T cells/kg, 27% in patients given the dose of 1×106 cells/kg in the 68 mL formulation, and 22% in patients given the dose of 1×106 cells/kg in the 40 mL formulation; the percentages of patients experiencing grade ≥3 neurologic events were 21%, 25%, 27%, and 11% respectively. Overall complete remission rates (including complete remission with incomplete hematologic recovery) were 67% in all treated patients, 75% in patients given 2×106 CAR T cells/kg, 64% in patients given 1×106 cells/kg in the 68 mL formulation, and 67% in patients given 1×106 cells/kg in the 40 mL formulation. Overall minimal residual diseasenegativity rates were 100% among responders; 88% of responders underwent subsequent allogeneic stem-cell transplantation. In the 1×106 (40 mL) group (recommended phase II dose), the median duration of remission censored at allogeneic stem-cell transplantation and median overall survival were not reached. Pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia achieved high minimal residual disease-negative remission rates with a manageable safety profile after a single dose of KTE-X19. Phase II of the study is ongoing at the dose of 1×106 CAR T cells/kg in the 40 mL formulation. ClinicalTrials.gov: NCT02625480.

Two-year follow-up of KTE-X19 in patients with relapsed or refractory adult B-cell acute lymphoblastic leukemia in ZUMA-3 and its contextualization with SCHOLAR-3, an external historical control study.

BACKGROUND: Brexucabtagene autoleucel (KTE-X19) is an autologous anti-CD19 CAR T-cell therapy approved in the USA to treat adult patients with relapsed or refractory B-precursor acute lymphoblastic leukemia (R/R B-ALL) based on ZUMA-3 study results. We report updated ZUMA-3 outcomes with longer follow-up and an extended data set along with contextualization of outcomes to historical standard of care. METHODS: Adults with R/R B-ALL received a single infusion of KTE-X19 (1 × 106 CAR T cells/kg). Long-term post hoc subgroup assessments of ZUMA-3 were conducted. Outcomes from matched patients between historical clinical trials and ZUMA-3 patients were assessed in the retrospective historical control study SCHOLAR-3. RESULTS: After 26.8-months median follow-up, the overall complete remission (CR) rate (CR + CR with incomplete hematological recovery) among treated patients (N = 55) in phase 2 was 71% (56% CR rate); medians for duration of remission and overall survival (OS) were 14.6 and 25.4 months, respectively. Most patients responded to KTE-X19 regardless of age or baseline bone marrow blast percentage, but less so in patients with > 75% blasts. No new safety signals were observed. Similar outcomes were observed in a pooled analysis of phase 1 and 2 patients (N = 78). In SCHOLAR-3, the median OS for treated patients from ZUMA-3 (N = 49) and matched historical controls (N = 40) was 25.4 and 5.5 months, respectively. CONCLUSIONS: These data, representing the longest follow-up of CAR T-cell therapy in a multicenter study of adult R/R B-ALL, suggest that KTE-X19 provides a clinically meaningful survival benefit with manageable toxicity in this population. TRIAL REGISTRATION: NCT02614066.

Aberrant Lck Signal via CD28 Costimulation Augments Antigen-Specific Functionality and Tumor Control by Redirected T Cells with PD-1 Blockade in Humanized Mice.

Purpose: Combination therapy of adoptively transferred redirected T cells and checkpoint inhibitors aims for higher response rates in tumors poorly responsive to immunotherapy like malignant pleural mesothelioma (MPM). Only most recently the issue of an optimally active chimeric antigen receptor (CAR) and the combination with checkpoint inhibitors is starting to be addressed.Experimental Design: Fibroblast activation protein (FAP)-specific CARs with different costimulatory domains, including CD28, Δ-CD28 (lacking lck binding moiety), or 4-1BB were established. CAR-T cells were characterized in vitro and antitumor efficacy was tested in vivo in a humanized mouse model in combination with PD-1 blockade. Finally, the Δ-CD28 CAR was tested clinically in a patient with MPM.Results: All the three CARs demonstrated FAP-specific functionality in vitro Gene expression data indicated a distinct activity profile for the Δ-CD28 CAR, including higher expression of genes involved in cell division, glycolysis, fatty acid oxidation, and oxidative phosphorylation. In vivo, only T cells expressing the Δ-CD28 CAR in combination with PD-1 blockade controlled tumor growth. When injected into the pleural effusion of a patient with MPM, the Δ-CD28 CAR could be detected for up to 21 days and showed functionality.Conclusions: Overall, anti-FAP-Δ-CD28/CD3ζ CAR T cells revealed superior in vitro functionality, better tumor control in combination with PD-1 blockade in humanized mice, and persistence up to 21 days in a patient with MPM. Therefore, further clinical investigation of this optimized CAR is warranted. Clin Cancer Res; 24(16); 3981-93. ©2018 AACR.

Antigen-specific in vitro expansion of functional redirected NY-ESO-1-specific human CD8+ T-cells in a cell-free system.

BACKGROUND: Tumors can be targeted by the adoptive transfer of chimeric antigen receptor (CAR) redirected T-cells. Antigen-specific expansion protocols are needed to generate large quantities of redirected T-cells. We aimed to establish a protocol to expand functional active NY-ESO-1-specific redirected human CD8(+) T-cells. MATERIALS AND METHODS: The anti-idiotypic Fab antibody A4 with specificity for HLA-A 0201/NY-ESO-1157-165 was tested by competition assays using a HLA-A 0201/NY-ESO-1157-165 tetramer. HLA-A 0201/NY-ESO-1157-165 redirected T-cells were generated, expanded and tested for CAR expression, cytokine release, in vitro cytolysis and protection against xenografted HLA-A 0201/NY-ESO-1157-165-positive multiple myeloma cells. RESULTS: A4 demonstrated antigen-specific binding to HLA-A 0201/NY-ESO-1157-165 redirected T-cells. Expansion with A4 resulted in 98% of HLA-A 0201/NY-ESO-1157-165 redirected T-cells. A4 induced strong proliferation, resulting in a 300-fold increase of redirected T-cells. After expansion protocols, redirected T-cells secreted Interleukin-2, (IL-2), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) and lysed target cells in vitro and were protective in vivo. CONCLUSION: A4 expanded HLA-A 0201/NY-ESO-1157-165 redirected T-cells with preservation of antigen-specific function.

Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy. METHODS: To evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice. RESULTS: FAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice. CONCLUSION: FAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells.

Re-directed T cells for the treatment of fibroblast activation protein (FAP)-positive malignant pleural mesothelioma (FAPME-1).

BACKGROUND: Asbestos is the main cause of MPM in industrialized countries. Even since asbestos is banned in most developed countries, the peak wave of MPM incidence is anticipated for the next years due to the long latency of asbestos induced MPM. MPM patients not eligible for surgical procedures like decortication or pleuro-pneumectomie have a median survival of 12 months with palliative chemotherapy. Therefore, new therapeutic approaches are of crucial need in this clinical situation. METHODS/DESIGN: This is a phase I trial for patients with malignant pleural mesothelioma with pleural effusion testing the safety of a fixed single dose of 1x106 adoptively transferred FAP-specific re-directed T cells given directly in the pleural effusion. Lymphocytes will be taken 21 days before transfer from peripheral blood. CD8 positive T cells will be isolated and re-programmed by retroviral transfer of a chimeric antigen receptor recognizing FAP which serves as target structure in MPM. At day 0 of the protocol, re-directed T cells will be injected in the pleural effusion and patients will be monitored for 48h under intermediate care conditions. AE, SAE, SADR and SUSAR will be monitored for 35 days and evaluated by an independent safety board to define any dose limiting toxicity (DLT). No further patient can be treated before the previous patient passed day 14 after T cell transfer. The protocol will be judged as save when no DLT occurred in the first 3 patients, or 1 DLT in 6 patients. Secondary objectives are feasibility and immune monitoring. DISCUSSION: Adoptive T cell transfer is a new and rapidly expanding branch of immunotherapies focusing on cancer treatment. Recently, objective responses could be observed in patients with chronic lymphatic leukemia treated with adoptively transferred CD19-specific re-directed T cells. The choice of the target antigen determines the possible on-target off-tissue toxicity of such approaches. There are reports of severe toxicity in patients who received T cells intravenously due to unexpected expression of the target antigen (on-target) in other tissues than the tumor (off-tissue). To minimize the risk of on-target off-tissue toxicity and to maximize the on-target anti-tumor effect we propose a clinical protocol with loco-regional administration of re-directed T cells. FAP-specific T cells will be directly injected in the pleural effusion of patients with MPM. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01722149).

Induction of exosome release in primary B cells stimulated via CD40 and the IL-4 receptor.

Exosomes are lipid-bound nanovesicles formed by inward budding of the endosomal membrane and released following fusion of the endosomal limiting membrane with the plasma membrane. We show here that primary leukocytes do not release exosomes unless subjected to potent activation signals, such as cytokine or mitogen stimulation. In particular, high levels of exosomes were released when murine splenic B cells were stimulated via CD40 and the IL-4 receptor. This property was shared by B cells from different anatomic locations, as newly formed marginal zone and follicular B cells were capable of secreting exosomes upon CD40/IL-4 triggering. B cell exosomes expressed high levels of MHC class I, MHC class II, and CD45RA (B220), as well as components of the BCR complex, namely, surface Ig, CD19, and the tetraspanins CD9 and CD81. Ig on the plasma membrane of primary B cells was targeted to the exosome pathway, demonstrating a link between the BCR and this exocytic pathway. IgD and IgM were the predominant Ig isotypes associated with CD40/IL-4 elicited exosomes, though other isotypes (IgA, IgG1, IgG2a/2b, and IgG3) were also detected. Together, these results suggest that exosome release is not constitutive activity of B cells, but may be induced following cell: cell signaling.