Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

Factors associated with outcomes after a second CD19-targeted CAR T-cell infusion for refractory B-cell malignancies.

CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell therapy has shown significant efficacy for relapsed or refractory (R/R) B-cell malignancies. Yet, CD19 CAR T cells fail to induce durable responses in most patients. Second infusions of CD19 CAR T cells (CART2) have been considered as a possible approach to improve outcomes. We analyzed data from 44 patients with R/R B-cell malignancies (acute lymphoblastic leukemia [ALL], n = 14; chronic lymphocytic leukemia [CLL], n = 9; non-Hodgkin lymphoma [NHL], n = 21) who received CART2 on a phase 1/2 trial (NCT01865617) at our institution. Despite a CART2 dose increase in 82% of patients, we observed a low incidence of severe toxicity after CART2 (grade ≥3 cytokine release syndrome, 9%; grade ≥3 neurotoxicity, 11%). After CART2, complete response (CR) was achieved in 22% of CLL, 19% of NHL, and 21% of ALL patients. The median durations of response after CART2 in CLL, NHL, and ALL patients were 33, 6, and 4 months, respectively. Addition of fludarabine to cyclophosphamide-based lymphodepletion before the first CAR T-cell infusion (CART1) and an increase in the CART2 dose compared with CART1 were independently associated with higher overall response rates and longer progression-free survival after CART2. We observed durable CAR T-cell persistence after CART2 in patients who received cyclophosphamide and fludarabine (Cy-Flu) lymphodepletion before CART1 and a higher CART2 compared with CART1 cell dose. The identification of 2 modifiable pretreatment factors independently associated with better outcomes after CART2 suggests strategies to improve in vivo CAR T-cell kinetics and responses after repeat CAR T-cell infusions, and has implications for the design of trials of novel CAR T-cell products after failure of prior CAR T-cell immunotherapies.

Feasibility and efficacy of CD19-targeted CAR T cells with concurrent ibrutinib for CLL after ibrutinib failure.

We previously reported durable responses in relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) patients treated with CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell immunotherapy after ibrutinib failure. Because preclinical studies showed that ibrutinib could improve CAR T cell-antitumor efficacy and reduce cytokine release syndrome (CRS), we conducted a pilot study to evaluate the safety and feasibility of administering ibrutinib concurrently with CD19 CAR T-cell immunotherapy. Nineteen CLL patients were included. The median number of prior therapies was 5, and 17 patients (89%) had high-risk cytogenetics (17p deletion and/or complex karyotype). Ibrutinib was scheduled to begin ≥2 weeks before leukapheresis and continue for ≥3 months after CAR T-cell infusion. CD19 CAR T-cell therapy with concurrent ibrutinib was well tolerated; 13 patients (68%) received ibrutinib as planned without dose reduction. The 4-week overall response rate using 2018 International Workshop on CLL (iwCLL) criteria was 83%, and 61% achieved a minimal residual disease (MRD)-negative marrow response by IGH sequencing. In this subset, the 1-year overall survival and progression-free survival (PFS) probabilities were 86% and 59%, respectively. Compared with CLL patients treated with CAR T cells without ibrutinib, CAR T cells with concurrent ibrutinib were associated with lower CRS severity and lower serum concentrations of CRS-associated cytokines, despite equivalent in vivo CAR T-cell expansion. The 1-year PFS probabilities in all evaluable patients were 38% and 50% after CD19 CAR T-cell therapy, with and without concurrent ibrutinib, respectively (P = .91). CD19 CAR T cells with concurrent ibrutinib for R/R CLL were well tolerated, with low CRS severity, and led to high rates of MRD-negative response by IGH sequencing.

High rate of durable complete remission in follicular lymphoma after CD19 CAR-T cell immunotherapy.

Patients with follicular lymphoma (FL) with early relapse after initial chemoimmunotherapy, refractory disease, or histologic transformation (tFL) have limited progression-free and overall survival. We report efficacy and long-term follow-up of 21 patients with relapsed/refractory (R/R) FL (n = 8) and tFL (n = 13) treated on a phase 1/2 clinical trial with cyclophosphamide and fludarabine lymphodepletion followed by infusion of 2 × 106 CD19-directed chimeric antigen receptor-modified T (CAR-T) cells per kilogram. The complete remission (CR) rates by the Lugano criteria were 88% and 46% for patients with FL and tFL, respectively. All patients with FL who achieved CR remained in remission at a median follow-up of 24 months. The median duration of response for patients with tFL was 10.2 months at a median follow-up of 38 months. Cytokine release syndrome occurred in 50% and 39%, and neurotoxicity in 50% and 23% of patients with FL and tFL, respectively, with no severe adverse events (grade ≥3). No significant differences in CAR-T cell in vivo expansion/persistence were observed between FL and tFL patients. CD19 CAR-T cell immunotherapy is highly effective in adults with clinically aggressive R/R FL with or without transformation, with durable remission in a high proportion of FL patients. This trial was registered at clinicaltrials.gov as #NCT01865617.

Factors associated with durable EFS in adult B-cell ALL patients achieving MRD-negative CR after CD19 CAR T-cell therapy.

Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) have produced impressive minimal residual disease-negative (MRD-negative) complete remission (CR) rates in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the factors associated with durable remissions after CAR T-cell therapy have not been fully elucidated. We studied patients with relapsed/refractory B-ALL enrolled in a phase 1/2 clinical trial evaluating lymphodepletion chemotherapy followed by CD19 CAR T-cell therapy at our institution. Forty-five (85%) of 53 patients who received CD19 CAR T-cell therapy and were evaluable for response achieved MRD-negative CR by high-resolution flow cytometry. With a median follow-up of 30.9 months, event-free survival (EFS) and overall survival (OS) were significantly better in the patients who achieved MRD-negative CR compared with those who did not (median EFS, 7.6 vs 0.8 months; P < .0001; median OS, 20.0 vs 5.0 months; P = .014). In patients who achieved MRD-negative CR by flow cytometry, absence of the index malignant clone by IGH deep sequencing was associated with better EFS (P = .034). Stepwise multivariable modeling in patients achieving MRD-negative CR showed that lower prelymphodepletion lactate dehydrogenase concentration (hazard ratio [HR], 1.38 per 100 U/L increment increase), higher prelymphodepletion platelet count (HR, 0.74 per 50 000/µL increment increase), incorporation of fludarabine into the lymphodepletion regimen (HR, 0.25), and allogeneic hematopoietic cell transplantation (HCT) after CAR T-cell therapy (HR, 0.39) were associated with better EFS. These data allow identification of patients at higher risk of relapse after CAR T-cell immunotherapy who might benefit from consolidation strategies such as allogeneic HCT. This trial was registered at www.clinicaltrials.gov as #NCT01865617.

The response to lymphodepletion impacts PFS in patients with aggressive non-Hodgkin lymphoma treated with CD19 CAR T cells.

Factors associated with durable remission after CD19 chimeric antigen receptor (CAR)-modified T-cell immunotherapy for aggressive B-cell non-Hodgkin lymphoma (NHL) have not been identified. We report multivariable analyses of factors affecting response and progression-free survival (PFS) in patients with aggressive NHL treated with cyclophosphamide and fludarabine lymphodepletion followed by 2 × 106 CD19-directed CAR T cells/kg. The best overall response rate was 51%, with 40% of patients achieving complete remission. The median PFS of patients with aggressive NHL who achieved complete remission was 20.0 months (median follow-up, 26.9 months). Multivariable analysis of clinical and treatment characteristics, serum biomarkers, and CAR T-cell manufacturing and pharmacokinetic data showed that a lower pre-lymphodepletion serum lactate dehydrogenase (LDH) level and a favorable cytokine profile, defined as serum day 0 monocyte chemoattractant protein-1 (MCP-1) and peak interleukin-7 (IL-7) concentrations above the median, were associated with better PFS. MCP-1 and IL-7 concentrations increased after lymphodepletion, and higher intensity of cyclophosphamide and fludarabine lymphodepletion was associated with higher probability of a favorable cytokine profile. PFS was superior in patients who received high-intensity lymphodepletion and achieved a favorable cytokine profile compared with those who received the same intensity of lymphodepletion without achieving a favorable cytokine profile. Even in high-risk patients with pre-lymphodepletion serum LDH levels above normal, a favorable cytokine profile after lymphodepletion was associated with a low risk of a PFS event. Strategies to augment the cytokine response to lymphodepletion could be tested in future studies of CD19 CAR T-cell immunotherapy for aggressive B-cell NHL. This trial was registered at www.clinicaltrials.gov as #NCT01865617.

Overcoming resistance to single-agent therapy for oncogenic BRAF gene fusions via combinatorial targeting of MAPK and PI3K/mTOR signaling pathways.

Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.