Ruxolitinib mitigates steroid‐refractory CRS during CAR T therapy

Abstract Cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity are two major CAR T related toxicities. With the interventions of Tocilizumab and steroids, many patients can recover from severe CRS. However, some patients are refractory to steroids and develop life‐threatening consequences. Ruxolitinib is an oral JAKs inhibitor and promising drug in inflammatory diseases. In this pilot study, we evaluate the efficacy of Ruxolitinib in CRS. Of 14 r/r B‐ALL children who received CD19 or CD22 CAR T cell therapies, 4 patients developed severe (≥grade 3) CRS with symptoms that were not alleviated with high‐dose steroids and thus received ruxolitinib. Rapid resolution of CRS symptoms was observed in 4 patients after ruxolitinib treatment. Serum cytokines significantly decreased after ruxolitinib intervention. All patients achieved complete remission on day 30 after infusion, and we could still detect CAR T expansion in vivo despite usage of ruxolitinib. There were no obvious adverse events related to ruxolitinib. In vitro assays revealed that ruxolitinib could dampen CAR T expansion and cytotoxicity, suggesting that the timing and dosage of ruxolitinib should be carefully considered to avoid dampening anti‐leukaemia response. Our results suggest that ruxolitinib is active and well tolerated in steroid‐refractory and even life‐threatening CRS.


Study design and patients
1.1.1.1 We conducted a pilot study of ruxolitinib on steroid refractory CRS during anti-CD19 (mice derived) or anti-CD22 (humanized) chimeric antigen receptor T cells treating refractory or relapsed B acute lymphoblastic leukemia in Beijing Boren Hospital.The study was approved by the institutional review board of Beijing Boren Hospital, and informed consent was obtained in accordance with the Declaration of Helsinki.All these patients matched the diagnostic criteria for (r/r) B-ALL according to the WHO classification and completed morphological evaluation, immunophenotype analysis by flow cytometry (FCM), cytogenetic analysis by routine G-banding karyotype analysis and leukemia fusion gene screening by multiplex nested reverse transcriptase-polymerase chain reaction (PCR).Patients were eligible if they were heavily treated B-ALL who failed from re-induction chemotherapy after relapse or continued MRD + for more than three months, and patients had positive CD19 expression or CD22 expression on leukemia blasts by FCM (>95% CD19 or CD22).Patients are enrolled in ruxolitinib subgroup when they develop severe CRS (>3) or CRS related HLH and symptoms are not controlled by tocilizumab and high dose of steroids (1g/kg per day Methylprednisolone) within 24 hours.Enrolled patients received CD19 or CD22 between October 2019 and January 2020 and were evaluated for responses and adverse effects.After CAR T-cell infusion, clinical outcomes including overall survival (OS), leukemia-free survival (DFS), adverse effects and relapse were evaluated up to date as of August 31 th , 2020.

CAR construction, detection and in vitro cytotoxicity
Lentiviral vectors carrying second generation anti-CD19 or anti-CD22 CAR with 4-1BB co-stimulatory and CD3 signaling domains were constructed as previously described. 1,2Briefly, the CD19 recognition domain was composed of a single-chain fragment variable region derived from the FMC63 monoclonal antibody.The CD22 recognition domain was composed of a single-chain fragment variable region obtained from a human antibody phage display library.Cytotoxicity of CD19 or CD22 CAR T cells have been validated previously 1,3 .

Manufacture of CAR T cells
Peripheral blood (PB) mononuclear cells collected from patients or donor were stimulated with magnetic beads coated withanti-CD3/CD28 antibodies (Life Technologies, Carlsbad, CA, USA; now owned by Thermo Fisher Scientific, Waltham, MA, USA) overnight.The next day, transduction was performed at multiplicity of infection 1:10 ratio.Transduced cells were cultured in X-VIVO 15, a serum-free medium

Management of adverse effect according to symptoms
Cytokine releasing syndrome (CRS) and neurotoxicity were graded and managed according to ASTCT consensus grading system and the 2019 NCCN guideline about the management of immunotherapy-related toxicities. 3,4 atients are enrolled in ruxolitinib subgroup when they develop severe CRS (>3) or CRS related HLH and symptoms are not controlled by tocilizumab and high dose of steroids (1g/kg per day Methylprednisolone) within 24 hours.
Tocilizumab was given according to NCCN guideline, and steroids (2 to 15 mg/kg/d methylprednisolone) were given by intravenous injection (IV) in severe CRS patients (grade≥3).Mannitol (2.5 ml/kg/dose IV), furosemide (1 mg/kg/dose IV) and intrathecal injection of dexamethasone (2-5 mg) were used in patients with neurotoxicity (grade≥2).Other managements had been shown below: Serum cytokines were measured on days 0, 1, 7, 15, 30 after each cycle of infusion.To evaluate remission duration, BM biopsy was performed once a month or when the patients had any symptoms of relapse.

1. 2
Inclusion and exclusion criteria for enrolled patients 1.2.1 Inclusion Criteria 1.2.1.1Patients who were diagnosed as primary refractory or relapsed B-ALL.All the patients matched the diagnostic criteria of ALL according to the WHO classification and conducted morphological evaluation, immunophenotype analysis by flow cytometry (FCM), cytogenetic analysis by routine G-banding karyotype analysis, screen of 56 leukemia-related fusion genes by multiplex nested reverse transcriptase-polymerase chain reaction (RT-PCR), and quantification of fusion genes by real-time PCR with ABL1 as reference.Extramedullary diseases (EMDs) were confirmed CD19 + or CD22 + by FCM and evaluated by positron emission tomography/computed tomography (PET/CT), CT, MRI or ultrasonography.The patient relapsed during chemotherapy or failed from re-induction chemotherapy (including first and second-generation TKIs) after relapse or had a persistent positive minimal residual disease (MRD) for three months.Patients had positive CD19 or CD22 expression on leukemia blasts by FCM (>95% CD19 positive); 1.2.1.2Age from 0 to 70 years old;

(
Lonza) with 300 IU/ml interleukin-2, for the duration of cell culture.Transduction efficiency and cell viability were examined at the time of cell infusion.Transduction efficiency was defined as the ratio of CAR-T to CD3 + T cells, determined by FCM with a proprietary anti-CD19 or anti-CD22 CAR T-cell specific detection reagent.Cell viability was determined by Trypan blue exclusion.When the harvest of CD19 and CD22 CAR T-cells was less than 0.1 10 5 /kg, we defined it as CAR manufacture failure.The maximum infused dose of CD19 and CD22 CAR T-cells was 10 10 6 /kg.Clinical procedures 1.2.1.6CAR T-cells were manufactured from peripheral blood mononuclear cells collected by leukapheresis.Before each CAR T-cell infusion (day 0), patients received lymphodepleting chemotherapy composing of Fludarabine (30 mg/m 2 /day) and Cyclophosphamide (250 mg/m 2 /day) on days -5 to -3.Patients are enrolled in ruxolitinib subgroup when they develop severe CRS (>3) or CRS related HLH and symptoms are not controlled by tocilizumab and high dose of steroids (1g/kg per day Methylprednisolone) within 24 hours.All patients underwent bone marrow (BM) biopsy examination and radiology studies on days 30 and every month to determine the response and remission status.Bone biopsy, MRD status by FCM and RT-PCR (if the patient had fusion gene), and EMDs evaluation by CT/MRI/PET-CT were also conducted to determine the disease status.CAR detection by flow cytometryCD19 or CD22 CAR detection was performed by FCM using biotinylated goat anti-murine or human IgG Fab fragment (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania) as the first antibody, and Streptavidin conjugated with APC (BD Pharmingen, San Diego, California) for CAR detection.Alternatively, CD19 or CD22 CAR-T cell expansion in vitro and in vivo can be distinguished by flow cytometry assays with proprietary specific CD19 or CD22 CAR-T cell detection reagent (CD19-CAR-Green and CD22-CAR-Green, respectively from Shanghai YaKe Biotechnology Ltd., Shanghai, China).CAR T-cells in cerebrospinal fuild (CSF) were detected when the patients had central nervous system leukemia (CNSL) at enrollment or had signs of neurotoxicity.

1 2
Flowchart showing the inclusion of patients in this study.CAR T Therapy 15 days after CAR T Therapy before CAR T Therapy 15 days after CAR T Therapy 4 days after CAR T Therapy before CAR T Therapy 1 month after CAR T Therapy 15 days after CAR T Therapy before CAR T Therapy 15 days after CAR T Therapy 2 days after CAR T Therapy before CAR Chest CT scan images of all 4 patients in ruxolitinib subgroup before infusion (left panel), on site of CRS (middle panel) and restore from CRS (right panel).Red arrows represent pulmonary edema lesions. .Platelet counts of all 4 patients in roxolitinib subgroup after infusion.Black arrows represent platelet transfusion.Normal level is shown as dotted lines.(B differences of cytokines levels (IL-2, Granzyme A, IL-4, Perforin, IL-7a, sFasL and Granulysin) in supernatant liquid of 48-hour co-culture with CD19 CAR T cells, monocytes and Nalm6 cells (CD19 + ) under different drug concentrations (0, Ruxo 1uM, Ruxo 10uM, Dex 1uM, Dex 10uM) and T cells with pCDH vector, monocytes and Nalm6 cells (CD19 + ) were coculutre as negative control.The difference is compared by unpaired two-tailed Student's t-test.P-values of <0.05 were considered significant.Ruxo represents ruxolitinib and Dex represents dexamethasone.

Table S1 .
CAR T-cell treatment information of individual patient
). PPR and CCI before and after ruxolitinib treatment in each patient.Normal level is shown as dotted lines.The difference of PPR and CCI for platelet transfusion before and after ruxolitinib treatment is compared by unpaired two-tailed Student's t-test.P-values of <0.05 were considered significant.