Single-Cell Analysis of CD19-Specific CAR T Cell Treatment of Relapsed/Refractory CD19+ Acute Lymphoblastic Leukemia

By leveraging single-cell transcriptome and T cell receptor (TCR) sequencing, we aimed to track the transcriptional signatures of chimeric antigen receptor (CAR) T cell clonotypes throughout the course of treatment and furthermore identify molecular patterns leading to potent CAR T cell cytotoxicity. The data presented in this study encompass blood and bone marrow samples from patients ≤ 21 years of age with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) participating in the SJCAR19 phase I/II clinical trial (NCT03573700). In brief, patients enrolled in the clinical trial received either 1 x 10^6 (dose level 1) or 3 x 10^6 (dose level 2) per kilogram of body weight following successful generation of autologous CAR T cell products and lymphodepleting chemotherapy. Peripheral blood was drawn from each participant every week until week 4 post-infusion, at week 6 or 8, and month 3 or 6 if feasible. At week 4 post-infusion, blood marrow was also collected from participants. Total T cells (CD3+) were sorted from each post-infusion sample, as well as the pre-infusion CAR T cell products, and processed through 10x Genomics’ single-cell gene expression and V(D)J sequencing platform using the standard protocol. We identified a unique and unexpected transcriptional signature in a subset of pre-infusion CAR T cells that shared TCRs with post-infusion cytotoxic effector CAR T cells. Functional validation of cells with even a subset of these pre-effector markers demonstrated their immediate cytotoxic potential and resistance to exhaustion. The data deposited into dbGaP include all raw single-cell expression and V(D)J sequencing generated from samples obtained from 16 study participants. ]]>
This correlational study was conducted on samples obtained from an ongoing single institution Phase I/II clinical trial (NCT03573700). Inclusion Criteria:
Participants were deemed eligible to undergo autologous apheresis, manufacturing of the SJCAR19 product, and treatment with SJCAR19 if they met the following criteria:● Age ≤ 21 years old● ALL must be CD19+ with any of the following characteristics:
Minimal residual disease ≥ 1% at the end of up-front induction therapy
Hypodiploid CD19+ ALL with detectable disease at the end of up-front induction therapy
Increase in disease burden at any time after completion of up-front induction therapy
Primary refractory disease despite at least 2 cycles of an intensive chemotherapy regimen designed to induce remission
Refractory disease despite salvage therapy
First or greater relapse if patient requires an allogeneic HCT as part of the standard of care relapse therapy, but is found to be ineligible and/or unsuitable for HCT for any of the following reasons:
■ Patients that do not have an available allogeneic donor
■ Patients with refractory leukemia, for which allogeneic transplant is known to be less effective in the B-ALL population
■ Patients who are unable to receive myeloablative total body irradiation (TBI), which is included ins standard transplant regimens for patients with B-ALL
● Estimated life expectancy of ＞12 weeks for apheresis, > 8 weeks for treatment
● Detectable disease for treatment
● Karnofsky or Lansky (age-dependent performance score ≥ 50)● Patients with a history of prior allogeneic hematopoietic cell transplantation (HCT) must be clinically recovered from prior HCT therapy, have no evidence of active graft-versus-host disease (GVHD), and have not received a donor lymphocyte infusion (DLI) within the 28 days prior to apheresis
● Adequate cardiac function defined a left ventricular ejection fraction >40% or shortened fraction ≥ 25%
● EKG without evidence of clinically significant arrhythmia
● Adequate renal function defined as creatinine clearance or radioisotope GFR ≥ 50 ml/min/1.73m2 (glomerular filtration rate (GFR) 40 ml/min/1.73m2 if < 2 years of age)
● 3.2.1.1 Adequate pulmonary function defined as forced vital capacity (FVC) ≥ 50% of predicted value; or pulse oximetry ≥ 92% on room air if patient is unable to perform pulmonary function testing
● Total Bilirubin ≤ 3 times the upper limit of normal for age, except in subjects with Gilbert’s syndrome
● Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) ≤ 5 times the upper limit of normal for age
● Hemoglobin > 8 g/dl (can be transfused)
● Platelet count > 20,000/uL (can be transfused)
● Has recovered from all NCI CTA grade IIII-IV, non-hematologic acute toxicities from prior therapy
● For females of child bearing age:
Not lactating with intent to breastfeed
Not pregnant with negative serum pregnancy test within 7 days prior to enrollment
For treatment, if sexually active, agreement to use birth control until 6 months after T-cell infusion. Male partners should use a condom
● Available SJCAR19 product with ≥ 15% expression of CD19-CAR and killing of CD19+ targets ≥ 20% in an in vitro cytotoxicity assay
● Agree to participate in long-term follow-up
Exclusion Criteria:Participants were excluded from autologous apheresis and treatment if they met the following criteria:
● CNS-3 disease with or without neurologic changes
● CNS-1/CNS-2 disease with neurologic changes
● Known primary immunodeficiency
● History of HIV infection
● Severe intercurrent bacterial, viral, or fungal infection
● History of hypersensitivity reactions to murine protein-containing products
● Evidence of active, uncontrolled neurologic disease
● Severe, uncontrolled bacterial, viral or fungal infection
● Receiving systemic steroids therapy exceeding the equivalent of 0.5 mg/kg/day of Methylprednisolone, in the 7 days prior to CAR T-cell infusion
● Receiving systemic immunosuppressive therapy in the 14 days prior to CAR T-cell infusion, which will interfere with the activity of the SJCAR19 product in vivo (in the opinion of the study PI(s)).
● Receiving intrathecal chemotherapy in the 7 days prior to CAR T-cell infusion

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The single-cell gene expression and TCR sequencing dataset includes 16 pediatric and young adult individuals with relapsed or refractory B-ALL participating in the SJCAR19 CD19-CAR clinical trial from a single hospital. Patients were recruited into the study by the primary clinical service within the institution or referred from the patient's primary oncologist outside the institution. ]]>