Publisher's Note: There is an Inside Blood Commentary on this article in this issue.

BACKGROUND. Chimeric antigen receptor (CAR) T cells can induce remission in highly refractory leukemia and lymphoma subjects, yet the parameters for achieving sustained relapse-free survival are not fully delineated. METHODS. We analyzed 43 pediatric and young adult subjects participating in a phase I trial of defined composition CD19 CAR T cells (ClinicalTrials.gov, NCT02028455). CAR T cell phenotype, function, and expansion, as well as starting material T cell repertoire, were analyzed in relationship to therapeutic outcome (defined as achieving complete remission within 63 days) and duration of leukemia-free survival and B cell aplasia. RESULTS. These analyses reveal that initial therapeutic failures (n = 5) were associated with attenuated CAR T cell expansion and/or rapid attrition of functional CAR effector cells following adoptive transfer. The CAR T products were similar in phenotype and function when compared with products resulting in sustained remissions. However, the initial apheresed peripheral blood T cells could be distinguished by an increased frequency of LAG-3 + /TNF-α lo CD8 T cells and, following adoptive transfer, the rapid expression of exhaustion markers. For the 38 subjects who achieved an initial sustained minimal residual disease-negative remission, 15 are still in remission, 10 of whom underwent allogenic hematopoietic stem cell transplantation (alloHSCT) following CAR T treatment. Subsequent remission durability correlated with therapeutic products having increased frequencies of TNF-α-secreting CAR CD8 + T cells, but was dependent on a sufficiently high CD19 + antigen load at time of infusion to trigger CAR T cell proliferation. CONCLUSION. These parameters have the potential to prospectively identify patients at risk for therapeutic failure and support the development of approaches to boost CAR T cell activation and proliferation in patients with low levels of CD19 antigen. TRIAL REGISTRATION. ClinicalTrials.gov, NCT02028455.

Objective: To test whether systemic cytokine release is associated with central nervous system inflammatory responses and glial injury in immune effector cell-associated neurotoxicity syndrome (ICANS) after chimeric antigen receptor (CAR)-T cell therapy in children and young adults. Methods: We performed a prospective cohort study of clinical manifestations as well as imaging, pathology, CSF, and blood biomarkers on 43 subjects ages 1 to 25 who received CD19-directed CAR/T cells for acute lymphoblastic leukemia (ALL). Results: Neurotoxicity occurred in 19 of 43 (44%) subjects. Nine subjects (21%) had CTCAE grade 3 or 4 neurological symptoms, with no neurotoxicity-related deaths. Reversible delirium, headache, decreased level of consciousness, tremor, and seizures were most commonly observed. Cornell Assessment of Pediatric Delirium (CAPD) scores ≥9 had 94% sensitivity and 33% specificity for grade ≥3 neurotoxicity, and 91% sensitivity and 72% specificity for grade ≥2 neurotoxicity. Neurotoxicity correlated with severity of cytokine release syndrome, abnormal past brain magnetic resonance imaging (MRI), and higher peak CAR-T cell numbers in blood, but not cerebrospinal fluid (CSF). CSF levels of S100 calciumbinding protein B and glial fibrillary acidic protein increased during neurotoxicity, indicating astrocyte injury. There were concomitant increases in CSF white blood cells, protein, interferon-γ (IFNγ), interleukin (IL)-6, IL-10, and granzyme B (GzB), with concurrent elevation of serum IFNγ IL-10, GzB, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein 1 alpha, and tumor necrosis factor alpha, but not IL-6. We did not find direct evidence of endothelial activation. Interpretation: Our data are most consistent with ICANS as a syndrome of systemic inflammation, which affects the brain through compromise of the neurovascular unit and astrocyte injury.

Chimeric antigen receptor (CAR) T-cell immunotherapy has revolutionized the treatment of refractory leukemias and lymphomas, but is associated with significant toxicities, namely cytokine release syndrome (CRS) and neurotoxicity. A major barrier to developing therapeutics to prevent CAR T cell-mediated neurotoxicity is the lack of clinically relevant models. Accordingly, we developed a rhesus macaque (RM) model of neurotoxicity via adoptive transfer of autologous CD20-specific CAR T cells. Following cyclophosphamide lymphodepletion, CD20 CAR T cells expand to 272 to 4,450 cells/μL after 7 to 8 days and elicit CRS and neurotoxicity. Toxicities are associated with elevated serum IL6, IL8, IL1RA, MIG, and I-TAC levels, and disproportionately high cerebrospinal fluid (CSF) IL6, IL2, GM-CSF, and VEGF levels. During neurotoxicity, both CD20 CAR and non-CAR T cells accumulate in the CSF and in the brain parenchyma. This RM model demonstrates that CAR T cell-mediated neurotoxicity is associated with proinflammatory CSF cytokines and a pan-T cell encephalitis. We provide the first immunologically relevant, nonhuman primate model of B cell-directed CAR T-cell therapy-mediated CRS and neurotoxicity. We demonstrate CAR and non-CAR T-cell infiltration in the CSF and in the brain during neurotoxicity resulting in pan-encephalitis, accompanied by increased levels of proinflammatory cytokines in the CSF. .

Gardner et al report that early intervention with tocilizumab and steroids at the first signs of mild cytokine release syndrome (CRS) following CD19 chimeric antigen receptor (CAR) T-cell infusion for B-cell acute lymphocytic leukemia reduces the development of life-threatening severe CRS without having a negative impact on antileukemic effect.

Chimeric antigen receptor (CAR) redirected T cells can induce remission in highly refractory leukemia and lymphoma subjects. Despite the potential of this emerging therapeutic modality, treatment with CD19 CAR T cells is not uniformly effective for remission induction, and remissions can be short-lived in a significant proportion of treated subjects. Here, we analyzed 43 pediatric and young adult subjects participating in the phase 1 trial (NCT02028455) and correlated their outcomes with in vivo performances of SCRI-CAR19v1(a CD19 specific CAR T cell product), as well as starting material (SM) T cell repertoire- and final cell product (FP) -intrinsic attributes. Subjects were allocated to the dysfunctional response group defined as early treatment failure (no remission or early disease progression after remission while still having CAR engraftment) (n=5) and the functional response group defined as those who obtained an MRD-negative remission that was sustained beyond 63 days (n=37). We found the magnitude of absolute CAR T cell engraftment area under the curve (AUC) was attenuated in the dysfunctional response group (AUC 150.3, range 0.54-752.8, Mann-Whitney p=0.0033) as compared to the functional response group (median AUC 1309, range 5.23-9537). The absolute number of CD8+CAR+ cells and CD4+CAR+ cells at peak engraftment was also significantly higher in the functional response versus the dysfunctional response. The phenotype of the CAR+ cells was analyzed at peak engraftment by multiparameter flow cytometry. CAR+ CD8+ cells from both groups had similar frequencies of PD-1+ cells, whereas the dysfunctional response group showed a significantly higher frequency of LAG-3+ T cells, both in the CAR+CD8+ cells and the CAR+CD4+ cells. A similar trend was seen with the expression of TIM-3. In order to assess whether T cell intrinsic factors contributed to therapeutic failure in these subjects, we studied T cell repertoire status in apheresis products and final expanded CAR T cell products. Several reports have shown that using SM rich in terminally differentiated cells result in CD19 CAR-T cell products having limited replicative capacity and attenuated ability to transition to long lived memory cells . When comparing apheresis derived SM from the functional and dysfunctional response subject groups by flow cytometry for markers associated with functional exhaustion (LAG-3, TIM-3, PD-1), we observed a significantly higher percentage of CD8+ T cells expressing PD-1 and LAG-3 in the dysfunctional response group compared to the functional response subjects (p=0.0266 and p=0.0052). We also observed a higher frequency of CD4+ cells expressing PD-1 in the dysfunctional group. There was no difference in the frequency of cells expressing CD45RA, CD45RO, CCR7, CD27, or in the frequency of cells expressing TNF-α, IFN-γ or IL-2 in response to CD3/CD28 stimulation in both CD4 and CD8 SM T cells between the groups. Lastly, using classification and regression tree analysis, subjects could be classified by the frequency of SM CD8+ T cells expressing LAG-3 and the frequency of SM CD8+ T cells capable of secreting TNF-α upon CD3/CD28 bead activation (r2=0.636). Subjects with fewer than 0.745% SM CD8+ T cells expressing LAG-3 were all in the functional response group (n=26/43). Subjects with equal or more than 0.745% SM CD8+ T cells expressing LAG-3 (n=16) could be further sub-divided into two groups: subjects with equal or more than 25.283% of CD8+ T cells expressing TNF-α were also functional responders (n=8/8), while subjects with fewer than 25.283% of CD8+ T cells expressing TNF-α were in majority in the dysfunctional response group (n=5/8). Of the three subjects from the functional response group with high frequencies of LAG3+ cells and low frequencies of TNF- α -secreting cells, all three had short duration BCA and relapsed within 6 months. The combination of elevated frequency of cells expressing LAG-3 and a reduced capacity to secrete cytokines in response to stimulation potential serve as biomarkers for patients with perturbated T cell repertoires that generate CAR T cell products with attenuated anti-leukemic potency. The parameters identified herein, should they be validated in larger trial cohorts, have the potential to prospectively identify patients at risk for initial therapeutic failure thus requiring alternative therapies from those who have an excellent prognosis. Disclosures Li: Juno Therapeutics: Employment, Equity Ownership. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

<p>Absolute numbers of RM EGFRt- (non-CAR) T cells before and at indicated days after adoptive CD20 CAR T cell transfer in the blood.</p>

<p>Serum levels of 24 cytokines and chemokines measured before and at indicated days after adoptive GFP or CD20 CAR T cell transfer in the blood.</p>