Anti-CD19 chimeric antigen receptor (CAR)-expressing T cells are an effective treatment for B-cell lymphoma, but often cause neurologic toxicity. We treated 20 patients with B-cell lymphoma on a phase I, first-in-human clinical trial of T cells expressing the new anti-CD19 CAR Hu19-CD828Z (NCT02659943). The primary objective was to assess safety and feasibility of Hu19-CD828Z T-cell therapy. Secondary objectives included assessments of blood levels of CAR T cells, anti-lymphoma activity, second infusions and immunogenicity. All objectives were met. Fifty-five percent of patients who received Hu19-CD828Z T cells obtained complete remission. Hu19-CD828Z T cells had clinical anti-lymphoma activity similar to that of T cells expressing FMC63-28Z, an anti-CD19 CAR tested previously by our group, which contains murine binding domains and is used in axicabtagene ciloleucel. However, severe neurologic toxicity occurred in only 5% of patients who received Hu19-CD828Z T cells, whereas 50% of patients who received FMC63-28Z T cells experienced this degree of toxicity (P = 0.0017). T cells expressing Hu19-CD828Z released lower levels of cytokines than T cells expressing FMC63-28Z. Lower levels of cytokines were detected in blood from patients who received Hu19-CD828Z T cells than in blood from those who received FMC63-28Z T cells, which could explain the lower level of neurologic toxicity associated with Hu19-CD828Z. Levels of cytokines released by CAR-expressing T cells particularly depended on the hinge and transmembrane domains included in the CAR design.
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All requests for raw and analyzed data and materials are promptly reviewed by the National Cancer Institute Technology Transfer Center to verify whether the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the paper were generated as part of clinical trials and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement. All other data that support the findings of this study will be provided by the corresponding author upon reasonable request when possible. Raw data for all Figs. 1–5 and Extended Data Fig. 3 are in the submitted Source Data Excel file.
CAR sequences were all submitted to GenBank.
GenBank accession number for LSIN-Hu19-CD828Z: MN698642
GenBank accession number for LSIN-FMC63-CD828Z: MN702884
GenBank accession number for LSIN-Hu19-28Z: MN702882
GenBank accession number for MSGV-Hu19-CD828Z: MN702883
GenBank accession number for MSGV-FMC63-28Z: HM852952.1
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The clinical LSIN-Hu19-CD828Z vector was produced and gene-therapy monitoring was performed with assistance from the NHLBI-funded National Gene Vector Biorepository at Indiana University. We thank the following clinical units at the NIH clinical center for patient care: Experimental Transplantation and Immunotherapy Branch Clinical Service, the 3 Northeast Nursing Unit and the Department of Critical Care Medicine and the Intensive Care Unit staff. Funding for this work was from National Cancer Institute Intramural funding and Kite, a Gilead Company.
This work was supported by intramural funding of the Center for Cancer Research, NCI, NIH. In addition, the NCI has cooperative research and development agreements with Kite Pharma, a Gilead Company that supports development of anti-CD19 CAR T-cell therapies, and both J.N.K. and S.A.R. are NCI principal investigators of these research agreements. J.K. has a patent application for the Hu19-CD828Z CAR and has received royalty payments from Kite, a Gilead Company. A.B., J.R., A.X. and N.S. are all employees of Kite, a Gilead Company.
Peer review information Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Consort diagram of the Hu19-CD828Z clinical trial.
All Grade 4, 3, and 2 neurologic adverse events within the first month after CAR T-cell infusion are listed. Grading by National Cancer Institute Common Terminology Criteria for Adverse Events Version 3; all adverse events listed under “Neurologic” are included except syncope. Syncope was not included because it was associated with cytokine-release syndrome and hypotension. The highest grade of each adverse event experienced by each patient is listed. For example, if a patient had both Grade 2 and Grade 3 tremor at different times, tremor is only listed under Grade 3.
All Grade 4, 3, and 2 neurologic adverse events within the first month after CAR T-cell infusion are listed. Grading by National Cancer Institute Common Terminology Criteria for Adverse Events Version 3; all adverse events listed under “Neurologic” are included except syncope. Syncope was not included because it was associated with hypotension from cytokine-release syndrome. The highest grade of each adverse event experienced by each patient is listed. For example, if a patient had both Grade 2 and Grade 3 confusion at different times, confusion is only listed under Grade 3.
For all proteins, all 22 patients on the trial of FMC63-28Z T cells and all 20 patients on the trial of Hu19-CD828Z T cells were compared. Proteins were measured in serum samples by Luminex® assay between day 2 and 14 after CAR T-cell infusion. Statistics were by 2-tailed Mann-Whitney test.
Patient 3 was the only patient with Grade 3 or 4 neurologic toxicity on the Hu19-CD828Z trial. Peak serum levels of 9 immunological proteins are shown for patient 3. Peak levels were determined between day 2 and day 14 after CAR T-cell infusion. These 9 proteins are shown because they were found to be prominently different between the Hu19-CD828Z and FMC63-28Z clinical trials (Fig. 3). Proteins were measured by Luminex® assay. MCP-1, monocyte chemotactic protein-1; IL, interleukin; TNF-alpha, tumor necrosis factor-alpha; MIP-1-alpha, macrophage inflammatory protein-1-alpha; IFN-gamma, interferon-gamma. The red bars indicate the median protein levels for all 20 patients that received Hu19-CD828Z CAR T cells. Source data
For all proteins, all 22 patients on the trial of FMC63-28Z T cells and all 20 patients on the trial of Hu19-CD828Z T cells were compared. Proteins were measured in serum samples by Luminex® assay from days 2 to 14 after CAR T-cell infusion. Area under the curve (AUC) was calculated by trapezoidal method. Statistics were by 2-tailed Mann-Whitney test.
Top row: schematic representations of Hu19-CD828Z (left) and FMC63-28Z (right) CAR models are shown; scFv in blue; hinge in green; transmembrane domain in yellow; intracellular domain in red. The membrane position during molecular dynamics simulations is shown in grey. Bottom row: conformational flexibility for each corresponding CAR depicted as superimposed carbon-alpha traces for a set of 50 representative conformations observed during a 50 nanosecond molecular dynamics trajectory. The differences in flexibility originate in the very different structure and dynamic behavior of the corresponding hinge regions during the dynamics simulations. Transmembrane and scFv domains are affected by the hinge properties and display very different behaviors as well. A quantitative analysis of the molecular dynamics trajectories reveals that these behaviors affect the scFv mobility (assessed as molecular diffusibility) and the proper formation of a transmembrane dimer evaluated by the helix-helix occluded surface. All models assume a dimeric structure anchored by disulfide bonds. In short, Hu19-CD828Z exhibited less conformational flexibility than FMC63-28Z.
*Positive anti-CAR response was defined as 3x or greater increase in spot number from pretreatment to post-CAR T-cell infusion, and post-treatment spot number must have been 3x or more than the spot number of the media control. #Bin A contained peptides from the signal sequences, scFv linker, and hinge regions. Bin B contained peptides from the scFv light chain. Bin C contained peptides from the scFv heavy chain. Bin D contained peptides from transmembrane and intracellular domains. ^The increase in spots was the number of spots/400,000 total input PBMC at the positive time-point minus the number of spots/400,000 total input PBMC before CAR T-cell treatment.
Anti-CAR T-cell responses were assessed by ELISPOT analysis of PBMC before CAR T-cell treatment and at time-points within 6 weeks after CAR T-cell infusion as summarized in Extended Data Fig. 8. CAR+ cell levels in the blood were assessed by quantitative PCR. The top row shows peak blood CAR+ cell levels with results divided into patients with or without anti-CAR responses by ELISPOT. (a) Hu19-CD828Z (b) FMC63-28Z. The bottom row shows blood CAR+ cell levels 1-month after CAR T-cell infusion with results divided into patients with or without anti-CAR responses detected by ELISPOT: (c) Hu19-CD828Z, (d) FMC63-28Z. No statistically significant differences in blood CAR+ cell levels were found between patients with or without anti-CAR responses. All patients with adequate cell samples for both ELISPOT and qPCR are included. P values by Mann-Whitney test are shown on the plots; significance was defined as P<0.05. Of the 4 comparisons, the FMC63-28Z 1-month comparison was closest to statistical significance with P=0.061. Each symbol represents an individual patient. The number of unique patients analyzed were as follows: Hu19-CD828Z Peak, n=18; FMC63-28Z Peak, n=18; Hu19-CD828Z 1 month, n=18; FMC63-28Z 1 month n=13. Source data
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Brudno, J.N., Lam, N., Vanasse, D. et al. Safety and feasibility of anti-CD19 CAR T cells with fully human binding domains in patients with B-cell lymphoma. Nat Med 26, 270–280 (2020). https://doi.org/10.1038/s41591-019-0737-3
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