Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL

Abstract

After treatment with chimeric antigen receptor (CAR) T cells, interleukin-15 (IL-15) elevation and CAR T-cell expansion are associated with non-Hodgkin lymphoma (NHL) outcomes. However, the association of preinfusion CAR product T-cell functionality with clinical outcomes has not been reported. A single-cell analysis of the preinfusion CD19 CAR product from patients with NHL demonstrated that CAR products contain polyfunctional T-cell subsets capable of deploying multiple immune programs represented by cytokines and chemokines, including interferon-γ, IL-17A, IL-8, and macrophage inflammatory protein 1α. A prespecified T-cell polyfunctionality strength index (PSI) applied to preinfusion CAR product was significantly associated with clinical response, and PSI combined with CAR T-cell expansion or pretreatment serum IL-15 levels conferred additional significance. Within the total product cell population, associations with clinical outcomes were greater with polyfunctional CD4+ T cells compared with CD8+ cells. Grade ≥3 cytokine release syndrome was associated with polyfunctional T cells, and both grade ≥3 neurologic toxicity and antitumor efficacy were associated with polyfunctional IL-17A-producing T cells. The findings in this exploratory study show that a preinfusion CAR product T-cell subset with a definable polyfunctional profile has a major association with clinical outcomes of CAR T-cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT00924326.

Conflict of interest statement

Conflict-of-interest disclosure: J.R., Y.-W.S., and A.B. are employed by Kite, a Gilead Company, and have equity ownership in Gilead Sciences, Inc.; P.P. is employed by, has equity ownership in, and is a patent holder with IsoPlexis; K.M., B.F., A.K., T.C., and J.Z. are employed by and have equity ownership in IsoPlexis; C.N. and K.G. are employed by IsoPlexis; R.F. is cofounder and scientific advisor of IsoPlexis; S.M. is employed by, has equity ownership in, and is a patent holder with IsoPlexis and received honoraria from Kite, a Gilead Company; J.R.H. is a founder and board member of IsoPlexis; S.A.R. received research funding from Kite, a Gilead Company, and is a patent holder with National Cancer Institute Cooperative Research and Development Agreement/Kite; and J.N.K. received research funding from Kite, a Gilead Company and BlueBird Bio, is a patent holder with Kite, a Gilead Company BlueBird Bio, and Novartis, and has received travel expenses from Kite, a Gilead Company, and Celgene Corporation.

© 2018 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Schematic representation of the method used to evaluate T-cell polyfunctionality. (A-B) Schematic representations of CAR construct configuration and treatment protocol. (C-D) Product T-cell polyfunctionality was assessed by using enzyme-linked immunosorbent assay (ELISA) detection of proteins from each single-cell chamber after T-cell stimulation. (E-F) Polyfunctionality was measured through a PSI, spanning a prespecified panel of 32 key immunologically relevant molecules across major categories: homeostatic/proliferative, inflammatory, chemotactic, regulatory, and immune effector. cyt, cytokine; MIP-1α, macrophage inflammatory protein 1α; scFv, single-chain variable fragment.

Figure 2.

Association of PSI with OR and treatment-related adverse events. Single-cell product PSI was determined for 20 patient donors by using SCBC proteomic analysis of a panel of 32 secreted cytokines, chemokines, and cytotoxic molecules. Of the 20 patients, 14 had an OR to the CAR T-cell therapy. The products were ranked according to CAR T-cell PSI levels, and the PSI was associated with the OR (A,C) or grade ≥3 CRS (B,D) as indicated. The results are shown as patient-level PSI (A-B) and mean ± standard error PSI (C-D). All statistical values were computed using the Mann Whitney U test.

Figure 3.

Association of CAR product polyfunctionality with CD19 recognition and clinical outcome. (A) PSI of CAR T cells and CD4+ or CD8+ subset, ex vivo stimulated with CD19+ as compared with CD19− cells (NGFR transfected). (B-E) Association between OR and product PSI, IFN-γ measured in product coculture with CD19+ cells, or major product T-cell subsets defined by flow cytometry. T naïve, central memory (cm), effector memory (em), and effector (eff) cells were defined by staining for CD45RA and CCR7. (F) Individual P values (Mann Whitney U test) corresponding to the strength of association of major product attributes with clinical response.

Figure 4.

Major cytokines driving polyfunctional product CD4+and CD8+T cells by CD19 stimulation that distinguish responders to the therapy from nonresponders. (A-B) Single-cell proteomic analysis of a panel of 32 secreted cytokines, chemokines, and cytotoxic molecules was performed on product T cells from 20 patients treated with CAR T cells. The analysis was performed on all product cells or select CD4+ and CD8+ T cells. The product T cells were first stimulated with CD19-expressing target cells or control NGFR cells before the analysis. The graphs show PSI (mean ± standard error) with or without CD19 stimulation for all cells and for CD4+ and CD8+ subsets separately. The main cytokine drivers for each product T-cell subpopulation are also shown. (C-D) Product CD4+ and CD8+ T-cell PSI profiles were broken down per cytokine, between patient groups with no response and OR to CAR T-cell therapy. Only CD4 and CD8 cytokines that were upregulated relative to mock stimulation are shown. Each cytokine PSI level reflects its average secretion intensity in polyfunctional single cells. The diagram shows the cytokines that contribute to the polyfunctionality index in the CD8+ and CD4+ T-cell populations. (E-F) Polyfunctional strength of major cytokines and chemokines in CD4+ and CD8+ CAR T cells from nonresponders and responders. FD, fold difference; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Figure 5.

PSI in conjunction with CAR T-cell expansion in vivo or in conjunction with conditioning-driven IL-15 pre–CAR T-cell infusion correlates with OR. CAR T-cell levels in blood measured by quantitative PCR were correlated with clinical outcome. A composite index integrating PSI and CAR T-cell expansion in vivo was developed as detailed in the supplemental Methods and was associated with OR. The metrics were added to each other after each was first standardized to have unit variance. This standardization was achieved by dividing the metrics by their respective standard deviation to bring them to a common magnitude/scale. Whole product PSI alone (A), peak CAR levels in blood alone (B), and product PSI in conjunction with peak CAR levels (C) are shown in association with OR. Pre–CAR T-cell infusion (day 0) IL-15 serum levels alone (D) or in conjunction with product PSI (E) are shown in association with OR. Statistical values were computed using the Mann Whitney U test (P values were not adjusted for multiplicity).