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. 2020 Jun 30:18:571-581.
doi: 10.1016/j.omtm.2020.06.024. eCollection 2020 Sep 11.

The Art and Science of Selecting a CD123-Specific Chimeric Antigen Receptor for Clinical Testing

Janice M Riberdy  1 , Sheng Zhou  2 , Fei Zheng  2 , Young-In Kim  2 , Jennifer Moore  1 , Abishek Vaidya  1 , Robert E Throm  3 , April Sykes  4 , Natasha Sahr  4 , Challice L Bonifant  5 , Byoung Ryu  3 , Stephen Gottschalk  1 , Mireya Paulina Velasquez  1
Affiliations

The Art and Science of Selecting a CD123-Specific Chimeric Antigen Receptor for Clinical Testing

Janice M Riberdy et al. Mol Ther Methods Clin Dev. .

Abstract

Chimeric antigen receptor (CAR) T cells targeting CD123, an acute myeloid leukemia (AML) antigen, hold the promise of improving outcomes for patients with refractory/recurrent disease. We generated five lentiviral vectors encoding CD20, which may serve as a target for CAR T cell depletion, and 2nd or 3rd generation CD123-CARs since the benefit of two costimulatory domains is model dependent. Four CARs were based on the CD123-specific single-chain variable fragment (scFv) 26292 (292) and one CAR on the CD123-specific scFv 26716 (716), respectively. We designed CARs with different hinge/transmembrane (H/TM) domains and costimulatory domains, in combination with the zeta (z) signaling domain: 292.CD8aH/TM.41BBz (8.41BBz), 292.CD8aH/TM.CD28z (8.28z), 716.CD8aH/TM.CD28z (716.8.28z), 292.CD28H/TM. CD28z (28.28z), and 292.CD28H/TM.CD28.41BBz (28.28.41BBz). Transduction efficiency, expansion, phenotype, and target cell recognition of the generated CD123-CAR T cells did not significantly differ. CAR constructs were eliminated for the following reasons: (1) 8.41BBz CARs induced significant baseline signaling, (2) 716.8.28z CAR T cells had decreased anti-AML activity, and (3) CD28.41BBz CAR T cells had no improved effector function in comparison to CD28z CAR T cells. We selected the 28.28z CAR since CAR expression on the cell surface of transduced T cells was higher in comparison to 8.28z CARs. The clinical study (NCT04318678) evaluating 28.28z CAR T cells is now open for patient accrual.

Keywords: AML; CAR; CD123; T cell therapy; chimeric antigen receptor; immunotherapy; leukemia.

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Figures

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Graphical abstract
Figure 1
Figure 1
Generation of CD123-Specific CAR T Cells (A) Scheme of lentiviral vectors (LVs). Data throughout the figures are represented by the color code of the circles to the left of each construct. (B) Schematic for lentiviral transduction process. (C) Vector copy number (VCN) was determined by digital drop PCR analysis with primers within the lentiviral backbone (n = 5; p > 0.05 for all CAR T cell construct comparisons). (D–F) Flow cytometry evaluation was performed on day 8 after initial T cell activation. (D) CD123-CARCD20 expression (n = 5; p > 0.05). (E) CD20 transgene expression (n = 5; p > 0.05). (F) Coexpression of CD123-CAR and CD20. Representative histograms and dot plots are shown. The inset bar graph shows the mean and standard deviation values for cells expressing CD123-CAR and CD20 (n = 5; p > 0.05).
Figure 2
Figure 2
CD123-CARCD20 T Cells Have Similar Kinetics and Immunophenotypes (A) Fold expansion of the CD123-CARCD20 and non-transduced (NT) T cells (n = 5; p ≥ 0.05). (B) Viability of the indicated populations was determined by acridine orange/propidium iodide (AO/PI) exclusion (n = 5; p ≥ 0.05). (C and D) Immunophenotype and exhaustion phenotype was determined by flow cytometry on day 8. (C) T cell immunophenotypes (T cell subsets: naive, CCR7+CD45RO–; central memory (CM), CCR7+CD45RO+; terminally differentiated (TD), CCR7–CD45RO–; and effector memory (EM), CCR7–CD45RO+; n = 5. (D) CD4, CD8, Tim3, and PD1 expression (n = 5; p > 0.05 among CD123-CAR T cell groups).
Figure 3
Figure 3
CD123-CARCD20 T Cells Recognize and Kill CD123+ Targets in an Antigen-Specific Manner (A) Effector cells were grown in cocultures with media, K562 (CD123–), or Molm13 (CD123+) at an E:T ratio of 2:1 for 24 h. Supernatants were collected and evaluated for IFN-γ content by ELISA (n = 5; p < 0.0001 for NT versus CD123-CARCD20 T cell groups, and p > 0.05 for comparison among CD123-CARCD20 T cell groups). Scale magnification of data in (A; n = 5; p < 0.01 for comparison of 8.41BBz versus all other CD123-CARCD20 T cell groups). (B) Target cell populations were labeled with CFSE, incubated with effector T cells at the indicated ratios overnight and analyzed by flow cytometry by using absolute counting beads to determine cytotoxicity. n = 5; p > 0.05 for comparison on K562 targets and p < 0.0001 for CD123-CAR CD20, as compared with NT on Molm13.
Figure 4
Figure 4
Recognition of CD123+ Hematopoietic Precursor Cells by CD123-CARCD20 T Cells Effector cells were incubated with CD34+ HPCs for 4 h at E:T ratios of 5:1 and 1:1, plated on semisolid media, and evaluated 12–14 days later (n = 6 biological replicates; ∗p < 0.05; black asterisk: comparison to NT T cells; red asterisk: comparison among CAR constructs).
Figure 5
Figure 5
CD123-CARCD20 T Cells Have Potent Antitumor Activity In Vivo (A) Schematic of experimental design. (B–E) Animals were intravenously injected with Molm13-expressing luciferase, followed by infusion of either 3 × 106 or 1 × 107 effector T cells and in vivo imaging for evaluation of tumor burden. (B) Bioluminescence signal over time (total flux in photons/s) of group receiving 1 × 107 T cells. (C) Kaplan-Meier survival curves for animal groups receiving 1 × 107 T cells. Statistical significance was determined with a Wilcoxon rank-sum test (p < 0.05; blue shading in table). (D) Bioluminescence signal over time (total flux in photons/s) of group receiving 3 × 106 T cells. (E) Kaplan-Meier survival curves for animal groups receiving 3 × 106 T cells. Statistical significance was determined by Wilcoxon rank-sum test (p < 0.05; blue shading in table).
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