Chimeric antigen receptor (CAR) modified T cells have been an important immunotherapeutic strategy. Efficacy of CAR-T cells may be influenced by the structure of CAR, particularly the costimulator component of CAR. To improve the function of CAR-T cells, different costimulators were incorporated into CAR structures. Studies have shown that CAR-T cells containing 4-1BB costimulator have superior antileukemic efficacy and improved persistence compared with that containing CD28 costimulator. But how costimulators affect CAR-T cell function remained to be further studied. In this study, three different CARs with costimulators CD28, 4-1BB, and both CD28 and 4-1BB targeted the myeloid antigen CD33 (referred to as CD33 28z.CAR-T, CD33 BBz.CAR-T and CD33 28BBz.CAR-T, respectively) were constructed and the antileukemic functions of corresponding CD33 CAR-T cells were reported. In addition, the characteristics related to persistence and function of CAR-T cells were further analyzed.

To get CAR-T cells specific for CD33, the murine anti-human CD33 scFv derived from mouse hybridoma cells (clone HIM3-4,which was established in Institute of Hematology and Blood Diseases Hospital, CAMS&PUMC) was cloned into our previously constructed pCDH-CAR plasmids, and transfected into 293T/17 packaging cells with the packaging plasmids to produce lentiviruses. Then T cells from peripheral blood of healthy donors were isolated, stimulated and infected with lentiviruses to get CD33 28z.CAR-T, CD33 BBz.CAR-T and CD33 28BBz.CAR-T, respectively.

In vitro antileukemic function of CD33 CAR-T cells was detected by cytotoxic effect on target cells, CAR-T cells degranulation reflected by CD107a expression and cytokines release by ELISA. It showed that all three CD33 CAR-T cells exhibited profound proliferation and potent effector functions against AML cell lines, including specific cytotoxicity at low E:T ratios of 1:8 or even 1:20, LDH release by target cells, distinct degranulation and robust cytokine production. For in vivo effect, human CD33+ leukemia mouse model were established, and then treated with CD33 CAR-T cells, followed by observation of the body weight and overall survival of mouse. Three type CD33 CAR-T cells could all significantly prolong mouse survival without obvious weight loss.

Finally, the influences of different costimulators on CAR-T cells efficacy and persistence were explored, phenotypes of three CAR-T cells were assessed by flow cytometry based on memory markers CCR7 and CD45RA and exhaustion markers PD-1, LAG-3, Tim-3 et al. Distinctions among three types CAR-T cells emerged during in vitro expansion. CD33 BBz.CAR-T cells had an increased central memory compartment, which was related to T cell persistence. While CD33 28z.CAR-T cells were effector memory T cells predominant, which were the terminally differentiated T cells. In addition, CD33 28z.CAR-T cells were more inclined to become exhausted compared to CD33 BBz.CAR-T cells, indicated by higher expression of PD-1, LAG-3 and Tim-3.

In conclusion, this study reported the high specific cytotoxicity of the CAR-T cells for CD33+ AML cells. Furthermore, this study demonstrated that 4-1BB signaling domain in CARs could endowed CAR-T cells with anti-exhaustion capacity and increased central memory compartment, uncovered the reason that CAR-T cells with 4-1BB performed better in antileukemic function, and further illustrated 4-1BB CAR may be superior to incorporate into T cells to get persistent anti-tumor efficacy especially when transfused to the human body environment.

Keywords: Chimeric antigen receptor, CD33, Acute myeloid leukemia, Immunotherapy, Costimulator


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.