Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts

M Cartellieri, A Feldmann, S Koristka, C Arndt, S Loff, A Ehninger, M von Bonin, E P Bejestani, G Ehninger, M P Bachmann, M Cartellieri, A Feldmann, S Koristka, C Arndt, S Loff, A Ehninger, M von Bonin, E P Bejestani, G Ehninger, M P Bachmann

Abstract

The adoptive transfer of CD19-specific chimeric antigen receptor engineered T cells (CAR T cells) resulted in encouraging clinical trials in indolent B-cell malignancies. However, they also show the limitations of this fascinating technology: CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells crossreact with healthy tissues. Here, we describe a novel modular universal CAR platform technology termed UniCAR that reduces the risk of on-target side effects by a rapid and reversible control of CAR T-cell reactivity. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner. UniCAR T cells can be armed against different tumor targets simply by replacement of the respective TM for (1) targeting more than one antigen simultaneously or subsequently to enhance efficacy and (2) reducing the risk for development of antigen-loss tumor variants under treatment. Here we provide 'proof of concept' for retargeting of UniCAR T cells to CD33- and/or CD123-positive acute myeloid leukemia blasts in vitro and in vivo.

Conflict of interest statement

MPB, AE and GE have filed patents and patent applications related to mAbs directed to CD33, La and the UniCAR platform technology. AE, SL and MC are employed by GEMoaB and CPT, respectively. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
UniCAR T cells eradicate AML cells upon redirection with CD33-specific (αCD33 TM) or CD123-specific (αCD123 TM) targeting modules. (a) Schematic representation of T cells engineered with a conventional CAR (left panel, cCAR) or a UniCAR (right panel). For explanation, see text. (b) UniCAR surface expression was detected as described in the Materials and methods section. For all experiments, transduced T cells were sorted to >90% purity to allow comparison between different human donors. (c) Reduction of leukemic cells from three AML cell lines after 24 h of incubation with 2 × 104 engineered human T cells isolated from healthy donors in the presence (+) or absence (−) of 0.1 nM TM in an effector-to-target (e/t) ratio of 1:1. Samples were normalized to a target cell control without any T cells. Engineered T cells expressed either UniCARs containing a dual CD28/CD3-ζ signaling domain (open and closed circles), UniCARs lacking any signaling domain (head up triangle), enhanced green fluorescent protein (EGFP) marker protein (head down triangle) or where not genetically modified (rhombus). (d, e) Experimental set-up was similar to (c) but a lower e/t ratio of 1:5 was chosen. (d) The number of living MOLM-13 target cells was normalized to a control sample without any T cells. (e) T-cell expansion was calculated as the ratio of T cells present in the samples after 120 h (d5) to the number of cells seeded at the start of the experiment (d0). (f) Effective TM concentration required for lysis of MOLM-13 AML cells was determined after 24 h of cultivation with UniCAR T cells. TMs were added at the indicated concentrations. Statistical analysis for (c) was performed using nonparametric one-way analysis of variance (ANOVA; Kruskal–Wallis test) and post hoc Dunn's multiple comparison test. Results are indicated for UniCAR modified T cells plus TMs versus other samples (*P<0.05, **P<0.01, ***P<0.001).
Figure 2
Figure 2
Dual retargeting of UniCAR modified T cells against CD33 and CD123 enhances leukemic cell lysis. (a) Because of its modular nature, the UniCAR technology allows a redirection of UniCAR modified T cells against two antigens simultaneously or consecutively using either two monospecific TMs (TM 1+TM 2, left side) or combined dual-specific TMs (TM1-2, right side). (b) Concentration–response curves for combined CD33- and CD123-specific retargeting of UniCAR T cells. UniCAR T cells were incubated in an effector-to-target (e/t) cell ratio of 1:1 with 51Cr-labeled MOLM-13 (n=4, open squares) and OCI-AML3 (n=2, open rhombus) for 24 h. EC50 values were determined for anti-CD33 (αCD33 TM)+anti-CD123 TM (αCD123 TM): EC50 MOLM-13=70.2 pM, EC50 OCI-AML3=80.2 pM, for anti-CD123-anti-CD33 TM (αCD123-CD33 TM): EC50 MOLM-13=2.9 pM, EC50 OCI-AML3=11.7 pM. (c, d) Human engineered T cells from healthy donors or AML patients were incubated with 5 × 104 CD3-CD19- leukemic cells from AML patients in the presence (+) or absence (−) of anti-CD123-CD33 TM at the indicated concentrations and an e/t ratio of 1:1. T cells expressed either UniCARs containing a dual CD28/CD3-ζ signaling domain (open and closed circles), UniCARs lacking any signaling domain (head up triangle) or enhanced green fluorescent protein (EGFP) marker protein (head down triangle). (c) The number of living target cells after 48 h (left panel) and 120 h (right panel) is shown and compared with control AML samples without adding T cells (open squares). (d) T-cell expansion was calculated as the ratio of T cells present in the samples after 120 h (d5) to the number of cells seeded at the start of the experiment (d0). (e) T cell-specific cytokine secretion was determined from supernatants taken after 48 h from the experiments shown in (c). (f) Engineered T cells from AML patients were incubated with 2 × 104 AML target cells in the presence (+) or absence (−) of 0.5 nM TMs in an e/t ratio of 1:1. Number of living target cells was determined by flow cytometry after 24 h and compared with a control sample with target cells and TM but without T cells. Results from one representative donor are shown. Statistical analysis for (c) was performed using nonparametric one-way analysis of variance (ANOVA; Kruskal–Wallis test) and post hoc Dunn's multiple comparison test. Results are indicated for UniCAR modified T cells plus TMs versus other samples (*P<0.05).
Figure 3
Figure 3
UniCAR engineered T cells delay AML engraftment in a TM-dependent manner in vivo. (a) Pharmacokinetics of the dual-specific anti-CD123/CD33 TM (αCD123-CD33 TM) in peripheral blood of NSG mice upon i.v. injection via the tail vain or i.p. injection. Concentration of TM in blood samples was determined by ELISA. (b, c) Short-term treatment with anti-CD123-CD33 TM enhances the survival of NSG mice in an aggressive AML model. 1 × 106 human T cells engineered to express functional UniCARs (28/ζ+TM, red line, n=5), UniCARs lacking any signaling domain (Stop+TM, blue line, n=5) or expressing only enhanced green fluorescent protein (EGFP) marker protein (vc+TM, green line, n=3) were i.v. injected into NSG mice. After 28 days, 5 × 105 MOLM-13 were transferred into NSG mice via i.v. injection (Tx) and treatment with anti-CD123-CD33 TM (T) was started 5 days later. For this purpose, 250 ng TM per g mouse body weight was injected i.p. twice a day over 2 consecutive days. As additional controls, one group of mice was transplanted only with MOLM-13 and treated with anti-CD123-CD33 TM (w/o+TM, black line, n=4), and another group of mice was transplanted with functional UniCAR T cells and MOLM-13, but not treated with anti-CD123-CD33 TM (28/ζ w/o TM, light red line, n=4). (b) Survival curves of experimental groups and (c) percentage of CD3+ T cells, CD33+ MOLM-13 cells and human CD45+CD3-CD33- cells in the bone marrow of killed mice are shown. (d) To evaluate in vivo toxicity of UniCAR modified T cells, 1 × 106 engineered human T cells were i.v. injected into NSG mice. Body weight of mice was monitored in weekly intervals and expressed as percentage body weight change over time.

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