Development of A Chimeric Antigen Receptor Targeting C-Type Lectin-Like Molecule-1 for Human Acute Myeloid Leukemia

Abstract

The treatment of patients with acute myeloid leukemia (AML) with targeted immunotherapy is challenged by the heterogeneity of the disease and a lack of tumor-exclusive antigens. Conventional immunotherapy targets for AML such as CD33 and CD123 have been proposed as targets for chimeric antigen receptor (CAR)-engineered T-cells (CAR-T-cells), a therapy that has been highly successful in the treatment of B-cell leukemia and lymphoma. However, CD33 and CD123 are present on hematopoietic stem cells, and targeting with CAR-T-cells has the potential to elicit long-term myelosuppression. C-type lectin-like molecule-1 (CLL1 or CLEC12A) is a myeloid lineage antigen that is expressed by malignant cells in more than 90% of AML patients. CLL1 is not expressed by healthy Hematopoietic Stem Cells (HSCs), and is therefore a promising target for CAR-T-cell therapy. Here, we describe the development and optimization of an anti-CLL1 CAR-T-cell with potent activity on both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Furthermore, in a disseminated mouse xenograft model using the CLL1-positive HL60 cell line, these CAR-T-cells completely eradicated tumor, thus supporting CLL1 as a promising target for CAR-T-cells to treat AML while limiting myelosuppressive toxicity.

Keywords: AML; CAR-T-cell; CLL-1; hematopoiesis; optimization.

Figure 1

Validation of C-type lectin-like molecule-1 (CLL1) as a target for acute myeloid leukemia (AML) and the optimization of CLL1 chimeric antigen receptor (CAR) engineered T-cells (CAR-T-cells). CLL1 antigen is a valid AML target for CAR-T-cell therapy. (a) Anti-CLL1 CAR-T construct (CD8VLVH) cytotoxicity on AML cell lines at different Effector to Target (E:T) ratios. Data represents the mean values of triplicates +/− standard deviation (SD). (b) The percentage of cytotoxicity (E:T = 1) induced by anti-CLL1 CAR-T (CD8VLVH) strongly correlates with target cell antigen density. Antigen density was determined using a BD Quantibrite™ Beads PE Fluorescence Quantitation Kit. (c) Schematic of the CAR constructs designed and lentivirally transduced into healthy-donor derived activated T-cells (average transduction 50%). All constructs were sorted to normalize CAR expression prior to assays (final CAR expression: 90–98%). (d) In vitro cytotoxicity of each construct against the AML cell line HL60 at indicated E:T ratios. (e) Cytokine production for each construct from the cytotoxicity assay shown in (d) with E:T = 1:1. Data represents the mean values of triplicates +/− SD. Results shown are representative of two different donors. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to CD8VLVH, one-way ANOVA, and Dunnet’s multiple comparison post-test.

Figure 2

Anti-CLL1 CAR-T efficiently targets AML blasts in vitro and eradicates HL60 tumors in vivo while sparing hematopoiesis. Anti-CLL1 CAR-T (IgG4SVLVH) does not affect hematopoietic stem cell colony formation while inducing robust cytotoxicity on patient-derived AML blasts in vitro, and eradicates HL60 xenografts in vivo. (a) CLL1 Median Fluorescence Intensity (MFI) of cord-blood derived CD34+CD38− and CD34+CD38+ subsets. Results are representative of two different donors. Background MFI from an unstained control was subtracted from all test samples. (b) Anti-CLL1 CAR-T cytotoxicity on CD34+CD38− and CD34+CD38+ cells. Freshly isolated CD34+ cells were incubated with CAR-T-cells labeled with PKH67 at E:T = 10:1. After 4 h, cells were stained with anti-CD34, anti-CD38, and 7-AAD, and count bright absolute counting beads (Thermo Fisher Sci., Waltham, MA, USA) were added prior to acquisition by flow cytometry. (c) Colony-Forming Unit assay (CFU) of Hematopoietic Stem Cell (HSC) co-incubated for 4 h with CAR-T-cells. HSC were isolated and cultured with CAR-T-cells as per (b). The mixture was plated (500 HSC/plate) in semi-solid media and CFU were scored for each condition. For (a–c) data represent the mean values of triplicates +/− SD of two different donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, one-way ANOVA, Tukey’s multiple comparison post-test. BFU-E: burst-forming unit-erythroid; CFU-GM: granulocyte/macrophage progenitor cells; CFU-GEMM: granulocyte, erythroid, macrophage, megakaryocyte progenitor cells. (d) Anti-CLL1 CAR-T cytotoxicity on blasts from four different AML patients at an indicated E:T ratio. Data represent the mean values of triplicates +/− SD. (e) Anti-tumor activity of anti-CLL1 CAR-T in an HL60 xenograft model. Graph shows quantified tumor burden over time by bioluminescent imaging plotted as individual mice. IgG4SVLVH construct or a control CAR-T were transduced into T-cells of two different donors (CAR expression on day of injection >75–80%) and assayed under the same protocol (n = 3 for each donor). Mice injected with PBS or control CAR-T were sacrificed after three weeks due to disease progression.