Efficient combinatorial adaptor-mediated targeting of acute myeloid leukemia with CAR T-cells

Abstract

CAR T-cell products targeting lineage-specific cell-of-origin antigens, thereby eliminating both tumor and healthy counterpart cells, are currently clinically approved therapeutics in B- and plasma-cell malignancies. While they represent a major clinical improvement, they are still limited in terms of efficacy by e.g. single, sometimes low-expressed antigen targeting, and in terms of safety by e.g., lack of on-off activity. Successful cell-of-origin non-discriminative targeting of heterogeneous hematopoietic stem and progenitor cell malignancies, such as acute myeloid leukemia (AML), will require antigen-versatile targeting and off-switching of effectors in order to then allow rescue by hematopoietic stem cell transplantation (HSCT), preventing permanent myeloablation. To address this, we developed adaptor-CAR (AdFITC-CAR) T-cells targeting fluoresceinated AML antigen-binding diabody adaptors. This platform enables the use of adaptors matching the AML-antigen-expression profile and conditional activity modulation. Combining adaptors significantly improved lysis of AML cells in vitro. In therapeutic xenogeneic mouse models, AdFITC-CAR T-cells co-administered with single diabody adaptors were as efficient as direct CAR T-cells, and combinatorial use of adaptors further enhanced therapeutic efficacy against both, cell lines and primary AML. Collectively, this study provides proof-of-concept that AdFITC-CAR T-cells and combinations of adaptors can efficiently enhance immune-targeting of AML.

Fig. 1. Generation of AdFITC-CAR T-cells and fluorescein-labeled antibody constructs.

A Illustration of an AdFITC-CAR T-cell displaying anti-fluorescein scFv domain, targeting a generic tumor-associated antigen on a cell via a fluorescently labeled diabody adaptor as linking molecule. The surface co-expression of RQR8, which serves as a selection and depletion marker for AdFITC-CAR T-cells, is also depicted. B Schematic representation of second-generation CAR vector, presenting an anti-fluorescein scFv (clone E2). C Representative flow cytometry plots reflecting the transduction rate of healthy donor-derived T-cells are shown after staining with either AlexaFluor546-PEG₂-FITC (AF546-FITC) in green or anti-CD34 antibody in red. Non-transduced T-cells served as control (black). D Plasmid map of the diabodies (Db) targeting CD33 and CD117 engineered to express a C-terminal cysteine. E Dbs were expressed and coupled with fluorescein-maleimide after a reduction step, as indicated in the protein conjugation scheme, yielding site-specifically labeled diabody (Db-FM). CD33 Db-FM (F) and CD117 Db-FM (G) were characterized by size exclusion chromatography (left), SDS-PAGE under non-reducing (NR) and reducing (R) conditions (middle; in addition, UV-light illuminated SDS-PAGE gels are also shown), and by mass spectrometry (right). M=Protein ladder indicating the kDa. Surface Plasmon Resonance analysis of CD33 Db-FM (H) and CD117 Db-FM (I) on a CM-5 chip coated with the extracellular domains of CD33 and CD117 His-tagged proteins, respectively. Dissociation constants K_D are indicated. J Binding activity of the CD33 Db-FM and CD117 Db-FM was determined by dose titration on MOLM14-CD117^highGFP⁺Luc⁺ cells and detected by means of an APC-conjugated anti-FITC antibody. MFI was normalized to account for the higher level of CD117 antigen expression. Mean ± SD from triplicates. Resulting K_D,app are also indicated.

Fig. 2. Db-FM adaptors elicit AdFITC-CAR T-cell cytotoxicity against various human AML cell lines in a concentration, time, and antigen density-dependent manner.

A Representative histograms showing Kasumi-1 GFP⁺ cells stained with 10 nM CD33 Db-FM (left) and CD117 Db-FM (right), detected by APC-conjugated anti-FITC antibodies. MFI is indicated. B Kasumi-1 and AdFITC-CAR T-cells were co-cultured at an effector-to-target ratio E:T-1:1, tumor cell lysis was assessed via flow cytometry after 24, 48, and 72 h of co-culture. Percentage specific lysis of Kasumi-1 cells is shown relative to Db-FM concentration, represented as mean ± SD from three healthy-donor-derived AdFITC-CAR T-cell donors, each plated in duplicate wells. C Representative histograms showing HL-60 GFP⁺ cells transduced to express CD117 at high, medium, and low levels, labeled with 10 nM CD33 Db-FM (top) and CD117 Db-FM (bottom) as described in (A). D Percentage specific lysis of HL-60 cells was assessed over time after co-culture with AdFITC-CAR T-cells at E:T-1:1 and increasing levels of Db-FM. Mean ± SD from three healthy-donor AdFITC-CAR T-cell donors, each plated in duplicates. E Representative histograms showing MOLM14 GFP⁺ cells transduced to display various levels of CD117, stained and analyzed as described in (C). F MOLM14 cell lysis mediated by AdFITC-CAR T-cells at E:T-1:1 in culture with Db-FM. Percentage specific lysis of MOLM14 cells as a function of Db-FM concentration is represented as mean ± SD from three healthy-donor-derived AdFITC-CAR T-cell donors plated in duplicate wells. Quantification of IL-2 (G) and IFN-γ (H) in the supernatant from the same co-culture experiment indicated in (F) at 24 h.

Fig. 3. Combination of diabody adaptors enhances cytotoxicity against AML cell lines compared to single adaptors.

A Experimental setup to evaluate adaptor-mediated AdFITC-CAR T-cell activity against MOLM14 cells, double positive for the targeted antigens CD33 and CD117. B MOLM14 GFP^-CD117^midCD33⁺ cells were stained with 10 nM of either CD33 Db-FM, CD117 Db-FM, or both Db-FM (10 nM each). Unstained cells served as negative control. C Equal ratio of MOLM14-CD117^midCD33⁺ cells and AdFITC-CAR T-cells were cultured with increasing concentrations of CD33 and CD117 Db-FM, alone or in combination. Percentage specific lysis after 24 h is shown. Statistical analysis at the indicated EC₅₀ was performed with one-way ANOVA, ***p < 0.001. D, E Time-lapse imaging to assess MOLM14-CD117^midCD33⁺ cell lysis by AdFITC-CAR T-cells in combination with CD33 and/or CD117 Db-FM. Time to PI influx was significantly shorter with the combinatorial adaptor approach when compared to single adaptor targeting (results from two healthy donors, statistical analysis conducted using one-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). F Experimental setup to evaluate adaptor-mediated AdFITC-CAR T-cell activity against MOLM14 cells, being either single or double positive for the targeted antigens. G, H MOLM14-CD117^negCD33⁺, MOLM14-CD117^highCD33⁺, and MOLM14-CD117^highCD33^KO cells were mixed at equal ratios and co-cultured with AdFITC-CAR T-cells at an E:T = 1:1. CD33 and CD117 Db-FM were added at 1 nM as single agents or in combination. G Percentage specific lysis of the target cells was measured at indicated time points. Statistical analysis was conducted using two-way ANOVA; ***p < 0.001; ****p < 0.0001. H Representative flow cytometry plots showing residual MOLM14 subpopulations at 24 h. I–K MOLM14-CD117^negCD33⁺, MOLM14-CD117^highCD33⁺, and MOLM14-CD117^highCD33^KO cells were mixed at equal ratios and co-cultured with healthy donor-derived AdFITC-CAR T-cells were mixed as in (G, H). The combination of CD33 and CD117 Db-FM was added at 1 nM, and target-cell lysis was measured at indicated time points. Percentage specific lysis of the overall target cells (I) and subpopulations (J) at the indicated time points. Statistical analysis was conducted using two-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. K Representative flow cytometry plots showing residual MOLM14 subpopulations at 0, 4, 8, and 24 h. A–K Unless otherwise indicated, data are presented as mean ± SD from three healthy donor-derived AdFITC-CAR T-cells, each plated in duplicate wells.

Fig. 4. Combinatorial targeting of primary patient AML blasts is more efficient than single-adaptor targeting.

A Surface expression of target antigens (CD33 and CD117) on CD45^dimCD3^- cells from three AML patient samples. Representative flow cytometry analysis upon thawing, before and after the addition of healthy donor AdFITC-CAR T-cells at E:T = 1:1 ratio and after 24 h incubation in the presence of the indicated concentrations of diabody adaptors is shown. Percentage specific lysis of CD45^dimCD3^- AML blast cells after 24 h (B) and 48 h (C) in culture with Db-FM targeting CD33 and CD117, alone or in combination. Data from 2 independent experiments with AML cells from three patients and AdFITC-CAR T-cells derived from 3 different healthy donors plated in duplicates (mean ± SD). Statistical analysis was conducted using two-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) properties of Db-FM adaptors.

A Schematic representation of PK experimental set-up in mice not carrying target cells. NSG mice received a single i.v. or i.p. injection of 25 μg CD117 Db-FM (n = 2 mice per time point per administration route). Blood was collected from all mice at 1 min post-injection and terminally at the indicated time points. B Db-FM concentration in serum was determined by ELISA (mean ± SD of technical duplicates per mouse). Calculated t_1/2 and AUC are indicated. C Experimental set-up to study on-tumor residence time of CD117 Db-FM on MOLM14-CD117^high GFP⁺Luc⁺ cells in xenografted mice. NSG mice were sublethally irradiated and injected i.v. with 10⁵ MOLM14 cells. On day 10, 50 μg of CD117 Db-FM were injected i.v. or i.p. (n = 1 mouse per time point per administration route). D At the indicated time points, live MOLM14 cells (hCD45⁺GFP⁺) isolated from BM were analyzed by flow cytometry (representative dot plot on the left), and target-bound CD117 Db-FM was detected by APC-conjugated anti-FITC antibody staining (histograms on the right). MOLM14 cells from mice engrafted but not subsequently injected with Db-FM served as negative (−) and positive controls after ex vivo staining with 10 nM CD117 Db-FM (+). E Sublethally irradiated NSG mice were injected i.v. with 10⁵ MOLM14-CD117^highGFP⁺Luc⁺ cells. On day 10, 50 μg of CD117 or CD33 Db-FM were injected i.v. and femora were collected after 1 and 6 h to determine the clearance of Db-FM from the tumor cell surface by flow cytometry (n = 1 mouse per time point per Db-FM). A mouse engrafted with MOLM14 but not injected with adaptors served as negative control. F, G After ex vivo staining of cells isolated from the control mouse with titration of Db-FM, the surface labeling levels obtained from known Db-FM concentrations were used to interpolate the concentrations of Db-FM on tumor cells retrieved from mice injected with Db-FM. MFI resulting from target-bound Db-FM was detected by APC-conjugated anti-FITC antibody. F Interpolated on-tumor CD33 Db-FM concentration was estimated at 0.99 nM after 1 h injection and 0.22 nM after 6 h (each indicated in red). G Interpolated on-tumor CD117 Db-FM concentration was determined to be 4.37 nM after 1 h injection and 1.37 nM after 6 h (each indicated in blue).

Fig. 6. In vivo therapeutic efficiency of direct CAR T-cells and AdFITC-CAR T-cells in combination with CD33 or CD117 Db-FM adaptors against MOLM14-CD117^highAML cells.

A Schematic outline of the experimental setup. Sub-lethally irradiated mice were engrafted with 10⁵ MOLM14-CD117^highGFP⁺Luc⁺ cells. Seven days later, engraftment was confirmed by bioluminescence analysis (BLI), and mice were injected with either 10⁷ direct CAR T-cells targeting CD33 or CD117 (positive controls) or AdFITC-CAR T-cells (n = 3/4 mice per group). Mice from the latter group subsequently received 25 μg CD33 Db-FM or CD117 Db-FM (i.p. every 12 h) or no adaptors (negative controls). B Bioluminescence analysis of MOLM14 cell engraftment at days 7, 14, and 19 in mice treated with PBS, AdFITC-CAR T-cells in the absence or presence of adaptor, or direct CAR T-cells (CD33-CAR). C Quantification of the bioluminescence flux of whole-body imaging of mice in (B). D Absolute counts of hCD45⁺GFP⁺ MOLM14 cells (mean ± SD) in the BM of a single femur per mouse at terminal analysis. Statistical analysis was conducted using two-way ANOVA; *p < 0.05, **p < 0.01. E Bioluminescence analysis at indicated time points of mice treated with PBS, AdFITC-CAR T-cells with or without adaptor, or direct CAR T-cells (CD117-CAR). F Corresponding quantification of the bioluminescence flux of mice showed in (E). G Absolute hCD45⁺GFP⁺ MOLM14 cell count (mean ± SD) in the BM of a single femur per mouse at the end of the study, statistical analysis performed as indicated in (D). B–G Data shown from two independent experiments. H Representative flow cytometry plots of live cells from BM single-cell suspensions receiving the above-indicated treatment. I HE staining of the contralateral femora from the mice depicted in (H), as well as from healthy age-matched control mice (20x magnification, solid line indicates 100 μm).

Fig. 7. Combinatorial use of adaptors improved in vivo therapeutic efficacy of AdFITC-CAR T-cells against MOLM14-CD117^high AML cells.

A Sublethally irradiated NSG mice were i.v. injected with 10⁵ MOLM14-CD117^highGFP⁺Luc⁺ cells. On day 10, 50μg of CD117 or CD33 Db-FM were administered i.v. alone or in combination, and BM was collected 6 h post injection (n = 1 mouse per condition). B Flow cytometry histograms showing APC signal from anti-fluorescein antibodies targeting the Db-FM bound to MOLM14 cells at terminal analysis. MFIs indicated on the histograms show increased staining, obtained by combinatorial administration of adaptors. C MOLM14 cells from engrafted mice not receiving Db-FM injection were used as negative control and labeled ex vivo with 10 nM Db-FM alone or in combination to compare with in vivo labeling in (B). MFI is reported on the histograms. D Schematic experimental setup. NSG mice were sublethally irradiated and engrafted with 10⁵ MOLM14-CD117^highGFP⁺Luc⁺ cells. After seven days, mice were injected with 10⁷ AdFITC-CAR T-cells i.v. and subsequently with 12.5 μg CD33 and CD117 Db-FM i.p. alone or in combination every 12 h for three consecutive weeks (n = 3/4 mice per group). E In vivo tumor burden evaluation by bioluminescence imaging at indicated time points. On day 28, placeholders indicate mice previously terminated due to high tumor load. F Quantification of BLI flux signal over time (mean ± SD). G Absolute counts of hCD45⁺GFP⁺ MOLM14 cells (mean ± SD) isolated from BM of one femur each and from 150 μl of blood at terminal analysis. Counts refer to a final resuspension volume of 1 ml. H Percentages of MOLM14 cells, T-cells, and AdFITC-CAR T-cell fraction within total hCD45⁺ cells (mean ± SD) in BM and blood, identified based on surface expression of CD3, CD117, and RQR8 by flow cytometry.

Fig. 8. Combinatorial use of adaptors enhances the therapeutic efficacy of AdFITC-CAR T-cells against primary patient AML cells in murine xenograft models.

A Schematic experimental setup. Seven days after engraftment with 5 × 10⁶ CD3/CD19 double-depleted PB cells isolated from AML patient PID20, sublethally irradiated NSG mice were injected i.v. with either 10⁷ direct anti-CD117 or direct anti-CD33 CAR T-cells (alone or 5×10⁶ each in combination), or 10⁷ AdFITC-CAR T-cells. Subsequently, 12.5 μg CD33 and/or CD117 Db-FM were injected i.p. every 12 h for three weeks (n = 5 mice per group). B–E Absolute counts of residual AML cells (hCD45^dimCD3^-CD19^-) and respective subpopulations expressing the target antigens CD33 and CD117 (mean ± SD) in the BM of a single femur per mouse at terminal analysis. Statistical analysis was conducted using one-way ANOVA; *p < 0.05, **p < 0.01. F Schematic experimental setup. Seven days after engraftment with 5×10⁶ PID20 AML cells, mice were injected with either 10⁷ AdFITC-CAR T-cells i.v. followed by i.p. administration of 12.5 μg CD33 and CD117 Db-FM, alone or in combination, at 12 h intervals for one week (n = 4 mice per group). G Representative flow cytometry plots of live cells from BM single-cell suspensions at terminal analysis for each experimental group. H Representative flow cytometry histograms showing the target antigen levels in mice receiving the indicated treatments. MFIs are reported on the histograms. I, J Absolute counts of residual AML cells (hCD45^dimCD3^-CD19^-) and respective subpopulations expressing CD33 and CD117 (mean ± SD) in the BM of a single femur per mouse. Statistical analysis was conducted using one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001.