Balance of Anti-CD123 Chimeric Antigen Receptor Binding Affinity and Density for the Targeting of Acute Myeloid Leukemia

Abstract

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

Keywords: AML; CAR; CD123; affinity; immunotherapy.

Graphical abstract

Figure 1

Three-Dimensional Model of the Anti-CD123/CD123 Complex and SPR Binding Analysis (Top) Cartoon visualization of CD123 domain 1 (blue, with the region important for anti-CD123 binding in cyan) and anti-CD123 antibody (heavy and light chain in dark and light gray, respectively). Single mutations, shown as stick, were introduced in the antigen binding loops to affect CAR affinity. (Bottom) SPR sensogram showing the binding of immobilized WT single-chain antibody, CAM-L, and CAM-M to CD123. Raw data are shown in gray, whereas the fit used to calculate the binding properties is in color, with gradation indicating different concentrations. Association, dissociation, and binding affinity are shown.

Figure 2

Later Effector Functions, and Not Early Cytotoxic Profile, Are Impaired in CAR-CIK Cells Displaying Identical Binding Affinity but Reduced Expression (A and B) CAR expression (A) and MFI values (B) at day 21 of anti-CD123 CAR-CIK cells and NO DNA control. Data represented are the result of mean ± SEM. (A) n = 6 (**p < 0.01; one-way ANOVA, Bonferroni test); (B) n = 9 (**p < 0.01; ***p < 0.001; one-way ANOVA, Dunn’s multiple comparison test). (C–F) Short-term AnnV-7AAD assay by means of the cytotoxic double-target challenge at an effector-target (E:T) ratio of 5:1 after a 4 hr co-culture with both THP-1 and MHH-CALL-4 cell lines (C). Long-term proliferation assay after a 72 hr co-culture of the effector cells with THP-1 and MHH-CALL-4 cell lines (D). Intracellular cytokine staining of IFN-γ (E) and IL-2 (F) after a 5 hr co-culture between effector cells and both THP-1 and MHH-CALL-4 targets. Data represented are the result of mean ± SEM. n = 4 (***p < 0.001; two-way ANOVA, Bonferroni test).

Figure 3

Reduced CAR Binding Affinity Does Not Affect CIK Cell Cytotoxic Functions in Response to Highly CD123⁺ Target Cells (A and B) CAR expression (A) and MFI values (B) at day 21 of anti-CD123 CAR-CIK cells and NO DNA control. Data represented are the result of mean ± SEM. (A) WT CAR, CAM-H1, and CAM-L: n = 18; CAM-3: n = 7; (B) n = 7 (one-way ANOVA, Bonferroni test). (C–F) AnnV-7AAD assay through double-target challenge after 4 hr co-culture with both THP-1 and MHH-CALL-4 cell lines and primary AML and healthy bone marrow samples (C). E:T ratio 5:1, n = 4. Long-term proliferation assay after the co-culture of the effector cells with THP-1 and MHH-CALL-4 cell lines for 72 hr (D). Intracellular cytokine staining of IFN-γ (E) and IL-2 (F) after 5 hr co-culture between effector cells and THP-1, MHH-CALL-4 targets, primary AML, and healthy BM samples. Data represented are the result of mean ± SEM. WT CAR, CAM-H1, and CAM-L: n = 7; CAM-M: n = 4 (*p < 0.05, ***p < 0.001; two-way ANOVA, Bonferroni test).

Figure 4

Short-Term Cytotoxic Double-Target Challenge AnnV-7AAD assay, E:T ratio 5:1, (A) THP-1/MHH-CALL-4 (n = 5; CAM-H1: n = 4), (B) THP-1/U937 (n = 5; CAM-H1: n = 4), (C) THP-1/TIME (n = 6), (D) U937/MHH-CALL-4 (n = 5; CAM-H1: n = 4), and (E) TIME/MHH-CALL-4 (n = 6) double-target combinations. Data represented are the result of mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA, Bonferroni test).

Figure 5

Target Antigen Density, and Not CAR Binding Affinity, Affects Later Effector Functions (A) Long-term proliferation assay after the co-culture of the effector cells with the indicated cell lines for 72 hr. (B–E) Intracellular cytokine staining of IFN-γ (B), TNF-α (C), IL-2 (D), and IL-6 (E). Effector cells were co-cultured for 5 hr with THP-1, KG-1, U937, TIME, and MHH-CALL-4 targets. Data represented are the result of mean ± SEM. n = 12 (ALONE condition); n = 9 (THP-1, U937, and TIME experimental cell conditions); n = 3 for all CARs tested against the KG-1 cell line (*p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA, Bonferroni test).

Figure 6

TIME Cell Vessels Are Not Hampered by CAR-CIK Cells (A) Representative 5× and 10× magnifications of TIME cell vessels after a 4 hr culture on Matrigel (TIME cells alone) or with NO DNA and CAR-CIK cells (WT CAR and CAM-L), respectively. (B) Analysis of TIME cell branching point number, alone or after a 4 hr co-culture with unmanipulated and CAR-CIK cells, as a measure of TIME cell tissue integrity and vessel spreading. Data represented are the result of mean ± SEM. n = 3 (***p < 0.001; one-way ANOVA, Bonferroni test).

Figure 7

CAR Downmodulation in CAR-CIK Cells (A and B) Percentage of CAR-CIK cell downmodulation (A) and percentage of IFN-γ production by CAR-CIK cells (B) in response to THP-1 cells. (A) WT CAR, CAM-H1, and CAM-L: n = 11; CAM-M: n = 9; CAM-H2: n = 7 (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA, Bonferroni test). (B) WT CAR, CAM-H1, and CAM-L: n = 11; CAM-H2 and CAM-M: n = 4. (C) Plot of CAR downmodulation as a function of killing activity against both THP-1 and MHH-CALL-4 targets. (D) CAR-CIK cell downmodulation as a function of target antigen density. Data represented are the result of mean ± SEM, n = 11 for the stimulation with MHH-CALL-4, TIME, U937, and THP-1 cell lines; n = 7 for the stimulation with KG-1 cells.