Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia that affects B lymphocytes in adults. Natural killer (NK) cells in CLL patients are intrinsically potent but display poor in situ effector functions. NKG2D is an activating receptor found on NK and CD8⁺ T cells and plays a role in immunosurveillance of CLL. In this study, we developed mono- and dual-targeting triplebodies utilizing a natural ligand for human NKG2D receptor (ULBP2) to retarget NK cells against tumor cells. Triplebodies in both formats showed better ability to induce NK-cell-dependent killing of target cells compared to bispecific counterparts. A mono-targeting triplebody ULBP2-aCD19-aCD19 successfully triggered NK cell effector functions against CLL cell line MEC1 and primary tumor cells in allogenic and autologous settings. Additionally, a dual-targeting triplebody ULBP2-aCD19-aCD33 specific for two distinct tumor-associated antigens was developed to target antigen loss variants, such as mixed lineage leukemia (MLL). Of note, this triplebody exhibited cytotoxic activity against CD19/CD33 double positive cells and retained its binding features even in the absence of one of the tumor antigens. Further, ULBP2-aCD19-aCD19 showed significant in vivo activity in immune-deficient (NSG) mouse model transplanted with CLL cell line as target cells and human immune cells as an effector population providing a proof-of-principle for this therapeutic concept.

Keywords: CD19; CLL; NK cells; NKG2D; ULBP2; immunoligand; triplebody.

Figure 1.

Schematic representation and expression of ULBP2-aCD19, ULBP2-aCD33, ULBP2-aCD19-aCD33 and ULBP2-aCD19-aCD19 immunoligands. (A) Cartoon illustrating bispecific immunoligands ULBP2-aCD19 (U-19) and ULBP2-aCD33 (U-33) and triplebodies ULBP2-aCD19-aCD33 (U-19-33) and ULBP2-aCD19-aCD19 (U-19-19) as well as control constructs: aCD19scFv and aCD33scFv. All four “test” immunoligands contain 15mer Gly-Ser linker (GGGGS)_3x to provide flexibility and a c-Myc tag and 6xHis tag for purification/detection. An N-terminal part of ULBP2 facilitated secretion of all four immunoligands into the s/n of transfected cells. In contrast, Igκ leader sequence was placed 3′ of both control constructs (aCD19scFv and aCD33scFv) to facilitate their secretion and 6xHis tag was used for purification. (B and C) All constructs were expressed in eukaryotic cell line HEK293T and purified from the supernatant by affinity chromatography utilizing their 6xHis tag. All four (U-19, U-33, U-19-33 and U-19-19) immunoligands were separated on SDS-PAGE for protein gel blot staining (B) using anti c-Myc tag antibody or coomassie staining (C) to confirm the size and purity. Coomassie staining was also done to check the size and purity of both control constructs aCD19scFv and aCD33scFv (Data not shown).

Figure 2.

Specificity of ULBP2-aCD19, ULBP2-aCD33, ULBP2-aCD19-aCD33 and ULBP2-aCD19-aCD19 immunoligands to their respective target moieties. (A) CD19⁺ (MEC1) and CD19⁻ (HL60) cell lines were incubated with 10 µg/mL of ULBP2-aCD19 or ULBP2-aCD19-aCD19 and binding was detected by either anti c-Myc tag antibody (upper panel—“c-Myc”) or recombinant human NKG2D receptor (lower panel—“NKG2D”) where the latter confirms that immunoligands can bind to CD19 and NKG2D simultaneously. Gray-filled area depicts the background staining. Pre-blocking of CD19 antigen by anti-CD19scFv (20 µg/mL) inhibited the binding of immunoligands (dashed line—“MEC1”). (B) CD19⁺CD33⁺ cell line BV173 was incubated with 10 µg/mL of ULBP2-aCD19, ULBP2-aCD33 and ULBP2-aCD19-aCD33 and binding was detected by recombinant human NKG2D receptor (“No blocking”). Pre-blocking of CD19 or CD33 antigen by respective blocking construct (20 µg/mL) prevented the binding of ULBP2-aCD19 and ULBP2-aCD33, respectively (“Block + Bispecific ILs”) but not of ULBP2-aCD19-aCD33 (“Block + ULBP2-aCD19-aCD33”). Only simultaneous blocking of both CD19 and CD33 antigens on BV173 cells could completely inhibit the binding of ULBP2-aCD19-aCD33 (“Block + ULBP2-aCD19-aCD33”).

Figure 3.

Simultaneous and specific antigen binding of a triplebody ULBP2-aCD19-aCD33. Simultaneous binding of ULBP2-aCD19-aCD33 to all three target moieties CD19, CD33 and NKG2D was detected. CD19⁺ MEC1 cell line was incubated with ULBP2-aCD19-aCD33 and its CD19 specific binding to MEC1 cells was detected using recombinant human NKG2D-Fc and CD33-FLAG receptors followed by AF647 labeled anti-Fc and PE labeled anti-FLAG antibodies, respectively (U-19-33). Binding of ULBP2-aCD19 to CD19 on MEC1 cells was detected only using NKG2D-Fc as it lacked anti-CD33scFv (U-19). ULBP2-aCD33 was used as a negative control that failed to bind CD19⁺ MEC1 cells (U-33) whereas NKG2D-Fc and CD33-FLAG background binding was minimal (No IL).

Figure 4.

Enhancement of primary NK cell effector functions by bispecific immunoligands and triplebodies. Cytotox assays. Primary NK cells were purified from peripheral blood mononuclear cells (PBMC) of healthy donors by negative selection and were cultured with IL-2 (200 U/mL) and IL-15 (10 ng/mL) overnight before the experiments on the following day. For all experiments, each N represents independent healthy donor. (A, Upper panel) Purified NK cells were co-incubated with DiR labeled CD19⁺ (MEC1) and CD19⁻ (HL60) cell lines at indicated effector to target (E:T) ratios either alone (•) or in presence of 10 nM ULBP2-aCD19 (▪) or ULBP2-aCD19-aCD19 (▴) immunoligand for 3 h and dead target cells were measured by 7-AAD staining on FACS. (A, lower lane) Purified NK cells were co-incubated with DiR labeled CD19⁺CD33⁺ cell lines (BV173 and SEM) at indicated effector to target (E:T) ratios either alone (•) or in presence of 100 nM ULBP2-aCD19 (▪), ULBP2-aCD33 (▾) or ULBP2-aCD19-aCD33 (▴) immunoligand for 3 h and dead target cells were measured by 7-AAD staining on FACS. For simplicity, selected statistical significances are shown in comparison with “No construct” group (*p < 0.05; **p < 0.01). Error bars indicate SEM (MEC1 (N = 4), HL60 (N = 3), BV173 (N = 5) and SEM (N = 4)). (B) Degranulation assay. Purified NK cells were co-incubated with MEC1 and SEM cells at E:T ratio of 2.5:1 either alone or in presence of indicated immunoligand for 6 h and CD107a/LAMP-1 staining within NK cells (stained and gated with anti-CD56 and anti-NKp46 antibodies) were measured to determine degranulated NK cell population. Error bars indicate SEM and ** represents p < 0.01; *** represents p < 0.001 (MEC1 (N = 3) and SEM (N = 5)) (C) ELISA-based IFNγ assay. Left: Purified NK cells were co-incubated with MEC1 cells at E:T ratio of 1:1 either alone or in presence of 10 nM immunoligand for 24 h and supernatant was collected for IFNγ detection by ELISA. IFNγ secretion by MEC1 cells (with or without immunoligand) was carefully controlled and was found to be negative (data not shown). Experiments were conducted with two independent NK donors and one example is shown where error bars indicate SEM of duplicates. (C, right) Purified NK cells were cultured in plate pre-coated with indicated immunoligands for 48 h and supernatant was collected for IFNγ detection by ELISA. Experiments were conducted with three independent NK cell donors and one example is shown where error bars indicate SEM of duplicates.

Figure 5.

ULBP2-aCD19-aCD19 mediates NK-cell-dependent killing of primary CLL cells in allogenic and autologous settings. (A) Allogeneic setting. Purified NK cells from healthy donors were co-incubated with DiR labeled primary CLL cells either alone (•) or in presence of 10 nM ULBP2-aCD19 (▪) or ULBP2-aCD19-aCD19 (▴) immunoligands for 3 h and dead target cells were measured by 7-AAD staining on FACS. Two representative results (Pt #1 and Pt #2) from four independent experiments with different CLL patients are shown. NK cells for each experiment were obtained from different healthy donors. (B) Autologous setting. NK cells were purified from PBMC of CLL patients (Pt #3 and Pt #4) by negative selection and were cultured with IL-2 (200 U/mL) and IL-15 (10 ng/mL) overnight and on the next day were co-incubated with DiR labeled CLL cells from the same patient either alone (•) or in presence of 10 nM ULBP2-aCD19-aCD19 (▴) for 3 h before measuring 7-AAD staining on FACS. (C) Innate NK:CLL ratio. Isolated PBMC from CLL patients (Pt #3 and Pt #4) were cultured alone or in presence of 10 nM ULBP2-aCD19-aCD19 for 48 h and CLL cell count was determined by gating for CD20⁺/CD5⁺ CLL cells.

Figure 6.

Antitumor activity of ULBP2-aCD19-aCD19 in an immunodeficient (NSG) mouse model. 5 × 10⁶ MEC1 cells were subcutaneously (s.c.) transplanted into 21 NSG mice. Two days later, 4 × 10⁵ PBMC from a healthy donor together with 15 µg of ULBP2-aCD19-aCD19 were injected (i.v.) into seven mice while another seven mice were left untreated (control group). Remaining seven mice were either treated with 4 × 10⁵ PBMC alone (four mice) or in combination with 15 µg of a non-specific immunoligand ULBP2-aPSMA (three mice). (A) Tumor growth for each mouse was measured at regular interval using the formula for tumor volume (L × W × H/2). Results show the measurements until day 28 (when all the mice were alive) and are depicted as mean (SEM) of seven mice in “no treatment” (•), “PBMC ± U-PSMA” (▪) and “ULBP2 + U-19-19” (▴) group, respectively. For simplicity, statistical significance between “No treatment” and “PBMC + U-19-19” groups are shown. (*p < 0.05; **p < 0.01). (B) Mice were sacrificed when the tumor volume reached 1,000 mm³. The study was continued for 51 d since the tumor challenge with no signs of tumor development in “U-19-19” treatment group and mice in this group were censored at the end of the study. Survival curve of “PBMC + U-19-19” group was significantly different from “PBMC ± U-PSMA” (p = 0.0003) and “No treatment” group (p = 0.0002) while there was no significant difference between “PBMC ± U-PSMA” and “No treatment” groups (p = 0.568). (Two tailed p value < 0.0167 was considered as significant; 0.0167 was Bonferroni-corrected threshold). (C) Tumor size in individual mouse at day 32 showing late onset of tumor growth in mice treated with PBMC + U-PSMA (non-specific immunoligand; ▴) compared to PBMC (▪) treated mice (*p < 0.05; **p < 0.01). (D) Days required for tumor volume to reach 1,000 mm³ in individual mouse in all control groups. Despite late onset of tumor growth in three mice within “PBMC + U-PSMA” (▴) group, tumor in these mice progressed much quicker and required similar time-point as “no treatment” (•) and “PBMC” (▪) groups to reach 1,000 mm³.