NKG2D Ligand-Targeted Bispecific T-Cell Engagers Lead to Robust Antitumor Activity against Diverse Human Tumors

Abstract

Two new bispecific T-cell engaging (BiTE) molecules with specificity for NKG2D ligands were developed and functionally characterized. One, huNKG2D-OKT3, was derived from the extracellular portion of the human NKG2D receptor fused to a CD3ε binding single-chain variable fragment (scFv), known as OKT3. NKG2D has multiple ligands, including MICA, which are expressed by a variety of malignant cells. A second molecule, B2-OKT3, was created in the tandem scFv BiTE format that targets MICA on tumor cells and CD3ε on human T cells. Both BiTEs specifically activated T cells to kill human tumor cell lines. Cytotoxicity by B2-OKT3, but not huNKG2D-OKT3, is blocked by soluble rMICA. The huNKG2D-OKT3 induced greater T-cell cytokine production in comparison with B2-OKT3. No T-cell pretreatment was required for IFNγ production upon coculture of B2-OKT3 or huNKG2D-OKT3 with T cells and target cells. The effector memory T-cell compartment was the primary source of IFNγ, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density-dependent production of IFNγ from both CD4⁺ and CD8⁺ T cells. There was 2-fold more IFNγ produced per CD8⁺ T cell and 5-fold greater percentage of CD8⁺ T cells producing IFNγ compared with CD4⁺ T cells. In addition, both BiTEs elicited significant antitumor responses against human metastatic melanoma tumor samples using autologous or healthy donor T cells. These data demonstrate the robust antitumor activity of these NKG2D ligand-binding bispecific proteins and support their further development for clinical use. Mol Cancer Ther; 16(7); 1335-46. ©2017 AACR.

Figure 1. BiTEs specifically activate T cells and induce killing of MICA⁺ cell lines

(A) Cytotoxicity of human T cells induced by BiTEs against MICA⁺ human cell lines over a range of effector: target (E:T) cell ratios. Cultured T cells from four healthy donors were incubated with K562-Luc (left) or Panc1-Luc (right) tumor cells with 250 ng/mL B2-OKT3 or huNKG2D-OKT3, or no BiTE in triplicate wells. Relative light units (RLU) were measured after overnight culture. Representative plots with averaged RLU are shown. Error bars reflect the standard deviation. *p≤0.05 compared to no BiTE treatment. (B) Dose response of cytotoxicity induced by BiTE. Cultured T cells from two healthy donors were incubated with K562-Luc (left) or Panc1-Luc (right) cells at an E:T of 10:1 with the indicated concentrations of B2-OKT3 or huNKG2D-OKT3 in triplicate wells. Representative plots with averaged RLU are shown. Error bars reflect the standard deviation. *p≤0.05 compared to 0ng/ml BiTE. T cell IFNγ production when cultured with huNKG2D-OKT3 (C) or B2-OKT3 (D) against different tumor cell lines. Cultured T cells from four to six healthy donors (K, S, X, CC, DD, or EE) were incubated with human tumor cell lines, K562, PC3, Panc1, MCF7, or T47D. IFNγ was measured in cell-free medium after 24 hours. Error bars reflect the standard deviation. (E) Representative flow plots showing expression of MICA/B (black) compared to unstained controls (grey) for the cell lines used in C and D. (F) T cell IFNγ production (from healthy donors Q, S, W, U) when cultured with huNKG2D-OKT3 (left) B2-OKT3 (middle) or a TZ47-OKT3 (control BiTE; right) against either MICA^- cell line B16F10 or B16F10 cells transduced to express MICA (B16F10-MICA). (G) Flow plots showing expression of MICA/B (black) compared to unstained controls (grey) for B16F10 and B16F10-MICA.

Figure 2. B2-OKT3 and huNKG2D-OKT3 BiTEs bind different epitopes on MICA and B2-OKT3 cytotoxic activity is blocked at supraphysiological concentrations of rMICA

B2-OKT3 and huNKG2D-OKT3 binding to rMICA was measured by biolayer interferometry. Streptavidin biosensors were loaded with biotinylated rMICA, saturated with the blocking BiTE (A) huNKG2D-OKT3 or (B) B2-OKT3 and then dipped into the test analytes: anti-MICA/B, huNKG2D-OKT3, B2-OKT3, and buffer only. Data are representative of at least two independent experiments. Cultured human T cells from four different donors were plated with either K562-Luc or Panc1-Luc at a 4:1 ratio. (C) huNKG2D-OKT3 or (D) B2-OKT3 was added at 50ng/ml. rMICA was at the indicated concentrations. ** Indicates concentrations where the cytotoxicity was significantly lower than no rMICA controls, p < 0.01.

Figure 3. After BiTE treatment, IFNγ producing T cells are enriched for effector memory T cells

A) Gating strategy used to identify functionally active T cell subsets based on IFNγ⁺ intracellular staining. In the indicated treatment groups, PBMCs and target K562 cells were cultured at an E:T ratio of 4:1, and BiTE was used at a dose of 250 ng/ml. Treatment with PMA and Ionomycin (Ion) was used as a positive control. Samples were stained with CD3, CD45RO, CCR7 and IFNγ. T cell subsets were designated as follows: Naïve (T_N) CD3⁺CD45RO^-CCR7⁺, Central memory (T_CM) CD3⁺CD45RO⁺CCR7⁺, Effector memory (T_EM) CD3⁺CD45RO⁺CCR7^-, and Terminal Effector T cells (T_EMRA) CD3⁺CD45RO^-CCR7^-. Data are representative of experiments performed with four different PBMC Donors. B) Distribution of cells in each T cell subset in the treatment groups described in (A). Data are shown for mean value of the four donors + standard deviation. Statistical significance determined by one-way ANOVA with Newman-Keuls multiple comparisons analysis. The color of the asterisk indicates the population that is statistically different. * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 4. Antigen density directly impacts T cell activation by B2-OKT3 and huNKG2D-OKT3 BiTEs

A) Activated T cells from ten donors were incubated with immobilized rMICA at the indicated concentrations and either huNKG2D-OKT3 (left), B2-OKT3 (center) or negative control BiTE (right) at 250 ng/mL for 24 hours. IFNγ from cell free supernatant was measured by ELISA. (B) Cells from 2-4 donors were treated as in A, but after a six hour incubation in the presence of brefeldin A, cells were stained for expression of CD4, CD8 and intracellular IFNγ, and data were acquired by flow cytometry. Percentage of activated cells as indicated by IFNγ⁺ staining are shown in B, while MFI for each T cell subset over the range of antigen densities tested is shown in C. MFI were compared to 0 ng/ml group by paired t test (* p ≤ 0.05, **p ≤ 0.01). Error bars represent SD of triplicates in (A) and between donors in (C). Blood donors are designated by letters (K, S, P, X, Y, Z, CC, DD, EE, FF, GG).

Figure 5. huNKG2D-OKT3 and B2-OKT3 BiTEs are able to activate healthy donor T cells, PBMCs and donor matched T cells against human melanoma tumor cell samples

T cells from two different healthy donors and autologous T cells were plated with each melanoma tumor cell sample at an E:T ratio of 4:1 and A) huNKG2D-OKT3 or B) B2-OKT3 BiTE at 250 ng/ml and incubated for 24 hours. IFNγ production was measured by ELISA. C) Unstimulated PBMCs from two healthy donors were incubated with each primary melanoma sample as in A and B, but IFNγ production in the supernatants was measured after a 48 hour incubation. Treatment with huNKG2D-OKT3 is shown on the left, and B2-OKT3 on the right. Each sample was run in triplicate and data are shown + SD of triplicates. A * indicates p ≤ 0.05 or ** indicates p ≤ 0.01 compared to all control groups. Auto = autologous; tumors are designated E8-13, blood donors are designated by letters (S, Q, P, X, Y, Z).