Dual targeting ovarian cancer by Muc16 CAR-T cells secreting a bispecific T cell engager antibody for an intracellular tumor antigen WT1

Abstract

Epithelial ovarian cancer is the most lethal of gynecological cancers. The therapeutic efficacy of chimeric antigen receptor (CAR) T cell directed against single antigens is limited by the heterogeneous target antigen expression in epithelial ovarian tumors. To overcome this limitation, we describe an engineered cell with both dual targeting and orthogonal cytotoxic modalities directed against two tumor antigens that are highly expressed on ovarian cancer cells: cell surface Muc16 and intracellular WT1. Muc16-specific CAR-T cells (4H11) were engineered to secrete a bispecific T cell engager (BiTE) constructed from a TCR mimic antibody (ESK1) reactive with the WT1-derived epitope RMFPNAPYL (RMF) presented by HLA-A2 molecules. The secreted ESK1 BiTE recruited and redirected other T cells to WT1 on the tumor cells. We show that ESK1 BiTE-secreting 4H11 CAR-T cells exhibited enhanced anticancer activity against cancer cells with low Muc16 expression, compared to 4H11 CAR-T cells alone, both in vitro and in mouse tumor models. Dual orthogonal cytotoxic modalities with different specificities targeting both surface and intracellular tumor-associated antigens present a promising strategy to overcome resistance to CAR-T cell therapy in epithelial ovarian cancer and other cancers.

Figure 1

Cytolytic activity of ESK1-BiTE against ovarian cancer cells. (A.) Expression of WT1 protein was measured by intracellular staining of the SKOV3 Muc16 low cells using mAb to WT1 protein. (B). Expression of the cell surface WT1 epitope RMF/HLA-A2 complex was measured by staining the cells with mouse ESK1 mAb or its control at 3ug/ml and analyzed by flow cytometry. (C and D) ESK1.BiTE-mediated T cell cytotoxicity against SKOV3/A2 (C) or HL-60 (D). PBMCs from a healthy donor were incubated with SKOV3 Muc16 low target cells at a E:T ratio of 30:1 in the presence or absence of BiTEs at the indicated concentrations for 5 hr. The BiTE-mediated T cell cytotoxicity was measured by standard ⁵¹Cr-release assay. The HL-60 AML cell line (HLA-A2−) was used as a control. (E) Binding of ESK1 to fresh tumor cells in ascites from an ovarian cancer patient. Ascites cells were stained with mouse ESK1 vs anti-CD45 or its isotype control, to distinguish lymphocytes from tumor cells. Smaller lymphocytes and large tumor cells were gated and the CD45 vs ESK1 staining was shown in each gate (lower panels).

Figure 2

Therapeutic activity of ESK1-BiTE against ovarian cancer cells in vivo. SKOV3 Muc16low (2.5 million cells) were ip injected into NSG mice and tumor engraftment was confirmed on day 3 by bioluminescent imaging. PBMCs (10 million/mouse) from a healthy donor were injected ip into mice. A few hours later, ESK1 or control BiTE was injected ip into mice (5ug/mouse) and the BiTE dosing was repeated in a daily basis for total nine days. Tumor growth was monitored by BLI after 4 and 9 injections (A). Tumor burden was calculated by summing the luminescent signal of each mouse and average signal for each group (n = 5 per group) plus/minus SD is plotted (B). Total flux on day 14 post-therapy was shown for each experimental group and p Value is determined by 2 way ANOVA test (C).

Figure 3. Generation of 4H11 CAR T cells secreting ESK1-BiTE.

(A) Schematic of retroviral vectors encoding the 4H11BBz CAR alone (top) and ESK1-4H11BBz CAR constructs encoding a secreted ESK1 bite (bottom). A 6xHis-tag was added at the C-terminal of ESK1 bite for easy detection and affinity purification. A P2A element sequence was used to link ESK1 bite with MUC16 CAR sequences. (B) The (His)6-tag ESK1 bites were pulled down from the medium supernatants of WT, 4H11BBζ, and ESK1-4H11BBζ producer cells using Ni-NTA agarose beads, separated using SDS-PAGE, and analyzed by immunoblotting using an anti-His-tag antibody. The ESK1 bite appeared at the predicted molecular weight (∽58 kDa) in the conditioned media of ESK1-4H11BBζ Galv9 producer cells. (C-E). Supernatant fluids from 4H11, ESK1.BiTE-4H11 or Mock-T cells were concentrated 30-40 fold and used to stain AML-14 (C), HL-60 (D), or Jurkat (E) cells, followed by anti-His tag mAb staining to detect BiTE. Recombinant ESK1 BiTE or its control was used at 10ug/ml. (F). Jurkat cells were blocked by the supernatant fluids from Mock-T, 4H11 or ESK1.BiTE-4H11 CAR T cells (1:1 ratio) on ice for 30 minutes and followed by staining with anti-human CD3 mAb.

Figure 4

Functional activity of 4H11 CART cells secreting ESK1-BiTE. Expression of Muc16 on SKOV3 wild type (HLA-A2−) or HLA-A2+transduced cells was measured on SKOV3 wild type (Muc16 low) (A and C) or Muc16-transduced cells (Muc16 high) (B and D), using mAb to Muc16. Mouse ESK1 or its isotype control (3ug/ml) was used to measure the expression of WT1 RMF/HLA-A2 complex. (E and F): CAR T cells (10,000/well were serially diluted and incubated for one day to ensure BiTE secretion in the wells. After 1 day of incubation, SKOV3 target cells (10,000 cells/well) and (5,000 Mock T from same donor) were added to all wells for a total of 48 hours of incubation. (G). Supernatants from 4H11 CAR T or ESK1 BITE-secreting CART cells were pooled and concentrated for 16-fold and then serially diluted and incubated with PBMCs as effectors and target SKOV3 cells, at an E:T ratio of 10:1 for 48 hours. ESK1-BiTE and its control BiTE (1ug/ml) was used as control, in a similar serial dilution. The mean shown is the average of triplicate microwell cultures plus minus SD. The data are representative of six separate experiments.

Figure 5

Anti-tumor activity of 4H11 CAR T cells secreting ESK1-BiTE in vivo. (A) Muc16 low SKOV3 cells (2 million cells) were injected ip into NSG mice and tumor engraftment was confirmed on day 5 by bioluminescent imaging. 4H11 CAR T cells, ESK1 BiTE-secreting 4H11 CAR T cells, or control mock-T cells (2 million/mouse) were injected ip, followed by injection ip of two million activated T cells (mock-T from the same donor) on the next day. Tumor burden was monitored by bioluminescent imaging on day 12, 19 and 26. (B). Tumor burden of mice in panel A was calculated by summing the luminescent signal of each mouse, and average signal for each group (n = 4 per group, except for tumor alone group n = 3) plus minus SD, is plotted. Survival of mice from panel A experimental groups was monitored and the significance was evaluated by Mantel-Cox P test. * = <0.05; ** = <0.01 (C).