Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

Abstract

Background: Bispecific T-cell engagers (BiTEs) are recombinant bispecific proteins designed to stimulate polyclonal T-cell immunity. In recent years, B7H3, a pan-cancer antigen, has been considered a promising target for future immunotherapy. However, the B7H3-targeting BiTE faces the challenge of systemic toxicity. Oncolytic viruses (OVs) represent a new class of cancer immunotherapeutics and serve as an appropriate platform for locoregional delivery of therapeutic genes. In this study, we designed an oncolytic adenovirus (OAd) encoding BiTE targeting human B7H3. We hypothesized that OVs encoding B7H3 BiTE deliver this molecule persistently to the tumor site while mediating polyclonal T-cell activation and redirecting it to tumor cells.

Methods: B7H3-targeting BiTE was constructed by linking a single-chain variable fragment (scFv) that recognizes human B7H3 to an scFv that recognizes human CD3. B7H3 BiTE was inserted into OAd to construct OAd-B7H3-BiTE. The function of the OV-delivered B7H3 BiTE was detected via co-culturing B7H3⁺ target cells and peripheral blood mononuclear cells. A humanized immune system mouse model was used to evaluate the therapeutic effects in vivo.

Results: B7H3 is highly expressed in a high proportion of human malignancies. OV-delivered BiTEs bind to T cells and target cells. We observed a series of phenomena reflecting T-cell activation induced by OAd-B7H3-BiTE, including cell clustering, cell size, activation markers, cytokine secretion, and proliferation. Furthermore, T-cell activation was mirrored by the corresponding cytotoxicity against B7H3⁺ tumor cells. In vivo, B7H3 BiTE was persistently expressed in tumors and enhanced the antitumor T-cell immune response.

Conclusions: Using an OV for the local expression of B7H3 BiTE maximizes the local concentration of BiTE while reducing systemic exposure. OV also provides a relatively "hot" T-cell immune environment for the function of BiTE. Because of its capacity to activate polyclonal T cells, BiTE has the potential to redirect virus-specific T cells to tumors. Our study provides new opportunities for the exploitation of B7H3-BiTE-armed OVs as therapeutic agents for the treatment of B7H3-positive malignancies.

Keywords: Bispecific T cell engager - BiTE; Immunotherapy; Oncolytic virus; T cell.

Figure 1. Expression of B7H3 in human malignancies and generation of an OV encoding B7H3 BiTE (OAd-B7H3-BiTE). (A) B7H3 (CD276) expression between tumor and normal samples analyzed by TIMER2.0 (http://timer.cistrome.org/). (B–C) Representative immunohistochemistry images (B) and quantification (C) of B7H3 staining in peritumoral and cancerous tissue from patients with lung adenocarcinoma (LUAD) (n=3), breast cancer (BC) (n=6) or colon adenocarcinoma (COAD) (n=2). Each pair of dots represents a patient. (D) Schematic of recombinant OAd-B7H3-BiTE genomes. (E) Cytotoxic effects of OAd-B7H3-BiTE. MGC-803 and U87 cells were infected with serial dilutions of OAd or OAd-B7H3-BiTE. Four days after infection, cell viability was examined. (F) Viral replication of OAd-B7H3-BiTE. MGC-803 and U87 cells were infected with OAd or OAd-B7H3-BiTE and then harvested at different time points. DNA extraction and quantitative PCR were performed to detect the copy number of the E1a gene. Fold change to 0 hour was calculated. (G) Serial volumes of anti-CD3 scFv were detected with anti–anti-CD3-scFv antibody by western blot. (H) HEK293 cells were infected with OAd or OAd-B7H3-BiTE at multiplicity of infection of 0.5. 48 hours after infection, supernatants were collected. B7H3 BiTE was detected with anti–anti-CD3-scFv antibody by western blot. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; OAd, oncolytic adenovirus; scFv, single-chain variable fragment.

Figure 2. B7H3 expression in cell lines and the binding of delivered B7H3 BiTE to T and target cell. (A) B7H3 expression on MGC-803, SH-SY5Y, U87, SGC-7901 and Raji cells were detected using flow cytometry. (B) Binding of delivered B7H3 BiTE to target cells. Several cell lines were incubated with supernatants of the OAd-B7H3-BiTE-infected HEK293 cells. B7H3 BiTE binding was detected via flow cytometry with anti–anti-CD3-scFv antibody. (C) CD3 expression on Jurkat cells was detected using flow cytometry. (D) Binding of delivered B7H3 BiTE to T cells. PBMCs were incubated with supernatants of the OAd-B7H3-BiTE-infected HEK293 cells. B7H3 BiTE binding was detected via flow cytometry with anti–anti-CD3-scFv antibody. (E) Competition between delivered B7H3 BiTE and OKT3. PBMCs were incubated with supernatants of OAd or OAd-B7H3-BiTE-infected HEK293 cells. Then, cells were stained with fluorescein-conjugated OKT3 and examined throughflow cytometry. (F) Cell-bridging assay. A mixture of MGC-803/U87 and PBMCs was incubated with the supernatants of the OAd or OAd-B7H3-BiTE-infected HEK293 cells. The percentage of T/target cell pairs was quantified using flow cytometry. Statistical analysis was performed by unpaired Student’s t-test (n=3). NS: not significant; **p<0.01 and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; OAd, oncolytic adenovirus; PBMC, peripheral blood mononuclear cell; scFv, single-chain variable fragment.

Figure 3. T-cell activation mediated by oncolytic viruses-delivered B7H3 BiTE. (A–D) PBMCs were cultured alone or co-cultured with the indicated cell lines in the absence (Mock) or presence of the supernatants of OAd or OAd-B7H3-BiTE-infected cells for 48 hours. (A) Representative brightfield images are shown. (B) Representative flow cytometry histograms showing FSC-A of CD4⁺ and CD8⁺ T cells. (C) Cumulative data of flow cytometry showing CD69 or CD107a expression in CD4⁺ and CD8⁺ T cells. (D) The levels of IFN-γ and IL-2 in supernatants at 48 hours measured by ELISA. (E) T-cell proliferation assay. CFSE-stained PBMCs were co-cultured with MGC-803 cells and incubated with or without the supernatants of OAd or OAd-B7H3-BiTE-infected cells. Proliferation was determined after incubation for 4 days. Statistical analysis was performed by unpaired Student’s t-test (n=3). NS: not significant; **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester; IFN, interferon; IL, interleukin; OAd, oncolytic adenovirus; PBMC, peripheral blood mononuclear cell.

Figure 4. T-cell cytotoxicity mediated by OAd-B7H3-BiTE. (A–C) After labeling with CFSE, MGC-803 (A), U87 (B) or Raji (C) cells were infected with OAd or OAd-B7H3-BiTE; uninfected cells (Mock) were used as a control. After 24 hours, peripheral blood mononuclear cells were added and co-cultured for 48 hours. Representative flow cytometry plots (left) and cumulative data (right) are shown. Dead target cells are defined as CFSE⁺ Zombie Dye⁺. The percentage of dead target cells is calculated as follows: CFSE⁺ Zombie-Dye⁺/CFSE⁺. Statistical analysis was performed by unpaired Student’s t-test (n=3). **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester; OAd, oncolytic adenovirus.

Figure 5. OAd-B7H3-BiTE treatment elicits potent therapeutic effects in vivo. (A) Schematic diagram of treatment schedule for MGC-803 bearing mice (B–G). (B) Representative flow cytometry plots showing human CD45⁺ CD3⁺ T cells in tumor and spleen of untreated mice. Flow cytometry plots gated on live-cell population. (C) Summary data for average tumor growth (left) and body weight changes (right) for all treatment groups. (D) Tumor pictures and weights for all treatment groups. (E–F) Primary tumors were collected and analyzed with reverse transcription-quantitative PCR to examine the expression of E1a (E) and B7H3 BiTE (F). (G) Representative H&E staining of the major organs for all treatment groups. Statistical analysis was performed by unpaired two-way analysis of variance (C) or unpaired Student’s t-test (D) (n=5). ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; mRNA, messenger RNA; OAd, oncolytic adenovirus; PBS, phosphate-buffered saline; PBMC, peripheral blood mononuclear cell; s.c., subcutaneous injection.

Figure 6. OAd-B7H3-BiTE increases T-cell infiltration and activation. Flow cytometry analysis of T cells in the tumor xenografts model treated as described in figure 5A. (A) Proportion of CD4⁺ and CD8⁺ TILs. (B–D) The expression of CD69 (B), Tim-3 (C) and PD-1 (D) on TILs. (E) Expression of CD69 on CD4⁺ and CD8⁺ T cells in the spleen. Statistical analysis was performed by unpaired Student’s t-test (n=5). NS: not significant; *p<0.05, **p<0.01. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; OAd, oncolytic adenovirus; PD-1, programmed cell death-1; TILs, tumor-infiltrating lymphocytes; Tim-3, T cell immunoglobulin mucin-3.