Table 2 Preclinical studies investigating oncolytic viruses encoding bispecific T cell engagers

Yu et al. (2014)

Oncolytic Vaccinia virus (VV), derived from Western Reserve vaccine strain

EphA2

First OV-BiTE agent described in the literature

In vitro: T cell effector cytokine production and cytotoxicity

In vivo: T cell effector cytokine production

T cell proliferation requires exogenous IL-2

SCID mice with A549 xenografts

s.c. tumors pre-mixed with PBMCs, virus i.p.: no tumor engraftment

i.v. lung colonization model, PBMC and virus mix i.v.: delayed tumor progression and prolonged survival

Fajardo et al. (2017)

Oncolytic adenovirus (AdV), derived from ICOVIR-15 K

EGFR

In vitro: T cell activation, proliferation, cytotoxicity, effector, and proinflammatory cytokine production

In vivo: transient increase in intratumoral T cell abundance (HCT116 model)

no T cell-mediated depletion of virus

SCID/beige mice with s.c. xenografts

A549 tumors, virus i.t., PBMCs i.v.: delayed tumor progression

HCT116 tumors, virus i.v., pre-activated T cells i.v., IL-2 i.p.: reduced tumor growth

Barlabé et al. (2019)

AdV (Fajardo et al. 2017)

EGFR

OV delivery via menstrual blood-derived mesenchymal stem cells (MenSCs)

In vitro: T cell cytotoxicity

In vivo: reduced viral load vs. unmodified virus

NSG mice with s.c. A549 xenografts

i.v. PBMCs, i.p. virus/virus-infected MenSCs: delayed tumor growth vs. OV-BiTE application without MenSCs/MenSCs carrying unmodified virus

Freedman et al. (2017)

AdV, derived from enadenotucirev (EnAd)

EpCAM, FHA (control)

First OV-BiTE study to include efficacy studies in primary, patient-derived model systems

In vitro: CD4⁺ and CD8⁺ T cell activation, proliferation, effector and inflammatory cytokine production, degranulation, cytotoxicity (recombinant BiTE from transfected cells); T cell activation and cytotoxicity via apoptosis induction (OV-BiTE)

Ex vivo: T cell activation, proliferation, degranulation, cytotoxicity

Tumor cell depletion in ex vivo malignant peritoneal ascites and pleural effusions containing tumor cells, immune cells, stromal cells, and soluble immunosuppressive factors

Speck et al. (2018)

Oncolytic measles viruses, derived from Edmonston B vaccine strain

CEA, CD20

BiTEs engineered to target human and murine CD3ε, respectively, for use in complementary mouse models and as controls; first study to show superiority of OV-BiTE to purified BiTE

In vitro: T cell cytotoxicity, effector and inflammatory cytokine production

In vivo: no negative selection of BiTE target antigen, no BiTE detected in serum following i.t. injection (PDX model); increased intratumoral mT cell levels and effector-to-regulatory T cell ratio; increased expression of T cell activation, differentiation, proliferation, and exhaustion markers (B16 model)

NSG mice with s.c. patient-derived xenografts, PBMCs i.t., virus i.t.: delayed tumor progression and prolonged survival

C57BL/6J mice with s.c. MC38/B16 tumors expressing human antigens, endogenous mT cells, virus i.t.: Delayed tumor progression, prolonged survival, long-term remissions with immune protection; efficacious also in MV-immune animals; no significant difference in efficacy compared to UV-inactivated, i.e., non-replicative, virus

Wing et al. (2018)

AdV (Fajardo et al. 2017)

EGFR

First study describing combination of OV-BiTE with CAR T cells

In vitro: CAR T cell cytotoxicity toward BiTE-targeted tumor cells, T cell activation, effector cytokine production and proliferation

In vivo: increased intratumoral abundance of CAR T cells, CAR T cell activation and proliferation (Panc-1 model)

NSG mice with s.c. xenografts, virus i.t., FRα-CAR T cells i.v

HCT116 (CAR target high) tumors: delayed tumor growth, prolonged survival

Panc-1 (CAR target low): delayed tumor growth

Porter et al. (2020)

AdV plus helper-dependent adenovirus encoding immunomodulators

CD44v6, CD19 (control)

Additional transgenes IL-12p70, PD-L1 inhibitor

In vitro: T cell activation, differentiation (T_H1), exhaustion

In vivo: CAR T cell activation, lower CAR levels at the tumor site

NSG mice with xenografts

FaDu/CAPAN-1 tumors s.c., virus i.t., HER2-/PSCA-CAR T cells i.v.: Similar efficacy for immunomodulatory vectors with and without BiTE transgene

Orthotopic FaDu/FaDu-HER2^−/− xenografts, virus i.t., HER2-CAR T cells: Trends toward reduced tumor load and prolonged survival

Yu et al. (2017)

VV

FAP

BiTE targeting CAFs instead of tumor cells; first study describing TME targeting via BiTE-encoding OV

In vitro: T cell effector cytokine production, cytotoxicity

In vivo: Increased intratumoral T cell infiltration, effector cytokine production, B16-specific T cell responses (ELISpot)

C57BL/6J mice with B16 tumors

s.c. model with virus i.t. and uninjected contralateral tumors: Correlation of FAP⁺ cell depletion with increased viral load in injected tumors; delayed tumor progression

i.v. B16F10 lung colonization model with virus i.v.: Reduced number of tumor nodules

Freedman et al. (2018)

EnAd AdV

FAP

CAF-targeting BiTE

In vitro: CD4⁺ and CD8⁺ T cell activation, degranulation and cytotoxicity, T cell proliferation, effector cytokine production (recombinant BiTE from transfected cells), T cell activation and cytotoxicity via induction of apoptosis (OV-BiTE)

Ex vivo: T cell activation, effector cytokine production, proliferation, cytotoxicity, reduction in TGF-β levels, differential gene expression—upregulation of T cell-associated genes and chemokines and antigen-presenting machinery, downregulation of fibroblast-associated genes and chemokines, and shift from M2 to M1 macrophage markers (malignant exudates), T cell activation, effector cytokine production, cytotoxicity via induction of apoptosis (prostate tumor biopsies)

Ex vivo malignant peritoneal ascites and pleural effusions: Reduction in FAP⁺ cells

Ex vivo thin tissue slices from prostate cancer samples: Stromal degradation

Sostoa et al. (2019)

ICOVIR-15K AdV

FAP

CAF-targeting BiTE; recognizes both human and mouse FAP

In vitro: CD4⁺ and CD8⁺ T cell proliferation, T cell activation, effector cytokine production, cytotoxicity

In vivo: Increased intratumoral T cell accumulation (A549 model)

NSG mice with s.c. A549/HPAC xenografts, virus i.t., T cells i.v.: FAP depletion, delayed tumor progression, prolonged survival

Scott et al. (2019)

EnAd AdV

FRβ, FHA (control)

BiTE targeting TAMs instead of tumor cells; comparison of different scFv orders; study also reports on trispecific T cell engagers

In vitro: T cell activation and cytotoxicity, also in presence of ascites fluid (recombinant BiTE from transfected cells)

Ex vivo: CD4⁺ and CD8⁺ T cell activation and proliferation, T cell effector cytokine production and cytotoxicity (for both recombinant BiTE from transfected cells and OV-BiTE)

CD11b⁺ CD64⁺ target cell reduction in ex vivo malignant peritoneal ascites and pleural effusions containing tumor cells, immune cells, stromal cells, and soluble immunosuppressive factors