Viro-antibody therapy: engineering oncolytic viruses for genetic delivery of diverse antibody-based biotherapeutics

Figures & data

Table 1. Overview of developed therapeutic antibody-encoding OVs: OVs expressing recombinant IgGs/monoclonal antibodies (Viro-MAb therapy) sorted according to targets and publication date

Virus	Antibody gene expression strategy	Antibody format	Target	Key results for antibody-encoding OV^a	Reference
NDV (wt mesogenic strain)	IgG heavy and light chains as separate, adjacent additional transcription cassettes with gene stop and gene start signal for viral transcription	IgG	Fibronectin extradomain B (tumor-specific vascular marker)	OV-encoded antibody is produced and binds to antigen after infection of tumor cells in vitro and after intratumoral injection in a xenograft mouse model	[121]
Ad (Ad5/3-E1AΔ24)	Replacement of early genes (E3) by Ig chains linked via IRES	IgG2	Human CTLA-4	Subcutaneous xenograft mouse tumor model/intratumoral virus injection: OV-encoded antibody detected in xenografts; 43-fold higher antibody concentration in tumor versus plasma 81-fold higher antibody concentration detected in tumors after injection of antibody-encoding OV compared with antibody-encoding replication-deficient control virus OV-encoded antibody activates T cells from cancer patients, which are more susceptible than T cells from healthy donors	[106]
Ad (E2F promoter driving viral E1A)	Replacement of early genes (E3) by Ig chains linked via 2A	IgG2	Murine CTLA-4	Antibody-encoding OV results in tumor-specific antibody expression in a panel of cell cultures in vitro Subcutaneous syngeneic mouse tumor model/intratumoral OV injection: tumor growth inhibition by antibody-encoding OV	[107]
Influenza A virus (IAV)	Heavy chain in PB1 segment downstream of PB1 gene via 2A; light chain in PA segment downstream of PA gene via 2A; scFv cloned into both segments	IgG and scFv	Proof-of-concept (IgG) and murine CTLA-4 (scFv)	Antibody insertion reduces titer, replication and in vivo morbidity and mortality of IAV Functions of OV-produced IgG similar to hybridoma-produced ab Subcutaneous syngeneic bilateral mouse tumor model/intratumoral OV application: scFv-encoding OV shows superior tumor growth inhibition (both flanks) and prolonged survival compared with parental virus	[117]
NDV (wt velogenic Italien strain)	IgG heavy and light chains as separate, adjacent additional transcription cassettes with gene stop and gene start signal for viral transcription	IgG	CD147 (metuximab)	Orthotopic xenograft mouse tumor model/intravenous OV application: antibody-encoding OV results in Antibody expression in tumors and tumor necrosis, Reduced intrahepatic metastasis and prolonged survival compared with parental virus	[122]
Vaccinia virus (Western Reserve strain, TK^− and RR^−)	Separate transcription unit with different viral promoters at TK locus	IgG, Fab, scFv	murine PD-1	Optimal promoter choice is required to avoid imbalance in expression of light versus heavy chain (IgG, Fab) OV-encoded IgG, Fab, and scFv are expressed and functional Subcutaneous syngeneic mouse tumor model/intratumoral virus application: OV-encoded IgG peaks at d 5 in tumor and serum mirroring virus replication IgG concentration and tumor/serum ratio higher for antibody-encoding OV than after i.t. injection of 10 μg mAb Therapeutic activity of IgG- and scFv-encoding OV in one of two tumor models superior to parental virus and similar to parental virus plus repeated large dose systemic antibody therapy	[103]
HSV-2 (ICP34.5^− and ICP47^−)	Expression of heavy and light chains as separate transcription units from CMV and RSV promoters, respectively	IgG	Human PD-1	Syngeneic mouse tumor model with humanized PD-1 mouse and tumor cells with recombinant HSV receptor/intratumoral OV injection: Antibody-encoding OV compared with parental OV: superior tumor growth inhibition, numbers and activation of T cells and induction of tumor-specific T cells in spleen Tumor growth inhibition by antibody-encoding OV superior to systemic antibody application and similar to combined treatment with parental virus and systemic antibody application Induction of memory response (protection from tumor cell rechallenge) by antibody-encoding OV	[92]
Ad (Ad5/3-E1AΔ24)	Replacement of early genes (E3) by Ig chains linked via IRES	IgG1	Human HER2 (Trastuzu-mab)	OV-encoded antibody shows direct antitumor activity and triggers ADCC in vitro Subcutaneous xenograft mouse tumor model/intratumoral virus injection: Enhanced antitumor efficacy of antibody-encoding OV compared with parental virus or trastuzumab for Her2-positive xenografts Higher tumor-to-blood antibody concentrations (for endpoint tumors) by antibody-encoding OV compared with conventional antibody application NK cell–dependent DC activation in draining lymph nodes by antibody-encoding OV	[108]
Ad (EnAd, chimeric type B Ad)	Insertion into late transcription unit with splice acceptor sequence (expression from major late promoter), Ig chains linked via IRES	IgG1	Human VEGF	tumor cell-specific replication and antibody production in vitro with >2000-fold window of selectivity orthotopic xenograft mouse tumor model/intravenous OV application: strongly reduced tumor burden by antibody-encoding OV, superior to parental virus, but no significance (antibody is specific for human VEGF)	[111]
Vaccinia virus (Western Reserve strain)	Expression of heavy and light chains fused via 2A peptide and fused to luciferase from separate transcription unit with viral early/late promoter	IgG	Murine TIGIT	Syngeneic subcutaneous or intraperitoneal mouse tumor models/intratumoral or intraperitoneal OV injection: Antibody-encoding OV shows stronger tumor growth inhibition and increased survival compared with parental virus CD8^+-T cell infiltration induced by OV application, but not further increased by antibody expression; reduced fraction of TIGIT^+ T cells and increased IFN-γ by antibody expression therapeutic effect of antibody-encoding OV abrogated by CD8 + T cell depletion, but not by NK cell depletion Mice cured by antibody-encoding OV reject tumor rechallenge	[105]

^aExpression of functional antibody in vitro shown; virus replicative and/or oncolytic features usually not or only slightly attenuated as analyzed in vitro.

Table 2. Overview of developed therapeutic antibody-encoding OVs: OVs expressing BiTEs (viro-BiTE therapy), sorted according to target and publication date

Virus	Antibody gene expression strategy	Antibody format	Target	Key results for antibody-encoding OV^a	Reference
Vaccinia virus (vvDD, Western Reserve strain, TK^− and VGF^−)	Separate transcription unit, late viral promoter	BiTE	Human EphA2 × human CD3	Co-cultures of infected tumor and unstimulated T cells or PBMCs: BiTE-encoding OV, not control virus, induces T cell activation, which depends on presence of EphA2-positive cells, and T cell-dependent bystander tumor cell killing Subcutaneous xenograft mouse tumor model with mixed tumor cells and PBMCs/intraperitoneal virus application (immediately after cell injection): improved tumor growth inhibition (i.e., prevention of tumor growth), survival and PBMC activation compared with control virus Lung metastasis xenograft mouse tumor model/intravenous OV and/or PBMC injection: BiTE-encoding OV shows significantly delayed tumor growth compared with controls	[98]
Ad (ICOVIR-15K: E1AΔ24, E2F binding site in E1A promoter, RGDK in fiber shaft)	Insertion into late transcription unit with splice acceptor sequence (expression from major late promoter)	BiTE	Human EGFR (cetuximab-derived scFv) × human CD3	Tumor cell-PBMC co-cultures: infection with BiTE-encoding OV but not parental virus, triggers T cell activation, proliferation and bystander target cell killing Subcutaneous xenograft tumor mouse model/intravenous human PBMC or pre-activated T cells and intratumoral OV injection: Increased accumulation and persistence of T cells observed for BiTE-encoding OV compared with parental virus PBMC-dependent improvement of therapeutic outcome for BiTE-encoding OV compared with parental virus injection of PBMC does not affect persistence of the virus	[110]
Ad (ICOVIR-15K: E1AΔ24, E2F binding site in E1A promoter, RGDK in fiber shaft)	Insertion into late transcription unit with splice acceptor sequence (expression from major late promoter)	BiTE	Human EGFR (cetuximab-derived scFv) × human CD3	Combination therapy with folate receptor (FR)-targeted CAR-T cells Co-cultures of infected tumor cells and CAR-T cells in vitro: Efficacy and specificity of target cell killing by EGFR-BiTE-encoding OV + FR-targeted CAR-T superior to CAR-T alone or combination of EGFR-targeted CAR-T and FR-targeted CAR-T BiTE-encoding OV triggers activation of CAR-T cells in absence of FR and activation of CAR^− T cell fraction in CAR-T cell preparations Subcutaneous xenograft mouse tumor model/intratumoral OV and intravenous T cell injection: BiTE-encoding OV + CAR-T combination therapy significantly decreased tumor growth and increased survival compared with monotherapies or combinations containing control T cells or OVs Increase in CAR-T infiltration and activation for xenografts weakly expressing CAR-T target	[112]
Ad (ICOVIR-15K: E1AΔ24, E2F binding site in E1A promoter, RGDK in fiber shaft)	Insertion into late transcription unit with splice acceptor sequence (expression from major late promoter)	BiTE	Human EGFR (cetuximab-derived scFv) × human CD3	Subcutaneous xenograft mouse tumor model/intravenous injection of allogeneic PBMCs and intraperitoneal injection of OV or menstrual blood-derived mesenchymal stem cells (MenSC) infected with OV: MenSC+BiTE-encoding OV treatment results in significant tumor growth inhibition compared with MenSC+parental virus or with BiTE-encoding OV Reduced virus genome copies and viral protein immunostaining at end of experiment for MenSC+BiTE-encoding OV compared with MenSC+parental virus Superior BiTE mRNA expression for MenSC+BiTE-encoding OV compared with BiTE-encoding OV	[114]
Ad (EnAd, chimeric type B Ad)	Inserted as separate transcription unit with CMV promoter (constitutive) or inserted in late viral transcription unit with splice acceptor sequence (expression from viral major late promoter, replication-dependent)	BiTE	Human EpCAM × human CD3	Co-cultures of infected tumor cells and PBMC-derived T cells: BiTE-encoding OVs trigger superior activation of PBMC-derived T cells and tumor-specific cell killing compared with control viruses Ex vivo tumor exudates (containing tumor and immune cells) from patients with EpCAM+ malignancies in 50% immunosuppressive exudate fluid: BiTE-encoding OVs induce proliferation and activation of endogenous T cells and tumor cell depletion superior to control viruses and similar to BiTE protein Splice acceptor sequence-driven BiTE expression, but not CMV promoter-driven BiTE expression restricted to tumor cells	[74]
MeV (Edmonston B vaccine strain)	Additional transcription unit downstream of H gene	BiTE	Human CEA × murine or human CD3, human CD20 × murine CD3	Subcutaneous syngeneic mouse tumor models/intratumoral OV injection: BiTE-encoding OV results in (1) prolonged survival, in one of two models superior to control virus or direct BiTE injection; (ii) increased T cell infiltration and activation; and (3) protective immunity (to parental tumor cells not expressing the BiTE-target. Thus indicative of antigen spread, i.e., activation of endogenous T cells specific for tumor antigens) Patient-derived subcutaneous xenograft mouse tumor model/intratumoral OV and PBMC injection: BiTE-encoding OV in combination with intratumoral PBMC injection shows superior survival compared with BiTE-encoding OV or PBMC alone	[125]
Vaccinia virus (Double-deleted VV vvDD, Western Reserve strain, TK^− and VGF^−)	Separate transcription unit, viral late promoter	BiTE	Murine FAP × murine CD3	Co-cultures of infected FAP-encoding tumor cells and splenocytes: BiTE-encoding OV (but not control virus) results in T cell activation and enhanced cytotoxicity for (bystander) FAP-expressing cancer cells Subcutaneous syngeneic mouse tumor model/intratumoral OV injection: BiTE-encoding OV (compared with control virus) results in reduction of FAP+ cells, an increase in virus titer per g tumor tissue, increased infiltration of CD4+ and CD8 + T cells in injected tumors, increased CD8- and CD4-response to tumor antigen (but not to FAP), and superior tumor growth inhibition of injected and contralateral tumors (in which infectious VVs were found) Syngeneic lung metastases mouse model/intravenous OV injection: BiTE-encoding OV reduces number of lung surface nodules compared with control virus BiTE not detectable in blood, no systemic α-FAP activity after infection with BiTE-encoding OV	[104]
Ad (EnAd, chimeric type B Ad)	Inserted as separate transcription unit with CMV promoter (constitutive) or inserted in late viral transcription unit with splice acceptor sequence (expression from viral major late promoter, replication-dependent)	BiTE	human FAP × human CD3	Co-cultures with PBMC-derived CD3^+ cells + cancer cell lines mixed with normal fibroblasts: BiTE-encoding OVs but not control viruses mediate T cell activation and target cell killing OV with BiTE transgene in the late transcription unit requires infection of cancer cells for replication-dependent (thus tumor-specific) antibody expression Ex vivo ascites model with total ascites cells (including cancer cells, cancer-associated fibroblasts, immune cells): BiTE-encoding OVs, but not control viruses, trigger T cell activation and proliferation, depletion of FAP^+ fibroblasts, increase in inflammatory signature and repolarization of macrophages to M1 phenotype Ex vivo culture of thin sections of prostate cancer biopsies: BiTE-encoding OV increases T cell activation and LDH release superior to control virus with infected cells surrounded by apoptotic cells	[75]
Ad (ICOVIR-15K: E1AΔ24, E2F binding site in E1A promoter, RGDK in fiber shaft)	Insertion into late transcription unit with splice acceptor sequence (expression from major late promoter)	BiTE	Human/murine FAP × human CD3	Co-cultures of infected tumor cells + stained FAP^+ cells + T cells: BiTE-encoding OV, not parental virus, induces (bystander) target cell killing Subcutaneous xenograft mouse tumor model (inducing FAP^+ stroma)/intratumoral OV injection and intravenous injection of T cells: BiTE-encoding OV compared with parental virus shows Increased accumulation and persistence of intravenously injected pre-activated T cells in tumors sSronger tumor growth inhibition and increased survival Similar intratumoral Ad genome copy numbers, but reduced mFAP RNA copy numbers and protein staining	[113]
Ad (EnAd, chimeric type B Ad)	Inserted as separate transcription unit with CMV promoter	BiTE	Human folate receptor-β × human CD3	Ex vivo ascites model with total ascites cells: BiTE-encoding OV induces T cell activation and expansion, depletion of macrophages, and increase of M1 markers on remaining macrophages (repolarization) superior to parental and control viruses Order of scFvs in BiTE construct is critical for activity of BiTE-encoding OV	[76]
HSV-1 G207 (ICP6^− and ICP34.5^−)	Inserted as separate transcription unit with CMV promoter	BiTE or nanobody-scFv fusion	Human PD-L1 (scFv or nanobody) × human CD3 (scFv)	PD-L1-positivity of T cells does not prevent expansion or effector functions after activation by purified BiTE Co-cultures of infected tumor cell line, PBMC-derived T cells and immunosuppressive ascites fluid: BiTE-encoding OVs, not control virus, induce depletion of tumor cells	[93]
				Co-cultures of in vitro-differentiated and infected M2-like macrophages, PBMC-derived T cells and immunosuppressive ascites fluid: BiTE-encoding OVs, not control virus, induce expansion and activation of T cells and reduced expression of M2 marker in surviving cells Co-cultures of tumor cell line, in vitro-differentiated M2-like macrophages, and PBMC-derived T cells were infected: BiTE-encoding OVs, not parental virus, induce increase in T cell number and killing of both tumor cells and macrophages Ex vivo ascites model with total ascites cells: BiTE-encoding OVs, not parental or control viruses, induce T cell activation, which is stronger in presence of immunosuppressive ascites fluid, and reduced expression of PD-L1 and M2 markers in surviving cells

^aExpression of functional antibody in vitro shown; virus replicative and/or oncolytic features usually not or only slightly attenuated as analyzed in vitro.

Table 3. Overview of developed therapeutic antibody-encoding OVs: OVs expressing immune checkpoint inhibitors (viro-CHECKin therapy), sorted according to target and publication date

Virus	Antibody gene expression strategy	Antibody format	Target	Key results for antibody-encoding OV^a	Reference
Ad (Ad5/3-E1AΔ24)	Replacement of early genes (E3) by Ig chains linked via IRES	IgG2	Human CTLA-4	See Table 1	[106]
Ad (Ad E2F promoter E1A)	Replacement of early genes (E3) by Ig chains linked via 2A	IgG2	Mouse CTLA-4	See Table 1	[107]
Influenza A virus (IAV)	Heavy chain in PB1 segment downstream of PB1 gene via 2A; light chain in PA segment downstream of PA gene via 2A; scFv cloned into both segments	IgG and scFv	Proof-of-concept (IgG) and murine CTLA4 (scFv)	See Table 1	[117]
HSV-1 (bioselected clinical strain, ICP34.5^− and ICP47^−, also encoding murine GM-CSF and a highly fusogenic glycoprotein)	Separate transcription unit, MMLV LTR promoter	scFv fused to mouse IgG1	Murine CTLA-4	Bilateral subcutaneous syngeneic mouse tumor model/low dose intratumoral OV injection of right flank tumor: antibody-encoding OV increases tumor growth inhibition of injected and not injected tumors (although significance not reached)	[90]
NDV lentogenic strain	Additional transcription unit downstream of P gene	scFv	Murine CTLA-4	Intradermal syngeneic mouse tumor model/irradiation/intratumoral OV injection: antibody-encoding OV + X-ray shows similarly increased survival and tumor growth inhibition than parental virus + X-ray + systemic α-CTLA-4 when compared with α-CTLA-4 alone	[135]
MeV (attenuated vaccine strain)	Separate transcription unit downstream of H gene	scFv-IgG1 Fc fusion	Murine CTLA-4, murine PD-L1	Subcutaneous syngeneic mouse tumor model/intratumoral OV injection: α-CTLA-4-encoding OV reduces tumor progression, whereas α-PD-L1-encoding OV prolongs survival both compared with control virus Both antibody-encoding OVs increase T cell infiltration, decrease Treg infiltration and result in splenocyte activation (early after infection for the α-CTLA-4 virus or late for the α-PD-L1 virus) For targeting CTLA-4, but not PD-L1 the combination treatment of systemically applied checkpoint inhibitor antibody with parental OV was significantly superior to the antibody-encoding OV Subcutaneous xenograft mouse tumor model/intratumoral OV injection: antibody-encoding OVs are as effective as control virus	[133]
VSV (M51R mutant)	Additional transcription unit between G and L genes	scFv	Human PD-L1 (avelumab-derived)	Subcutaneous syngeneic mouse tumor model with hPD-L1-expressing mouse tumor cells/intratumoral OV injection: Antibody-encoding-OV or combination of parental OV + intraperitoneal scFv reduce tumor growth and improve survival in comparison to monotherapies, 5/6 mice cured with antibody-encoding-OV resist rechallenge with tumor cells Increase of activated CD8^+ T cells in spleen of mice cured after treatment with antibody-encoding-OV compared with normal mice	[134]
Vaccinia virus (CF33: chimera derived by recombination between 9 strains)	Additional transcription unit with viral H5 promoter	scFv	Human PD-L1	Intraperitoneal xenograft mouse tumor model/intravenous or intraperitoneal injection of antibody-encoding OV: Detection of scFv expression stronger after intraperitoneal application of antibody-encoding OV virus compared with intravenous application scFv-encoding OV shows therapeutic activity which is superior after intraperitoneal application (no comparison to parental virus)	[131]
Vaccinia virus (CF33: chimera derived by recombination between 9 strains; also encoding hNIS)	Additional transcription unit with viral H5 promoter	scFv	Human PD-L1	Co-cultures of PDAC cell lines and activated T cells were infected: parental OV results in translocation of PD-L1 to cell surface in cancer cells; antibody-encoding OV delivers sufficient α-PD-L1 scFv to block cell surface detection of PD-L1 on cancer cells; OV-encoded scFv increases granzyme B production and prevents OV-induced decrease in perforin release by T cells	[137]
NDV (lentogenic strain)	Additional transcription unit downstream of P gene	scFvs and scFv-cytokine (scmIL-12) fusions (immunocytokines)	Murine PD-1, PD-L1, CD28	Unilateral subcutaneous syngeneic mouse tumor model/intratumoral OV injection: α-PD-1- and α-PD-L1-encoding OVs show superior therapeutic activity compared with parental virus, esp. when combined with systemic α-CTLA-4 >50% complete remission for α-CD28-mIL12-encoding OV, α-PD-L1-encoding OV and α-PD-L1-mIL12-encoding OV, each combined with systemic α-CTLA-4 Bilateral subcutaneous syngeneic mouse tumor model/intratumoral OV injection in one flank: Tumor growth inhibition observed in contralateral tumors Strongest survival benefit for α-PD-1-, α-PD-1-mIL-12- and α-PD-L1-mIL-12-encoding OV, each combined with systemic α-CTLA-4 Strongest upregulation of immune markers in tumors for α-CD28-mIL12-encoding OV and α-PD-L1-mIL12-encoding OV, both combined with systemic α-CTLA-4	[132]
Vaccinia virus (Western Reserve strain, TK^− and RR^−)	Separate transcription unit with different viral promoters at TK locus	IgG, Fab, scFv	Murine PD-1	See Table 1	[103]
HSV-2 (ICP34.5^− and ICP47^−)	Expression of HC and LC as separate transcription units from CMV and RSV promoters, respectively	IgG	Human PD-1	see Table 1	[92]
HSV-1 NG34 (ICP6^− and ICP34.5^−, also expression of human GADD34 gene from nestin-hsp68 promoter)	Separate transcription unit with CMV promoter	scFv	Human/murine PD-1	Orthotopic syngeneic mouse tumor model/intratumoral OV application: Increased survival of antibody-encoding OV versus mock (significant) and parental OV (not significant), rechallenge of cured mice with tumor cells rejected (in 2 mouse models) Detection of antibody and viral mRNA at 16 h post-infection, strongly reduced at 36 h, active virus not recoverable (but recoverable in orthotopic human xenograft) → inefficient OV replication in mouse tumors	[89]
HSV (ICP34.5^−, ICP0^−, ICP27 promoter replaced by hTERT promoter)	Separate transcription unit with CMV promoter	scFv	Murine PD-1	In vitro phagocytosis assay with DCs and cancer cells: antibody-encoding OV increases phagocytosis compared with parental virus Subcutaneous syngeneic mouse tumor model/intratumoral OV injection: antibody-encoding OV Results in increasing scFv expression over 72 h Increases cross-presentation of model antigen compared with control virus Results in injected and distal tumor growth inhibition similar to combination of control OV with intratumoral scFv injection and superior to control OV alone, long-term regressors rejected tumor cell rechallenge Triggers T cell infiltration in injected and non-injected tumors similar to control OV, but with higher activation status Triggers MDSC infiltration in injected and non-injected tumors more than control OV In combination with systemic α-TIGIT therapy results in superior tumor growth inhibition and infiltration of model tumor antigen-specific CD8^+-T cells compared with combination of control OV with α-TIGIT; tumor growth inhibition was dependent on CD8^+- and CD4^+-T cells	[91]
Vaccinia virus (Western Reserve strain)	Expression of HC and LC fused via 2A peptide and fused to luciferase from as separate transcription unit from viral early/late promoter	IgG	Mouse TIGIT	See Table 1	[105]

^aExpression of functional antibody in vitro shown; virus replicative and/or oncolytic features usually not or only slightly attenuated as analyzed in vitro.

Table 4. Overview of developed therapeutic antibody-encoding OVs: OVs expressing anti-angiogenic antibodies (viro-ANGin therapy), sorted according to target and publication date

Virus	Antibody gene expression strategy	Antibody format	Target	Key results for antibody-encoding OV^a	Reference
Vaccinia virus (GLV-1h68: Lister vaccine strain, triple mutant)	Separate transcription unit, one of 3 viral promoters (synthetic early [SE], synthetic early/late [SEL], or synthetic late [SL])	scFv	Mouse and human VEGF	Promoter determines strength of antibody expression (SEL, SL > SE) Subcutaneous xenograft mouse tumor model/intravenous OV injection: Antibody detected in serum at 7, 21 and 37 d post-infection (SEL, SL viruses) Antibody concentration in infected areas of tumors >10-fold higher than in sera at 7 d p.i. (SEL, SL viruses) Antibody-encoding OVs result in stronger tumor growth inhibition compared with control virus (significant for SEL, SL viruses) Negative correlation of late tumor size and early antibody concentration in blood Reduced blood vessel density in infected areas of tumors for antibody-encoding OV	[94]
Vaccinia virus (GLV-1h68: Lister vaccine strain, triple mutant)	Separate transcription unit, synthetic late promoter	scFv	Mouse and human VEGF (binds canine VEGF)	OVs replicate productively in canine cancer cells Subcutaneous canine cancer xenograft mouse models/intravenous OV injection: Tumor-specific virus biodistribution and tumor growth inhibition in 1 of 2 models (not compared with parental virus) Reduced blood vessel density in infected but not noninfected tumor areas for antibody-encoding OV compared with control virus	[95]
Vaccinia virus (GLV-1h100: Lister vaccine strain, triple mutant)	Separate transcription unit, synthetic late promoter	scFv	Mouse and human VEGF	OV-encoded α-VEGF scFv reverses VEGF-induced radioresistance of endothelial cells, but not tumor cells Subcutaneous xenograft mouse tumor model/systemic virus application + irradiation: Enhanced tumor growth inhibition and reduced intratumoral VEGF concentration and vessel number for antibody-encoding OV + irradiation compared with monotherapies or combination with control virus, durable responses for combination, only Irradiation enhances OV spread in xenograft as measured by reporter expression	[96]
Vaccinia virus (GLV-1h68: Lister vaccine strain, triple mutant)	Separate transcription unit, synthetic late promoter	scFv	Mouse and human VEGF	Subcutaneous xenograft and malignant effusion mouse lung cancer model/intravenous OV application: Parental OV results in infection of tumor cells in vessels and at tumor borders, tumor growth inhibition and less, delayed or disappearing malignant effusion, but also in an increase in vascular density, CD31 expression and accumulation of leukocytes Antibody-encoding OV results in stronger tumor growth inhibition and stronger reduction of malignant effusion compared with control virus	[97]
Vaccinia virus (GLV-1h100: Lister vaccine strain, triple mutant)	Separate transcription unit, synthetic late promoter	scFv	Mouse and human VEGF	Orthotopic xenograft mouse breast cancer model/intratumoral OV injection: antibody-encoding OV shows significantly superior therapeutic activity, noticeable decreased vascular flow by in vivo Doppler ultrasonography and decreased vasculature by immunostaining compared with control virus	[99]
Vaccinia virus (Lister vaccine strain 6.1.1)	Separate transcription unit, synthetic early/late promoter	scFv	Mouse and human VEGF, binds feline VEGF	Subcutaneous feline cancer xenograft mouse model/intravenous OV injection: antibody-encoding OV results in tumor growth inhibition similar to parental virus, reduced intratumoral functional VEGF and reduced blood vessel density in infected areas compared with parental virus	[100]
Vaccinia virus (Lister vaccine strain 6.1.1)	Separate transcription unit, synthetic early/late promoter	scFv	Mouse and human VEGF, binds canine VEGF	Subcutaneous canine cancer xenograft mouse model/intravenous OV injection: antibody-encoding OV regresses tumor growth and reduces blood vessel density more than parental virus	[101]
Vaccinia virus (GLV-1h68: Lister vaccine strain, triple mutant)	Separate transcription unit, viral promoters (SEL, SL [VEGF] or SEL+SL)	scFv, nanobody	VEGF (scFv) + EGFR (nanobody); VEGF (scFv) + cross-species FAP (scFv)	Subcutaneous xenograft mouse tumor model/intravenous OV injection: OVs encoding single antibodies (targeting EGFR, VEGF, or FAP) inhibit tumor growth more rapidly (one xenograft model) or stronger (other xenograft model) than control virus OVs encoding two antibodies result in strongest tumor growth inhibition, significantly superior to control virus, significance not reached in comparison to single antibody-encoding OVs Therapeutic activity of OV encoding antibodies targeting EGFR and VEGF is similar to combination of control virus with systemic Avastin and Erbitux OVs encoding the EGFR-targeted antibody show superior suppression of cell proliferation in noninfected tumor areas compared with control virus reduced blood vessel density in infected areas of OVs encoding the VEGF-targeted antibody in comparison to control virus	[102]
Ad (EnAd, chimeric type B Ad)	Insertion into late transcription unit with splice acceptor sequence (expression from major late promoter), Ig chains linked via IRES	IgG1	Human VEGF	See Table 1	[111]

^aExpression of functional antibody in vitro shown; virus replicative and/or oncolytic features usually not or only slightly attenuated as analyzed in vitro.

Table 5. Overview of the developed therapeutic antibody-encoding OVs: OVs expressing cytotoxic immunofusions (viro-iTOX therapy)

Virus	Antibody gene expression strategy	Antibody format	Target	Key results for antibody-encoding OV	Reference
Ad (Ad5/3 [serotype 3 cell-binding knob] E1AΔ24)	Insertion into late transcription unit with splice acceptor sequence (expression from major late promoter)	scFv-RNase fusion (immunoRNase)	Human EGFR (cetuximab-derived)	Optimized gene expression control for strictly late expression of immunoRNase required to avoid adverse effects on OV infectivity/replication Potent and specific bystander killing of target cells, including cetuximab-resistant tumor cells (distinct mode of action) Antibody-encoding OV induces strong increase in cytotoxicity for target cells compared with control virus in vitro and in vivo (subcutaneous xenograft mouse tumor model/intratumoral OV injection)	[109]
HSV-2	Separate transcription unit with RSV-LTR promoter	scFv-trimerization domain-human sFasL fusion	Human Her2 + human Fas	Reduced virus replication of antibody-encoding OV compared with parental virus Subcutaneous xenograft mouse tumor model/low dose intratumoral OV injection: antibody-encoding OV shows stronger tumor growth inhibition than parental virus, transient halt of tumor growth Subcutaneous syngeneic mouse tumor model/high dose intratumoral OV injection: antibody-encoding OV (pre-passaged in vivo for improved replication) shows prolonged tumor regression compared with parental virus	[88]

Figure 1. Viro-antibody therapy – an overview. Transgenes encoding recombinant therapeutic antibodies are inserted into the genome of oncolytic viruses (OVs) by exploitation of diverse strategies for transgene expression (see Figure 2). OV-encoded antibodies are produced locally in the tumor by infected cancer cells and expression lasts as long as active OV infection and spread is ongoing. The produced antibodies, dependent on their format, valency and size (see Figure 3), perfuse the tumor, bind their target on (noninfected) cancer cells or cancer-associated cells, and trigger their direct or indirect killing via diverse modes of action (see Figure 4)

Figure 2. Viruses and modes of antibody expression exploited for viro-antibody therapy. A panel of viruses with or without envelope and with DNA (double-strand) or RNA genomes (negative strand, continuous for MeV, NDV, VSV or segmented for influenza virus) have been exploited for viro-antibody therapy (left panel). Dependent on the virus chosen, different strategies have been pursued for the insertion of antibody genes into the viral genomes toward efficient and/or replication-dependent gene expression and minimizing the required genomic space (right panel). ab, antibody; Env, envelope; HSV, herpes simplex virus; MeV, measles virus; NDV, Newcastle disease virus; ORF, open reading frame; VSV, vesicular stomatitis virus

Figure 3. Antibody formats utilized in viro-antibody therapy. BiTE, bispecific T cell engager; C_H/C_L, constant domain of heavy/light chain; Fab, antigen-binding fragment; Fc, constant fragment of antibody; IgG, immunoglobulin G; RNase, ribonuclease; scFv, single chain variable fragment; sdAb, single domain antibody (variable fragment of camelid antibody heavy chain); V_H/V_L, variable domain of heavy/light chain. For the scFv fusion proteins, proteins might be fused at the C-terminus of the scFv (cytokine, trimerization peptide and FasL) or at the N-terminus (RNase); scFvs might be in V_H-V_L or V_L-V_H configuration

Figure 4. Target cells and modes of action of OV-encoded antibodies. The depicted cancer targets and direct or immune- or stroma-mediated modes of action have been reported. For some of the depicted modes of action only examples of the explored antibody formats are depicted (see Tables 1 – 5 for a comprehensive list). CAF, cancer-associated fibroblast; TAM, tumor-associated macrophage; TECs, tumor endothelial cells

Pühler F, Willuda J, Puhlmann J, Mumberg D, Römer-Oberdörfer A, Beier R. Generation of a recombinant oncolytic Newcastle disease virus and expression of a full IgG antibody from two transgenes. Gene Ther. 2008;15(5):371–83. doi:10.1038/sj.gt.3303095.

 PubMed Web of Science ®Google Scholar

Dias JD, Hemminki O, Diaconu I, Hirvinen M, Bonetti A, Guse K, Escutenaire S, Kanerva A, Pesonen S, Löskog A, et al. Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4. Gene Ther. 2012;19(10):988–98. doi:10.1038/gt.2011.176.

 PubMed Web of Science ®Google Scholar

Du T, Shi G, Li YM, Zhang JF, Tian HW, Wei YQ, Deng H, and Yu DC. Tumor-specific oncolytic adenoviruses expressing granulocyte macrophage colony-stimulating factor or anti-CTLA4 antibody for the treatment of cancers. Cancer Gene Ther. 2014;21(8):340–48. doi:10.1038/cgt.2014.34.

 PubMed Web of Science ®Google Scholar

Hamilton JR, Vijayakumar G, Palese P. A recombinant antibody-expressing influenza virus delays tumor growth in a mouse model. Cell Rep. 2018;22(1):1–7. doi:10.1016/j.celrep.2017.12.025.

 PubMed Web of Science ®Google Scholar

Wei D, Li Q, Wang XL, Wang Y, Xu J, Feng F, Nan G, Wang B, Li C, Guo T, et al. Oncolytic Newcastle disease virus expressing chimeric antibody enhanced anti-tumor efficacy in orthotopic hepatoma-bearing mice. J Exp Clin Cancer Res. 2015;34(1):153. doi:10.1186/s13046-015-0271-1.

 PubMedGoogle Scholar

Kleinpeter P, Fend L, Thioudellet C, Geist M, Sfrontato N, Koerper V, Fahrner C, Schmitt D, Gantzer M, Remy-Ziller C, et al. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death −1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition. Oncoimmunology. 2016;5(10):e1220467. doi:10.1080/2162402X.2016.1220467.

 PubMed Web of Science ®Google Scholar

Zhu Y, Hu X, Feng L, Yang Z, Zhou L, Duan X, Cheng S, Zhang W, Liu B, and Zhang K. Enhanced Therapeutic Efficacy Of A Novel Oncolytic Herpes Simplex Virus Type 2 encoding an antibody against programmed cell death 1. Mol Ther Oncolytics. 2019;15:201–13. doi:10.1016/j.omto.2019.10.003.

 PubMed Web of Science ®Google Scholar

Liikanen I, Tähtinen S, Guse K, Gutmann T, Savola P, Oksanen M, Kanerva A, and Hemminki A. Oncolytic adenovirus expressing monoclonal antibody trastuzumab for treatment of HER2-positive cancer. Mol Cancer Ther. 2016;15(9):2259–69. doi:10.1158/1535-7163.MCT-15-0819.

 PubMed Web of Science ®Google Scholar

Marino N, Illingworth S, Kodialbail P, Patel A, Calderon H, Lear R, Fisher KD, Champion BR, and Brown ACN. Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes. PLoS One. 2017;12(5):e0177810. doi:10.1371/journal.pone.0177810.

 PubMed Web of Science ®Google Scholar

Zuo S, Wei M, He B, Chen A, Wang S, Kong L, Zhang Y, Meng G, Xu T, Wu J, et al. Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT). EBioMedicine. 2021;64:103240. doi:10.1016/j.ebiom.2021.103240.

 PubMed Web of Science ®Google Scholar

Yu F, Wang X, Guo ZS, Bartlett DL, Gottschalk SM, Song XT. T-cell engager-armed oncolytic vaccinia virus significantly enhances antitumor therapy. Mol Ther. 2014;22(1):102–11. doi:10.1038/mt.2013.240.

 PubMed Web of Science ®Google Scholar

Fajardo CA, Guedan S, Rojas LA, Moreno R, Arias-Badia M, de Sostoa J, June CH, and Alemany R. Oncolytic adenoviral delivery of an EGFR-targeting T-cell engager improves antitumor efficacy. Cancer Res. 2017;77(8):2052–63. doi:10.1158/0008-5472.CAN-16-1708.

 PubMed Web of Science ®Google Scholar

Wing A, Fajardo CA, Posey AD, Jr., Shaw C, Da T, Young RM, Alemany R, June CH, and Guedan S. Improving CART-cell therapy of solid tumors with oncolytic virus-driven production of a bispecific T-cell engager. Cancer Immunol Res. 2018;6(5):605–16. doi:10.1158/2326-6066.CIR-17-0314.

 PubMed Web of Science ®Google Scholar

Barlabé P, Sostoa J, Fajardo CA, Alemany R, Moreno R. Enhanced antitumor efficacy of an oncolytic adenovirus armed with an EGFR-targeted BiTE using menstrual blood-derived mesenchymal stem cells as carriers. Cancer Gene Ther. 2020;27(5):383–88. doi:10.1038/s41417-019-0110-1.

 PubMed Web of Science ®Google Scholar

Freedman JD, Hagel J, Scott EM, Psallidas I, Gupta A, Spiers L, Miller P, Kanellakis N, Ashfield R, Fisher KD, et al. Oncolytic adenovirus expressing bispecific antibody targets T-cell cytotoxicity in cancer biopsies. EMBO Mol Med. 2017;9(8):1067–87. doi:10.15252/emmm.201707567.

 PubMed Web of Science ®Google Scholar

Speck T, Heidbuechel JPW, Veinalde R, Jaeger D, von Kalle C, Ball CR, Ungerechts G, and Engeland CE. Targeted BiTE expression by an oncolytic vector augments therapeutic efficacy against solid tumors. Clin Cancer Res. 2018;24(9):2128–37. doi:10.1158/1078-0432.CCR-17-2651.

 PubMed Web of Science ®Google Scholar

Yu F, Hong B, Song X-T. A T-cell engager-armed oncolytic vaccinia virus to target the tumor stroma. Cancer Transl Med. 2017;3(4):122–32.

 Google Scholar

Freedman JD, Duffy MR, Lei-Rossmann J, Muntzer A, Scott EM, Hagel J, Campo L, Bryant RJ, Verrill C, Lambert A, et al. An oncolytic virus expressing a T-cell engager simultaneously targets cancer and immunosuppressive stromal cells. Cancer Res. 2018;78(24):6852–65. doi:10.1158/0008-5472.CAN-18-1750.

 PubMed Web of Science ®Google Scholar

De Sostoa J, Fajardo CA, Moreno R, Ramos MD, Farrera-Sal M, Alemany R. Targeting the tumor stroma with an oncolytic adenovirus secreting a fibroblast activation protein-targeted bispecific T-cell engager. J Immunother Cancer. 2019;7(1):19. doi:10.1186/s40425-019-0505-4.

 PubMedGoogle Scholar

Scott EM, Jacobus EJ, Lyons B, Frost S, Freedman JD, Dyer A, Khalique H, Taverner WK, Carr A, Champion BR, et al. Bi- and tri-valent T cell engagers deplete tumour-associated macrophages in cancer patient samples. J Immunother Cancer. 2019;7(1):320. doi:10.1186/s40425-019-0807-6.

 PubMed Web of Science ®Google Scholar

Khalique H, Baugh R, Dyer A, Scott EM, Frost S, Larkin S, Lei-Rossmann J, and Seymour LW. Oncolytic herpesvirus expressing PD-L1 BiTE for cancer therapy: exploiting tumor immune suppression as an opportunity for targeted immunotherapy. J Immunother Cancer. 2021;9(4). doi:10.1136/jitc-2020-001292.

 Web of Science ®Google Scholar

Thomas S, Kuncheria L, Roulstone V, Kyula JN, Mansfield D, Bommareddy PK, et al.Thomas S, Kuncheria L, Roulstone V, Kyula JN, Mansfield D, Bommareddy PK, Smith H, Kaufman HL, Harrington KJ, and Coffin RS. Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1. J Immunother Cancer. 2019;7(1):214. doi:10.1186/s40425-019-0682-1.

 PubMed Web of Science ®Google Scholar

Vijayakumar G, Palese P, Goff PH. Oncolytic Newcastle disease virus expressing a checkpoint inhibitor as a radioenhancing agent for murine melanoma. EBioMedicine. 2019;49:96–105. doi:10.1016/j.ebiom.2019.10.032.

 PubMed Web of Science ®Google Scholar

Engeland CE, Grossardt C, Veinalde R, Bossow S, Lutz D, Kaufmann JK, Shevchenko I, Umansky V, Nettelbeck DM, Weichert W, et al., CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic measles virus therapy. Mol Ther. 2014;22(11):1949–59. doi:10.1038/mt.2014.160.

 PubMed Web of Science ®Google Scholar

Wu C, Wu M, Liang M, Xiong S, Dong C. A novel oncolytic virus engineered with PD-L1 scFv effectively inhibits tumor growth in a mouse model. Cell Mol Immunol. 2019;16(9):780–82. doi:10.1038/s41423-019-0264-7.

 PubMed Web of Science ®Google Scholar

Woo Y, Zhang Z, Yang A, Chaurasiya S, Park AK, Lu J, Kim SI, Warner SG, Von Hoff D, and Fong Y. Novel chimeric immuno-oncolytic virus CF33-hNIS-antiPDL1 for the treatment of pancreatic cancer. J Am Coll Surg. 2020;230(4):709–17. doi:10.1016/j.jamcollsurg.2019.12.027.

 PubMed Web of Science ®Google Scholar

Zhang Z, Yang A, Chaurasiya S, Park AK, Lu J, Kim SI, et al. CF33-hNIS-antiPDL1 virus primes pancreatic ductal adenocarcinoma for enhanced anti-PD-L1 therapy. Cancer Gene Ther; 2021. doi: 10.1038/s41417-021-00350-4

 Google Scholar

Vijayakumar G, McCroskery S, Palese P. Engineering Newcastle disease virus as an oncolytic vector for intratumoral delivery of immune checkpoint inhibitors and immunocytokines. J Virol. 2020;94(3). doi:10.1128/JVI.01677-19.

 PubMed Web of Science ®Google Scholar

Passaro C, Alayo Q, De Laura I, McNulty J, Grauwet K, Ito H, Bhaskaran V, Mineo M, Lawler SE, Shah K, et al. Arming an oncolytic herpes simplex virus type 1 with a single-chain fragment variable antibody against PD-1 for experimental glioblastoma therapy. Clin Cancer Res. 2019;25(1):290–99. doi:10.1158/1078-0432.CCR-18-2311.

 PubMed Web of Science ®Google Scholar

Lin C, Ren W, Luo Y, Li S, Chang Y, Li L, Xiong D, Huang X, Xu Z, Yu Z, et al. Intratumoral delivery of a PD-1-blocking scFv encoded in oncolytic HSV-1 promotes antitumor immunity and synergizes with TIGIT blockade. Cancer Immunol Res. 2020;8(5):632–47. doi:10.1158/2326-6066.CIR-19-0628.

 PubMed Web of Science ®Google Scholar

Frentzen A, Yu YA, Chen N, Zhang Q, Weibel S, Raab V, and Szalay AA. Anti-VEGF single-chain antibody GLAF-1 encoded by oncolytic vaccinia virus significantly enhances antitumor therapy. Proc Natl Acad Sci U S A. 2009;106(31):12915–20. doi:10.1073/pnas.0900660106.

 PubMed Web of Science ®Google Scholar

Patil SS, Gentschev I, Adelfinger M, Donat U, Hess M, Weibel S, Nolte I, Frentzen A, and Szalay AA. Virotherapy of canine tumors with oncolytic vaccinia virus GLV-1h109 expressing an anti-VEGF single-chain antibody. PLoS One. 2012;7(10):e47472. doi:10.1371/journal.pone.0047472.

 PubMed Web of Science ®Google Scholar

Buckel L, Advani SJ, Frentzen A, Zhang Q, Yu YA, Chen NG, Ehrig K, Stritzker J, Mundt AJ, and Szalay AA. Combination of fractionated irradiation with anti-VEGF expressing vaccinia virus therapy enhances tumor control by simultaneous radiosensitization of tumor associated endothelium. Int J Cancer. 2013;133(12):2989–99.

 PubMed Web of Science ®Google Scholar

Weibel S, Hofmann E, Basse-Luesebrink TC, Donat U, Seubert C, Adelfinger M, Gnamlin P, Kober C, Frentzen A, Gentschev I, et al., Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer. J Transl Med. 2013;11(1):106. doi:10.1186/1479-5876-11-106.

 PubMedGoogle Scholar

Gholami S, Marano A, Chen NG, Aguilar RJ, Frentzen A, Chen CH, Lou E, Fujisawa S, Eveno C, Belin L, et al. A novel vaccinia virus with dual oncolytic and anti-angiogenic therapeutic effects against triple-negative breast cancer. Breast Cancer Res Treat. 2014;148(3):489–99. doi:10.1007/s10549-014-3180-7.

 PubMed Web of Science ®Google Scholar

Adelfinger M, Gentschev I, Grimm de Guibert J, Weibel S, Langbein-Laugwitz J, Härtl B, Murua Escobar H, Nolte I, Chen NG, Aguilar RJ, et al., Evaluation of a new recombinant oncolytic vaccinia virus strain GLV-5b451 for feline mammary carcinoma therapy. PLoS One. 2014;9(8):e104337. doi:10.1371/journal.pone.0104337.

 PubMed Web of Science ®Google Scholar

Adelfinger M, Bessler S, Frentzen A, Cecil A, Langbein-Laugwitz J, Gentschev I, and Szalay AA. Preclinical testing oncolytic vaccinia virus strain GLV-5b451 expressing an anti-VEGF single-chain antibody for canine cancer therapy. Viruses. 2015;7(7):4075–92. doi:10.3390/v7072811.

 PubMed Web of Science ®Google Scholar

Huang T, Wang H, Chen NG, Frentzen A, Minev B, Szalay AA. Expression of anti-VEGF antibody together with anti-EGFR or anti-FAP enhances tumor regression as a result of vaccinia virotherapy. Mol Ther Oncolytics. 2015;2:15003. doi:10.1038/mto.2015.3.

 PubMed Web of Science ®Google Scholar

Fernández-Ulibarri I, Hammer K, Arndt MA, Kaufmann JK, Dorer D, Engelhardt S, Kontermann RE, Hess J, Allgayer H, Krauss J, et al. Genetic delivery of an immunoRNase by an oncolytic adenovirus enhances anticancer activity. Int J Cancer. 2015;136(9):2228–40. doi:10.1002/ijc.29258.

 PubMed Web of Science ®Google Scholar

Loya SM, Zhang X. Enhancing the bystander killing effect of an oncolytic HSV by arming it with a secretable apoptosis activator. Gene Ther. 2015;22(3):237–46. doi:10.1038/gt.2014.113.

 PubMed Web of Science ®Google Scholar