Bispecific Adapter-Mediated Retargeting of a Receptor-Restricted HSV-1 Vector to CEA-Bearing Tumor Cells
Authors: Hyunjung Baek, Hiroaki Uchida, Kyungok Jun, Jae-Hong Kim, Masahide Kuroki, Justus B Cohen, Joseph C Glorioso, Heechung Kwon
Journal: Molecular Therapy (2010)

The safety and efficacy of viral therapies for solid tumors can be enhanced by redirecting the virus infection to tumor-specific cell-surface markers. Successful retargeting of herpes simplex virus type 1 (HSV-1) has been achieved using vectors that carry a modified envelope glycoprotein D (gD) engineered to interact directly with novel receptors. In addition, soluble bridging molecules (adapters) have been used to link gD indirectly to cell-specific receptors. Here, we describe the development of an adapter connecting gD to the common tumor antigen carcinoembryonic antigen (CEA). The adapter consisted of a CEA-specific single-chain antibody fused to the gD-binding region of the gD receptor, herpes virus entry mediator (HVEM). We used this adapter in combination with a vector that is detargeted for recognition of the widely expressed gD receptor nectin-1, but retains an intact binding region for the less common HVEM. We show that the adapter enabled infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic CEA and nectin-1/CEA-bearing human gastric carcinoma cells that are resistant to the vector alone. We observed cell-to-cell spread following adapter-mediated infection in vitro and reduced tumor growth in vivo, indicating that this method of vector retargeting may provide a novel strategy for tumor-specific delivery of tumoricidal HSV. The safety and efficacy of viral therapies for solid tumors can be enhanced by redirecting the virus infection to tumor-specific cell-surface markers. Successful retargeting of herpes simplex virus type 1 (HSV-1) has been achieved using vectors that carry a modified envelope glycoprotein D (gD) engineered to interact directly with novel receptors. In addition, soluble bridging molecules (adapters) have been used to link gD indirectly to cell-specific receptors. Here, we describe the development of an adapter connecting gD to the common tumor antigen carcinoembryonic antigen (CEA). The adapter consisted of a CEA-specific single-chain antibody fused to the gD-binding region of the gD receptor, herpes virus entry mediator (HVEM). We used this adapter in combination with a vector that is detargeted for recognition of the widely expressed gD receptor nectin-1, but retains an intact binding region for the less common HVEM. We show that the adapter enabled infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic CEA and nectin-1/CEA-bearing human gastric carcinoma cells that are resistant to the vector alone. We observed cell-to-cell spread following adapter-mediated infection in vitro and reduced tumor growth in vivo, indicating that this method of vector retargeting may provide a novel strategy for tumor-specific delivery of tumoricidal HSV. IntroductionThe goal of this study was to develop a soluble bridging molecule (adapter) to redirect herpes simplex virus type 1 (HSV-1) infection to cells that express the tumor marker carcinoembryonic antigen (CEA). Normal HSV-1 infection requires the binding of viral envelope glycoprotein D (gD) to one of several specific entry receptors, predominantly nectin-1/HveC, a widely expressed intercellular adhesion molecule, and herpes virus entry mediator (HVEM)/HveA, expressed mainly on cells of the immune system.1Spear PG Herpes simplex virus: receptors and ligands for cell entry.Cell Microbiol. 2004; 6: 401-410Crossref PubMed Scopus (446) Google Scholar Receptor binding activates the latent effector ("profusion") function of gD, which in turn is believed to activate gB and gH, the two key mediators of viral capsid delivery to the cytoplasm via fusion of the viral envelope with cellular membranes.2Campadelli-Fiume G Amasio M Avitabile E Cerretani A Forghieri C Gianni T et al.The multipartite system that mediates entry of herpes simplex virus into the cell.Rev Med Virol. 2007; 17: 313-326Crossref PubMed Scopus (113) Google Scholar Thus, because gD binding to its natural receptors launches the infection cascade, redirecting HSV infection requires (i) the elimination of these natural receptor-binding activities and (ii) insertion of a ligand for an alternate receptor in such a manner that the new binding interaction causes activation of the gD profusion function.Accumulating knowledge of the structure–function relationship of gD acquired over many years has recently enabled the rational construction of detargeted and retargeted versions of gD.3Zhou G Roizman B Construction and properties of a herpes simplex virus 1 designed to enter cells solely via the IL-13α2 receptor.Proc Natl Acad Sci USA. 2006; 103: 5508-5513Crossref PubMed Scopus (73) Google Scholar,4Menotti L Cerretani A Hengel H Campadelli-Fiume G Construction of a fully retargeted herpes simplex virus 1 recombinant capable of entering cells solely via human epidermal growth factor receptor 2.J Virol. 2008; 82: 10153-10161Crossref PubMed Scopus (90) Google Scholar,5Menotti L Nicoletti G Gatta V Croci S Landuzzi L De Giovanni C et al.Inhibition of human tumor growth in mice by an oncolytic herpes simplex virus designed to target solely HER-2-positive cells.Proc Natl Acad Sci USA. 2009; 106: 9039-9044Crossref PubMed Scopus (74) Google Scholar These constructs hold great promise for highly specific HSV targeting to a variety of cell types and tissues although their ability to function with diverse ligands is yet unknown.An alternate retargeting strategy that does not require target-specific engineering of viral gD involves the use of bispecific adapters to promote virus interaction with novel receptors.6Waehler R Russell SJ Curiel DT Engineering targeted viral vectors for gene therapy.Nat Rev Genet. 2007; 8: 573-587Crossref PubMed Scopus (539) Google Scholar,7Schaffer DV Koerber JT Lim KI Molecular engineering of viral gene delivery vehicles.Annu Rev Biomed Eng. 2008; 10: 169-194Crossref PubMed Scopus (123) Google Scholar This strategy is based in part on the repeated demonstration that HSV infection through HVEM and nectin-1 is blocked by soluble versions of these receptors,8Montgomery RI Warner MS Lum BJ Spear PG Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (998) Google Scholar,9Geraghty RJ Krummenacher C Cohen GH Eisenberg RJ Spear PG Entry of α-herpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.Science. 1998; 280: 1618-1620Crossref PubMed Scopus (768) Google Scholar,10Krummenacher C Rux AH Whitbeck JC Ponce-de-Leon M Lou H Baribaud I et al.The first immunoglobulin-like domain of HveC is sufficient to bind herpes simplex virus gD with full affinity, while the third domain is involved in oligomerization of HveC.J Virol. 1999; 73: 8127-8137PubMed Google Scholar,11Lopez M Cocchi F Avitabile E Leclerc A Adelaide J Campadelli-Fiume G et al.Novel, soluble isoform of the herpes simplex virus (HSV) receptor nectin1 (or PRR1-HIgR-HveC) modulates positively and negatively susceptibility to HSV infection.J Virol. 2001; 75: 5684-5691Crossref PubMed Scopus (38) Google Scholar,12Whitbeck JC Peng C Lou H Xu R Willis SH Ponce de Leon M et al.Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry.J Virol. 1997; 71: 6083-6093PubMed Google Scholar suggesting that adapters composed of the gD-binding domain of either receptor combined with a targeting ligand would obviate the need to detarget gD from both receptors. Our previous study using a nectin-1-based adapter targeted by a single-chain antibody (scFv) against the human epidermal growth factor receptor demonstrated efficient adapter-mediated infection of gD receptor–deficient cells expressing ectopic epidermal growth factor receptor, but infection via nectin-1 was not blocked.13Nakano K Asano R Tsumoto K Kwon H Goins WF Kumagai I et al.Herpes simplex virus targeting to the EGF receptor by a gD-specific soluble bridging molecule.Mol Ther. 2005; 11: 617-626Abstract Full Text Full Text PDF PubMed Scopus (35) Google ScholarHere, we combined a novel HVEM-based adapter with a nectin-1-detargeted virus to promote CEA-dependent infection of cancer cells. The rationale for this design was that functional HVEM expression beyond the immune system is relatively limited8Montgomery RI Warner MS Lum BJ Spear PG Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (998) Google Scholar,14Kwon BS Tan KB Ni J Oh KO Lee ZH Kim KK et al.A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation.J Biol Chem. 1997; 272: 14272-14276Crossref PubMed Scopus (252) Google Scholar,15Marsters SA Ayres TM Skubatch M Gray CL Rothe M Ashkenazi A Herpesvirus entry mediator, a member of the tumor necrosis factor receptor (TNFR) family, interacts with members of the TNFR-associated factor family and activates the transcription factors NF-κB and AP-1.J Biol Chem. 1997; 272: 14029-14032Crossref PubMed Scopus (263) Google Scholar,16Manoj S Jogger CR Myscofski D Yoon M Spear PG Mutations in herpes simplex virus glycoprotein D that prevent cell entry via nectins and alter cell tropism.Proc Natl Acad Sci USA. 2004; 101: 12414-12421Crossref PubMed Scopus (75) Google Scholar and thus that organs such as stomach17Hsu H Solovyev I Colombero A Elliott R Kelley M Boyle WJ ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.J Biol Chem. 1997; 272: 13471-13474Crossref PubMed Scopus (154) Google Scholar should be largely resistant to nectin-1-detargeted viruses. Because these viruses remain capable of binding to HVEM, tumors in these organs may be specifically targeted with HVEM-based adapters. CEA is an attractive antigen for therapeutic targeting because it is expressed in a high percentage of certain cancers,18Hammarström S The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues.Semin Cancer Biol. 1999; 9: 67-81Crossref PubMed Scopus (909) Google Scholar but is rare in normal adult tissues. We show that the adapters promoted the CEA-dependent HSV-1 infection of HSV-resistant Chinese hamster ovary (CHO)-K1 cells and increased the infection of CEA-bearing human gastric carcinoma MKN45 cells by a nectin-1-detargeted mutant virus. Lateral virus spread was detectable in vitro following adapter-enhanced infection. In vivo experiments demonstrated an adapter-dependent infection and growth inhibition of MKN45 tumors. Our results indicate that adapters may be useful not only for the cell-specific delivery of nonreplicating HSV vectors, but also to target replication-competent vectors for specific tumor destruction.ResultsscCEA-HveA adapter-mediated HSV infection of CEA-expressing CHO cellsTo promote HSV-1 interaction with CEA, we generated a bispecific adapter consisting of, in sequence, a scFv against human CEA, a Gly4–Ser linker, the gD-binding 102 N-terminal amino acids of HVEM/HveA [cysteine-rich pseudorepeats (CRPs) 1 and 2],19Whitbeck JC Connolly SA Willis SH Hou W Krummenacher C Ponce de Leon M et al.Localization of the gD-binding region of the human herpes simplex virus receptor, HveA.J Virol. 2001; 75: 171-180Crossref PubMed Scopus (44) Google Scholar and a C-terminal histidine (His6) tag (scCEA-HveA; Figure 1a). The coding sequence was assembled in a mammalian expression plasmid and the protein expressed by transfection of 293T cells. The adapter protein was affinity-purified from the culture media and identified by immunoblotting with His6- and HVEM-specific antibodies (data not shown), as described earlier.20Kwon H Bai Q Baek HJ Felmet K Burton EA Goins WF et al.Soluble V domain of Nectin-1/HveC enables entry of herpes simplex virus type 1 (HSV-1) into HSV-resistant cells by binding to viral glycoprotein D.J Virol. 2006; 80: 138-148Crossref PubMed Scopus (41) Google ScholarCHO-K1 cells are resistant to HSV-1 infection because of the absence of gD receptors.8Montgomery RI Warner MS Lum BJ Spear PG Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (998) Google Scholar,9Geraghty RJ Krummenacher C Cohen GH Eisenberg RJ Spear PG Entry of α-herpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.Science. 1998; 280: 1618-1620Crossref PubMed Scopus (768) Google Scholar We tested the activity of the scCEA-HveA adapter by infection of CEA-expressing (CHO-CEA)21Kuroki M Arakawa F Matsuo Y Oikawa S Misumi Y Nakazato H et al.Molecular cloning of nonspecific cross-reacting antigens in human granulocytes.J Biol Chem. 1991; 266: 11810-11817Abstract Full Text PDF PubMed Google Scholar and parental CHO-K1 cells with a replication-defective reporter virus, QOZHG,22Chen X Li J Mata M Goss J Wolfe D Glorioso JC et al.Herpes simplex virus type 1 ICP0 protein does not accumulate in the nucleus of primary neurons in culture.J Virol. 2000; 74: 10132-10141Crossref PubMed Scopus (57) Google Scholar in the presence of increasing amounts of adapter. QOZHG contains a lacZ expression cassette enabling detection of infected cells by X-gal staining and quantification of β-galactosidase activity by o-nitrophenyl-β-D-galactopyranoside assay. As shown in Figure 2a,b, the adapters increased the infection of CHO-CEA cells at all concentrations tested, whereas infection of CHO-K1 cells remained minimal throughout. The level of adapter-mediated infection of CHO-CEA cells increased with the multiplicity of infection (MOI; established on complementing Vero-7b cells23Krisky DM Marconi PC Oligino T Rouse RJ Fink DJ Glorioso JC Rapid method for construction of recombinant HSV gene transfer vectors.Gene Ther. 1997; 4: 1120-1125Crossref PubMed Scopus (86) Google Scholar), but reached a plateau at ~250 nmol/l adapter at MOIs of 3–30 (Figure 2c), suggesting that this concentration of adapter was saturating for the receptor. We compared the activity of scCEA-HveA to three related adapters (Figure 1a,b) that had (i) the positions of the scFv and HVEM portions of scCEA-HveA switched such that the HVEM CRPs were N-terminal to the scFv (HveA-scCEA); (ii) the HVEM portion of scCEA-HveA replaced by the gD-binding V domain of nectin-1/HveC (scCEA-HveC); or (iii) the nectin-1 V domain positioned N-terminally to scFv-His6 (HveC-scCEA). We observed little activity with the two C-terminal scFv adapters (HveA-scCEA and HveC-scCEA) and reduced activity with the N-terminal scFv-nectin-1 fusion (scCEA-HveC) (Figure 2d). These results established that the relative positions of the specificity elements of the adapter are important activity determinants and that the gD-binding region of HVEM as an adapter component is at least as effective as the previously used V domain of nectin-1.13Nakano K Asano R Tsumoto K Kwon H Goins WF Kumagai I et al.Herpes simplex virus targeting to the EGF receptor by a gD-specific soluble bridging molecule.Mol Ther. 2005; 11: 617-626Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Together, these observations demonstrated that scCEA-HveA is an efficient mediator of CEA-targeted HSV infection.Figure 2Adapter-mediated infection of Chinese hamster ovary-carcinoembryonic antigen (CHO-CEA) cells. (a,b) Infection of CHO-K1 and CHO-CEA cells with QOZHG (MOI = 10) in the presence of increasing amounts of scCEA-HveA. Cells were stained with (a) X-gal or (b) processed for o-nitrophenyl-β-D-galactoside (ONPG) assay at 16 hours postinfection. (c) CHO-CEA cells were infected with QOZHG at different MOIs in the presence of increasing concentrations of scCEA-HveA adapter. Virus entry was determined by ONPG assay, as above. (d) CHO-K1 (filled bars) and CHO-CEA (open bars) cells were infected with QOZHG (MOI = 10) in the absence of adapter or in the presence of HveC-scCEA, scCEA-HveC, HveA-scCEA, or scCEA-HveA, abbreviated as HC-sc, sc-HC, HA-sc, and sc-HA, respectively. Virus entry was determined at 16 hours postinfection by ONPG assay. Each value in b–d represents the mean of three determinations ± SD. MOI, multiplicity of infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Specificity of adapter-mediated CHO-CEA infectionTo confirm that scCEA-HveA functioned by interacting with both viral gD and cellular CEA, we performed blocking experiments with the soluble ectodomain of gD (sgD287)24Tsvitov M Frampton Jr, AR Shah WA Wendell SK Ozuer A Kapacee Z et al.Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection.Virology. 2007; 360: 477-491Crossref PubMed Scopus (15) Google Scholar and with anti-CEA antibodies. As shown in Figure 3a, preincubation of QOZHG and scCEA-HveA (250 nmol/l) with increasing amounts of sgD287 inhibited adapter-mediated infection of CHO-CEA cells in a dose-dependent manner. Likewise, preincubation of the cells with various concentrations of CEA-specific monoclonal antibody reduced scCEA-HveA-mediated infection (Figure 3b); the antibody had no effect on QOZHG infection of Vero cells (data not shown). Together, these results indicated that the adapters acted by connecting virion gD to cell-surface CEA, thereby initiating virus entry into the cells.Figure 3Competitive inhibition of scCEA-HveA-mediated infection. Chinese hamster ovary (CHO)-CEA cells were infected with QOZHG (multiplicity of infection = 10) in the presence of 250 nmol/l scCEA-HveA and increasing concentrations of (a) soluble gD ectodomain (sgD287) or (b) anti-CEA monoclonal antibody F11-39. Infection without adapter was used as a control. Virus entry at 16 hours postinfection was determined by o-nitrophenyl-β-D-galactoside assay. Values represent the means of three determinations ± SD. CEA, carcinoembryonic antigen.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Adapter-mediated infection of CEA-expressing human cancer cells by nectin-1-detargeted HSVAmong the principal HSV-1 entry receptors, nectin-1 is widely expressed,25Takai Y Nakanishi H Nectin and afadin: novel organizers of intercellular junctions.J Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (471) Google Scholar while HVEM is found predominantly on cells of the immune system.8Montgomery RI Warner MS Lum BJ Spear PG Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (998) Google Scholar,14Kwon BS Tan KB Ni J Oh KO Lee ZH Kim KK et al.A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation.J Biol Chem. 1997; 272: 14272-14276Crossref PubMed Scopus (252) Google Scholar,15Marsters SA Ayres TM Skubatch M Gray CL Rothe M Ashkenazi A Herpesvirus entry mediator, a member of the tumor necrosis factor receptor (TNFR) family, interacts with members of the TNFR-associated factor family and activates the transcription factors NF-κB and AP-1.J Biol Chem. 1997; 272: 14029-14032Crossref PubMed Scopus (263) Google Scholar,17Hsu H Solovyev I Colombero A Elliott R Kelley M Boyle WJ ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.J Biol Chem. 1997; 272: 13471-13474Crossref PubMed Scopus (154) Google Scholar Although HVEM has also been detected on various nonimmune cell lines, several published reports have shown that its expression on these cells is often insufficient for HSV-1 infection.16Manoj S Jogger CR Myscofski D Yoon M Spear PG Mutations in herpes simplex virus glycoprotein D that prevent cell entry via nectins and alter cell tropism.Proc Natl Acad Sci USA. 2004; 101: 12414-12421Crossref PubMed Scopus (75) Google Scholar,26Simpson SA Manchak MD Hager EJ Krummenacher C Whitbeck JC Levin MJ et al.Nectin-1/HveC mediates herpes simplex virus type 1 entry into primary human sensory neurons and fibroblasts.J Neurovirol. 2005; 11: 208-218Crossref PubMed Scopus (46) Google Scholar,27Uchida H Shah WA Ozuer A Frampton Jr, AR Goins WF Grandi P et al.Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.J Virol. 2009; 83: 2951-2961Crossref PubMed Scopus (31) Google Scholar Hence, although HSV detargeting from nectin-1 is likely a universal requirement to limit infection to the target cell or tissue, preservation of the HVEM-binding activity of the virus may not dramatically increase off-target infection, depending on the site of virus application. Accordingly, we used mutant viruses detargeted for HVEM or nectin-1 to assess the contribution of HVEM to the HSV susceptibility of CEA-positive MKN45 and CEA-negative MKN74 (ref. 28Tanaka T Huang J Hirai S Kuroki M Kuroki M Watanabe N et al.Carcinoembryonic antigen-targeted selective gene therapy for gastric cancer through FZ33 fiber-modified adenovirus vectors.Clin Cancer Res. 2006; 12: 3803-3813Crossref PubMed Scopus (42) Google Scholar) gastric carcinoma cells. K-d5-28V (K26-gD:d5-28V in ref. 27Uchida H Shah WA Ozuer A Frampton Jr, AR Goins WF Grandi P et al.Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.J Virol. 2009; 83: 2951-2961Crossref PubMed Scopus (31) Google Scholar) is a mutant virus that is highly impaired for HVEM-mediated infection, while K-222/3NI (K26-gD:R222N/F223I in ref. 27Uchida H Shah WA Ozuer A Frampton Jr, AR Goins WF Grandi P et al.Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.J Virol. 2009; 83: 2951-2961Crossref PubMed Scopus (31) Google Scholar) has an ~750-fold reduced ability to infect cells via nectin-1. We infected MKN45 and MKN74 cells with equal numbers of virions of the two receptor-restricted viruses and their unrestricted parent, K26GFP,29Desai P Person S Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid.J Virol. 1998; 72: 7563-7568Crossref PubMed Google Scholar and visualized infection at 8 hours postinfection by anti-VP16 immunostaining. As illustrated in Figure 4, both cell lines were infected efficiently by K26GFP and K-d5-28V, while infection by K-222/3NI was almost undetectable, indicating that nectin-1 is the principal HSV-1 entry receptor on these cells. We then compared K-222/3NI infection in the presence and absence of the scCEA-HveA adapter. Increased infection was observed on MKN45 cells in the presence of the adapter, while infection of MKN74 cells was essentially unaltered (Figure 5). The adapter had no effect on infection of MKN45 cells by K26GFP (data not shown).Figure 4Susceptibility of carcinoembryonic antigen (CEA)-positive MKN45 and CEA-negative MKN74 cells to wild-type gD herpes simplex virus type 1 and receptor-restricted derivatives. Cells were infected with K26GFP, nectin-1-restricted K-d5-28V, or HVEM-restricted K-222/3NI at 100 genome copies/cell and stained at 8 hours postinfection with anti-VP16 antibody and Cy3-conjugated secondary antibody. The lower panels for each cell line show phase-contrast images of the same fields.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Adapter-mediated K-222/3NI infection of MKN45 cells. (a) MKN45 and MKN74 cells were infected with K-222/3NI (MOI = 0.5) in the presence or absence of scEA-HveA (500 nmol/l). Infection at 8 hours postinfection was detected by anti-VP16 immunostaining (Alexa Fluor 594-conjugated secondary antibody) and fluorescence microscopy (dark panels). Phase-contrast images of the same fields are included for reference (light panels). (b) Mean fluorescent signals from triplicate wells ± SD as determined by Thermo Labsystems Fluoroskan Ascent (Thermo Scientific, Waltham, MA). eGFP, enhanced green fluorescent protein; rfu, relative fluorescent units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Spread of HVEM-restricted virus following adapter-mediated infectionWe previously showed that K-222/3NI has a residual ability to use nectin-1 for lateral spread.27Uchida H Shah WA Ozuer A Frampton Jr, AR Goins WF Grandi P et al.Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.J Virol. 2009; 83: 2951-2961Crossref PubMed Scopus (31) Google Scholar To determine whether this ability is sufficient to mediate cell-to-cell spread of the virus on MKN45 cells, we infected these cells at low MOI (0.01) in the presence of scCEA-HveA adapter, inactivated extracellular virus at 1 hour postinfection, and incubated the monolayer under 0.5% methylcellulose overlay. Anti-VP16 staining at 72 hours postinfection showed multiple clusters of infected cells indicative of lateral virus spread (Figure 6). Infection in the absence of adapter also yielded an occasional multicellular infected center (Figure 6), indicating that spread was not dependent on the presence of the adapter during the initial infection. As a control, the figure also shows a typical plaque formed by receptor-unrestricted K26GFP (MOI = 0.005). Together, these results indicated that K-222/3NI offers an attractive balance between impaired primary infection via nectin-1 and residual ability to spread via the same receptor.Figure 6Lateral spread of K-222/3NI following adapter-mediated infection. MKN45 monolayers were infected with K-222/3NI (MOI = 0.01) in the presence or absence of 500 nmol/l scCEA-HveA, treated with acidic buffer to remove extracellular virus, and left to recover under fresh medium for 8 hours at 37 °C. The cells were then incubated with high-density medium for 2.5 days and stained with anti-VP16 primary antibody and Alexa Fluor 594-conjugated secondary antibody. The panel at the right shows a typical plaque formed by parallel infection with K26GFP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Adapter-dependent infection of tumor tissue and inhibition of tumor growthWe explored the ability of the adapter to promote infection of established MKN45 tumors in nude mice. Subcutaneous tumors were injected with K-222/3NI virus with or without adapter and thin sections stained for VP16 at 2 or 6 days after virus inoculation. Representative results for two animals per time point are shown in Figure 7a. No staining was observed in MKN45 tumors without adapter or MKN74 tumors with adapter. In contrast, VP16 was detected in each of the MKN45 tumors inoculated with virus plus adapter. The infected area appeared to increase with time after vector administration, suggesting virus spread, but the sample size, including one animal examined after 10 days (Figure 7a), was too small to draw conclusions in this regard.Figure 7Adapter effect on tumor infection and growth. (a) MKN45 and MKN74 tumors grown subcutaneously in nude mice to a size of 200 mm3 were inoculated with K-222/3NI virus (1 × 108 plaque-forming units) with or without scCEA-HveA adapter (1.2 µmol/l; n = 2/group). Tumors were removed at 2, 6, or 10 days after vector administration and sections were immunoperoxidase stained for VP16. Each image represents a separate tumor. No infection was observed in MKN74 tumors inoculated with virus alone (data not shown). (b) Established MKN45 flank tumors in nude mice were inoculated with K-222/3NI with or without adapter as above on days 0, 3, 6, and 9. Tumor diameters were measured every other day and the calculated volumes (tumor size) plotted as mean ± SD. Statistical analysis by t-test yielded a P value of 0.0033.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the effect of the virus–adapter combination on tumor growth, subcutaneous MKN45 tumors were injected 4 times at 3-day intervals with K-222/3NI virus plus adapter or K-222/3NI alone. Measurement of tumor volumes every other day over a period of 30 days showed a significant reduction in tumor growth in the presence of the adapter (P < 0.05, t-test; Figure 7b). Together, these in vivo results indicated that the CEA-specific adapter combined with an unattenuated, HVEM-selective HSV vector can mediate specific infection of CEA-expressing tumor cells and reduce tumor growth.DiscussionResearch into viral therapies for solid tumors has long focused on the development of replication-impaired mutant vectors whose defects are selectively complemented by certain or all tumor ce