|Year : 2017 | Volume
| Issue : 4 | Page : 122-132
A T-cell engager-armed oncolytic vaccinia virus to target the tumor stroma
Feng Yu1, Bangxing Hong1, Xiao-Tong Song2
1 Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, TX, USA
2 Center for Cell and Gene Therapy, Texas Children’s Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, TX; Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX; Texas Children’s Cancer Center, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA
|Date of Submission||29-Mar-2017|
|Date of Acceptance||01-Jun-2017|
|Date of Web Publication||14-Aug-2017|
Center for Cell and Gene Therapy, Baylor College of Medicine, 1102 Bates Street, Suite 1770, Houston, TX 77030-2316
Source of Support: None, Conflict of Interest: None
Aim: Cancer-associated fibroblasts (CAFs) are the key cellular components of the tumor stroma. CAFs express fibroblast activation protein (FAP) and FAP-targeted immunotherapies have shown potent antitumor effects in preclinical mouse studies, highlighting their central role in tumorigenesis. However, safety concerns have been raised in regard to FAP-targeted immunotherapies since bone marrow failure and cachexia were observed in transgenic models and preclinical studies. Here, we describe a novel oncolytic virotherapy by locally targeting FAP within tumor tissue.
Methods: T-cell engager-armed oncolytic vaccinia virus (TEA-VV) that encodes a secretory bi-specific T-cell engager consisting of two single-chain variable fragments specific for murine CD3 and fibroblast activation protein (mFAP-TEA-VV) was generated. The antitumor effects of mFAP-TEA-VV were compared to unmodified VVs using standard in vitro immunological assays and an immunocompetent B16 melanoma mouse model.
Results: In vitro, the ability of mFAP-TEA-VV to replicate within tumor cells and induce oncolysis was similar to that of unmodified VVs. However, in co-culture assays, only mFAP-TEA-VV induced bystander killing of noninfected FAP-expressing cells in the presence of murine T-cells. In vivo, mFAP-TEA-VV enhanced viral titer within the tumor and had potent antitumor activity in comparison to control VVs in an immunocompetent B16 melanoma mouse model. Importantly, the improved viral spread of mFAP-TEA-VV correlated with the destruction of tumor stroma.
Conclusion: Arming oncolytic VVs with an FAP-targeted T-cell engager may be a promising improvement to oncolytic virus therapy for solid tumors.
Keywords: Cancer-associated fibroblast, fibroblast activation protein, oncolytic virus, T-cell engager, tumor stroma
|How to cite this article:|
Yu F, Hong B, Song XT. A T-cell engager-armed oncolytic vaccinia virus to target the tumor stroma. Cancer Transl Med 2017;3:122-32
| Introduction|| |
Cancer-associated fibroblasts (CAFs) are the key cellular components of the tumor stroma and present in majority of common epithelial cancers, sarcomas, and melanomas.,,, CAFs mediate resistance to chemo-, radio- and immuno-therapy, and CAF tumor content or genes expressed in CAFs correlate with outcome.,,, CAFs express fibroblast activation protein (FAP), and the targeted deletion of FAP-positive CAFs in transgenic mouse models has potent antitumor effects highlighting their central role in tumorigenesis and their prospect as a target for cancer therapies.
Several immunotherapeutic strategies are being developed to target FAP-positive CAFs.,,, For example, clinical studies targeting FAP with a humanized monoclonal antibody, sibrotuzumab, showed that the antibody preferentially localized to metastatic tumor sites although no objective clinical responses were observed., Several groups have shown that Salmonella More Details typhimurium-, plasmid-, or dendritic cell-based FAP vaccines have antitumor activity in mouse models as they modulate the tumor microenvironment.,,, Recently, we and others have explored T-cells genetically modified to express FAP-specific chimeric antigen receptors to target CAFs and have shown that these cells have antitumor effects by themselves or increase the antitumor activity of tumor-specific T-cells.,, However, safety concerns have been raised in regard to immune-based targeting of FAP since bone marrow failure and cachexia were observed in one transgenic model and one preclinical T-cell therapy study.,
Thus, it would be desirable to develop a local therapy to limit the potential toxicity of targeting FAP. Oncolytic vaccinia viruses (VVs) have been an appealing addition to the current treatment options for solid tumors because they are safe and can selectively infect, replicate in, and lyse tumor cells.,,, We recently developed an approach to locally redirect T-cells to antigens within tumors by genetically engineering oncolytic VVs to express secretory bispecific T-cell engagers that consist of two single-chain variable fragments, one being specific for CD3 expressed on all T-cells and the other being specific for tumor-associated antigen (TAA)., These so-called T-cell engager-armed VVs (TEA-VVs) activated T-cells in a TAA-dependent manner, redirected T-cells to uninfected tumor cells, and had improved antitumor activity in preclinical xenograft models in comparison to unarmed VVs.
Here, we report the development of TEA-VV encoding secretory T-cell engagers that bind both to murine CD3 and FAP (mFAP-TEA-VV). In vitro, mFAP-TEA-VVs activate T-cells in an FAP-specific manner and induce T-cell-mediated bystander killing of uninfected FAP-expressing cells. In vivo, mFAP-TEA-VVs induce destruction of FAP-positive stromal cells, have a greater capacity to replicate, increase the infiltration of activated T-cells, and have greater antitumor activity as compared to unarmed VVs in the immunocompetent B16 melanoma mouse model without overt toxicity.
| Methods|| |
The B16F1 (CRL-6323, melanoma, C57BL/6), CT26 (CRL-2638, colon cancer, BALB/c), CV-1 (CCL-70, kidney, nonhuman primate), and HuTK-143B (CRL-8303, osteosarcoma, human) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The GL261 (glioma, C57BL/6) cell line was kindly provided by Dr. Hideho Okada (University of San Francisco, School of Medicine, San Francisco, CA, USA), and the MC-38 (breast cancer, C57BL/6) cell line by Dr. Sheng Guo (University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA). B16F1, CV-1, HuTK-143B, GL261, CT26, and MC-38 were grown in Dulbecco's modified Eagle's medium with high glucose (HyClone, Logan, UT, USA) containing 10% fetal calf serum (FCS; HyClone, Logan, UT, USA) and 2 mmol/L GlutaMAX-I (Invitrogen, Carlsbad, CA, USA). Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. All other cell lines were cultured in RPMI1640 medium.
Preparation of mouse splenocytes
C57BL/6 mouse splenocytes were prepared and cultured in RPMI1640 medium (Thermo Scientific HyClone, Waltham, MA, USA; Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated FCS and 2 mmol/L GlutaMAX, followed by stimulated with 5 μM concanavalin A (Con A; Sigma, St. Louis, MO, USA) for 72 h.
Construction of vaccinia viruses
The recombinant double-deleted VVs (vvDD; Western Reserve strain) encoding T-cell engagers or granulocyte-macrophage-colony-stimulating factor (GM-CSF) were generated as previously described. The FAP-specific T-cell engager consisted of an N-terminal leader sequence, the murine CD3-specific scFv 145.2C1,, a short linker consisting of four glycines and one serine (G4S), and the murine FAP-specific scFv MO36. The EphA2-specific T-cell engager consisted of an N-terminal leader sequence, the murine CD3-specific scFv 145.2C1, a short G4S linker, and the Ephrin type-A receptor 2 (EphA2)-specific scFv 4H5.,, mFAP-TEA-VV, EphA2-TEA-VV, or GM-CSF-VV was produced by recombination with a pSEL shuttle plasmid encoding the FAP- or EphA2-specific T-cell engager or GM-CSF into the TK gene locus of the VSC20 strain of western reserve VV [Figure 1]. The inserted transgenes were expressed under the transcriptional control of the F17R late promoter., Viral stocks were prepared as previously reported. Recombinant VVs were amplified in Hela cells for 3 days, purified by ultracentrifugation over a sucrose cushion, and titered on confluent CV1 cells as described previously.
|Figure 1: Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses express fibroblast activation protein-T-cell engagers. (a) Scheme of expression cassettes encoding fibroblast activation protein-scFv-CD3-scFv (murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus), Ephrin type-A receptor 2-scFv-CD3-scFv (Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus), or granulocyte-macrophage-colony-stimulating factor (granulocyte-macrophage-colony-stimulating factor-vaccinia virus). (b) Expression of fibroblast activation protein-CD3 engager and Ephrin type-A receptor 2-CD3 engager by recombinant vaccinia virus. MC-38 cells were infected with murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus or Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus at multiplicity of infection 5 supplied with 2% fetal bovine serum. Supernatant was collected 24 h postinfection and determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ephrin type-A receptor 2-CD3 engager protein served as positive control and green fluorescent protein-vaccinia virus as negative control. (c) Expression of granulocyte-macrophage-colony-stimulating factor by granulocyte-macrophage-colony-stimulating factor-vaccinia virus. B16 and GL-261 cells were infected with granulocyte-macrophage-colony-stimulating factor-vaccinia virus at increasing multiplicity of infections (0, 0.1, 1, and 5). Twenty-four hours postinfection, supernatant was collected for ELISA assay|
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For detection of TE-redirected T-cell killing of tumor cells, spleen cells were stimulated with Con A and interleukin (IL)-2 for 3 days before coculture with green fluorescent protein (GFP)-positive tumor cells for 24 h supplied with the supernatant collected from EphA2-TEA-VV- or mFAP-TEA-VV-infected tumor cells. Tumor cells and T-cells were collected and washed once with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA; FACS buffer) before analysis. For each sample, 10,000 cells were analyzed using FACSCalibur instrument (BD, Becton Dickinson, Mountain View, CA, USA) with the CellQuest Software (Becton Dickinson) or with FCS Express Software (De Novo Software, Los Angeles, CA, USA). For detection of murine IL (mIL-2) or murine interferon gamma (mIFN-γ) expression in mouse experiments, tumor tissue was dissected and single cell suspension was prepared. For FACS analysis, cells were stained with anti-CD4 or -CD8 antibody followed by intracellular staining with anti-IL-2 or anti-IFN-γ antibody.
Enzyme-linked immunosorbent assay
For detection of mIL-2 or mIFN-γ, tumor cells were cocultured with Con A-stimulated spleen T-cells with the supernatant collected from MC-38 cells infected with either EphA2-TEA-VV or mFAP-TEA-VV at multiplicity of infection (MOI) 0.1 or 1 for 48 h. After 24 h coculture, supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) with DuoSet ELISA Development Kit (R and D Systems, Minneapolis, MN, USA) according to the manufacturer's guidelines. To detect the expression of GM-CSF, murine tumor cells B16 or GL-261 were infected with GM-CSF-VV at MOI 0, 0.1, 1, and 5 for 48 h, and supernatant was collected for ELISA analysis (R&D Systems, Minneapolis, MN, USA).
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium assay
Tumor cells were plated in a 96-well tissue culture plate at 1 × 104 cells per well (100 μL) and incubated overnight at 37°C. The tumor cells were infected with VVs at indicated MOIs in 2.5% FBS medium for 2 h, followed by culturing in complete medium for 48 h. All samples were measured in triplicate. The viability of tumor cells was then determined using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) formazan viability assay (Promega, Madison, WI, USA). Optical density of the wells was read at 490 nm on a plate reader (Molecular Devices VERSAmax, Molecular Devices, Sunnyvale, CA, USA). The mean viability of the virus-infected tumor cells for each viral dilution was calculated as a percentage relative to the control wells treated with media alone (100% survival) ± standard error mean.
B16 tumor models
This study was approved by the Institutional Animal Care and Use Committees of Baylor College of Medicine (BCM). C57BL/6J mice which were 6–8 weeks old were purchased from Jackson Laboratories and maintained in a pathogen-free mouse facility at BCM according to the institutional guidelines. For B16 subcutaneous (s.c.) tumor model, intradermal tumors were implanted by inoculation of 1 × 105 cells in the right flank on day 0 and 5 × 104 in the left flank on day 4. On days 7, 10, 13, and 16, mice were injected intratumorally with 1 × 108 plaque-forming units (PFUs) of VV. To determine tumor volume by external caliper, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined. Tumor volume based on caliper measurements was calculated by the modified ellipsoidal formula: tumor volume = 1/2 (length × width 2). For B16-F10 lung model, 2 × 105 tumor cells were injected intravenously (i.v.) on day 0. On day 1 and 3, 1 × 108 PFUs of VVs were injected through tail vein. Control groups were injected with equal volume of PBS. On day 15, subsets of mice were sacrificed to enumerate the number of lung tumor nodules.
For FAP and CD3 immunodetection to test the targeted elimination by mFAP-TEA-VV and T-cell infiltration in tumors, mice were injected with of 1 × 105 cells in the right flank s.c. on day 0 and 5 × 104 in the left flank on day 4, followed by with 1 × 108 PFU of VV in PBS or PBS as control on days 7, 10, and 13. Tumors were collected for immunofluorescence analysis with anti-FAP antibody (ab53066; Abcam, Cambridge, MA, USA) or anti-CD3 antibody (ab5690; Abcam). An Alexa Fluor 488-conjugated goat anti-rabbit antibody was used as the secondary antibody (ab150077; Abcam).
All in vitro experiments were performed in triplicate. Measurement data were presented as mean ± standard deviation. The differences between means were tested by nonparametric Mann–Whitney U-test or Bonferroni's multiple comparison t-test. The significance level used was P < 0.05. The relationship of two variables was tested by correlation coefficient r-test. The strong negative relationship level used was r < −0.7.
| Results|| |
Generation of murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus, Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus, and granulocyte-macrophage-colony-stimulating factor-vaccinia virus
We generated vvDD (Western Reserve strain) expressing FAP-scFv-CD3-scFv (mFAP-TEA-VV), EphA2-scFv-CD3-scFv (EphA2 precursor, EphA2-TEA-VV), GM-CSF (GM-CSF-VV), or GFP (GFP-VV) [Figure 1]a. EphA2-TEA-VV was used as a control virus since EphA2 is a non-relevant tumor antigen that is not expressed by either B16 tumor cell line or tumor stromal cells. GM-CSF-VV was used as a control since recently several GM-CSF-armed oncolytic viruses, such as T-VEC (GM-CSF-armed HSV) or JX594 (GM-CSF-armed VV), had been widely evaluated in clinical studies. To investigate if TEA-VV-infected tumor cells expressed secretory bi-specific T-cell engagers, MC-38 cells were transduced with mFAP-TEA-VV, EphA2-TEA-VV, or GFP-VV at an MOI of 5 with 2% FBS. After 24 h, supernatant was applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to test the production of T-cell engagers. Specific bands were seen for cells infected with mFAP-TEA-VV or EphA2-TEA-VV. Compared to the molecular weight of the EphA2-CD3 protein standard, mFAP-TEA-VV produced > 1 μg/mL FAP-T-cell engagers in 24 h [Figure 1]b. In addition, B16 and GL261 cells were transduced with GM-CSF-VV at an MOI of 0.1, 1, or 5, and after 24 h, cell culture media were collected for ELISA detection of GM-CSF. GM-CSF-VV-infected B16 and GL261 cells effectively expressed GM-CSF at MOI of 0.1 in vitro [Figure 1]c.
Fibroblast activation protein-T-cell engager expression does not impair ability of vaccinia viruses to replicate and induce tumor cell lysis
To demonstrate that FAP-T-cell engagers do not impair the ability of mFAP-TEA-VV to replicate and kill tumors, CV-1 cells and tumor cell lines GL261, MC38, B16, and CT26 were infected with mFAP-TEA-VV, EphA2-TEA-VV, GM-CSF-VV, GFP-VV, or parental VV (VSC20) at MOI of 0.1 [Supplementary Figure 1]a [Additional file 1]. Infection of all these cells with mFAP-TEA-VV and control VVs yielded similar amounts of virus at various time points. Thus, mFAP-T-cell engagers do not interfere with VV replication. Next, we compared the ability of mFAP-TEA-VV, EphA2-TEA-VV, GM-CSF-VV, or GFP-VV to induce tumor cell lysis in the absence of murine T-cells. GL261, MC38, B16, and CT26 mouse tumor cells were transduced with mFAP-TEA-VV, EphA2-TEA-VV, GM-CSF-VV, or GFP-VV at increasing MOIs (0, 0.01, 0.1, 1, 5, or 10). Forty-eight hours postinfection, tumor cell viability was determined by MTS assay. All four sets of murine tumor cells were killed with increasing MOIs regardless of the VVs used [Supplementary Figure 1]b. There was no difference between mFAP-TEA-VV and control VVs, indicating that the expression of FAP-T-cell engagers does not interfere with the ability of VVs to induce tumor cell lysis.
Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses redirect murine T-cells to fibroblast activation protein-positive cells
To determine if mFAP-TEA-VVs redirect mouse T-cells to FAP-positive cells, GL261 cell line was first transduced with a lentiviral vector encoding a full-length murine FAP and GFP. GL261-mFAP-GFP cells were infected with mFAP-TEA-VV or EphA2-TEA-VV at MOI of 1 or 0.1. Next, Con A-stimulated mouse splenocytes were added to GL261-mFAP-GFP cells at a splenocyte to GL261-mFAP-GFP ratio of 3:1. After 24 or 48 h of coculture, GL261-mFAP-GFP viability was determined by flow cytometry. Non-infected or EphA2-TEA-VV-infected GL261-mFAP-GFP served as controls. While EphA2-TEA-VV induced only moderate killing at MOI of 1, mFAP-TEA-VV induced > 98% of cell killing within 24 or 48 h of coculture (P < 0.05, EphA2-TEA-VV vs. mFAP-TEA-VV, 24 h: 30.4% vs. 1.67%; 48 h: 16.4% vs. 0.67%) [Figure 2]a.
|Figure 2: Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses redirect murine T-cells to fibroblast activation protein-positive cells. (a and b) GL261-fibroblast activation protein-green fluorescent protein cells were infected with Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus or murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus at multiplicity of infection 0.1 or 1. Infected GL261-fibroblast activation protein-green fluorescent protein cells were cocultured with concanavalin A-activated splenocytes at an E: T of 3:1. Cells were harvested at 24 and 48 h for FACS analysis (a) and supernatants were collected for detecting interleukin-2 and interferon gamma production by ELISA assay (b). (c and e) MC38-fibroblast activation protein-green fluorescent protein cells were infected with Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus or murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus at multiplicity of infection 5, and supernatants were collected for co-culture assays with concanavalin A-stimulated mouse spleen T-cell and MC38-murine CD3 and fibroblast activation protein-green fluorescent protein or GL261-fibroblast activation protein-green fluorescent protein. Medium served as negative control. After 48 h, killing efficacy was measured by (c) FACS analysis for green fluorescent protein-positive tumor cells. (d) Bar graph of killing efficacy (data normalized to the medium-treated samples). (e) Immunofluorescence microscopy of GL261-fibroblast activation protein-green fluorescent protein at 24 h post coculture|
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To determine if FAP-T-cell engagers also activate mouse T-cells, GL261-mFAP-GFP cells were infected with mFAP-TEA-VV or EphA2-TEA-VV at an MOI of 1 or 0.1. Con A-stimulated mouse splenocytes were added as described above, and 24 h postvirus infection, cell culture media were collected to determine the presence of pro-inflammatory cytokines by ELISA. Con A-stimulated mouse splenocytes were activated by FAP-T-cell-engagers as judged by the production of pro-inflammatory cytokines such as IFN-γ and IL-2 in the cell culture supernatant of mFAP-TEA-VV-infected GL261-mFAP-GFP and T-cells. T-cells produced little to no IFN-γ and IL-2 in response to EphA2-TEA-VV-infected GL261-mFAP-GFP (P < 0.05) [Figure 2]b, indicating that mouse T-cell activation depends on the expression of FAP-T-cell engagers by tumor cells.
Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses induce bystander killing of non-infected fibroblast activation protein-positive cells in vitro
To investigate the ability of mFAP-TEA-VVs to induce bystander killing of tumor cells, media from GL261-mFAP-GFP cells, infected with mFAP-TEA-VV, EphA2-TEA-VVs, or GFP-VVs, were mixed with GL261-mFAP-GFP or MC38-mFAP-GFP cells and Con A-stimulated mouse splenocytes. After 48 h, tumor killing was measured using flow cytometry [Figure 2]c and [Figure 2]d. In addition, tumor cell killing was confirmed by fluorescence microscopy for GL261-mFAP-GFP [Figure 2]e. MC38-mFAP-GFP or GL261-mFAP-GFP cells were only killed in the presence of cell culture medium from mFAP-TEA-VV-infected GL261-mFAP-GFP cells, demonstrating bystander killing of tumor cells.
Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses destroy fibroblast activation protein-positive stroma and have an improved capacity to replicate in vivo
To characterize the in vivo activity of mFAP-TEA-VV, we focused on the B16 model since B16 is FAP-negative but induces an FAP-positive stroma. To establish B16 tumors, 1 × 105 B16 cells were inoculated s.c. into the right flank of C57 mice, followed by an s.c. inoculation of 5 × 104 B16 cells into the left flank on day 4. Besides, 1 × 108 PFUs of mFAP-TEA-VV, EphA2-TEA-VV, or GM-CSF-VV was injected into tumors on the right flank on day 7, 10, and 13. Two days after the last injection, tumor tissues on the right flank were collected and one part of each tumor was processed for histological analysis, and from the other part, a tumor lysate was prepared. mFAP-TEA-VVs significantly reduced the number of FAP-positive cells in tumors that were directly injected with virus as judged by immunofluorescence microscopy, demonstrating the ability of mFAP-TEA-VVs to target FAP-positive stroma in vivo [Figure 3]a and [Figure 3]b.
|Figure 3: Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses destroy fibroblast activation protein-positive stroma and had an improved capacity to replicate in vivo. 1 × 105 B16 cells were inoculated subcutaneous on the right flank of C57/BL6 mice. On day 4, 5 × 104 B16 cells were subcutaneous inoculated on the left flank. Tumor-bearing mice were injected with 1 × 108 plaque-forming units of vaccinia viruses or phosphate buffered saline on days 7, 10, and 13. On day 15, tumors were harvested for detection fibroblast activation protein expression and assessments of the vaccinia virus titers. (a) Immunofluorescence staining for fibroblast activation protein expression in B16 tumors (right flank) injected with vaccinia viruses or phosphate buffered saline. (b) Mean number of fibroblast activation protein-positive cells in each field (n = 10). ** P < 0.01. (c) Vaccinia viruses titer per gram in B16 tumor tissue (n = 10) on the right flank. *P < 0.05. (d) Linear relationship between titers of different vaccinia viruses and fibroblast activation protein percentage (r = 0.7846). (e) Vaccinia viruses titer per gram in B16 tumor tissue (n = 10 per group) on left flank|
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Since tumor stroma hinders viral spread within tumors,, we next determined if the destruction of FAP-positive stroma increased the ability of VVs to replicate within tumors. As a surrogate marker for viral replication, we determine the viral titer of the prepared tumor lysates using CV-1 cells. The viral titer was significantly higher (mean: 3 × 104; range 2.5–3.5 × 104) in tumors that were directly injected with mFAP-TEA-VV in comparison to EphA2-TEA-VV- or GM-CSF-VV-injected tumors [Figure 3]c. In addition, there was a strong inverse correlation between viral titer and the presence of FAP-positive stromal cells (r< −0.7) [Figure 3]d. Viral titers for all three VVs were ~ 4 logs lower in the noninjected tumor, and there were no significant differences between VVs, indicating that mFAP-TEA-VVs do not have a greater ability to spread to distant tumor sites [Figure 3]e.
Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses induce the intratumoral infiltration of activated T-cells in vivo
To determine if mFAP-TEA-VVs induce the infiltration of activated T-cells within tumors, mice were injected with B16 cells on the right flank followed by intratumoral injection of VVs as described above. Tumor-infiltrating cells were isolated on day 15 and subjected to FACS analysis. Injection of all VVs increased the frequency of CD4- and CD8-positive T-cells within tumor in comparison to PBS-injected tumors [Figure 4]a and [Figure 4]b. However, the frequency of CD4- and CD8-positive T-cells was significantly higher in mFAP-TEA-VV-injected tumor in comparison to EphA2-TEA- or GFP-VV-injected tumors. The increased frequency of T-cells in VV-treated tumors was confirmed by immunofluorescence staining of CD3+ T-cells [Figure 4]c. We next determined the activation status of intratumoral T-cells using IL-2 production as an activation marker for CD4-positive T-cells and IFN-γ production as an activation marker for CD8-positive T-cells. mFAP-TEA-VVs induced CD4+ T-cells activation in comparison to other injected tumors as judged by intracellular IL2 staining (n = 5, mFAP-TEA-VV vs. PBS vs. GM-CSF-VV vs. EphA2-TEA-VV: 12.3% vs. 0% vs. 6.34% vs. 1.14%) [Figure 4]d and [Figure 4]e. In contrast, all three VVs activated CD8-positive T-cells as judged by intracellular IFN-γ staining (n = 5, mFAP-TEA-VV vs. PBS vs. GM-CSF-VV vs. EphA2-TEA-VV: 9.58% vs. 0% vs. 10.9% vs. 7.35%) [Figure 4]d and [Figure 4]e.
|Figure 4: Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses activate intratumoral T-cells and induced systemic B16-specific T-cell responses. B16 cells of 1 × 105 were inoculated subcutaneous on the right flank of C57/BL6 mice. Tumor-bearing mice were injected with 1 × 108 plaque-forming units of vaccinia viruses or phosphate buffered saline on days 7, 10, and 13. On day 15, tumors and spleens were harvested and subjected to analysis. CD8+ (a) and CD4+ (b) T-cell infiltration determined by FACS analysis (n = 5 per group). (c) Immunofluorescence staining for detection of CD3-positive T-cells within tumor tissues. (d) The presence of activated CD4- and CD8-positive T-cells was determined by intercellular staining for interleukin-2 (CD4) or interferon gamma (CD8). (e) Spleen CD4+ and CD8+ were isolated and subjected to interferon gamma ELISpot assay. (n = 5, murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus vs. Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus or granulocyte-macrophage-colony-stimulating factor-vaccinia virus: 235 + 25 vs. 68 + 12 or 158 + 22) or CD4+ (n = 5, murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus vs. Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus or granulocyte-macrophage-colony-stimulating factor-vaccinia virus: 136 + 15 vs. 45 + 12 or 110 + 14)|
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Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses induce systemic B16-specific T-cell responses
To determine whether VVs induced systemic T-cell responses, mice were injected with B16 cells on the right flank followed by intratumoral injection of VVs as described above. Spleens were collected and single cell suspension was subjected to ELISpot detection. Bone marrow-derived dendritic cells (BMDCs) were made as antigen presenting cells. BMDCs were transduced with lentivirus expressing FAP or TRP2 and matured with LPS. TRP2, tyrosinase-related protein 2, is a well-studied tumor antigen that is expressed by B16 tumor cell. TRP2-specific immune responses were measured to monitor the oncolytic virus-mediated immune responses against B16 tumor cells. To do this, CD4+ or CD8+ splenocytes from experimental mice were cocultured with DC-FAP-Lv, DC-TRP2-Lv, DC-Lv-control, or medium for 20 h before determining IFN-γ secreting spots. The results suggested that mFAP-TEA-VVs induced potent systemic CD8+ (n = 5, mFAP-TEA-VV vs. EphA2-TEA-VV or GM-CSF-VV: 235 + 25 vs. 68 + 12 or 158 + 22) or CD4+ (n = 5, mFAP-TEA-VV vs. EphA2-TEA-VV or GM-CSF-VV: 136 + 15 vs.45 + 12 or 110 + 14) T-cell responses against B16 in spleen tissue, compared to EphA2-TEA-VV and GM-CSF-VV. However, none of the VVs induced potent FAP-specific T-cell responses [Figure 4]e.
Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses have enhanced antitumor activity in immunocompetent B16 melanoma mouse model
Having shown that mFAP-TEA-VVs destroy FAP-positive stroma, have an increased capacity to replicate in vivo, and induce the infiltration of activated T-cells, we finally wanted to investigate the antitumor activity of mFAP-TEA-VV in the local and systemic B16 tumor model. B16 cells of 1 × 105 were inoculated s.c. into the right flank of C57 mice, followed by an s.c. inoculation of 5 × 104 B16 cells into the left flank of the C57 mice on day 4. Moreover, 1 × 108 PFUs of mFAP-TEA-VV, EphA2-TEA-VV, or GM-CSF-VV was injected into the tumor on the right flank on day 7, 10, 13, and 16 [Figure 5]a. Mice receiving PBS served as control. EphA2-TEA-VV or GM-CSF-VV moderately inhibited tumor growth on both sides as compared to control mice. In contrast, mice that received mFAP-TEA-VVs showed a significant decrease in tumor growth on both sides, compared to mice that received EphA2-TEA-VV, GM-CSF-VV, or PBS (mFAP-TEA-VV vs. EphA2-TEA-VV or GM-CSF-VV; P < 0.05) [Figure 5]b and [Figure 5]c.
|Figure 5: Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses have antitumor activity in subcutaneous B16 melanoma model. (a) Treatment scheme of subcutaneous B16 model (n = 5). Tumor volume of the right (b) (n = 5, murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus vs. phosphate buffered saline or Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus or granulocyte-macrophage-colony-stimulating factor-vaccinia virus, P < 0.05) and left flank (c) (n = 5, murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus vs. phosphate buffered saline or Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus or granulocyte-macrophage-colony-stimulating factor-vaccinia virus, P < 0.05) was monitored longitudinally|
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To determine mFAP-TEA-VV's antitumor activity in the systemic B16 mouse model, 2 × 105 B16 cells were injected i.v. into the tail vain, followed by i.v. injections of 1 × 108 PFUs of mFAP-TEA-VV, EphA2-TEA-VV, GM-CSF-VV, or PBS on days 1 and 3 (n = 3 per group; [Figure 6]a). Lung tissues were collected from three mice from each group on day 15 to count the surface tumor nodules [Figure 6]b. mFAP-TEA-VV significantly decreased the number of tumor nodules compared to PBS, EPhA2-TEA-VV, or GM-CSF-VV [Figure 6]c.
|Figure 6: Murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia viruses have antitumor activity in systemic B16 melanoma model. (a) Treatment scheme of B16 systemic metastases model. (b and c) The mice were sacrificed after 15 days treatment. Lung metastases from control (phosphate buffered saline) or vaccinia viruses treated mice were counted (n = 3). Asterix * indicates P < 0.05 and is significantly different than phosphate buffered saline group, granulocyte-macrophage-colony-stimulating factor-vaccinia virus and Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus groups (n = 3, mean ± standard deviation, murine CD3 and fibroblast activation protein-T-cell engager-armed vaccinia virus vs. phosphate buffered saline or Ephrin type-A receptor 2-T-cell engager-armed vaccinia virus or granulocyte-macrophage-colony-stimulating factor-vaccinia virus-vaccinia virus: 8 + 1 vs. 105 + 52 or 46 + 5 or 24 + 8)|
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We then compared the toxicity of mFAP-TEA-VV to EphA2-TEA-VV, GM-CSF-VV, or PBS in vivo. First, 1 × 105 B16 cells were s.c. inoculated on the right flank of C57/BL6 mice (n = 5). On day 4, 5 × 104 B16 cells were s.c. inoculated on the left flank. The mice were injected 1 × 108 VV on day 7, 10, and 13 described as above. On day 15, bone marrow was collected for counting of total bone marrow cells and bone marrow cell numbers from different groups show no difference [Supplementary Figure 2]a [Additional file 2]. The body weight of mice was monitored every 2–3 days. We observed that mFAP-TEA-VVs did not affect mouse weight, compared to EphA2-TEA-VV, GM-CSF-VV, or PBS [Supplementary Figure 2]b. Ruffled fur, anorexia, cachexia, skin tenting (due to dehydration), and skin ulcerations were monitored, and no difference was observed between mFAP-TEA-VV and control virus. These results indicated that mFAP-TEA-VV does not target FAP in normal tissues in this s.c. B16 model.
Next, 2 × 105 B16 cells were i.v. injected through tail vein of C57/BL6 mice (n = 5). The mice were injected 1 × 108 VV through tail vein on days 1 and 3 described as above. On day 15, bone marrow was collected for counting of total bone marrow cell numbers and bone marrow cell numbers from different groups show no difference [Supplementary Figure 3]a [Additional file 3]. The body weight of mice was monitored every 2–3 days. We observed that FAP-TEA-VVs did not affect mouse weight, compared to EphA2-TEA-VV, GM-CSF-VV, or PBS [Supplementary Figure 3]b, indicating mFAP-TEA-VVs do not target FAP in normal tissues in the systemic B16 model. In addition, ruffled fur, anorexia, cachexia, skin tenting (due to dehydration), and skin ulcerations were monitored, and no difference was observed between mFAP-TEA-VV and control virus.
At last, we determined the concentration of FAP T-cell engager in the peripheral blood and if mFAP-TEA-VVs have systematic anti-FAP activity. In the B16 s.c. or systemic models described as above, two days after the last VV injection, serum was collected from mice treated with PBS, GFP-VV, EphA2-TEA-VV, or mFAP-TEA-VV (n = 5). The supernatant was collected from MC-38 cells infected with GFP-VV, EphA2-TEA-VV, or mFAP-TEA-VV at MOI of 5 for 24 h as positive control. GL261-FAP-GFP cells were cocultured with Con A-activated mouse splenocytes in the presence of supernatant or serum for 24 h. FACS analysis demonstrated that FAP T-cell engager is not detectable in peripheral blood, and mFAP-TEA-VVs have no systemic anti-FAP activity [Supplementary Figure 4] [Additional file 4].
| Discussion|| |
In this study, we developed a TEA-VV expressing an anti-FAP engager and evaluated its efficacy in an immunocompetent B16 melanoma mouse model. We demonstrated that mFAP-TEA-VV can effectively redirect murine T-cells to target murine FAP-positive target cells leading to their cytolysis in vitro. The mFAP-TEA-VV also targeted murine FAP-positive CAFs in B16 model diminishing the growth of FAP-negative tumors.
CAFs produce collagens, laminins, fibronectins, proteoglycans, and hyaluronan, forming a dense extracellular matrix (ECM) around tumor cells. CAFs combined with the overproduction of ECM provide physical barriers to oncolytic viruses, forming a shield surrounding tumor nests and preventing oncolytic viruses from reaching and infecting tumor cells., Oncolytic viruses have limited autonomous motility and can only be spread through cell-to-cell contact or soluble diffusion across concentration gradients, both of which can be blocked by CAF and ECM. First, viruses could potentially adhere to any surfaces of the cells where they are first administered and then fail to spread since CAFs are rather resistant to virus infection. Second, oncolytic VV has a diameter > 200 nm and physically does not fit through the strands of the ECM, preventing passive diffusion across concentration gradients to reach the tumor. A study of HSV (over 100 nm) showed that coinjection of matrix-degrading collagenase improved viral spread within the tumor. Using matrix-degrading enzymes to remove physical ECM barriers is currently the foremost option. However, CAFs could produce ECMs continuously. Thus, the efficacy of matrix-degrading collagenase could rely on the speed of production and degradation of ECMs.
In this study, we aimed to destroy CAFs and remove physical barriers to oncolytic virus in a more permanent manner. We observed that administration of mFAP-TEA-VV significantly enhanced viral titer within tumors. The increase of viral titer is correlated with the destruction of FAP-positive stromal cells. We observed no difference in the viral titer within the tumors of the mice injected with EphA2-TEA-VV or GM-CSF-VV, indicating the activation of innate immunity has no significant effect on viral spread within tumors. These results suggest that viral spread within tumors is limited by FAP-positive tumor stromal cells and targeting FAP by oncolytic VV could effectively destroy FAP-positive tumor stromal cells, highlighting the importance of targeting stroma to promote antitumor efficacy in immunotherapies.
We also evaluated T-cell responses to mFAP-TEA-VVs in an immunocompetent B16 melanoma mouse model. mFAP-TEA-VVs significantly promoted tumor infiltration and activation of CD8+ and CD4+ T-cells [Figure 4]a,[Figure 4]b,[Figure 4]c,[Figure 4]d, compared to EphA2-TEA-VV and GM-CSF-VV. Interestingly, mFAP-TEA-VVs induced potent systemic T-cell responses against B16 in spleen tissues, compared to EphA2-TEA-VV and GM-CSF-VV. However, while all of these VVs are capable of inducing a systemic T-cell response against B16 tumor cells, none induced potent splenic T-cell responses against FAP [Figure 4]e, likely due to the fact that VVs infect tumor cells rather than fibroblasts. Tumor cells are mainly lysed by oncolytic virus, while fibroblasts are lysed by T-cell engager-mediated T-cells.
In conclusion, this study provides preclinical evidence for the therapeutic potential of TEA-VVs targeting FAP on CAF. In this report, our mFAP-TEA-VVs enhanced viral spread within tumors and antitumor activity in an immunocompetent B16 melanoma mouse model. Our FAP-targeted T-cell engager arming strategy may be applicable to the broad range of solid tumors in which FAP are generally overexpressed.
Financial support and sponsorship
This work was funded by NIH R01 CA148748.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Cirri P, Chiarugi P. Cancer associated fibroblasts: the dark side of the coin. Am J Cancer Res
2011; 1 (4): 482–97.
Marx J. Cancer biology. All in the stroma: cancer's Cosa Nostra. Science
2008; 320 (5872): 38–41.
Dolznig H, Schweifer N, Puri C, Kraut N, Rettig WJ, Kerjaschki D, Garin-Chesa P. Characterization of cancer stroma markers: in silico
analysis of an mRNA expression database for fibroblast activation protein and endosialin. Cancer Immun
2005; 5: 10.
Navab R, Strumpf D, Bandarchi B, Zhu CQ, Pintilie M, Ramnarine VR, Ibrahimov E, Radulovich N, Leung L, Barczyk M, Panchal D, To C, Yun JJ, Der S, Shepherd FA, Jurisica I, Tsao MS. Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer. Proc Natl Acad Sci U S A
2011; 108 (17): 7160–5.
Chometon G, Jendrossek V. Targeting the tumour stroma to increase efficacy of chemo-and radiotherapy. Clin Transl Oncol
2009; 11 (2): 75–81.
Loeffler M, Krüger JA, Niethammer AG, Reisfeld RA. Targeting tumor-associated fibroblasts improves cancer chemotherapy by increasing intratumoral drug uptake. J Clin Invest
2006; 116 (7): 1955–62.
Liao D, Luo Y, Markowitz D, Xiang R, Reisfeld RA. Cancer associated fibroblasts promote tumor growth and metastasis by modulating the tumor immune microenvironment in a 4T1 murine breast cancer model. PLoS One
2009; 4 (11): e7965.
Zhang B, Bowerman NA, Salama JK, Schmidt H, Spiotto MT, Schietinger A, Yu P, Fu YX, Weichselbaum RR, Rowley DA, Kranz DM, Schreiber H. Induced sensitization of tumor stroma leads to eradication of established cancer by T cells. J Exp Med
2007; 204 (1): 49–55.
Kraman M, Bambrough PJ, Arnold JN, Roberts EW, Magiera L, Jones JO, Gopinathan A, Tuveson DA, Fearon DT. Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha. Science
2010; 330 (6005): 827–30.
Kakarla S, Song XT, Gottschalk S. Cancer-associated fibroblasts as targets for immunotherapy. Immunotherapy
2012; 4 (11): 1129–38.
Scott AM, Wiseman G, Welt S, Adjei A, Lee FT, Hopkins W, Divgi CR, Hanson LH, Mitchell P, Gansen DN, Larson SM, Ingle JN, Hoffman EW, Tanswell P, Ritter G, Cohen LS, Bette P, Arvay L, Amelsberg A, Vlock D, Rettig WJ, Old LJ. A phase I dose-escalation study of sibrotuzumab in patients with advanced or metastatic fibroblast activation protein-positive cancer. Clin Cancer Res
2003; 9 (5): 1639–47.
Hofheinz RD, al-Batran SE, Hartmann F, Hartung G, Jäger D, Renner C, Tanswell P, Kunz U, Amelsberg A, Kuthan H, Stehle G. Stromal antigen targeting by a humanised monoclonal antibody: an early phase II trial of sibrotuzumab in patients with metastatic colorectal cancer. Onkologie
2003; 26 (1): 44–8.
Welt S, Divgi CR, Scott AM, Garin-Chesa P, Finn RD, Graham M, Carswell EA, Cohen A, Larson SM, Old LJ. Antibody targeting in metastatic colon cancer: a phase I study of monoclonal antibody F19 against a cell-surface protein of reactive tumor stromal fibroblasts. J Clin Oncol
1994; 12 (6): 1193–203.
Santos AM, Jung J, Aziz N, Kissil JL, Puré E. Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice. J Clin Invest
2009; 119 (12): 3613–25.
Gottschalk S, Yu F, Ji M, Kakarla S, Song XT. A vaccine that co-targets tumor cells and cancer associated fibroblasts results in enhanced antitumor activity by inducing antigen spreading. PLoS One
2013; 8 (12): e82658.
Wen Y, Wang CT, Ma TT, Li ZY, Zhou LN, Mu B, Leng F, Shi HS, Li YO, Wei YQ. Immunotherapy targeting fibroblast activation protein inhibits tumor growth and increases survival in a murine colon cancer model. Cancer Sci
2010; 101 (11): 2325–32.
Li X, Wang Y, Zhao Y, Yang H, Tong A, Zhao C, Shi H, Li Y, Wang Z, Wei Y. Immunotherapy of tumor with vaccine based on basic fibroblast growth factor-activated fibroblasts. J Cancer Res Clin Oncol
2014; 140 (2): 271–80.
Kakarla S, Chow KK, Mata M, Shaffer DR, Song XT, Wu MF, Liu H, Wang LL, Rowley DR, Pfizenmaier K, Gottschalk S. Antitumor effects of chimeric receptor engineered human T cells directed to tumor stroma. Mol Ther
2013; 21 (8): 1611–20.
Wang LC, Lo A, Scholler J, Sun J, Majumdar RS, Kapoor V, Antzis M, Cotner CE, Johnson LA, Durham AC, Solomides CC, June CH, Puré E, Albelda SM. Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity. Cancer Immunol Res
2014; 2 (2): 154–66.
Schuberth PC, Hagedorn C, Jensen SM, Gulati P, van den Broek M, Mischo A, Soltermann A, Jüngel A, Marroquin Belaunzaran O, Stahel R, Renner C, Petrausch U. Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells. J Transl Med
2013; 11: 187.
Tran E, Chinnasamy D, Yu Z, Morgan RA, Lee CC, Restifo NP, Rosenberg SA. Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia. J Exp Med
2013; 210 (6): 1125–35.
Roberts EW, Deonarine A, Jones JO, Denton AE, Feig C, Lyons SK, Espeli M, Kraman M, McKenna B, Wells RJ, Zhao Q, Caballero OL, Larder R, Coll AP, O'Rahilly S, Brindle KM, Teichmann SA, Tuveson DA, Fearon DT. Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia. J Exp Med
2013; 210 (6): 1137–51.
Russell SJ, Peng KW, Bell JC. Oncolytic virotherapy. Nat Biotechnol
2012; 30 (7): 658–70.
Heo J, Reid T, Ruo L, Breitbach CJ, Rose S, Bloomston M, Cho M, Lim HY, Chung HC, Kim CW, Burke J, Lencioni R, Hickman T, Moon A, Lee YS, Kim MK, Daneshmand M, Dubois K, Longpre L, Ngo M, Rooney C, Bell JC, Rhee BG, Patt R, Hwang TH, Kirn DH. Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer. Nat Med
2013; 19 (3): 329–36.
Breitbach CJ, Burke J, Jonker D, Stephenson J, Haas AR, Chow LQ, Nieva J, Hwang TH, Moon A, Patt R, Pelusio A, Le Boeuf F, Burns J, Evgin L, De Silva N, Cvancic S, Robertson T, Je JE, Lee YS, Parato K, Diallo JS, Fenster A, Daneshmand M, Bell JC, Kirn DH. Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature
2011; 477 (7362): 99–102.
Adair RA, Roulstone V, Scott KJ, Morgan R, Nuovo GJ, Fuller M, Beirne D, West EJ, Jennings VA, Rose A, Kyula J, Fraser S, Dave R, Anthoney DA, Merrick A, Prestwich R, Aldouri A, Donnelly O, Pandha H, Coffey M, Selby P, Vile R, Toogood G, Harrington K, Melcher AA. Cell carriage, delivery, and selective replication of an oncolytic virus in tumor in patients. Sci Transl Med
2012; 4 (138): 138ra77.
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.
Song XT. Combination of virotherapy and T-cell therapy: arming oncolytic virus with T-cell engagers. Discov Med
2013; 16 (90): 261–6.
Leo O, Foo M, Sachs DH, Samelson LE, Bluestone JA. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc Natl Acad Sci U S A
1987; 84 (5): 1374–8.
Mottram PL, Murray-Segal LJ, Han W, Maguire J, Stein-Oakley AN. Remission and pancreas isograft survival in recent onset diabetic NOD mice after treatment with low-dose anti-CD3 monoclonal antibodies. Transpl Immunol
2002; 10 (1): 63–72.
Brocks B, Garin-Chesa P, Behrle E, Park JE, Rettig WJ, Pfizenmaier K, Moosmayer D. Species-crossreactive scFv against the tumor stroma marker “fibroblast activation protein” selected by phage display from an immunized FAP-/-knock-out mouse. Mol Med
2001; 7 (7): 461–9.
Damschroder MM, Widjaja L, Gill PS, Krasnoperov V, Jiang W, Dall'Acqua WF, Wu H. Framework shuffling of antibodies to reduce immunogenicity and manipulate functional and biophysical properties. Mol Immunol
2007; 44 (11): 3049–60.
Coffman KT, Hu M, Carles-Kinch K, Tice D, Donacki N, Munyon K, Kifle G, Woods R, Langermann S, Kiener PA, Kinch MS. Differential EphA2 epitope display on normal versus malignant cells. Cancer Res
2003; 63 (22): 7907–12.
Chow KK, Naik S, Kakarla S, Brawley VS, Shaffer DR, Yi Z, Rainusso N, Wu MF, Liu H, Kew Y, Grossman RG, Powell S, Lee D, Ahmed N, Gottschalk S. T cells redirected to EphA2 for the immunotherapy of glioblastoma. Mol Ther
2013; 21 (3): 629–37.
Broyles SS, Kremer M, Knutson BA. Antiviral activity of distamycin A against vaccinia virus is the result of inhibition of postreplicative mRNA synthesis. J Virol
2004; 78 (4): 2137–41.
Dower K, Rubins KH, Hensley LE, Connor JH. Development of Vaccinia reporter viruses for rapid, high content analysis of viral function at all stages of gene expression. Antiviral Res
2011; 91 (1): 72–80.
Choi IK, Strauss R, Richter M, Yun CO, Lieber A. Strategies to increase drug penetration in solid tumors. Front Oncol
2013; 3: 193.
Wojton J, Kaur B. Impact of tumor microenvironment on oncolytic viral therapy. Cytokine Growth Factor Rev
2010; 21 (2-3): 127–34.
McKee TD, Grandi P, Mok W, Alexandrakis G, Insin N, Zimmer JP, Bawendi MG, Boucher Y, Breakefield XO, Jain RK. Degradation of fibrillar collagen in a human melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector. Cancer Res
2006; 66 (5): 2509–13.
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