Curative one-shot systemic virotherapy in murine myeloma

Abstract

Current therapy for multiple myeloma is complex and prolonged. Antimyeloma drugs are combined in induction, consolidation and/or maintenance protocols to destroy bulky disease, then suppress or eradicate residual disease. Oncolytic viruses have the potential to mediate both tumor debulking and residual disease elimination, but this curative paradigm remains unproven. Here, we engineered an oncolytic vesicular stomatitis virus to minimize its neurotoxicity, enhance induction of antimyeloma immunity and facilitate noninvasive monitoring of its intratumoral spread. Using high-resolution imaging, autoradiography and immunohistochemistry, we demonstrate that the intravenously administered virus extravasates from tumor blood vessels in immunocompetent myeloma-bearing mice, nucleating multiple intratumoral infectious centers that expand rapidly and necrose at their centers, ultimately coalescing to cause extensive tumor destruction. This oncolytic tumor debulking phase lasts only for 72 h after virus administration, and is completed before antiviral antibodies become detectable in the bloodstream. Antimyeloma T cells, cross-primed as the virus-infected cells provoke an antiviral immune response, then eliminate residual uninfected myeloma cells. The study establishes a curative oncolytic paradigm for multiple myeloma where direct tumor debulking and immune eradication of minimal disease are mediated by a single intravenous dose of a single therapeutic agent. Clinical translation is underway.

Figure 1. Generation and characterization of VSV expressing IFNβ and NIS

(A) Schematic of VSV-IFNβ-NIS. Two viruses were constructed, one encoding mouse IFNβ, the other human IFNβ. (B) One-step virus growth curves on BHK cells infected with VSV-GFP, VSV-mIFNβ-NIS or VSV-hIFNβ-NIS at MOI 1.0; (C) secretion of murine or human IFNβ by VSV-IFNβ-NIS-infected BHK cells, measured by ELISA. n.d. is not detectable. (D) Radioiodine uptakes by BHK cells infected with VSV-GFP, VSV-mIFNβ-NIS or VSV-hIFNβ-NIS at MOI 1.0. Uptakes were determined with (black symbols) or without (grey symbols) potassium perchlorate (KClO₄), a specific inhibitor of NIS-mediated radioiodine uptake (E) Killing of myeloma cell lines by VSV. Viability of mouse IFNβ-treated (100U/ml for 12 hours) or untreated 5TGM1 and MPC-11 murine myeloma cells and B-16 murine melanoma cells was assessed by MTT assay at 48h after infection with VSV-GFP (MOI 1.0) and plotted as % viability compared to untreated cells. Significant differences were measured by t-test and P values are shown. (F) Timecourse of 5TGM1 and MPC-11 cell killing was monitored following infection with VSV-mIFNβ-NIS or VSV-hIFNβ-NIS (MOI 1.0) by measuring cell viability at 12h intervals by MTT assay. MPC-11 was killed more rapidly than 5TGM1. Error bars indicate Standard error of the mean (SEM)

Figure 2. Monitoring intratumoral spread of intravenously administered VSV-IFN-NIS

Female, 6-10 week old C57Bl6/KaLwRij mice bearing subcutaneous syngeneic 5TGM1 myeloma tumors or Balb/c mice bearing subcutaneous MPC-11 myeloma tumors were treated with a single intravenous (IV) dose of 100ul PBS, or 1×10⁸TCID₅₀ VSV-mIFNβ-NIS. SPECT-CT imaging was performed at 24h intervals, each image being obtained one hour after intraperitoneal administration of ^99mTcO₄ (500μCi). Serial day 1, 2, 3 and 4 SPECT/CT images from one representative animal (right panel) bearing (A) 5TGM1 myeloma or (B) MPC-11 myeloma show rapid radioiodine uptake following virus administration. Radioisotope uptake is seen in the thyroid gland (Th), and stomach (St), with slight excreted radioisotope visible in the bladder (Bl). Tumors from control PBS-treated animals (on left) show only background ^99mTcO₄ uptake. Semi-quantitative monitoring of intratumoral virus spread in subcutaneous (C) 5TGM1 and (D) MPC-11 tumor models. SPECT/CT images from n=5 VSV-mIFNβ-NIS-treated and n=2 control (PBS-treated) animals were analyzed to quantify ^99mTcO₄ radioisotope uptake by tumors days 1 through 5 following virus therapy. Mean group values are plotted for each timepoint (errors bars indicate SEM)

Figure 3. Intratumoral extravasation, spread and cell killing by intravenously administered VSV-IFNβ-NIS

(A) Intratumoral distribution of virus-infected cells was analyzed 1, 2 and 3 days after virus administration by harvesting tumors immediately following SPECT-CT imaging and subjecting adjacent tumor sections to autoradiography and immunofluorescence staining to detect VSV antigens (red) as well as TUNEL-positive dead and dying cells (green). Note the increase in VSV-infected cells and TUNEL positive cells at 72 hours, with associated reduction of radioisotope uptake due to loss of cell viability. (B) VSV and TUNEL positivity were quantified using Image J software to determine percent positive cells averaged for 4 sections from n=3 analyzed tumors at the 24 and 48 hour timepoints, or n=2 tumors at the 72 hour timepoint. There was a significant increase in both VSV and TUNEL positivity between 24 and 48h post treatment using t-test (P=0.0455 and P=0.0163 respectively). (C) 5TGM1 tumors from virus-treated mice were harvested at 6 hour intervals following intravenous administration of VSV-mIFNβ-NIS, and were analyzed by immunofluorescent staining to detect VSV-infected cells (green) and CD31-positive blood vessels (red). Representative images are shown at (i) 6h, (ii) 12h, (iii) 18h and (iv-v) 24h following virus administration, magnification 100×.

Figure 4. Therapeutic efficacy of systemically administered VSV-IFNβ-NIS

Mice bearing subcutaneous 5TGM1 tumors were treated with a single intravenous dose of (i) PBS, (ii) VSV-mIFNβ-NIS or (iii) VSV-hIFNβ-NIS. (A) Tumor burden was measured by serial caliper measurements which were used to calculate tumor volume over time. (B) Tumor responses are categorized as tumor progression, early death (≤ 3 days post treatment), complete tumor regression, or regression followed by relapse. NI: no incidence. Statistical difference in rate of tumor relapse within mice with sustained tumor regression was measured by Fischer Exact test indicating significantly higher rate of tumor relapse in VSV-hIFNβ-NIS treated mice vs. VSV-mIFNβ-NIS treated mice (P=0.026). (C) Generation of VSV neutralizing antibodies is measured in serum of PBS treated (n=2) and VSV-IFNβ-NIS treated (n=3 for each virus) mice in the first 5 days post treatment and plotted as the minimum fold dilution that protects BHK cells from infection with 500 TCID₅₀ VSV-GFP.

Figure 5. Immune mediated elimination of tumor cells prevents tumor relapse

(A) Quantification of murine IFNβ in serum of mice bearing subcutaneous 5TGM1 tumors treated intravenously with PBS, VSV-mIFNβ-NIS or VSV-hIFNβ-NIS, measured by ELISA. nd: not detectable (B) C57Bl6/KaLwRij mice that had complete tumor regression after intravenous therapy with VSV-mIFNβ-NIS treatment (n=6) and naïve age-matched syngeneic mice (n=6) were subsequently challenged subcutaneously with 1×10⁷ 5TGM1 cells. Tumor occurrence was recorded on day 21 post-challenge. NI is no incidence. (C) Immunotherapeutic efficacy of a single subcutaneous immunization with VSV-infected 5TGM1 cells. 1×10⁷ 5TGM1 cells were infected at MOI 10.0 with VSV-mIFNβ-NIS and 12 hours later implantated on the left flank. One day or five days later, 5×10⁶ uninfected 5TGM1 tumor cells were implanted subcutaneously on the right flank. Log rank survival analysis comparison shows that day(-5) vaccination prolongs survival of mice following tumor implantation compared to unvaccinated mice (P=0.0253). (D) Mice bearing subcutaneous 5TGM1 tumors were treated with a single intravenous dose of (i) PBS, (ii) VSV-mIFNβ-NIS or (iii) VSV-mIFNβ-NIS combined with antibodies to deplete CD4⁺ and CD8+ T cells. Tumor burden was measured by serial caliper measurements. (E) Relapse rates between T-depleted and control groups were compared by Fischer Exact test indicating a higher rate of tumor relapse when VSV-mIFNβ-NIS was combined with T-cell depletion (P=0.0498).