Systemic therapy of disseminated myeloma in passively immunized mice using measles virus-infected cell carriers

Abstract

Multiple myeloma (MM) is bone marrow plasma cell malignancy. A clinical trial utilizing intravenous administration of oncolytic measles virus (MV) encoding the human sodium-iodide symporter (MV-NIS) is ongoing in myeloma patients. However, intravenously administered MV-NIS is rapidly neutralized by antiviral antibodies. Because myeloma cell lines retain bone marrow tropism, they may be ideal as carriers for delivery of MV-NIS to myeloma deposits. A disseminated human myeloma (KAS 6/1) model was established. Biodistribution of MM1, a myeloma cell line, was determined after intravenous infusion. MM1 cells were found in the spine, femurs, and mandibles of tumor-bearing mice. Lethally irradiated MM1 cells remained susceptible to measles infection and transferred MV to KAS 6/1 cells in the presence of measles immune sera. Mice-bearing disseminated myeloma and passively immunized with measles immune serum were given MV-NIS or lethally irradiated MV-NIS-infected MM1 carriers. The antitumor activity of MV-NIS was evident only in measles naive mice and not in passively immunized mice. In contrast, survivals of both measles naive and immune mice were extended using MV-NIS-infected MM1 cell carriers. Hence, we demonstrate for the first time that systemically administered cells can serve as MV carriers and prolonged survival of mice with pre-existing antimeasles antibodies.

Figure 1

Characterization of the KAS 6/1 disseminated human multiple myeloma SCID mouse model. ICR-SCID mice were injected intravenously via the tail vein with 6 × 10⁶ KAS 6/1-Gluc-CFP cells. (a) Tumor growth in mice from three independent experiments was monitored by measuring Gaussia luciferase (Gluc) activity in 5 µl of whole blood (n = 5–8 mice per batch). P < 0.05 denotes significant increase in tumor burden compared to the previous time point. (b) Bioluminescent imaging for firefly luciferase activity showing distribution of human myeloma disease in bone marrow of mice. (c) Images of explanted tissues showing presence of CFP⁺ tumors predominantly in the spine, legs (femur), lower jaw bones (mandible). (d) X-ray images taken of the skeleton of mice with advanced myeloma disease indicate presence osteolytic lesions or fractures (white arrows) in the spine, legs, or mandible due to myeloma growth. CFP, cyan fluorescent protein; SCID, severe combined immunodeficiency.

Figure 2

Feasibility of lethally irradiated MM1 cells as a vehicle for MV delivery. (a) 10 Gy lethally irradiated MM1 cells remained susceptible to MV-GFP infection and expressed viral proteins, resulting in syncytia formation 48 hours postinfection. (b) Quantitation of viral infection and expression in irradiated cells by measuring percentage of single cells expressing GFP using flow cytometry. (c) Clonogenic assays showed that irradiation (RT) resulted in significant loss of MM1 cell viability and clonogenicity at 14 days after cell plating (n = 6 replicates per RT condition). RT0, mock irradiated. (d) Bioluminescent images showing tumor growth in mice injected intravenously with nonirradiated (RT0 Gy) MM1-Fluc cells. In contrast, no tumor growth was seen in mice given lethally irradiated (RT10 or RT40 Gy) MM1-Fluc cells at 42 days after cell infusion. Fluc, firefly luciferase; GFP, green fluorescent protein; MM, multiple myeloma; MOI, multiplicities of infection; MV, measles virus.

Figure 3

Biodistribution of MM1 cell carriers after intravenous delivery in SCID mice-bearing disseminated KAS 6/1 myeloma disease. (a) Percentage of ¹¹¹Indium oxine-labeled MM1 or KAS 6/1 cells in organs harvested from tumor-free SCID mice (CTL) or mice with disseminated KAS 6/1 disease (KAS). (b) Whole body planar gamma camera image of tumor free or tumor-bearing mice given ¹¹¹Indium oxine-labeled MM1 cells. The hot spot in the gamma camera image (circled) correlated with the presence of a large CFP⁺ KAS 6/1 tumor in the mandible. (c) Dosimetry measurements (counts per minute) showing the amount of radiolabeled MM1 cells in a portion of the spine, legs, or mandibles of KAS 6/1-bearing mice as compared to normal control (CTL) mice. (d) Fluorescence microscopy confirmed presence of DiI-labeled (red) MM1 cells in the bone marrow flushings obtained from the femurs and lower jaw bones (LBJ) of tumor free and KAS 6/1 (CFP⁺)-bearing mice. CFP, cyan fluorescent protein; IN, indium; MM, multiple myeloma; SCID, SCID, severe combined immunodeficiency.

Figure 4

High-resolution analysis of heterofusion between green MM1 and red KAS 6/1 cells which resulted in observation of dual colored syncytia. At 18 hours postinfection, MV-Luc infected (MOI 1.0) MM1 cells (labeled green with CellTracker Green CMFDA) were mixed with KAS 6/1 target cells (labeled red with CellTracker Red CMPTX) at a 1:1 ratio. At (a) 2 hours, (b) 48 hours, and (c) 72 hours after mixing, the cells were photographed using a fluorescence microscope at (original magnification ×200) under blue and green light separately and the photographs were merged, yielding “orange colored” syncytia. (d) Coculture of uninfected MM1 cells (green) with KAS 6/1 cells (red) did not result in heterofusion of cells or syncytia. Bars = 50 µm. MM, multiple myeloma; MOI, multiplicities of infection; MV, measles virus.

Figure 5

Transfer of MV-NIS infection from irradiated MM1 cell carriers to target cells in the presence of antimeasles antibodies. (a) Coculture of CMFDA green labeled nonirradiated or 10 Gy irradiated MM1 cells with DsRed-expressing KAS 6/1 cells. Extensive cell fusion was observed between MV-NIS infected RT-MM1 cells (MOI 1.0) and KAS 6/1 cells after 48 hours of coculture. (b) Production of MV progeny in RT10-MM1 cells over time. Cell-associated virus titer peaked at 48 hours postinfection. (c) Crystal violet staining for syncytia in Vero cell monolayer at 48 hours postinfection with cell-free MV-NIS or cocultured with MV-NIS infected RT10-MM1 cells in the presence of increasing dilutions of measles immune sera. (d) Quantitation of the numbers of syncytia induced by MV-NIS or cell-associated virus (RT10-MM1/MV) in the Vero monolayer in the absence (Ab 0) or presence of measles immune sera (dilution 1:8 to 1:1,024). MM, multiple myeloma; MOI, multiplicities of infection; MV, measles virus.

Figure 6

KAS 6/1 cell killing by MV-NIS or MM1 cell-associated MV-NIS in the presence of antimeasles antibodies. (a) KAS 6/1 cells stably expressing Fluc cells were infected with MV-NIS or cocultured with MV-NIS infected, (b) nonirradiated MM1, (c) 10 Gy irradiated, or (d) 40 Gy irradiated MM1 cells. Various multiplicities of infection (MOI) of MV-NIS were tested. Cell killing was measured by quantitation of firefly luciferase activity in viable KAS 6/1-Fluc cells at 48 hours after mixing. Fluc, firefly luciferase; MM, multiple myeloma; MV, measles virus.

Figure 7

Systemic delivery and treatment outcome using MV-NIS infected RT10-MM1 cell carries in measles naive and passively immunized SCID mice-bearing disseminated KAS 6/1 myeloma disease. (a) Bioluminescent images showing amount and location of myeloma disease in mice over time. (b) Quantitation of Fluc activity as a measurement of disease burden, and (c) Kaplan–Meier survival curves of mice in the various treatment groups. Fluc, firefly luciferase; MM, multiple myeloma; MV, measles virus; SCID, severe combined immunodeficiency.