Murine mesothelin: characterization, expression, and inhibition of tumor growth in a murine model of pancreatic cancer

Background Mesothelin has attracted much interest as a tumor specific antigen; it has been reported to promote tumor development and to be a good target for cancer treatment. Most studies to date have used human mesothelin in immunocompromised mice. Since these models do not allow for study of the natural immune response to mesothelin expressing tumors, we have undertaken the characterization of mouse mesothelin so the effects of this protein can be assessed in immunocompetent mouse strains. Methods We analyzed mouse mesothelin expression, tissue distribution, shedding and biochemistry. In addition we constructed stable mesothelin overexpressing lines of the pancreatic cancer line Panc02 by two methods and tested them for growth and tumorigencity in vitro and in vivo. Results We show here that mouse mesothelin is similar to human mesothelin in biochemical characteristics, tumor expression and tissue distribution, suggesting the mouse may be a suitable model for study of mesothelin. Stable overexpression of mesothelin in a pancreatic cancer cell line did not increase cell proliferation or anchorage-independent growth in vitro, suggesting that mesothelin is not necessarily a tumor progression factor. Surprisingly overexpression of mesothelin inhibited tumor formation in vivo in immunocompetent mice. Conclusion The mouse may be a good model for studying mesothelin in the context of an intact immune response. Mesothelin is not necessarily a tumor progression factor, and indeed mesothelin overexpression inhibited tumor growth in immunocompetent mice

Effects of Modified Vaccinia Virus Ankara Expressing Mesothelin and the Poxvirus Immunoregulatory A35R gene in the Treatment of Murine Pancreatic Cancer

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Pancreatic tumors over-express the protein mesothelin, and therefore, it is used as a tumor target antigen for vaccine development. Our current research uses an improved Modified Vaccinia Ankara poxvirus that we developed to express mesothelin to the murine immune system to treat pancreatic tumors and create an anti-cancer vaccine. PCR, Flow Cytometry, and Western Blot confirmed virus construction, purity, and protein expression. Our hypothesis was that the A35R[delta] recombinant virus would be more efficient at stimulating the immune system and better protect against tumors caused by the Panc02 pancreatic adenocarcinoma cell line. MVA was found to both kill and replicate in Panc02 cells, showing it is an oncolytic virus. However, multiple schemes of infection/vaccination and boosts were used without any significant protection from tumor challenge in mice. Assays to determine immune response against mesothelin suggested that mice vaccinated with the mesothelin-expressing vaccine virus did not generate a strong immune response to mesothelin as expected, however the mice had robust immunity to the poxvirus. Together these results suggest that native mesothelin may not be a good vaccine target antigen for cancer treatment, possibly because immune cells responding to this self-protein are deleted and/or suppressed during development of the immune system as a protection against autoimmunity. Strategies for improving anti-mesothelin immunity are being explored.M.S

Effects of Modified Vaccinia Virus Ankara Expressing Mesothelin and the Poxvirus Immunoregulatory A35R gene in the Treatment of Murine Pancreatic Cancer

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Pancreatic tumors over-express the protein mesothelin and therefore it is used as a tumor target antigen for vaccine development. Our current research uses an improved Modified Vaccinia Ankara poxvirus that we developed to express mesothelin to the murine immune system to treat pancreatic tumors and create an anti-cancer vaccine. PCR Flow Cytometry and Western Blot confirmed virus construction purity and protein expression. Our hypothesis was that the A35R[delta] recombinant virus would be more efficient at stimulating the immune system and better protect against tumors caused by the Panc02 pancreatic adenocarcinoma cell line. MVA was found to both kill and replicate in Panc02 cells showing it is an oncolytic virus. However multiple schemes of infection/vaccination and boosts were used without any significant protection from tumor challenge in mice. Assays to determine immune response against mesothelin suggested that mice vaccinated with the mesothelin-expressing vaccine virus did not generate a strong immune response to mesothelin as expected however the mice had robust immunity to the poxvirus. Together these results suggest that native mesothelin may not be a good vaccine target antigen for cancer treatment possibly because immune cells responding to this self-protein are deleted and/or suppressed during development of the immune system as a protection against autoimmunity. Strategies for improving anti-mesothelin immunity are being explored

Murine mesothelin: characterization, expression, and inhibition of tumor growth in a murine model of pancreatic cancer

Background: Mesothelin has attracted much interest as a tumor specific antigen; it has been reported to promotetumor development and to be a good target for cancer treatment. Most studies to date have used humanmesothelin in immunocompromised mice. Since these models do not allow for study of the natural immuneresponse to mesothelin expressing tumors, we have undertaken the characterization of mouse mesothelin so theeffects of this protein can be assessed in immunocompetent mouse strains.Methods: We analyzed mouse mesothelin expression, tissue distribution, shedding and biochemistry. In addition we constructed stable mesothelin overexpressing lines of the pancreatic cancer line Panc02 by two methods and tested them for growth and tumorigencity in vitro and in vivo.Results: We show here that mouse mesothelin is similar to human mesothelin in biochemical characteristics, tumor expression and tissue distribution, suggesting the mouse may be a suitable model for study of mesothelin. Stable overexpression of mesothelin in a pancreatic cancer cell line did not increase cell proliferation or anchorageindependent growth in vitro, suggesting that mesothelin is not necessarily a tumor progression factor. Surprisingly overexpression of mesothelin inhibited tumor formation in vivo in immunocompetent mice.Conclusion: The mouse may be a good model for studying mesothelin in the context of an intact immune response. Mesothelin is not necessarily a tumor progression factor, and indeed mesothelin overexpression inhibited tumor growth in immunocompetent mice

Treatment of tumor bearing mice.

<p>C57BL/6 mice were injected s.c. in one flank with 640,000 wild type Panc02 cells. At 11, 18, and 25 days (arrows) post tumor injection, mice were injected i.m. contralaterally with PBS or 7.6 x 10⁷ pfu/mouse of MVA, MVAmeso (Meso) or MVAmesoA35Del (A35D) viruses in 25 ul. (A) Tumor volumes were measured 3 times per week and survival (B) was monitored.</p

MVA kills Panc02 cells.

<p>Panc02 cells (10,000 cells/well) were plated in triplicate in a 96-well plate. MVA was added to the wells at an MOI of 0.5 or 5.0 pfu/cell. After 24 h, 10 ul MTS/PMS was added to each well, and the absorbance was read at 492 nm. Averages <u>+</u> SD error bars are shown. * p< 0.005. Media with no cells was used to define the background control level.</p

Mesothelin is expressed from MVAmeso viruses.

<p>BHK cells (2x10⁶ cells/well) were uninfected or infected with MVA, MVAmeso or MVAmesoA35Del. Cells were lysed and samples were loaded onto a pre-cast 4–20% gradient gel. Rabbit anti-mesothelin antibody and anti-rabbit IgG (Fc) AP Conjugate (Promega) detected mesothelin protein (arrow). HEK transected cells, previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193131#pone.0193131.ref007" target="_blank">7</a>], were used as control.</p

MVA meso virus injections are safe.

<p>Female C57BL/6 mice (n = 5) were injected i.m. with PBS, MVA, MVAmeso or MVAmesoA35Del and monitored daily by body score condition (no change) and weight up to 125 days. No adverse effects were noted. Average weights are shown <u>+</u> SEM.</p

MVA replicates in Panc02 cells.

<p>350,000 BHK or Panc02 cells were plated in a 24-well plate and infected with MVA at an MOI of 5. Supernatants and cells were harvested separately at six time points: 0, 12, 16, 24, 36, and 48 h post-infection. These samples were frozen and thawed three times before being titered in BHK cells in 6-well plates.</p

Development of improved therapeutic mesothelin-based vaccines for pancreatic cancer

Pancreatic cancer is the 5th leading cause of cancer deaths, and there are no effective treatments. We developed a poxvirus platform vaccine with improved immunogenicity and inserted the mesothelin gene to create an anti-mesothelin cancer vaccine. Mesothelin expression is mostly restricted to tumors in adult mammals and thus may be a good target for cancer treatment. We show here that the modified vaccinia virus Ankara (MVA) virus expressing mesothelin and the enhanced MVA virus missing the immunosuppressive A35 gene and expressing mesothelin were both safe in mice and were able to induce IFN-gamma secreting T cells in response to mesothelin expressing tumor cells. In addition, the MVA virus has oncolytic properties in vitro as it can replicate in and kill Panc02 pancreatic adenocarcinoma cell line tumor cells, even though it is unable to replicate in most mammalian cells. Deletion of the A35 gene in MVA improved T cell responses as expected. However, we were unable to demonstrate inhibition of Panc02 tumor growth in immunocompetent mice with pre-vaccination of mice, boosts, or even intratumoral injections of the recombinant viruses. Vaccine efficacy may be limited by shedding of mesothelin from tumor cells thus creating a protective screen from the immune system