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Author: The Wistar Institute

Nikon Small World: An Up-close Look at the Unseen World

PHILADELPHIA — (Dec. 13, 2018) — Are we looking at the masked exoskeleton of cinema’s latest superhero, a firework and an amoeba? No, these images are the eye of a beetle, the central region of the retina and a single human tear, and each of them represent just three of the captivating images from the 2018 Nikon Small World competition of photomicrography. These photographs taken through microscopes will be on display at The Wistar Institute, with an opening reception on Jan. 18, 2019.

At Wistar, researchers look through microscopes and hypothesize over microscopic images with the goal of advancing cancer and infectious disease research to develop future therapeutics. Winning photographers this year are both scientists and artists possessing the consummate skill, scientific discipline and creativity for which the Nikon Small World competition is known. Small World spans 44 years as a leading, global competition for photomicrography. This year’s images were chosen from more than 2,500 entries from 89 countries. Wistar has been hosting the exhibit for more than 15 years.

Opportunities at Wistar’s opening reception include:

  • Top 20 honoree images will be on view.
  • A feature wall of 15 high-definition TV screens that projects all 2018 Nikon Small World in Motion winners and Photomicrography Competition winners.
  • A hands-on microscope demonstration by Wistar scientists.
  • A self-guided tour of cell photographs created by Wistar scientists and other pieces of Wistar history.
  • And brief talks from Image of Distinction awardee James E. Hayden, Wistar Imaging Facility managing director; 7th place winner Norm Barker, professor of pathology & art as applied to medicine at Johns Hopkins School of Medicine; Image of Distinction awardee Michael Much; and Nikon Instruments Inc. communications manager Eric Flem.

From Jan. 21 through April 5, 2019, the top-20 images will be on view at Wistar and the exhibit is FREE to the public. The Wistar Institute is the only Pennsylvania venue to host these remarkable works of art.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer and infectious disease research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Engineered DNA-encoded PCSK9 Inhibitors May Provide an Effective Alternative for Treating High Cholesterol

PHILADELPHIA — (Nov. 15, 2018) — Researchers at The Wistar Institute have developed novel synthetic DNA-encoded monoclonal antibodies (DMAbs) directed against PCSK9, a protein key to regulating cholesterol levels in the bloodstream. Results of preclinical studies showed a significant cholesterol decrease, opening the door for further development of this approach as a simple, less frequent and cost-effective therapy, as reported in a paper published online in Molecular Therapy.

Elevated, low-density lipoprotein cholesterol (LDL-C) is a major risk factor for cardiovascular disease, the leading cause of death in the U.S. and worldwide. Statins are effective and widely used cholesterol-lowering medications, but have been associated with a number of side effects that have prompted development of alternative treatment strategies, including monoclonal antibodies targeting the PSCK9 protein that result in reduced degradation of LDL-C receptors on liver cells and increased cholesterol clearance from blood circulation.

“Any therapy based on recombinant monoclonal antibodies faces challenges of production among other issues as molecules may be difficult to manufacture and require multiple administrations,” said lead researcher David B. Weiner, Ph.D., executive vice president, director of Wistar’s Vaccine & Immunotherapy Center, and the W.W. Smith Charitable Trust Professor in Cancer Research at The Wistar Institute. “Anti-PCSK9 therapy presents an important opportunity for development of alternative approaches, possibly expanding options for such therapies.”

Weiner and collaborators engineered synthetic DNA constructs that are delivered by intramuscular injection and encode the genetic instructions for the body to make its own functional monoclonal antibodies, entirely bypassing bioprocess and manufacturing factory approaches. This study provides the first proof of principle that such engineered DMAbs may be developed as a new option for coronary artery disease.

The researchers tested expression and activity of the DMAbs targeting PCSK9 in mice. A single intramuscular administration drove robust antibody expression within days and for up to two months, resulting in a substantial increase in the presence of LDL-C receptors on liver cells. This in turn resulted in a significant decrease in total cholesterol and non-high-density lipoprotein cholesterol (non-HDL-C), an important parameter for evaluating cardiovascular risk.

“We are excited about these findings that support the flexibility and versatility of the DMAb platform as a next generation approach that can be optimized for a wide host of applications,” said Makan Khoshnejad, Ph.D., first author on the study and a postdoctoral fellow in the Weiner Lab.

Co-authors of this study from The Wistar Institute include Ami Patel, Krzysztof Wojtak, Sagar B. Kudchodkar, and Kar Muthumani; other co-authors include Laurent Humeau from Inovio Pharmaceuticals, Inc.; and Nicholas N. Lyssenko and Daniel J. Rader from the University of Pennsylvania.

This work was supported by funding from Inovio Pharmaceuticals, Inc.

Development of Novel DNA-encoded PCSK9 Monoclonal Antibodies as Lipid-lowering Therapeutics,
Molecular Therapy (2018). Advance online publication.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Synthetic DNA-delivered Antibodies Protect Against Ebola in Preclinical Studies Representing a Novel Platform for Antibody Therapies for Outbreak Infections

PHILADELPHIA — (Nov. 13, 2018) — Scientists at The Wistar Institute and collaborators have successfully engineered novel DNA-encoded monoclonal antibodies (DMAbs) targeting Zaire Ebolavirus that were effective in preclinical models. Study results, published online in Cell Reports, showed that DMAbs were expressed over a wide window of time and offered complete and long-term protection against lethal virus challenges. DMAbs may also provide a novel powerful platform for rapid screening of monoclonal antibodies enhancing preclinical development.

Ebola virus infection causes a devastating disease, known as Ebola virus disease, for which no licensed vaccine or treatment are available. The 2014-2016 Zaire Ebola virus epidemic in West Africa was the most severe reported to date, with more than 28,600 cases and 11,325 deaths according to the Center for Disease Control. A new outbreak is ongoing in the Democratic Republic of Congo, with a death toll of more than 200 people since August. One of the experimental avenues scientists are pursuing is evaluating the safety and efficacy of monoclonal antibodies isolated from survivors as promising candidates for further development as therapeutics against Ebola virus infection. However, this approach requires high doses and repeated administration of recombinant monoclonal antibodies that are complex and expensive to manufacture, so meeting the global demand while keeping the cost affordable is challenging.

“Our studies show deployment of a novel platform that rapidly combines aspects of monoclonal antibody discovery and development technology with the revolutionary properties of synthetic DNA technology,” said lead researcher David B. Weiner, Ph.D., executive vice president and director of Wistar’s Vaccine & Immunotherapy Center, and W.W. Smith Charitable Trust Professor in Cancer Research.

Weiner’s lab designed and enhanced optimized DMAbs that, when injected locally, provide the genetic blueprint for the body to make functional and protective Ebola virus-specific antibodies, circumventing multiple steps in the antibody development and manufacturing process. Dozens of DMAbs were tested in mice and the best-performing ones were selected for further studies. These proved to be highly effective for providing complete protection from disease in challenge studies.

“Due to intrinsic biochemical properties, some monoclonal antibodies might be difficult and slow to develop or even impossible to manufacture, falling out of the development process and causing loss of potentially effective molecules,” added Weiner. “The DMAb platform allows us to collect protective antibodies from protected persons and engineer and compare them rapidly and then deliver them in vivo to protect against infectious challenge. Such an approach could be important during an outbreak, when we need to design, evaluate and deliver life-saving therapeutics in a time-sensitive manner.”

“We started with antibodies isolated from survivors and compared the activity of anti-Ebola virus DMAbs and recombinant monoclonal antibodies over time,” said Ami Patel, Ph.D., first author on the study and associate staff scientist in the Wistar Vaccine and Immunotherapy Center. “We showed that in vivo expression of DMAbs supports extended protection over traditional antibody approaches.”

The researchers also looked at how DMAbs physically interact with their Ebola virus targets, called epitopes, and confirmed that DMAbs bind to identical epitopes as the corresponding recombinant monoclonal antibodies made in traditional bioprocess facilities.

The Weiner Laboratory is also developing an anti-Ebola virus DNA vaccine. Preclinical results from this efforts were published recently in the Journal of Infectious Diseases.

Co-authors of this study from The Wistar Institute include Daniel H. Park, Marguerite E. Gorman, Sarah T.C. Elliott, Rianne Esquivel, and Kar Muthumani. Other co-authors include Carl W. Davis and Rafi Ahmed from Emory University; Trevor R.F. Smith, Charles Reed, Megan C. Wise, Jian Yan, Jing Chen, Kate E. Broderick, Laurent Humeau, and Niranjan Y. Sardesai from Inovio Pharmaceuticals; Anders Leung, Kevin Tierney, Trina Racine, Shihua He, Xiangguo Qiu, and Darwyn Kobasa from Public Health Agency of Canada; Aubrey Bryan, Edgar Davidson and Benjamin J. Doranz from Integral Molecular; Xiaoying Yu and Erica Ollmann Saphire from The Scripps Research Institute, La Jolla; James E. Crowe from Vanderbilt University; and Gary P. Kobinger from Université Laval, Canada.

This work was supported by a grant from the Defense Advanced Research Projects Agency (DARPA) awarded to Inovio Pharmaceuticals and by National Institutes of Health contract HHSN272201400058C.

In Vivo-delivered Synthetic Human DMAbs Protect Against Ebolavirus Infection in a Mouse Model,
Cell Reports (2018). Advanced online publication.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Novel Mechanism of Immune Activation in HIV-exposed, Seronegative People who Inject Drugs

PHILADELPHIA — (Nov. 13, 2018) — According to new research from The Wistar Institute, the S100A14 protein is expressed at higher levels in people who inject drugs and remain uninfected despite many years of high-risk, needle sharing behavior in areas with high HIV prevalence. The protein mediates activation of a type of immune cell called natural killer cells (NK), which play a key role in the host immune defense during the earliest phases of viral infection. These results were published in the journal JAIDS.

A small percentage of individuals who are at high risk of HIV infection because of their continued exposure to the virus remain uninfected. Previous research from Wistar has shown that these individuals, referred to as HIV-1 exposed seronegative (HESN), have a higher level of innate immune activation, including enhanced activity of NK cells and dendritic cells.

“Understanding the basis of natural resistance to HIV is important because it may instruct the design of novel approaches for prevention of HIV infection based on manipulating the host immune defense,” said corresponding author Luis J. Montaner, D.V.M., D.Phil., director of the HIV-1 Immunopathogenesis Unit at The Wistar Institute Vaccine & Immunotherapy Center and the Herbert Kean, M.D., Family Endowed Chair Professor. “We investigated the mechanism of immune activation in a specific group of HESN individuals who inject drugs (HESN-PWID) and are at high risk of HIV infection because of needle sharing.”

Using proteomic analysis, the team found that HESN-PWID individuals have a significantly higher expression of interferon-related proteins and S100 family proteins in their blood and within isolated NK cells compared with control donors. Montaner and colleagues focused on the S100A14 protein and showed that it potently activates a population of immune cells in the blood called monocytes, which in turn activate NK cells.

“Our findings suggest that S100A14 represents a novel element in the host defense against HIV infection,” said Costin Tomescu, Ph.D., senior staff scientist in the Montaner Lab and co-corresponding author on the study. “Further studies will explore the potential role of this protein in augmenting the ability of NK cells to inhibit HIV replication and eliminate infected cells and could help develop novel approaches for prevention of HIV infection.”

Other authors from The Wistar Institute include first author Krystal Colón and David W. Speicher. Other co-authors include Peter Smith, Mack Taylor and David S. Metzger from University of Pennsylvania.

This work was supported by National Institutes of Health (NIH) grants R21 DA040554, T32 AI007632, UM1 AI126620, R01 AI094603. Additional funding was provided by The Philadelphia Foundation (Robert I. Jacobs Funds), the Kean Family Professorship, Henry S. Miller, Jr. and J. Kenneth Nimblett, and the Penn Center for AIDS research (grant P30AI045008).

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Function of Neutrophils During Tumor Progression Unraveled

PHILADELPHIA — (Oct. 15, 2018) — Researchers at The Wistar Institute have characterized the function of neutrophils, a type of white blood cells, during early stages of tumor progression, showing that they migrate from the bone marrow to distant sites and facilitate tumor cell seeding and establishment of metastasis. Importantly, these neutrophils don’t possess the immunosuppressive characteristics of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). This seminal study was published online in Nature Immunology.

PMN-MDSCs are pathologically activated neutrophils with the ability to suppress immune responses to cancer and to promote tumor progression by conditioning tumor cells at the primary site. The role of neutrophils in setting the stage for metastatic growth at distant sites was not clear.

“Our research shed light on the role of neutrophils in the early stages of tumor progression, when overt metastasis has not yet formed but the conditions for metastatic spread are being created,” said Dmitry I. Gabrilovich, M.D., Ph.D., Christopher M. Davis Professor and program leader of the Immunology, Microenvironment and Metastasis Program at Wistar. “Our study revealed that the activation of neutrophils in cancer is a two-phase process. We focused on the first phase and described the accumulation of a previously uncharacterized population of neutrophils that lack immunosuppressive activity but display a potent ability to spontaneously migrate, whereas the later phase is associated with accumulation of neutrophils with typical features of PMN-MDSCs.”

Gabrilovich and colleagues isolated neutrophils from the bone marrow of tumor bearing mice from different models of disease and at different stages of tumor development. The highly migratory population present in the early stages, which they designated as PMN-MDSC-like cells (PM-LCs), displays higher glucose uptake, increased metabolic activity and higher expression of genes associated with energy production. Further in vivo experiments demonstrated that PM-LCs promote seeding of tumor cells in distant sites and may favor metastatic dissemination.

The team validated the clinical relevance of these findings by describing the same spontaneous migratory behavior of neutrophils isolated from cancer patients.

“Our study elucidates the mechanism through which neutrophils contribute to early tumor dissemination,” said Jerome Mastio, Ph.D., postdoctoral fellow in the Gabrilovich Lab and co-first author of the study. “We describe the dynamic changes that neutrophils undergo in cancer, with PM-LCs representing the first step of pathologic activation.”

This work was supported by National Institutes of Health grant P01 CA140043 and T32 CA09171. Core support for The Wistar Institute was provided by the Cancer Center Support Grant P30 CA010815.

Sima Patel and Shuyu Fu from The Wistar Institute are co-first authors on this study. Other co-authors from Wistar include George Dominguez, Abhilasha Purohit, Andrew Kossenkov, Cindy Lin, Kevin Alicea-Torres, Mohit Sehgal, Yulia Nefedova, Dario C. Altieri, and Zachary Schug. Other co-authors include Jie Zhou from Sun Yat-sen University, China; Lucia R. Languino from Thomas Jefferson University; Cynthia Clendenin and Robert H. Vonderheide from University of Pennsylvania; Charles Mulligan, Brian Nam, Neil Hockstein, Gregory Masters, and Michael Guarino from Helen F Graham Cancer Center at Christiana Care Health System.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer and infectious disease research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Synthetic DNA Vaccine Against Ebola Virus Shows Potent and Long-term Efficacy in Preclinical Studies

PHILADELPHIA — (October 10, 2018) — A novel synthetic DNA vaccine developed based on technology pioneered by scientists at The Wistar Institute Vaccine & Immunotherapy Center offers complete protection from Zaire Ebolavirus (EBOV) infection in promising preclinical research. Study results were published online in the Journal of Infectious Diseases.

Ebola virus infection causes a severe hemorrhagic fever that has a 50% fatality rate, according to the World Health Organization. Recent advances have led to the development of promising experimental vaccine candidates that may be associated with side effects and/or may not be applicable in specific vulnerable populations, such as children, pregnant women and immunocompromised individuals. In addition, there is a need to boost these vaccines to provide long-term protection.

Using a unique approach, Wistar scientists designed optimized synthetic DNA vaccine candidates targeting a virus surface protein called glycoprotein. They demonstrated efficacy of the novel vaccine candidates and durability of the immune responses in animal models. Importantly, results showed strong immune responses one year after the last dose, supporting the long-term immunogenicity of the vaccine – a particularly challenging area for Ebola vaccines.

“Synthetic non-viral based DNA technology allows for rapid vaccine development by delivery directly into the skin, resulting in consistent, potent and rapid immunity compared to traditional vaccine approaches,” said lead researcher David B. Weiner, Ph.D., executive vice president and director of Wistar’s Vaccine & Immunotherapy Center, and W.W. Smith Charitable Trust Professor in Cancer Research. “An anti-Ebola virus DNA vaccine like this may provide an important new tool for protection, and we are excited to see what future studies will unveil.”

The researchers optimized a shorter, dose-sparing, immunization regimen and simplified vaccine administration directly into the skin. This new approach induced rapid and protective immunity from virus challenges. The detected antibody levels were equal or higher to those reported for other vaccines currently being evaluated in the clinic, according to the study.

“The success of intradermal delivery of a low-dose regimen is very encouraging,” said Ami Patel, Ph.D., associate staff scientist in the Weiner Lab. “The ultimate goal of our work is to create effective and safe vaccines that are optimized for field use in at-risk areas.”

This work was supported in part by a grant from the Defense Advanced Research Projects Agency (DARPA) to Inovio Pharmaceuticals and a subcontract to The Wistar Institute/University of Pennsylvania. Additional funding was provided by Inovio Pharmaceuticals.

Co-authors of this study from The Wistar Institute include Emma L. Reuschel, Daniel H. Park, Amelia A. Keaton, and Kar Muthumani. Other co-authors include Kimberly A. Kraynyak, Dinah Amante, Megan C. Wise, Jewell Walters, Jean Boyer, Kate E. Broderick, Jian Yan, Amir S. Khan, and Niranjan Y. Sardesai from Inovio Pharmaceuticals, Inc.; Trina Racine, Jonathan Audet, Gary Wong, Marc-Antoine de La Vega, Shane Jones, Alexander Bello, Geoff Soule, Kaylie N. Tran, Shihua He, Kevin Tierney, and Xiangguo Qiu from National Microbiology Laboratory, Public Health Agency of Canada; Veronica L. Scott from William Carey University; Daniel O. Villarreal, Devon J. Shedlock, and Ross Plyler from University of Pennsylvania; Gary P. Kobinger from Université Laval, Canada.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer and infectious disease research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Mechanism of Resistance to Novel Targeted Therapy for Ovarian Cancer Identified

PHILADELPHIA — (Oct. 8, 2018) — Scientists at The Wistar Institute have unraveled a mechanism of resistance to EZH2 inhibitors in ovarian cancers with mutations in the ARID1A gene. The study, published in Nature Communications, suggests that inhibition of the cell death regulator BCL2 may be used to circumvent or prevent ovarian cancer treatment resistance.

Mutations in the ARID1A gene are frequent in clear cell ovarian cancer and represent a known genetic driver in this type of malignancy. Previous Wistar research has shown that ARID1A-mutant ovarian cancers are sensitive to inhibition of EZH2, an enzyme that promotes compaction of the DNA, suggesting the use of EZH2 inhibitors, which are in clinical trials for the treatment of lymphoma, as a potential targeted therapy for ovarian clear cell carcinoma.

“Acquired resistance to targeted cancer therapies represents a substantial challenge and limits their utility. There is a pressing need to elucidate the molecular mechanisms underlying resistance so that we can design new strategies to circumvent it,” said lead researcher Rugang Zhang, Ph.D., deputy director of The Wistar Institute Cancer Center, and professor and co-leader of the Gene Expression and Regulation Program. “We report the first mechanism of resistance to EZH2 inhibition in the context of ARID1A-mutant cancers and a potential approach to bypass the issue.”

Zhang and colleagues discovered a molecular switch that happens in the SWI/SNF protein complex, of which ARID1A is a component, in ovarian cancer cells resistant to EZH2 pharmacologic inhibition. Because the SWI/SNF complex remodels chromatin and modulates gene transcription, this switch causes a shift in expression of a subset of genes and activation of factors that favor tumor cell survival.
The two proteins involved in the switch, namely SMARCA2 and SMARCA4, perform similar functions in the complex but do not work simultaneously, each being specific of certain conditions, like workers in different shifts. The researchers found that, while SMARCA4 is typically active in ovarian cancer cells, SMARCA2 takes on its job in EZH2 inhibitor-resistant cells. Consequently, several genes that are normally repressed by SMARCA4 are expressed at higher level and drive cell survival by inhibiting programmed cell death. The most relevant of these genes is BCL2, the Zhang Lab found.

Consistent with this finding, a small molecule inhibitor of BCL2 killed ovarian cancer cells resistant to EZH2 inhibition in vitro and caused shrinking of tumors established by injection of resistant cells in mice. This resulted in significant improvement in the survival of the tumor-bearing mice.

“We discovered a potential therapeutic strategy to revert resistance to EZH2 inhibition in ovarian clear cell carcinoma,” said Shuai Wu, Ph.D., first author of the study and a postdoctoral researcher in the Zhang Lab. Our study also suggests that BCL2 inhibition may be used in combination with EZH2 inhibitors to prevent the onset of resistance.”

The study identified a new therapeutic use for BCL2 inhibitors, which are approved for treatment of lymphoma.

Co-authors include co-first author Nail Fatkhutdinov, Takeshi Fukumoto, Benjamin G. Bitler, Pyoung Hwa Park, Andrew V. Kossenkov, Marco Trizzino, Hsin-Yao Tang, Alessandro Gardini, and David W. Speicher from Wistar, and Lin Zhang from University of Pennsylvania.

This work was supported by National Institutes of Health/National Cancer Institute grants R01CA160331, R01CA163377, R01CA202919, R00CA194318, R01CA131582, R50CA221838, and R50CA211199, and U.S. Department of Defense OC140632P1 and OC150446. Core support for The Wistar Institute was provided by the Cancer Center Support Grant P30 CA010815.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer and infectious disease research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Engineered Synthetic DNA-Encoded Checkpoint Inhibitor Antibodies Advance the Field of Cancer Immunotherapy

PHILADELPHIA — (Oct. 4, 2018) — Wistar scientists and collaborators demonstrate for the first time that through engineering constructs, they can express DNA-encoded monoclonal antibodies (DMAbs) targeting CTLA-4, an important cancer checkpoint molecule that blocks anti-cancer immunity. Using a synthetic DNA platform, they built versions of the anti-CTLA-4 molecule and were able to then deliver the DMAbs and have them generate fully functional anti-CTLA4 molecules in vivo. This proof-of-principle study opens new avenues for the design and delivery of therapeutic checkpoint inhibitors and suggests potentially novel applications of this technology in cancer treatment. Study results were published online in Cancer Research.

Treatment of cancer with checkpoint inhibitors has recently revolutionized cancer immunotherapy. Since the discovery of immune checkpoints, which was recognized as a groundbreaking development for cancer therapy and awarded the Nobel Prize in physiology or medicine this week, checkpoint inhibitors are becoming standard of care for various malignancies, showing unprecedented impact for patients.

Despite the tremendous advancement in cancer therapy brought by monoclonal antibodies targeting checkpoint molecules, manufacturing complexity and repeated dosing may limit a broader use of this technology.

“Our work provides the first demonstration that we can use synthetic DNA technology to produce checkpoint inhibitor molecules in vivo to impact tumor growth in a preclinical setting,” said lead researcher David B. Weiner, Ph.D., executive vice president and director of the Vaccine & Immunotherapy Center at The Wistar Institute, and W.W. Smith Charitable Trust Professor in Cancer Research. “We showed that DMAbs may represent a valuable addition to the cancer immunotherapy toolbox: In our preclinical studies, DMAbs achieved antitumor activity comparable to that of traditional monoclonal antibodies, while being delivered through a simpler formulation that may provide a bridge to expand target populations for checkpoint inhibitors.”

The team developed a synthetic, sequence-optimized DNA plasmid designed to encode anti-mouse CTLA-4 monoclonal antibodies. When injected in the muscle of mice with the aid of an electroporation device to enhance uptake, the anti-CTLA-4 DMAbs resulted in significant and prolonged antibody expression with even a single dose. Importantly, this approach stimulated robust CD8+ T-cell infiltration, achieving tumor clearance across multiple mouse tumor models. The researchers then went on to develop human checkpoint inhibitor molecules and demonstrated their production in mice and their ability to stimulate human T-cell responses associated with antitumor activity.

“Our results open the door for further applications of DMAbs in cancer immunotherapy,” said Elizabeth K. Duperret, Ph.D., postdoctoral fellow in the Weiner Lab and first author on the study. “This platform is rapid and flexible, allowing for further optimization of antibody sequences, including development of novel therapeutic approaches for which conventional monoclonal antibodies are not suitable.”

This work was supported by National Institutes of Health grants F32 CA213795 and SPORE P50CA174523, and funding from The W.W. Smith Charitable Trust and the Basser Foundation. Core support for The Wistar Institute was provided by the Cancer Center Support Grant P30 CA010815. Additional funding was provided by Inovio Pharmaceuticals.

Co-authors of this study from The Wistar Institute include Aspen Trautz, Regina Stoltz, Ami Patel, Alfredo Perales-Puchalt, and Kar Muthumani. Other co-authors include Megan C. Wise, Trevor Smith, Kate Broderick, Emma Masteller, J. Joseph Kim, and Laurent Humeau from Inovio Pharmaceuticals.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer and infectious disease research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Age-related Changes in Skin Structure and Lymphatic System Permeability Promote Melanoma Metastasis

PHILADELPHIA — (Oct. 2, 2018) — Changes in the structure of the skin and the lymphatic system that occur with the natural aging process create permissive conditions for melanoma metastasis, according to two studies by The Wistar Institute. These changes are caused by loss of the HAPLN1 protein, which is part of the extracellular matrix, during aging. The studies were published back-to-back in Cancer Discovery.

Older age is a negative prognostic factor for melanoma, associated with higher frequency of development of incurable distant metastasis. Ashani Weeraratna, Ph.D., Ira Brind Professor, and professor and co-leader of the Immunology, Microenvironment and Metastasis Program at Wistar, and team have a long-standing focus on how aging affects the melanoma microenvironment, or the tumor’s ecosystem that includes immune cells, fibroblasts, blood and lymphatic vessels, and signaling molecules, to understand how age-related changes contribute to tumor progression and therapy resistance.

In these new studies, the Weeraratna Lab and collaborators characterized architectural changes that occur in the extracellular matrix (ECM) in the skin and surrounding the lymphatic vessels, which promote the spread of melanoma cells to distant sites by influencing tumor cell and immune cell trafficking. They also discovered a fundamental role played by the HAPLN1 protein in the molecular mechanisms underlying these changes.

In one study, the researchers focused on ECM produced by fibroblasts in the dermal layer of the skin and observed dramatic changes in expression levels of many ECM proteins, particularly HAPLN1.

“The same structural changes that happen in our skin with aging and cause the appearance of wrinkles are also responsible for the increased risk of metastasis in older melanoma patients,” said Weeraratna, senior author in both studies. “With advancing age, the network of fibers that supports our skin loses the ‘basket weave’ organization that is characteristic of younger skin, and becomes looser. In a tumor setting, we think of it as a barrier that helps contain the tumor cells by inhibiting their motility while favoring infiltration of immune cells into the tumor mass. In older patients, due to the loss of HAPLN1, this barrier becomes less efficient. These age-related changes also affect the way immune cells enter the tumor, which may have important implications for immunotherapy.”

By manipulating expression levels of HAPLN1 in three-dimensional human skin reconstruct models and in mouse skin models, Weeraratna and colleagues showed that loss of HAPLN1 creates a permissive microenvironment that favors escape of tumor cells while hampering trafficking of antitumor immune cells, particularly CD8+ T cells. Accordingly, injection of recombinant HAPLN1 around the tumor in melanoma mouse models reduced the size and metastatic capability of the tumor.

In the second study, Weeraratna and colleagues showed that age-associated loss of lymphatic vessel integrity allows melanoma cells to escape more easily the lymphatic system and the proximal lymph nodes to reach distant sites. Results showed that this process, too, is associated with loss of HAPLN1, which causes a similar scenario to that described in the skin: degradation of the extracellular matrix in which the lymphatic vessels are embedded and reduced anchorage of lymphatic endothelial cells to their structural support, which results in increased permeability.

“It is known that older individuals with melanoma have a lower incidence of lymphatic metastasis than younger patients yet higher rates of distant visceral metastasis,” said Weeraratna. “Our observation that older lymphatic vessels and lymph nodes are less efficient as a barrier to contain the metastatic cells may underlie that observation.”

In this setting, injection of recombinant HAPLN1 into the draining lymph nodes of aged melanoma-bearing mice increased the rates of lymphatic micrometastases while reducing the frequency of lung metastasis, suggesting that containment of the metastatic cells in the local lymphatic system may have therapeutic implications when coupled to surgical resection of the sentinel lymph nodes.

Taken together, the two studies support a novel, fundamental role for HAPLN1 as a prognostic factor for long-term survival and a potential new therapeutic avenue.

These research studies were supported by National Institutes of Health grants R01CA174746, R01CA207935, RO1CA131582, P01CA114046, P50CA174523, K99CA208012, F99CA212437, T32CA009171. Additional support was provided by a research grant from the Melanoma Research Foundation and a Melanoma Research Alliance/L’Oréal Paris-USA Women in Science Team Science Award. Weeraratna is supported by the Ira Brind Professorship and the Wistar Science Discovery Fund. Core support for The Wistar Institute was provided by the Cancer Center Support Grant CA010815.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.

Targeting Multiple Members of a Family of Tumor Antigens with a Synthetic DNA Vaccine Shows Promise for Cancer Immunotherapy

PHILADELPHIA — (Sept. 27, 2018) — Scientists at The Wistar Institute have implemented a novel structurally designed synthetic DNA vaccine to simultaneously target multiple members of a family of proteins that are specifically overexpressed in several types of cancer. This approach addressed a difficult issue in cancer immunotherapy, specifically how to simultaneously drive antitumor immune responses against multiple tumor antigens in a single, easily delivered formulation. The new strategy could simplify immunotherapy treatment and may prevent cancer escape from immune pressure as the immune system could attack the cancer at multiple susceptible target points. The new vaccine, targeting the human cancer-associated MAGE-A family of proteins, is effective and safe in a melanoma preclinical model, as described in a paper published online in Clinical Cancer Research.

Because their expression is restricted to tumor cells, proteins belonging to the MAGE-A family represent promising targets for immunotherapy. Yet, cancer vaccines targeting the original MAGE-A3 member, which has the highest expression in several solid tumors, have thus far failed to demonstrate efficacy in clinical trials.

In an attempt to solve this conundrum and advance the clinical applications of this promising immunotherapy, researchers at Wistar performed a thorough analysis of the expression levels of all the twelve proteins in the MAGE-A family in human cancers. They observed that many of the MAGE-A members, and not just MAGE-A3, are highly expressed on tumor cells in several cancer types, some of them being present simultaneously in the same patient. These findings suggest that previous vaccines
with limited focus on one target were likely not effective in driving strong T-cell immunity because of the natural immune dampening system known as immune tolerance.

“The combination of structural design and synthetic DNA technology offers ample flexibility and specificity in the development of a designer target immunogen,” said lead researcher David B. Weiner, Ph.D., executive vice president of The Wistar Institute, director of The Wistar Institute Vaccine & Immunotherapy Center, and W.W. Smith Charitable Trust Professor in Cancer Research. “We amalgamated structurally relevant primary sequences from multiple MAGE-A members, obtaining an optimized consensus DNA vaccine capable of targeting seven MAGE-A family members simultaneously. This vaccine is recognized by the host immune system much more robustly, resulting in improved immune performance.”

Tested in mice, the vaccine induced immune cross-reaction with multiple MAGE-A proteins and induced a robust CD8+ T cell-mediated immune response.

“CD8+ T cells are the predominant effectors in the response to immunotherapy; we can think of them as the Navy Seals of cancer immunology,” added Weiner.

Importantly, the vaccine significantly slowed tumor growth and prolonged survival in a mouse model of melanoma. The researchers observed reduced invasion in the skin, which was associated with accumulation of CD8+ T cells into the tumors, demonstrating the ability of the vaccine to drive antitumor immunity of importance for melanoma therapy.

“Our cross-reactive vaccine has a significant advantage in preventing tumor escape compared to previously designed MAGE-A3-specific vaccines,” said Elizabeth K. Duperret, Ph.D., postdoctoral fellow in the Weiner Lab and first author on the study. “Patients whose tumors express multiple members of this family of antigens represent an important group to study the benefits of this immunotherapy approach.”

This work was supported by National Institutes of Health grants SPORE P50CA174523 and F32 CA213795. Additional funding was provided by the W.W. Smith Charitable Trust, the Basser Foundation and a grant from Inovio Pharmaceuticals, Inc. Core support for The Wistar Institute was provided by the Cancer Center Support Grant P30 CA010815.

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The Wistar Institute is an international leader in biomedical research with special expertise in cancer and infectious disease research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the United States, Wistar has held the prestigious Cancer Center designation from the National Cancer Institute since 1972. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. wistar.org.