- 2011 - Melville A. Hare Award for Distinction in Research
John Puskas was born in Rochester, New York, and graduated from John Marshall High School. He attended Monroe Community College from where he received an Associate of Science degree in Liberal Arts. He matriculated at the State University of New York College at Brockport where he graduated Cum Laude and received a Bachelor of Art degree in English with an emphasis in Creative writing. He later returned to school and earned an Associate of Applied Science degree in Biotechnology from Monroe community college. He subsequently worked in the Clinical Products division of Eastman Kodak under the supervision of Dr. John Findlay and Dr. David Sharkey on an automated PCR-based diagnostic assay for infectious diseases. He obtained a Bachelor of Science degree from the Rochester Institute of Technology in May of 1997. He held the position of Technical Associate at the University of Rochester from 1997 to 2003. He subsequently entered the graduate program in the Department of Microbiology and Immunology at the University of Rochester, and performed his doctoral research in the laboratory of Dr. John Frelinger, receiving his PhD degree in 2011. He currently is working with Dr. James J. Mulé, Ph.D. at the H. Lee Moffitt Cancer Center & Research Institute in Tampa Florida.
Immune responses to tumors have been shown not only in experimental animal models but also in cancer patients. Unfortunately, such responses are often weak and ineffective and new strategies to improve responses are needed. One approach has been the use of high dose cytokine therapy delivered systemically, which has in some cases brought about complete remission of tumors. Unfortunately, toxic side effects of cytokine immunotherapy often limited its use. We set out to develop a general approach in which cytokines could be functionally attenuated until activated locally at the tumor site, alleviating the negative side effects of systemic delivery. Here we report the development and initial characterization of a novel class of fusion proteins in which human or mouse interleukin-2 (IL-2), a potent growth factor for immune cells including T and NK cells, is joined via a protease site to an IL-2 inhibitory component. The rationale is that upon cleavage by a protease that is preferentially expressed at the tumor site the cytokine is free to dissociate from the inhibitory component and becomes biologically available to immune cells. We designed and characterized several fusion proteins employing distinct approaches for inhibiting cytokine function. The initial strategy investigated an approach. The initial strategy investigated an approach based on a steric hindrance. To test this approach we used the cytokine IL-2, a cleavage sequence specific for the protease Prostate Specific Antigen (PSA), and a chemokine (Mip-1a). The putative inhibitor in this model, Mip1a, relied on proximity as the mechanism of attenuation. The fusion protein could be expressed using the baculovirus system and could be cleaved by PSA. However, functional studies indicated that the cytokine was active prior to cleavage and that cleavage did not result in an increase in biologic activity. Therefore, alternative models of cytokine inhibition based on specific binding of inhibitory moieties were developed utilizing either a cytokine receptor or an antibody fragment (scFv) specific for the cytokine. Examination of these fusion proteins showed that they could be cleaved and this cleavage resulted in an increase in the amount of biologically available cytokine. We further showed the flexibility of this approach by modifying the cleavage sequence from PSA to sequences specific for Matrix Metalloproteinase2 (MMP2) and/or Matrix Metalloproteinase9 (MMP9). The functional activity of the cytokine in a fusion protein containing an MMP cleavage sequence was markedly inhibited (over 60 fold) compared to free IL-2 and, importantly, there was an increase in the amount of biologically available IL-2 after cleavage. Further, treatment with an IL-2 fusion protein with an MMP cleavage sequence reduced in vivo tumor growth in a mouse tumor model. This thesis work demonstrates the first proof of principle of novel attenuated cytokine fusion proteins activated by tumor-expressed proteases. Importantly, this is a general strategy, which could be applied to other proteases and immune modulators allowing site-specific activation while reducing unwanted side effects. These proteins represent a new class of compounds for immunotherapy for tumors.
- Hinkle PM, Puskas JA. (2004) Detection of G protein-coupled receptors by
immunofluorescence microscopy. Methods in Molecular Biology; 237:127-34.
- Mitra S,, Cassar SE, Niles DJ, Puskas JA, Frelinger JG, Foster TH. (2006) Photodynamic therapy mediates the oxygen-independent activation of hypoxia inducible factor 1alpha. Molecular Cancer Therapuetics. 5:3268-74.
- Puskas, J., Skrombolas, D., Sedlacek, A. Lord, E.M., Sullivan, M., Frelinger, J.G. (2011) Development of an attenuated IL-2 fusion protein that can be activated by tumor expressed proteases. Immunology 133: 206-220.