(7f) Multi-Scale Cellular and Protein Therapeutic Engineering for the Development of Novel Immunotherapies

John James Blazeck

Postdoctoral Fellow, University of Texas at Austin

Successful Proposals:

American Cancer Society Postdoctoral Fellowship

NIH F32 Ruth L. Kirschstein Postdoctoral NSRA

Research Interests:

Novel immunotherapies have the capacity to cure cancers by activating anti-tumor T cell-driven immune responses and have shown remarkable efficacy in the clinic. However, clinical response is limited to a subset of patients because these therapies employ a single intervention strategy that can be rendered ineffective by redundancies within the immune system. To account for this issue, my research will aim to develop tools that precisely control cellular and immunological outputs through distinct, easily combined mechanisms for rapid therapeutic development. More specifically, I have accrued expertise in the fields of cellular, metabolic, and immunological engineering to implement large-scale, multi-level engineering programs to develop tumor-inhibition-resistant and tumor-activated T cell therapies while concurrently engineering immunological proteins with novel functionalities.

My graduate work provided me with an in-depth tool kit for engineering cellular response at the transcriptional, pathway, and whole-cell level that will be invaluable towards designing and engineering cellular therapies. In particular, I developed techniques to precisely control transcriptional regulation in response to multiple environmental inputs, and I utilized widespread metabolic pathway perturbations to drastically alter cellular phenotype. For instance, I developed an array of novel synthetic promoters in multiple eukaryotic organisms [1-3], and I engineered a nonconventional yeast strain to accumulate lipids at the highest reported values when published [4]. To complete the foundation for my own research program, I cultivated an immunological protein engineering skill set during my postdoctoral work that I will couple with my cellular engineering tool kit. Specifically, I developed an anti-cancer enzyme therapeutic that degrades kynurenine, an immune-inhibitory small molecule produced by cancers. There are currently >20 clinical trials evaluating small molecule inhibition of kynurenine production, but efficacy is limited because kynurenine is synthesized in vivo by three enzymes. Thus, we hypothesized that enzyme-mediated clearance of tumoral kynurenine would be an ideal route to relieve immune suppression. To this end, I undertook a large-scale protein engineering effort that improved the activity of the Human Kynureninase enzyme by 600 fold - to pre-determined levels suitable for kynurenine clearance. Administration of engineered kynureninase enzymes to mouse models of cancer depleted kynurenine levels, allowed for a robust T cell-driven anti-cancer immune response, and drastically slowed tumor growth [manuscript under review]. In the future, I plan to combine my expertise engineering immunological proteins and controlling cellular phenotypes to enable translational research to effectively target and kill cancer cells.

Future Directions:

A major challenge of developing therapies for immunological applications is that the complexity of the immune system can prevent desired outcomes. To address this challenge, my research program will merge my expertise in cellular manipulation and immunological engineering to develop novel anti-cancer cellular and protein therapies by engineering control of the immune response through multiple mechanisms. Specifically, by engineering immune cells at the transcriptional, enzymatic, and pathway levels, I will create robust autologous T cell therapies that are impervious to tumor-mediated immune suppression and are activated by the tumor microenvironment. To this end, I will design novel transcriptional control units activated by tumors (e.g. by lactic acid, kynurenine, hypoxia etc.), and I will rewire cellular pathways to resist the nutrient-limited tumor metabolic environment. Similarly, T cell inhibitory receptors can be co-opted to invert tumor-mediated signaling towards stimulatory outcomes, and engineered immune cells could even be designed to act as cellular sinks for tumor-produced suppressive small molecules or for metabolites required for tumor anabolic growth.

Additionally, I will engineer the basic architecture of immune proteins, such as T cell receptors and antibodies, to introduce novel functionalities that will enable new applications as single-entity anti-cancer therapeutics or for efficient combinatorial targeting of cancer cell mutations. For instance, by engineering physically separable antigen-specific and constant (cell-bound) regions of the T cell receptor (TCR), it will be possible to combine a clonal T cell population expressing the cell-bound TCR-region with multiple antigen specific TCR fragments to generate a well-defined, multi-target, ‘drop-in’ autologous anti-cancer T cell therapy. I will further engineer antibody backbones by introducing novel functional sites to create new classes of Bi or Tri-specific binding proteins designed to be ideal partners for cellular therapy treatments. While my initial work will focus on establishing independent control of distinct cellular levels within T cells, my long term vision is to synergize these control mechanisms within single cellular therapies. The capacity to rewrite the T cell regulatory and metabolic network will allow me to exploit the interconnected nature of the immune response and direct it towards beneficial outcomes against cancer, and engineered T cell therapies can be further combined with protein therapeutics to activate immune cells to fight cancer. The tools developed to achieve these goals will also be generalizable to many other immune-altered states (e.g. autoimmune disorders) and other biological networks with similar complexity. My foundation in synthetic biology and pathway engineering leaves me well-situated to undertake this research program in immunological engineering.

Teaching Interests:

I was fortunate to have an excellent experience teaching undergraduate course material before beginning my graduate research program. Specifically, as an MCAT organic chemistry and biology instructor for the Princeton Review, I designed and taught over forty hours of fully participatory lecture material. I kept my students interested and actively engaged in my lectures by consistently guiding them towards conclusions and relying on them to supply key pieces of information as I taught, i.e., by using the Socratic Method. I also had my students work together on short sample questions to let them apply their new knowledge set and gain confidence in their ability to solve MCAT-styled problems. In short, I learned how to effectively create lecture material that both led and collaborated with my students in their intellectual pursuits.

During graduate school, I happily served as a teaching assistant for two chemical engineering courses, Thermodynamics and Chemical Reactor Analysis and Design. I was able to see first-hand the benefit that a passionate instructor could have towards motivating and engaging young, bright minds. I learned techniques to improve course quality and provide additional support as students tackled difficult subject matter. For instance, I will begin my lectures with a brief overview of the previous and current classes’ subject matter, employ peer-teaching opportunities and small-group efforts, and I will write my lecture notes by hand as class proceeds to pace delivery of material.

Core Courses: I am prepared to teach Kinetics/Reactor Design, Transport, and Mass/Energy Balances.

Advanced Courses: I am prepared to develop a Bioreactor and Cellular Engineering course that will be dedicated to understanding cellular conversion and metabolism at all scales, i.e. enzymatic, single-cell, and bioreactor levels.

Mentorship:

I have greatly enjoyed mentoring undergraduate and beginning graduate students as they start their research careers. To date, I have mentored more than ten undergraduate researchers, many of whom have gone onto graduate school, and three beginning graduate students. Six of my undergraduate mentees secured $1,000 Undergraduate Research Fellowships and three won 1^st place in department or university poster competitions.

I look forward to mentoring graduate students, postdoctoral researchers, and undergraduate students in my own lab. As an advisor, I will strive to (1) encourage the development of my mentee’s critical thinking and project development skills, (2) advance their understanding of their project and their field, and (3) cultivate their experimental expertise and communication skills. I will encourage my mentees’ development by granting them greater domain over their projects as they progress, and I will host weekly rapid-fire round-robin journal clubs to establish the importance of keeping up with scientific literature. All incoming graduate students will ‘intern’ with a senior lab member for 3-6 months. In this manner, senior lab members will develop valuable teaching skills as beginning researchers learn experimental techniques. To aid in the development of communication skills, I will strongly encourage all students and postdocs to apply for fellowships, involve mentees in the grant writing process, and expect mentees to author scientific manuscripts and to present at group meetings and scientific conferences. Furthermore, all mentees will author a review article relevant to their project in their first 1-2 years to hasted development of their writing skills and understanding of their field. I will passionately urge all mentees to mentor undergraduate researchers, as it has been of such personal and scientific benefit to me. Finally, I will meet weekly with all graduate students and postdocs and help them achieve goals through bi-annual progress reviews that include individualized career and lab-specific development plans.

Referenced Publications:

[1] Blazeck, J. and Alper, H. (2013). Biotechnology Journal. 8(1): 46-58.

Times Cited = 103.

[2] Blazeck, J., Garg, R., et al. (2012). Biotechnology and Bioengineering. 109(11) 2884-2995.

Times Cited = 122.

[3] Blazeck, J., Liu, L. et al. (2011). Applied and Environmental Microbiology. 77(22): 7905-7914.

Times Cited = 105.

[4] Blazeck, J., Hill, A., et al. (2014). Nature Communications. 5(3131) doi:10.1038/ncomms4131.

Times Cited = 138.