(6q) Engineering Protein Specificity: New Tools and Biologics to Remediate Human Diseases

Recombinant protein drugs and protein-based diagnostics have opened up new ways by which we understand, diagnose, and treat diseases. Antibodies are now prescribed for the treatment of cardiovascular and infectious diseases, cancers, autoimmune and inflammation. Enzyme replacement therapies (ERTs) have well-established clinical examples and enzymatic amino acid depletion strategies hold great promise in fighting several types of cancers. Nevertheless, in order to expand the pharmacopeia of protein drugs and overcome our toughest health challenges, existing protein and cell therapies need to be further improved or introduced and novel protein-based therapeutic approaches need to be developed.

Owing to recent advances in protein engineering, cellular engineering and synthetic biology, protease therapeutics have started to gain more attention for the treatment of diseases associated with protein dysregulation and misfolding. Unlike antibodies and small molecule drugs which bind their targets stoichiometrically, protease therapeutics offer a distinct mechanistic and therapeutic approach because they can catalytically degrade their targets. Another major advancement in protein therapeutics has been the emergence of targeted therapies enabled by protein-drug conjugates (PDCs) whereby a payload is conjugated to a targeted protein scaffold via a site-selective chemical or enzymatic post-translational modification (PTM). As of now, these two technologies are not yet fully established. Potential therapeutic proteases often fail in the clinic due to toxicity stemming from their off-target activities or inactivation by serum inhibitors. PDC scaffolds are currently mostly limited to antibodies and cell surface engineering techniques to produce cell-based therapies and diagnostics are only starting to be developed. This is in part because many enzymes that enable protein-drug conjugations (e.g. transpeptidases) suffer from low activity and a restricted substrate specificity which limits their general applicability.

Work in my laboratory aims to tackle the challenge of engineering proteases and protein-modifying enzymes to bring these two technologies to the foreground of biotechnology and biomedicine. I aim to redesign the specificity of proteases to (1) degrade proteins involved in neurodegenerative and autoimmune diseases and (2) to develop ERTs that treat rare monogenic disorders and thrombotic diseases. I will further evolve protein-modifying enzymes for site-specific protein and cell surface manipulations. As I seek these two broad objectives, I aim to develop novel high-throughput technologies that facilitate protein engineering. My work in the Huimin Zhao and Brent Iverson labs have provided with the necessary foundation to successfully pursue these goals. In the Huimin Zhao lab, I gathered expertise in directed protein evolution and synthetic biology, leveraged enzyme specificity to enable creative chemo-enzymatic processes for asymmetric synthesis and gained expertise in cellular engineering. In the Brent Iverson lab, I further enriched my experience in protein engineering and cellular engineering by building in vitro and in vivo tools that facilitate protease engineering, in particular through the development of high-throughput compartmentalization platforms. With this accumulated experience, my independent career will be dedicated to engineering enzymes for therapeutic and diagnostic applications, as well as fundamental biomedical research.

In the future of biomedicine, I envision targeted protein and cell-based therapies that are highly functionalized such as stimulus-responsive prodrugs with biochemical logic capable of both effector and biosensor functions. I also envision completely novel engineered therapeutic protein scaffolds entering the drug market, beyond antibodies.

Research experience

PhD research

My graduate work centered on the development of chemo-enzymatic one-pot reactions that combine chemical and biocatalysis in ways that capitalized on their underutilized cooperativity, to convert readily available starting materials into enantiopure products. Specifically, I developed catalytic routes to convert mixtures of alkenes into a single chiral product, a fundamental building block in synthetic chemistry. In a set of investigations, I combined for the first time an olefin cross- metathesis reaction that establishes an equilibrium of alkenes of different lengths and functional groups with a P450-catalyzed epoxidation that is selective for the cross-metathesis product. By engineering the enzyme and reaction conditions, up to 90% yield of the final epoxide product could be obtained, which is twice as high as the yields of two sequential reactions. The off-equilibrium chemo-enzymatic reactions I developed were an important addition to the bio-catalysis field, as it showed that these combinations of chemical and biocatalysts could not only be made possible, but also be made better than the sum of their parts. Throughout my PhD work, I applied a similar strategy to synthesize chiral 2-succinate derivatives in high yields and enantioselectivity. Lastly, using an immobilized glucose isomerase and a supported acid catalyst, I prepared hyroxymethylfurfural (HMF) selectively starting from glucose in a two-step cascade reaction. Importantly, at the center of my graduate work were my ability to work in multidisciplinary teams, as well as to introduce novel and creative approaches to tackle synthetic challenges. In addition, I gained a synthetic biology toolkit by engineering translational control of gene expression in mammalian cells. Through this set of investigations, I developed in-depth analytical, experimental, and problem-solving abilities, garnered expertise in a multitude of disciplines including chemical biology, bioengineering, enzyme engineering and immobilization, and firmly enriched my synthetic biology background.

Post-doctoral research

To complete the foundation of my research program, I wanted to couple my experience in reaction engineering and synthetic biology with more in-depth experience in protein evolution and high-throughput screening. I joined the Iverson Lab to develop state-of-the-art high-throughput screening platforms for protease engineering, in an effort to produce recombinant human protease drugs as treatments for autoimmune diseases.

I developed and optimized several tools for protease engineering. First, I improved an in vivo yeast endoplasmic reticulum sequestration system (YESS), an approach previously developed in our lab that enables both protease engineering and substrate specificity profiling using fluorescence-activated cell sorting (FACS). In particular, I restructured this platform to allow a fast, modular, and efficient assembly of genetic elements that control transcription, translation, spatial sequestration and reporter tags. This seamless design-build-test cycle greatly speeds up protease engineering and screening efforts. Second, I used YESS to narrow the substrate specificity of human neutrophil elastase (hne) towards the selective cleavage of soluble tumor necrosis factor-alpha, a target in inflammatory bowel disease treatment. Third, using Next-Generation sequencing (NGS), I have analyzed the degradome of proteases and their variants, including tissue kallikreins which are biomarkers in cancers and inflammation. This high-throughput substrate specificity profiling allows a truly comprehensive analysis of sequence space that is not provided by positional scanning and proteomics-based methods. This in-depth analysis will help gain further insight into the roles of proteases in diseases and aid in the design of more specific peptide inhibitors, fluorogenic substrates, and protease-activatable prodrugs. Lastly, I independently developed an in vitro compartmentalization directed evolution platform for the engineering protease substrate specificity. This tool offers the advantages of 1) bypassing protease-induced cytotoxicity, 2) enabling engineering on full proteins in the context of their 3D structures rather than peptide substrates and 3) offering a level of control over screening conditions not achievable in vivo. Using this platform, I am engineering human matrix metalloproteases (MMPs) to cleave IgG hinge regions as a treatment for IgG-mediated pathogenic conditions (rheumatoid arthritis, systemic lupus erythematosus, and multiple myeloma). Recent evidence has showed that ablating IgG levels through cleavage of IgG hinges by the bacterial enzyme IdeS (immunoglobulins degrading enzyme from Streptococcus pyogenes) alleviates autoimmune disease symptoms in a number of animal models of antibody-mediated disorders and reduces transplant rejection in humans. Although effective, we hypothesize that an engineered human protease would be preferred for repeated injections since IdeS inevitably triggers an immune reaction that diminishes or abrogates its efficacy.

My post-doctoral work allowed me to gather a new set of skills, namely in the in-depth analysis of DNA libraries using NGS, in creating large DNA libraries and manipulating the genome of S. cerevisiae. In particular, I learned how to create emulsion-based in vitro engineering platform, which will form the basis of my early independent career. In summary, my accumulated experience during both by PhD and post-doctoral work puts me in a unique position to establish a successful research program 1) building high-throughput directed evolution platforms to 2) engineer new and improved enzymes for therapeutic applications.

Teaching Interests:

Teaching experience. During my graduate studies, I was a teaching assistant (TA) for three chemical engineering classes. In Biomolecular Engineering (ChBE 473), I designed homework and quiz questions, formulated exam questions and developed grading rubrics. In particular, I was tasked to give three guest lectures, which provided me the opportunity to teach a class of about 50 students. I learned how to effectively prepare class material. I implemented many active learning methods during my guest lectures. In particular, I found that asking students to explain to a classmate how they understood the material was a valuable teaching tool. In Momentum and Heat Transfer (ChBE 421), I led weekly discussion sessions to a class of about 30 students. In these sessions, students and I went over homework and coursework. These discussion sessions became a more intimate setting that allowed students to obtain personalized help, which they wouldn’t have gotten in a 300-student class.

As a postdoctoral fellow, I attended a class on evidence-based teaching in order to sharpen my teaching skills, namely through the introduction of various teaching and assessment strategies. Through this class, I learned how to effectively plan lessons using backwards design, delineate learning objectives and design assessments that specifically address these objectives. I also learned a variety of learning strategies. Most importantly, evidence-based teaching guided and cemented my teaching philosophy.

Teaching philosophy. Throughout my undergraduate, graduate and postdoctoral studies, I have had the opportunity to observe, analyze and practice many different teaching styles. These formative years have provided me with a strong foundation to become an effective teacher at many educational levels. My core teaching philosophy is to build and foster an environment that promotes: (1) accountability and clear expectations from both teacher and students, (2) collaborative learning, (3) connection to the real world, (4) organized study, (5) an inclusive learning environment and lastly, (6) enthusiasm.

Teaching interests and goals. I am prepared to teach all core chemical engineering classes, as well as introductory classes such as biochemistry, molecular biology, and higher-level graduate-level applied courses in protein and cellular engineering and biocatalysis. Throughout my career, I would also like to develop postgraduate applied courses centered on the needs of the department and students while taking inspiration from my research interests and background. In undergraduate level classes, students will be expected to 1) demonstrate understanding of major concepts related to First Principles, 2) apply their knowledge to formulate solutions to real-life problems and 3) develop a strong foundation to tackle more advanced classes. In graduate-level classes, my goals are to instruct students to 1) grasp more advanced concepts, 2) competently evaluate the current literature of their respective fields, 3) build a set of tools which will help them become experts in their fields and 4) apply knowledge acquired in my classes to their research. To achieve these goals, I will apply several effective teaching methods.

First and foremost, the learning objectives and expectations will be clearly delineated at the beginning of each lecture or set of lectures. Second, as an effective teacher, I will structure my classes in a way that intersperses short lecturing times with student activities or short assessment questions. Activities can include discussion questions (open-ended, multiple-choice) that can be revisited at different points during a lecture to not only introduce concepts in a layered and organized manner, but also to instantly evaluate understanding. Third, I plan to pursue many additional formats to promote student engagement. To this end, I will illustrate text-book problems and provide historical or current problems related to the lecture content. In addition, I plan to engage students using problem-based learning (PBL). In the classroom, PBL will encourage peer-teaching through the formation of study groups and other active learning tools, including think-pair share activities, multiple choice questions, jigsaw discussions, literature reviews or semester-long projects. In particular, for introductory classes, I will implement two-stage exams where appropriate, whereby students first complete the exam individually, and then working in small groups, answer the exam questions again. During the group exam, students will receive immediate, targeted feedback on their solutions from their fellow classmates and learn about alternative solutions. This makes the exam itself a valuable learning experience. I will continue to encourage student learning outside the classroom, by posting videos on the class website that expands on concepts covered in class and set up a message board where I can have discussions with students on more elaborate problems and concepts. This will help me disseminate class material in different formats and provide students with many options to remain engaged. Lastly, I will ask students to evaluate my teaching, not at the end of the semester, but once during the semester. That way, I can identify knowledge gaps and misconceptions and quickly adjust my teaching methods.

Creating an inclusive learning environment. For efficient learning, creating an inclusive environment is very important. As part of my overall teaching philosophy, I guarantee that students in my classes will feel free to express themselves and ask questions, as I will treat them equally and respectfully. Students, especially freshmen and sophomores, may come in to challenging classes with many misconceptions, knowledge gaps, as well as different learning styles. Encouraging students, especially minority students who may otherwise hesitate before actively participating, is one of my primary goals in that respect.

Teaching through research mentorship. I have been fortunate to mentor six undergraduate students, as well as several graduate students. It has always pleased me to hear from them of the great things they have gone to accomplish and the awards they have won. Through these experiences and observing my own mentors, I learned that there is no one-size-fits-all mentoring style and each individual may require a different mentoring approach. During graduate studies, I learned various management skills that I will build on.

First, encouraging students in my lab to read literature and understand their field and the larger implications of their work is critical to their success. To maintain the awareness of my trainees beyond the scope of their project, they will be asked to lead literature review sessions and asked to write a review paper early in their graduate career. I will also encourage my trainees to think independently, to feel free to advance their own ideas, establish collaborations with their colleagues as well as colleagues from other labs. Second, to further improve communication and writing skills, graduate students and post-docs will be encouraged to apply for fellowships, participate in grant writing, and attend conferences yearly. Third, while my door will always be open to my mentees, I will hold a formal weekly meeting to discuss their progress, and I will ask graduate students and postdocs to turn in a yearly performance review that will include a self-assessment, an advisor evaluation and a refinement of their career goals. Such a process I believe is critical to their professional development.