(6n) Protein Engineering for Cell- and Ligand-Based Immunotherapy

Research Interests: Immunotherapy is a treatment method that harnesses a patient’s own immune system to fight disease. One such technology, chimeric antigen receptor (CAR) engineered T cells, has reinvigorated cancer immunotherapy with outstanding clinical success treating subsets of leukemia and lymphoma. A CAR is a synthetic molecule that directs a T cell to bind to a cancer-associated marker that it wouldn’t ordinarily recognize, causing the T cell to respond in a disease-targeted manner. Development of these receptors is often empirical with minimal optimization; previously established domains are fused to mediate cancer recognition and T cell response. Currently, the biggest challenge in this field is lack of potency when applying these therapies to diseases outside of hematologic malignancy. This stems from two disparate areas: 1) lack of optimization of new CARs can yield undesirable characteristics such as low expression, tonic signaling, or the inability to control tumors in vivo, and 2) the microenvironment of many tumors is suppressive to T cells and other immune cells, leading to a lack of therapeutic impact. My laboratory will address these challenges 1) by building a fundamental understanding of the impacts of domain-level alteration on CAR function via protein engineering directed by natural homology, structure-guided design, and macromolecular modeling and 2) by studying novel combination therapies – via hypothesis-driven experiments guided by protein and immune cell profiling in disease models and patient samples – for their ability to condition the tumor microenvironment to CAR T cell response.

The lab’s long-term goal is to apply our experience in protein engineering, immunotherapy development, and translational research to design new CARs that are optimized for therapeutic impact and pair these with engineered “companion” molecules to increase their impact on difficult-to-treat cancers. To meet these ends, we will employ protein engineering and high-throughput screening techniques to understand the impact of structure-function relationships in individual domains of CARs on therapeutic potential, leading to development of techniques for efficient optimization of these domains. Our experience in protein design and expression will be used to engineer and study companion molecules and their effects on the tumor microenvironment. Collaboration will be sought for clinical translation in the hope of turning our fundamental scientific advances into technologies with a positive impact on patients and their families.

Research in my group builds from the fundamentals of biology, chemistry, engineering, and immunology. This broad knowledge will allow us to adapt our approaches to diseases other than cancer, with potential applications in autoimmune disease and infectious disease.

Our initial investigations will focus on three primary aims:

1. Engineering CARs for unaddressed cancer biomarkers:

Although numerous CARs have been developed preclinically, substantial limitations remain and many diseases remain unaddressed. With our expertise in protein selection, we are uniquely positioned to develop new CARs from de novo approaches. We will engineer candidate antigen recognition domains with systematic variation in multiple design elements to study the interplay of affinity, antigen density, and bound epitope geometry on downstream signaling and anti-tumor effect in a variety of diseases. These studies will yield new therapeutic options, while also providing a blueprint for the field to break its reliance on previously discovered antigen recognition sequences.

2. Understanding and optimizing domains of CARs for potent, safe responses:

Previous CAR development has been largely empirical, relying on fusing a binding domain to known natural domains for membrane anchoring, signaling, and co-stimulation. We aim to understand how engineering the transmembrane, co-stimulatory, and stimulatory domains can lead to changes in CAR expression, potency and selectivity of response, and downstream signaling. Although initial studies will focus on engineering natural domains, future work will involve developing panels of synthetic domains capable of tuning CAR response for the unique challenges of individual diseases.

3. Engineering companion molecules for modification of the tumor microenvironment:

Suppression of CAR T cells and other immune populations in the tumor microenvironment poses a major challenge to immunotherapy. We aim to understand - via proteomic and immune cell profiling techniques - the combinations of suppressive molecules present in individual diseases and build proteins capable of antagonizing these axes. We will study these molecules alone and in combination with CAR T cells in appropriate mouse models to elicit effects of these treatments on immune cell recruitment and anti-tumor response.

Successful Proposals: Postdoctoral Fellowship Award, Tobacco-Related Disease Research Program (2018); Parker Institute for Cancer Immunotherapy Grant (Co-PI, PI: Xiuli Wang) (2017); NIGMS T32 Predoctoral Fellowship (2012)

Postdoctoral Project: Yeast Surface Display Techniques Enhance Development of Chimeric Antigen Receptors, Beckman Research Institute of the City of Hope (Advisor: Dr. Stephen J. Forman)

PhD Dissertation: Cellular Selections Aid Translational Binding in Ligand Discovery, Department of Chemical Engineering and Materials Science, University of Minnesota – Twin Cities (Advisor: Associate Professor Benjamin J. Hackel)

Research Experience: My research focused on the optimal development of proteins to meet diagnostic and therapeutic needs. These projects were driven by my education in chemical engineering and my training in protein engineering, molecular biology, and high-throughput screening. Much of this work was collaborative, allowing me the privilege of working with engineers, biologists, chemists, and clinicians to meet our goals. I believe these experiences position me well to work both as an independent investigator and as a member of a thriving research community.

My graduate work focused on a prevalent but rarely published problem in engineering protein-protein interactions: standard methods for in vitro development of binding ligands using recombinant target proteins often yield interactions that are not recapitulated with cell-expressed target proteins. To meet this challenge, I optimized and developed yeast surface display methods for selecting binding ligands directly against mammalian cell monolayers, reducing the prevalence of reagent binders. In addition to methodological contributions, I engineered ligands that are currently under study for molecular imaging and cancer therapeutics.

My postdoctoral research leverages my knowledge of protein engineering for the development and optimization of CARs with translational focus. Many CARs in development face challenges related to potential toxicities. One such toxicity is the formation of an immune response against the binding domain of the CAR, a molecule which is often of non-human origin. A second toxicity is off-tumor killing of healthy tissue that expresses molecules considered cancer biomarkers. To meet these challenges, I am currently demonstrating high-throughput methods for reduction in immunogenicity and optimization of CAR selectivity to spare healthy tissue while mediating potent anti-tumor response. In addition to developing candidates with translational intention, the parameters that I am studying will help develop a mechanistic understanding of CAR function that the field currently lacks.

Teaching Experience: During my PhD, I served as a teaching assistant for a core undergraduate Chemical Engineering course (Introduction to Biomolecular Engineering) and a core graduate course (Physical and Chemical Thermodynamics). My duties included grading assignments and exams, and holding weekly office hours to supplement lecture. As a senior graduate student, I became a recitation instructor for Introduction to Biomolecular Engineering. I led a weekly discussion section in supplementary lectures and problem sets. I also wrote and contributed to weekly problem sets and gave lectures to the full class in the instructor’s absence. In addition to my formal teaching experiences, I participated in education outreach opportunities including giving lectures on clinical diagnostics to Minnesota’s Profoundly Gifted Youth and to Honors Chemistry classes at a local high school, and leading laboratory tours for high school students. As a graduate student, I also had the honor of mentoring six excellent undergraduate students to success in the laboratory. As a postdoctoral fellow, I continue to mentor young scientists including one research associate and one summer undergraduate student.

Teaching Interests: My teaching interests encompass core areas of chemical engineering including kinetics, transport, and separations. I am also very interested in developing and teaching courses in biomolecular engineering and bioprocesses in manufacturing. In addition to teaching in the classroom, I will welcome undergraduate researchers into my laboratory with mentorship on independent research projects. Through these projects, I will aim to teach them the fundamentals of laboratory research while cultivating their curiosity and problem-solving skills.

Selected Publications:

1. Stern LA, Schrack IA, Johnson SM, Deshpande A, Bennett NR, Harasymiw LA, Gardner MK, Hackel BJ. “Geometry and Expression Enhance Enrichment of Functional Yeast-Displayed Ligands Via Cell Panning.” Biotechnology and Bioengineering, 2016, 113(11):2328-2341 (Highlighted as a “Spotlight”).
2. Stern LA, Csizmar CM, Woldring DR, Wagner CR, Hackel BJ. “Titratable Avidity Reduction Enhances Affinity Discrimination in Mammalian Cellular Selection of Yeast-Displayed Ligands.” ACS Combinatorial Science, 2017, 19(5):315-323.
3. Stern LA, Case BA, Hackel BJ. “Alternative Non-Antibody Protein Scaffolds for Molecular Imaging of Cancer.” Current Opinion in Chemical Engineering, 2013, 2:425-432.
4. Csizmar CM, Petersburg J, Hendricks A, Stern LA, Hackel BJ, Wagner CR. “Engineering Reversible Cell-Cell Interactions with Lipid Anchored Prosthetic Receptors.” Bioconjugate Chemistry, 2018, 29(4):1291-1301.
5. Woldring DR, Holec PV, Stern LA, Du Y, Hackel BJ. “Sitewise Diversity to Optimize Fitness of a Three-Helix Bundle Combinatorial Library.” Biochemistry, 2017, 56(11):1656-1671.
6. Case BA, Kruziki MA, Stern LA, Hackel BJ. “Evaluation of Affibody Charge Modification Identified by Synthetic Consensus Design in Molecular PET Imaging of Epidermal Growth Factor Receptor.” Molecular Systems Design & Engineering, 2018, 3:171-182.