(12ag) Structure-Guided Protein Engineering for Targeted Immunotherapy

Jamie Berta Spangler

Postdoctoral Fellow, Stanford University School of Medicine

Research Interests:

The repertoire of naturally occurring proteins is finite and many molecules induce multiple confounding effects, limiting their efficacy as therapeutics. Recently, there has been a growing interest in manipulating existing proteins or engineering new proteins to address the deficiencies of molecules found in nature. Researchers have traditionally taken an unbiased approach to protein engineering, but as our knowledge of protein structure-function relationships advances, we have the exciting opportunity to apply molecular principles to guide engineering. Leveraging cutting-edge tools and unique expertise from structural biology and molecular engineering, I aim to implement a structure-based engineering approach to elucidate the determinants of protein function in order to inform the development of novel protein therapeutics. I am particularly interested in engineering immunological proteins such as antibodies and cytokines to bias immune activity for clinically relevant applications including cancer, infectious diseases, and autoimmune disorders. Harnessing molecular insights and new engineering platforms I have developed in my postdoctoral research, I am poised to pursue a wealth of translational directions that will expand the scope of protein activity to address biomedical challenges.

Successful Proposals:

Leukemia & Lymphoma Society Career Development Postdoctoral Fellowship, Repligen Koch Institute for Integrative Cancer Research Graduate Fellowship, National Defense Science and Engineering Graduate Fellowship

Postdoctoral Project:

â??Engineering the interleukin-2 cytokine system to therapeutically modulate immune homeostasis.â?

Supervised by Prof. K. Christopher Garcia, Departments of Molecular & Cellular Physiology and Structural Biology, Stanford University School of Medicine

Ph.D. Dissertation:

â??Characterization and informed design of down-regulating epidermal growth factor receptor antibodies.â?

Supervised by Prof. K. Dane Wittrup, Department of Biological Engineering, Massachusetts Institute of Technology

Research Experience:

I have cultivated a longstanding interest in understanding and manipulating the molecular determinants of protein function in health and disease and have consequently pursued training in a combination of fundamental biophysical and engineering approaches to biochemistry. As an undergraduate in Biomedical Engineering at Johns Hopkins University, I conducted research in Kalina Hristovaâ??s lab, elucidating the mechanistic basis for pathogenicity of mutations in fibroblast growth factor receptor-3 (FGFR3). This work equipped me with essential biochemical and biophysical tools and piqued my interest in studying protein interactions relevant to disease. As a Ph.D. student in Biological Engineering at Massachusetts Institute of Technology, I worked under the supervision of Prof. K. Dane Wittrup. In my thesis research, I used epidermal growth factor receptor (EGFR) as a model antigen to establish antibody-mediated receptor down-regulation as an effective therapeutic strategy and designed multispecific antibody fusions that utilized this strategy to inhibit tumor growth in mice. Through this work I developed a vast knowledge of cell biology and biochemistry and gained experience with antibody engineering and in vivo models of cancer. As a postdoctoral researcher in Professor K. Christopher Garciaâ??s group at Stanford, I have adopted a structure-based approach to biochemistry, integrating new techniques in structural biology, cytokine engineering, and molecular and cellular immunology. My work has focused on interleukin-2 (IL- 2), a multifunctional cytokine that regulates immune homeostasis and that has been used therapeutically to treat diseases such as cancer and autoimmune disorders. The efficacy of IL-2 treatment has been limited by its concurrent promotion of both effector and regulatory immune cells, and I have implemented a multi-pronged approach toward engineering the IL-2 system to modulate the balance of its immunostimulatory versus immunosuppressive effects to achieve targeted therapy. As an independent investigator, I plan to combine my experience in both basic and applied biomedical sciences to build a translational research program at the interface of structural biology and molecular engineering.

Teaching Interests:

Throughout my academic research career, I have been fortunate to gain experience in teaching as well. As a teaching assistant for an introductory Biomedical Engineering course at Johns Hopkins University, I helped lead laboratory sessions and advised students on independent design projects, providing me with the opportunity to interactively train students in applying engineering and design approaches to solve medically relevant problems. As a Ph.D. student, I served as a teaching assistant for a graduate-level course in biomolecular kinetics and cellular dynamics at Massachusetts Institute of Technology. The course focused heavily on computer programming in MATLAB and I instructed several tutorials on this software and also led both lecture- and discussion-based recitation sessions.

In addition to classroom teaching, a formative experience in my pedagogical training has been the opportunity to mentor students. During my graduate career, I mentored six undergraduate students, many of whom have gone on to pursue biomedical careers: three are in top bioengineering Ph.D. programs and two are in medical school. I have also supervised three students and a technician during my postdoctoral tenure, and these opportunities to inspire the next generation of scientists have reinforced my desire to pursue a faculty career.

Future Directions:

The research program I will pursue in my own laboratory represents a culmination of the skills I have amassed through previous work, synthesizing engineering and design experience with structural and biophysical tools to create selective and efficacious molecular therapeutics for a broad range of immunological applications. The most prominent challenge to therapeutic development of cytokines is their pleiotropic nature, as stimulation of signaling on multiple immune cell subsets leads to dose-limiting toxicity and harmful off-target effects. By harnessing structural insights to isolate and selectively engineer the cellular activation properties of cytokines, I will construct a blueprint for biasing protein activity that can be tailored to a variety of disease objectives. In addition to affording unprecedented control over protein behavior, my research will deepen our understanding of the molecular mechanisms that underlie cellular responses and inform the design of candidate drugs for ready clinical translation. While initial work will focus on engineering antibodies to tune cytokine activity, my long term vision is to apply the modular engineering and therapeutic platforms I develop to other systems to allow for the direct manipulation of a diverse array of ligand-receptor interactions including hormone, growth factor, and membrane protein systems.

I recently devised structure-guided evolutionary strategies using yeast surface display methodology to establish two novel antibody-based platforms that modulate cytokine behavior: [1] Cytokine-targeted antibodies that allosterically disrupt cytokine-receptor interactions to bias activity; and [2] Agonistic antibody fragments that use a single binding site to bridge two receptor subunits and activate signaling in the absence of cytokine. Both platforms can be exploited to direct cytokine activity toward specific cell subsets based on surface receptor expression profiles, and their modularity allows for targeting of any protein interaction or cell population of interest to elicit either an immunostimulatory response (for treatment of cancer or chronic infection) or an immunosuppressive response (for engraftment or treatment of autoimmune disorders). In addition to building on platforms I have already established, I plan to develop new engineering platforms to isolate antibodies that act through alternative mechanisms such as modulation of internalization or molecular trafficking properties. The unique mechanisms recruited in these platforms will complement other activities inherent to the antibody scaffold such as competitive inhibition and Fc-mediated effector functions. Moreover, the stable and readily engineerable antibody construction will allow for development of bispecific molecules that synergistically combine antibodies with distinct modes of action, and structural feedback on molecules we develop will further inform design. My experience with mouse models of cancer and autoimmune disease and continued collaborations with leading T cell immunologists established during my postdoctoral tenure will facilitate pre-clinical evaluation and ultimate translation of engineered therapeutics. Through integration of expertise in molecular engineering and structural biology, I am uniquely positioned to make rapid progress in developing new approaches toward targeted therapy for immunomodulation and a host of other biomedical applications.

Selected Publications:

Spangler JB, Tomala J, Luca VC, Jude KM, Dong S, Ring AM, Votavova P, Pepper M, Kovar M, Garcia KC. Antibodies to interleukin-2 elicit selective T cell subset potentiation through distinct conformational mechanisms. Immunity. 2015;42(5):815-25. PMCID: 4439582.

Mitra S, Ring AM, Amarnath S, Spangler JB, Li P, Ju W, Fischer S, Oh J, Spolski R, Weiskopf K, Kohrt H, Foley JE, Rajagopalan S, Long EO, Fowler DH, Waldmann TA, Garcia KC, Leonard WJ. Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps. Immunity. 2015;42(5):826-38. PMCID: 4560365.

Spangler JB, Moraga I, Mendoza JL, Garcia KC. Insights into cytokine-receptor interactions from cytokine engineering. Annu Rev Immunol. 2015;33:139-67. PMCID: 4445396.

Moraga I, Spangler J, Mendoza JL, Garcia KC. Multifarious determinants of cytokine receptor signaling specificity. Adv Immunol. 2014;121:1-39. PMCID: 4449261.

Spangler JB, Manzari MT, Rosalia EK, Chen TF, Wittrup KD. Triepitopic antibody fusions inhibit cetuximab-resistant BRAF and KRAS mutant tumors via EGFR signal repression. J Mol Biol. 2012;422(4):532-44. PMCID: 4041985.

Spangler JB, Neil JR, Abramovitch S, Yarden Y, White FM, Lauffenburger DA, Wittrup KD. Combination antibody treatment down-regulates epidermal growth factor receptor by inhibiting endosomal recycling. Proc Natl Acad Sci U S A. 2010;107(30):13252-7. PMCID: 2922117.

You M*, Spangler J*, Li E, Han X, Ghosh P, Hristova K. Effect of pathogenic cysteine mutations on FGFR3 transmembrane domain dimerization in detergents and lipid bilayers. Biochemistry. 2007;46(39):11039-46.