Jennifer Rachel Cochran - US grants

DESCRIPTION (provided by applicant): The candidate has a multidisciplinary background in chemistry, biochemistry and engineering. These fields will be combined to develop stable, high affinity integrin binding proteins for mediating cell adhesion and angiogenesis in cancer. The candidate will spend a year in a mentored position in the laboratories of William F. DeGrade and Joel S. Bennett at the University of Pennsylvania School of Medicine. During this period, the applicant will develop skills in peptide and protein design, and organic synthesis while in the DeGrade lab. Experience in integrin receptor function and biological assays will be gained in the Bennett lab. This period will be important in establishing the groundwork for the unmentored portion of the award. The remainder of the award will be spent in an independent, preferrably tenure-track, academic position, where the applicant will build upon this additional expertise to develop biomolecules for cancer therapeutics and tumor imaging applications. The University of Pennsylvania School of Medicine has a strong commitment to research in health and human disease, and several of its faculty are conducting integrin and/or cancer related research. The mentors chosen by the applicant have well-established, productive collaborations in studying the structure and function of integrin proteins. In addition, Prof. DeGrade has much expertise in development of integrin-specific mimics through rational design and combinatorial library panning in collaboration with DuPont. Integrin-specific peptide mimics and antibodies have shown much therapeutic promise in the inhibition of angiogenesis and metastases in tumors, but can benefit greatly from stability and affinity maturation. The mentored period of the award will involve incorporation of integrin-specific peptide motifs into constrained molecular scaffolds to increase stability and binding affinity. These scaffolds will provide the framework for a portion of the unmentored period of the award, where integrin binding proteins will be engineered to even higher affinity using directed molecular evolution by yeast surface display. In addition, the mentored period of the award will focus on the design and synthesis of a series of novel peptide-based crosslinking reagents. These compounds will be used for creating multivalent integrin-specific peptides and proteins for high avidity binding and enhanced antagonism. In the unmentored period of the application, a nonimmune human library of scFv antibody fragments displayed on the surface of yeast will be used to isolate a panel of integrin-specific binders. These antibodies will be characterized for their ability to inhibit RGD-mediated binding and cell adhesion. Promising antibodies will be engineered to ultra high affinity and stability using yeast surface display to develop potent integrin antagonists.

DESCRIPTION (provided by applicant): The Met receptor tyrosine kinase is critical for mediating cell proliferation, survival and migration. The tendency for human tumors to invade and metastasize has been tied to the dysregulation of Met;therefore, Met is an extremely attractive target for therapeutic intervention. Interestingly, soluble Met extracellular domain (Met-ECD) acts as a potent antagonist of Met activation. Unfortunately, current methods for recombinant expression of Met-ECD have low yield, preventing both the full characterization of its biology and its deployment as a potential therapeutic. Our lab has robust and proven technology for the evolution, optimization, and expression of such challenging proteins, and Met-ECD is an ideal candidate for applying this technology. This work will accelerate biophysical characterization of Met ligand/receptor interactions, and applications of the Met receptor in cancer therapy and diagnostics. Aim 1: Engineer the full-length Met extracellular domain for high soluble expression levels in yeast. Directed evolution using yeast surface display provides us with a robust combinatorial platform to identify Met- ECD mutants that 1) possess the native receptor fold and 2) exhibit high soluble expression levels (mg/L) in yeast. Aim 2: Measure the ligand binding affinity and biological activity mediated by Met-ECD mutants. We will test the Met-ECD mutants isolated in Aim 1 to verify that engineered receptors with improved expression retain native ligand binding properties and biological function. We will fuse Met-ECD mutants to wild-type Met transmembrane and intracellular domains, and transfect these constructs into a mammalian cell line that expresses low levels of endogenous Met. We will test these cells lines for their ability to bind HGF ligand and induce cell signaling, comparing them to cells transfected with wild-type Met. These efforts will demonstrate the biological functions of Met-ECD mutants, validating them for future biophysical studies. Aim 3: Determine the ability of soluble Met-ECD mutants to bind to tumor cells and inhibit signaling. We will determine if soluble Met-ECD mutants function as receptor antagonists by measuring their ability to 1) bind to Met-expressing tumor cells and 2) inhibit ligand-dependent and ligand-independent activation of the Met kinase domain and downstream MAPK and PI3K signaling pathways. This Aim will validate Met-ECD mutants as receptor antagonists in a variety of cell types and motivate future research into their therapeutic potential. Project Narrative The availability of functional recombinant Met-ECD will profoundly impact future research into mechanisms of tumorigenesis, bacterial pathogenesis, and embryonic development, and will provide insight into how these processes can be manipulated. The fact that the Met-ECD itself could also be developed for applications in molecular imaging or cancer therapy is another example of this project's high impact.

Project Summary/Abstract Dysregulation of cell signaling pathways that mediate proliferation, survival, and migration are an underlying cause of cancer, and result in invasion and metastasis of many human tumors. In particular, dysregulation and over-expression of the Met tyrosine kinase receptor correlates to poor prognosis in many human tumors, making it an attractive target for therapeutic intervention. There are currently no FDA-approved therapeutics targeting the Met receptor; however, a few candidate molecules have recently entered early stage clinical trials. Therefore, molecules that potently inhibit Met receptor activation would have a significant clinical impact on cancer therapy. In addition, studies to develop Met-targeted molecular imaging agents for non- invasive visualization of Met expression in vivo have been extremely limited. The availability of such imaging agents would aid in cancer diagnosis, staging, and disease management, as well as help identify patients who would be good candidates for Met-targeted therapies. To develop robust Met-targeting agents we used the N- terminal and first Kringle domain (NK1) of the natural Met activating ligand, hepatocyte growth factor (HGF), as a basis for engineering potent Met receptor antagonists. Using directed evolution, we engineered NK1 mutants with significant improvements in Met binding affinity and thermal stability compared to wild-type NK1. Rationally-designed, site-directed mutations introduced into these NK1 proteins transformed them into Met receptor antagonists. In Aim 1 of the proposal, we will perform pre-clinical studies on fluorescently-labeled and radiolabeled NK1 mutants to test their ability to non-invasively image Met expression in living subjects, with the goal of developing them as in vivo molecular imaging agents. In Aim 2, we will perform pre-clinical studies to measure the effects of NK1 mutants on tumor growth, metastasis, and angiogenesis in several mouse tumor models. During the course of treatment, non-invasive imaging will be used to monitor growth and progression of the primary tumor and metastases, and to monitor changes in Met expression and metabolism at the tumor site. In Aim 3, we will fully characterize the binding of a larger panel of engineered NK1 mutants to Met-expressing tumor cells, and will determine their ability to dimerize and subsequently inhibit Met receptor activation. In Aim 4, we will use these engineered NK1 proteins to probe sequence-structure-function relationships of ligand- receptor interactions in the Met receptor system, and provide biochemical and biophysical insight into their mechanism of action. In all four aims, results will be compared to wild-type NK1 to determine the effects of Met receptor binding affinity and protein stability on biological activity in cell culture and animal models. Upon completion of this proposal, we will have evaluated the potential of engineered NK1 proteins as molecular imaging and therapeutic agents in pre-clinical models, an important step on the path to clinical translation.

DESCRIPTION (provided by applicant): Carbonic anhydrase IX (CA IX) is a membrane bound protein overexpressed on the surface of cancer cells in a hypoxic environment. CA IX is involved in tumor cell survival and metastasis, and increased expression correlates with poor clinical outcome, however there are no approved therapies against CA IX. Monoclonal antibodies have been used to target CA IX, but their large size limits penetration throughout a poorly vascularized tumor, and their slow blood clearance limits their use as tumor imaging agents or radiotherapeutics due to high background and toxicity. Small organic molecules that inhibit CA IX are highly non-specific, and can diffuse across cell membranes to bind to intracellular carbonic anhydrase isoforms abundant in healthy tissue. Here, we propose several strategies to engineer highly stable constrained peptides (knottins) and small molecule conjugates that selectively bind to the extracellular domain of CA IX with low nanomolar affinity. This work will identify novel CA-IX targeting peptides for clinical translation as diagnostic and therapeutic agents, and will also generate technology that could broadly be applied to target membrane receptors in cancer and other diseases. Aim 1: Develop tumor-targeting agents by engineering knottin peptides that bind to CA IX with high affinity. We will use yeast surface display to engineer knottin peptides that bind to CA IX with high affinities in the low nanomolar range. We will measure the relative binding affinities of engineered knottin peptides to CA IX expressed on the surface of tumor cells. Aim 2: Develop tumor-targeting agents by chemically conjugating small molecule CA IX inhibitors to knottin peptides. We will chemically couple small molecule CA IX inhibitors to knottin peptides to combine the CA IX targeting properties of known small molecules with the favorable tissue biodistribution afforded by knottin peptides. In addition to generating new CA IX targeting molecules, this aim will result in the development of a general technology platform to improve the biodistribution of small molecule tumor-targeting agents and will result in a novel approach for creating bi-specific tumor targeting agents. Aim 3: Measure biodistribution and tumor uptake of engineered CA IX targeting agents in living subjects. Engineered CA IX binding peptides will be tested for their ability to target hypoxic tumors in vivo. MicroPET imaging, biodistribution studies, and metabolite stability will be performed with 64Cu-labeled knottin peptides and small molecule conjugates in human tumor-bearing mouse xenograft models. This aim will further establish CA IX as a target for cancer diagnosis and therapy, and validate engineered CAIX-binding knottin peptides for additional clinical studies. PUBLIC HEALTH RELEVANCE: The availability of engineered peptides that target carbonic anhydrase IX (CA IX), a membrane bound protein overexpressed on the surface of cancer cells, will open up new research areas in tumorigenesis, cancer biology, molecular imaging, and structure-based drug design. The preclinical studies we are proposing will validate CA IX as a molecular target for cancer imaging to identify patients who would benefit most from targeted therapies and to monitor their disease progression. Moreover, this work will lay a foundation for future development of engineered CA IX-binding peptides as targeting agents for tumor-specific delivery of chemotherapeutics and radionuclides.

The urokinase-type plasminogen activator receptor (uPAR) has been found to be overexpressed on the surface of cancer cells, as well as their associated neovasculature, in virtually every cancer type tested, including primary, metastatic, and chemotherapy-resistant cancers. uPAR is an important master regulator that drives extracellular matrix degradation and angiogenesis, and cancer cell proliferation, adhesion, and migration. These processes in turn allow rapid tumor growth and metastasis to other sites in the body. Clinical studies indicate overexpression of uPAR and its soluble ligand uPA are independent predictors of low diseasefree and overall survival. Thus, uPAR is an extremely attractive target for therapeutic intervention; however, the absence of an FDA-approved drug that inhibits uPAR highlights a critical need for new approaches to effectively block the activity of this receptor. We will apply our expertise in molecular and cellular biology, biochemistry, and protein engineering to develop a novel first-in-class biologic capable of effectively blocking uPAR-mediated cancer growth, metastasis, and tumor-associated angiogenesis. Numerous uPAR antagonists have been developed over the last two decades, including small molecules, peptides, protein ligands, and antibodies. However, the efficacies of these inhibitors have been limited owing to their low affinity relative to the native uPAR ligands, and/or their inability to effectively block uPAR functions that drive cancer growth and metastasis. Protein engineering, combined with multispecific target binding, will generate in uPAR inhibitors with orders of magnitude higher binding affinity compared to previously developed antagonists. The resulting biologics have exciting potential to overcome critical barriers that have prevent development of successful uPAR-targeted therapeutics, and would represent a potential breakthrough for targeted therapy of aggressive cancers.

PROJECT SUMMARY We seek acceptance of our predoctoral biotechnology training program, which has a history of producing accomplished leaders in academia and industry. Biotechnology is broadening and evolving as a field with an expansion of knowledge, tools, and applications. In parallel, industry is shaping new efforts using biotechnology for prevention, diagnosis, and treatment of disease. Stanford provides an unusually rich environment, with co-located, nationally ranked schools devoted to basic science, engineering, and medicine, and a strong industrial presence from the surrounding Bay Area. We are leveraging this ecosystem to train talented students who are becoming the next-generation of interdisciplinary global biotechnology innovators, and who will lead and invent the future with integrity and rigor. We are seeking training funding for 10 trainees per year for a period of 5 years. The program fuses 39 investigators from 9 departments across at the university into a highly visible program that delivers a unique, applications-oriented training experience focused on health-related biotechnology. Differentiating features of this program include curricular offerings in biotechnology innovation and leadership, industrial internships, field trips, symposia, and deep interactions among trainees and academic and industrial mentors.

Lung cancer is the leading cause of cancer deaths worldwide. The most prevalent type of lung cancer is Non- Small Cell Lung Cancer (NSCLC). The goal of this application is to implement a collaborative effort involving preclinical models, bioengineering and functional genomics to further characterize and validate a novel ?first in class? therapeutic strategy for the treatment of lung cancer. Our prior published work and extensive preliminary data indicates that blockade of CLCF1-CNTFR signaling represents a unique and previously unexplored approach for lung cancer therapy. The Sweet-Cordero and Cochran laboratories have collaborated extensively over the past several years to validate this signaling axis, first with shRNA genetic approaches, and now through the development of an engineered CNTFR receptor decoy (eCNTFR). In Aim 1, we will further develop eCNTFR as a therapeutic candidate by measuring its thermal and proteolytic stability, potential for immunogenicity and toxicity, manufacturability, and pharmacokinetics, and will optimize these properties as needed. We will also perform structural analysis of eCNTFR in complex with CLCF1 to define high affinity binding characteristics. Lastly, we will test eCNTFR for therapeutic efficacy across a wide array of preclinical models of NSCLC including human cell lines and patient-derived xenografts. In Aim 2, we will perform in vitro biochemical assays to fully define the cell-autonomous mechanism of action of eCNTFR blockade. To further understand the mechanism of action of eCNTFR in vivo, we will complement xenograft models with a well-characterized genetically engineered mouse model of lung cancer. Importantly, this model will allow us to study the effects of eCNTFR not only on tumor cells but also on the microenvironment, and to assess whether eCNTFR has immunomodulatory effects. In Aim 3, we will identify the most effective combination approaches to enhance eCNTFR therapy. These efforts will be carried out in a rational and unbiased manner by leveraging CRISPR/CAS9 using a library directed specifically at the targets of FDA approved drugs. Candidate combination therapies will then be tested in animal models. Our studies will elucidate critical biological underpinnings of this ligand/receptor signaling axis, and will provide preclinical validation of an innovative strategy for targeting lung cancer to warrant its further clinical development.