Faculty Bibliography

Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation-containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design.

Manipulation of the ubiquitin-proteasome pathway to achieve targeted silencing of cellular proteins has emerged as a reliable and customizable strategy for remodeling the mammalian proteome. One such approach involves engineering bifunctional proteins called ubiquibodies that are comprised of a synthetic binding protein fused to an E3 ubiquitin ligase, thus enabling post-translational ubiquitination and degradation of a target protein independent of its function. Here, we have designed a panel of new ubiquibodies based on E3 ubiquitin ligase mimics from bacterial pathogens that are capable of effectively interfacing with the mammalian proteasomal degradation machinery for selective removal of proteins of interest. One of these, the Shigella flexneri effector protein IpaH9.8 fused to a fibronectin type III (FN3) monobody that specifically recognizes green fluorescent protein (GFP), was observed to potently eliminate GFP and its spectral derivatives as well as 15 different FP-tagged mammalian proteins that varied in size (27-179 kDa) and subcellular localization (cytoplasm, nucleus, membrane-associated, and transmembrane). To demonstrate therapeutically relevant delivery of ubiquibodies, we leveraged a bioinspired molecular assembly method whereby synthetic mRNA encoding the GFP-specific ubiquibody was coassembled with poly A binding proteins and packaged into nanosized complexes using biocompatible, structurally defined polypolypeptides bearing cationic amine side groups. The resulting nanoplexes delivered ubiquibody mRNA in a manner that caused efficient target depletion in cultured mammalian cells stably expressing GFP as well as in transgenic mice expressing GFP ubiquitously. Overall, our results suggest that IpaH9.8-based ubiquibodies are a highly modular proteome editing technology with the potential for pharmacologically modulating disease-causing proteins.

Background Current immunotherapies have shown promise in treating multiple cancer types but success remains elusive for cancers like pancreatic, the majority of colorectal cancers as well as cholangiocarcinoma. Poor prognosis in these tumor types is invariably linked to immune dysfunction. Galectin-9, which is a fundamental immune modulator and global immune suppressor, was previously identified as a molecule that plays a significant role in orchestrating and maintaining a tumor-permissive immune environment. In light of this, we have studied the relevance of galectin-9 as a biomarker and have developed LYT-200, a therapeutic, fully human IgG4 antibody to target galectin-9. Methods A synthetic, human antibody library was screened via phage display using purified carbohydrate binding domains (CRDs) of human and mouse galectin-9. Blockade of galectin-9-receptor binding and signaling by antibodies was assayed in both biochemical and cell-based formats. Antibody efficacy was assessed in orthotopic pancreatic cancer and subcutaneous melanoma mouse models as single agent and in combination with anti-PD1 and chemotherapy. Patient-derived samples (FFPE tissues, organotypic tumor spheroids (PDOTs)), which recapitulate complex tumor architecture, and patient blood samples were analyzed by immunohistochemistry, flow cytometry and ELISA to measure galectin-9 expression and compare with healthy controls across tumor types. Additionally, PDOTs were treated with clinical anti-galectin-9 antibodies and the resulting immune profile was analyzed. Results Significantly higher levels of galectin-9 were detected across patient blood/tissue samples (n >100 patients) compared with healthy controls. Analyses of PDOTs showed high levels of galectin-9 on tumor, myeloid and T cells. Its high expression, for example in pancreatic adenocarcinoma, suggest that galectin-9 is a promising therapeutic target and a biomarker that correlates with disease stage. From a therapeutic perspective, LYT-200 was selected out of numerous antibody candidates as the lead clinical clone based on its high affinity (dissociation constant < 1 nM) to galectin-9 across species, high specificity, stability, and blocking galectin-9 interactions with its ligands in biochemical and cellbased assays. Treatment of tumor bearing mice with LYT-200 significantly reduced in tumor size (p Conclusions LYT-200, a first-in-class human antibody that inhibits tumorpermitting activity of Galectin-9 was developed. The data collectively reveal novel galectin-9 biology and demonstrate therapeutic efficacy of LYT-200. Blockade of galectin-9 using LYT-200 is an innovative and promising strategy for treating aggressive solid tumors

Background Targeting and engineering gammadelta T cells has recently emerged as an orthogonal therapeutic approach in oncology with capacity to modulate both innate and adaptive immune properties. In solid tumors such as pancreatic ductal adenocarcinoma (PDA), melanoma, glioblastoma, ovarian, and breast cancer, gammadelta1 T cells express immunosuppression-related molecules and possess a protumorigenic capacity. We have shown that intra-tumoral gammadelta T cells from PDA, colorectal cancer (CRC) and hepatocellular carcinoma (HCC) potently suppress patients' alphabeta T cells. To harness the therapeutic potential of gammadelta1 T cell blockade, we developed highly specific, fully human antibodies against delta1-subset of gammadeltaT cell receptor (gammadelta1- TCR). Methods We determined the amino acid sequences of tumor specific delta1-TCR chains from primary PDA, CRC and gastric cancer samples. Multiple gammadelta-TCR proteins were produced and used to screen a proprietary synthetic, human antibody library using phage display. Surface plasmon resonance and bead-based assays were used to measure binding affinity and specificity. Affinity maturation was performed to improve cross-reactivity to monkey gammadelta1-TCR. Cell based assays were used to evaluate antibody-dependent cell cytotoxicity and phagocytosis (ADCC and ADCP). The levels of gammadelta1-T cell infiltration was measured in patient tumors. Efficacy in reversing immunesuppression was assayed using patient-derived organotypic tumor spheroids (PDOTS, n = 32), which recapitulate complex tumor architecture. Results Because the tumor-derived delta1 chains showed diverse CDR3 sequences, we developed antibodies that bind diverse delta1 TCRs. Our first-in-class anti-delta1 antibodies had low nanomolar affinity to delta1 TCRs and showed no binding to delta2 TCRs. Our lead clinical candidate showed no preference for the gamma chains of the TCR, which enables it to target diverse set of gammadelta1-TCRs. It had equivalent affinity for the human and cynomolgus monkey gammadelta1-TCRs and was potent in mediating cell based ADCC and ADCP. We showed that patient tumors can have a high delta1 T cell infiltration (up to 40% of the total T cell infiltrate). Our lead candidate achieved reproducible and robust efficacy in the up-regulation of pro-inflammatory T cell activation markers in PDOTS from a diverse set of gastrointestinal tumors. Furthermore, gammadelta knockout mice had an improved response to checkpoint inhibitors, anti-PD1 and anti-CTLA4, in melanoma and lung cancer models. Conclusions We have defined a novel therapeutic immuno-oncology strategy and translated it to develop a lead clinical candidate anti-delta1 monoclonal antibody. Our efficacious, novel immunotherapy has the potential to be transformative for the treatment of cancers where gammadelta1 T cells drive a pro-tumorigenic, immunosuppressive environment

Rapidly determining the biological effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, we describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. We developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-molecule inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochemical, structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.

Despite increasing demands for antibodies to post-translational modifications (PTMs), fundamental difficulties in molecular recognition of PTMs hinder the generation of highly functional anti-PTM antibodies using conventional methods. Recently, advanced approaches in protein engineering and design that have been established for biologics development were applied to successfully generating highly functional anti-PTM antibodies. Furthermore, structural analyses of anti-PTM antibodies revealed unprecedented binding modes that substantially increased the antigen-binding surface. These features deepen the understanding of mechanisms underlying specific recognition of PTMs, which may lead to more effective approaches for generating anti-PTM antibodies with exquisite specificity and high affinity.

Fluoride/proton antiporters of the CLC^F family combat F^- toxicity in bacteria by exporting this halide from the cytoplasm. These transporters belong to the widespread CLC superfamily but display transport properties different from those of the well-studied Cl^-/H⁺ antiporters. Here, we report a structural and functional investigation of these F^--transport proteins. Crystal structures of a CLC^F homolog from Enterococcus casseliflavus are captured in two conformations with simultaneous accessibility of F^- and H⁺ ions via separate pathways on opposite sides of the membrane. Manipulation of a key glutamate residue critical for H⁺ and F^- transport reverses the anion selectivity of transport; replacement of the glutamate with glutamine or alanine completely inhibits F^- and H⁺ transport while allowing for rapid uncoupled flux of Cl^-. The structural and functional results lead to a 'windmill' model of CLC antiport wherein F ^- and H⁺ simultaneously move through separate ion-specific pathways that switch sidedness during the transport cycle.

Controlling the catalytic properties of enzymes remain an important challenge in chemistry and biotechnology. We have recently established a strategy for altering enzyme specificity in which the addition of proxy monobodies, synthetic binding proteins, modulates the specificity of an otherwise unmodified enzyme. Here, in order to examine its broader applicability, we employed the strategy on Candida rugosa lipase 1 (CRL1), an enzyme with a tunnel-like substrate binding site. We successfully identified proxy monobodies that restricted the substrate specificity of CRL1 toward short-chain fatty acids. The successes with this enzyme system and a β-galactosidase used in the previous work suggest that our strategy can be applied to diverse enzymes with distinct architectures of substrate binding sites.