Faculty Bibliography

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.

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.

Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.

The maturation of RAS GTPases and approximately 200 other cellular CAAX proteins involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CAAX motif, proteolytic cleavage of the AAX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate. This final step is catalysed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryote-specific integral membrane enzyme that resides in the endoplasmic reticulum. ICMT is the only cellular enzyme that is known to methylate prenylcysteine substrates; methylation is important for the biological functions of these substrates, such as the membrane localization and subsequent activity of RAS, prelamin A and RAB. Inhibition of ICMT has potential for combating progeria and cancer. Here we present an X-ray structure of ICMT, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor, at 2.3 Å resolution. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for the cytosolic methyl donor, S-adenosyl-l-methionine, and for prenylcysteine substrates, which are associated with the endoplasmic reticulum membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localizations together in a membrane environment-a relatively uncharacterized but defining feature of many integral membrane enzymes.

The two isoforms of the Bcr-Abl tyrosine kinase, p210 and p190, are associated with different leukemias and have a dramatically different signaling network, despite similar kinase activity. To provide a molecular rationale for these observations, we study the Dbl-homology (DH) and Pleckstrin-homology (PH) domains of Bcr-Abl p210, which constitute the only structural differences to p190. Here we report high-resolution structures of the DH and PH domains and characterize conformations of the DH-PH unit in solution. Our structural and functional analyses show no evidence that the DH domain acts as a guanine nucleotide exchange factor, whereas the PH domain binds to various phosphatidylinositol-phosphates. PH-domain mutants alter subcellular localization and result in decreased interactions with p210-selective interaction partners. Hence, the PH domain, but not the DH domain, plays an important role in the formation of the differential p210 and p190 Bcr-Abl signaling networks.

Beta-arrestins (betaarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of betaarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of betaarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted betaarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting betaarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.