Faculty Bibliography

Intracellular oncoproteins can be inhibited with targeted therapy, but responses are not durable. Immune therapies can be curative, but most oncogene-driven tumors are unresponsive to these agents. Fragments of intracellular oncoproteins can act as neoantigens presented by the major histocompatibility complex (MHC) but recognizing minimal differences between oncoproteins and their normal counterparts is challenging. We have established a platform technology that exploits hapten-peptide conjugates generated by covalent inhibitors to create distinct neoantigens that selectively mark cancer cells. Using the FDA-approved covalent inhibitors sotorasib and osimertinib, we developed "HapImmuneTM" antibodies that bind to drug-peptide conjugate/MHC complexes but not to the free drugs. A HapImmuneTM-based bispecific T cell engager selectively and potently kills sotorasib-resistant lung cancer cells upon sotorasib treatment. Notably, it is effective against KRASG12C mutant cells with different HLA supertypes, HLA-A*02 and A*03/11, suggesting loosening of MHC restriction. Our strategy creates targetable neoantigens by design, unifying targeted and immune therapies.

Mutations in one of the three RAS genes (HRAS, KRAS, and NRAS) are present in nearly 20% of all human cancers. These mutations shift RAS to the GTP-loaded active state due to impairment in the intrinsic GTPase activity and disruption of GAP-mediated GTP hydrolysis, resulting in constitutive activation of effectors such as RAF. Because activation of RAF involves dimerization, RAS dimerization has been proposed as an important step in RAS-mediated activation of effectors. The α4-α5 allosteric lobe of RAS has been proposed as a RAS dimerization interface. Indeed, the NS1 monobody, which binds the α4-α5 region within the RAS G domain, inhibits RAS-dependent signaling and transformation as well as RAS nanoclustering at the plasma membrane. Although these results are consistent with a model in which the G domain dimerizes through the α4-α5 region, the isolated G domain of RAS lacks intrinsic dimerization capacity. Furthermore, prior studies analyzing α4-α5 point mutations have reported mixed effects on RAS function. Here, we evaluated the activity of a panel of single amino acid substitutions in the α4-α5 region implicated in RAS dimerization. We found that these proposed "dimerization-disrupting" mutations do not significantly impair self-association, signaling, or transformation of oncogenic RAS. These results are consistent with a model in which activated RAS protomers cluster in close proximity to promote the dimerization of their associated effector proteins (e.g., RAF) without physically associating into dimers mediated by specific molecular interactions. Our findings suggest the need for a nonconventional approach to developing therapeutics targeting the α4-α5 region.

RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.

Vascular endothelial cells (ECs) play a central role in the pathophysiology of many diseases. The use of targeted nanoparticles (NPs) to deliver therapeutics to ECs could dramatically improve efficacy by providing elevated and sustained intracellular drug levels. However, achieving sufficient levels of NP targeting in human settings remains elusive. Here, we overcome this barrier by engineering a monobody adapter that presents antibodies on the NP surface in a manner that fully preserves their antigen-binding function. This system improves targeting efficacy in cultured ECs under flow by >1000-fold over conventional antibody immobilization using amine coupling and enables robust delivery of NPs to the ECs of human kidneys undergoing ex vivo perfusion, a clinical setting used for organ transplant. Our monobody adapter also enables a simple plug-and-play capacity that facilitates the evaluation of a diverse array of targeted NPs. This technology has the potential to simplify and possibly accelerate both the development and clinical translation of EC-targeted nanomedicines.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease^1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium^8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

As of 8 August 2022, SARS-CoV-2, the causative agent of COVID-19, has infected over 585 million people and resulted in more than 6.42 million deaths worldwide. While approved SARS-CoV-2 spike (S) protein-based vaccines induce robust seroconversion in most individuals, dramatically reducing disease severity and the risk of hospitalization, poorer responses are observed in aged, immunocompromised individuals and patients with certain pre-existing health conditions. Further, it is difficult to predict the protection conferred through vaccination or previous infection against new viral variants of concern (VoC) as they emerge. In this context, a rapid quantitative point-of-care (POC) serological assay able to quantify circulating anti-SARS-CoV-2 antibodies would allow clinicians to make informed decisions on the timing of booster shots, permit researchers to measure the level of cross-reactive antibody against new VoC in a previously immunized and/or infected individual, and help assess appropriate convalescent plasma donors, among other applications. Utilizing a lab-on-a-chip ecosystem, we present proof of concept, optimization, and validation of a POC strategy to quantitate COVID-19 humoral protection. This platform covers the entire diagnostic timeline of the disease, seroconversion, and vaccination response spanning multiple doses of immunization in a single POC test. Our results demonstrate that this platform is rapid (~15 min) and quantitative for SARS-CoV-2-specific IgG detection.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here, we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Ã… structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Molecular display technologies have enabled the generation of synthetic binders with high affinities against a variety of antigens. However, engineering binders with high selectivity is still a challenging task. Here, we illustrate points to consider in developing highly selective binders against antigens of interest. We describe a systematic strategy for sorting selective binders using the yeast display technology. Using the approach described, our group has overcome molecular recognition challenges and developed a series of synthetic binders with exceptional selectivity against diverse antigens.