Faculty Bibliography

The HapImmune^TM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the V_H and V_L domains, binds HLAs in an unconventional, angled way, with V_L making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.

Synthetic binding proteins are human-made binding proteins that use non-antibody proteins as the starting scaffold. Molecular display technologies, such as phage display, enable the construction of large combinatorial libraries and their efficient sorting and, thus, are crucial for the development of synthetic binding proteins. Monobodies are the founding system of a set of synthetic binding proteins based on the fibronectin type III (FN3) domain. Since the original report in 1998, the monobody and related FN3-based systems have steadily been refined, and current methods are capable of rapidly generating potent and selective binding molecules to even challenging targets. The FN3 domain is small (∼90 amino acids) and autonomous and is structurally similar to the conventional immunoglobulin (Ig) domain. Unlike the Ig domain, however, the FN3 lacks a disulfide bond but is highly stable. These attributes of FN3 present unique opportunities and challenges in the design of phage and other display systems, combinatorial libraries, and library sorting strategies. This article reviews key technological innovations in the establishment of our monobody development pipeline, with an emphasis on phage display methodology. These give insights into the molecular mechanisms underlying molecular display technologies and protein-protein interactions, which should be broadly applicable to diverse systems intended for generating high-performance binding proteins.

Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.

Domain swapping is a process wherein a portion of a protein is exchanged with its counterpart in another copy of the molecule, resulting in the formation of homo-oligomers with concomitant repacking of a hydrophobic core. Here, we report domain swapping triggered upon modifying a β-hairpin sequence within a single-layer β-sheet (SLB) of a model protein, OspA that did not involve the formation of a reorganized hydrophobic core. The replacement of two β-hairpin sequences with a Gly-Gly and shorteing of a β-hairpin resulted in a protein that formed two distinct crystal structures under similar conditions: one was monomeric, similar to the parental molecule, whereas the other was a domain-swapped dimer, mediated by an intermolecular β-sheet in the SLB portion. Based on the dimer interface structure, we replaced the Gly-Gly sequence with three-residue sequences that enable the formation of a consecutive intermolecular β-sheet, including the Cys-Thr-Cys sequence that formed a stable disulfide-linked dimer. These results provide new insights into protein folding, evolution, and the designability of protein structure.

SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell analysis of blood from COVID-19 patients and healthy volunteers receiving the SARS-CoV-2 vaccine and booster. We profiled immune responses via transcriptional analysis and lymphocyte repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. B and T cell repertoire analysis revealed clonal expansion among effector cells in COVID-19 patients and memory cells in vaccine recipients. Furthermore, while clonal αβ T cell responses were observed in both COVID-19 patients and vaccine recipients, expansion of clonal γδ T cells was found only in infected individuals. Our dataset enables side-by-side comparison of immune responses to infection versus vaccination, including clonal B and T cell responses. Our comparative analysis shows that vaccination induces a robust, durable clonal B and T cell responses, without the severe inflammation associated with infection.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of β-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.

The necroptosis pathway is a lytic, pro-inflammatory mode of cell death that is widely implicated in human disease, including renal, pulmonary, gut and skin inflammatory pathologies. The precise mechanism of the terminal steps in the pathway, where the RIPK3 kinase phosphorylates and triggers a conformation change and oligomerization of the terminal pathway effector, MLKL, are only emerging. Here, we structurally identify RIPK3-mediated phosphorylation of the human MLKL activation loop as a cue for MLKL pseudokinase domain dimerization. MLKL pseudokinase domain dimerization subsequently drives formation of elongated homotetramers. Negative stain electron microscopy and modelling support nucleation of the MLKL tetramer assembly by a central coiled coil formed by the extended, ~80 Å brace helix that connects the pseudokinase and executioner four-helix bundle domains. Mutational data assert MLKL tetramerization as an essential prerequisite step to enable the release and reorganization of four-helix bundle domains for membrane permeabilization and cell death.

Both SARS-CoV-2 infection and vaccination elicit potent immune responses. A number of studies have described immune responses to SARS-CoV-2 infection. However, beyond antibody production, immune responses to COVID-19 vaccines remain largely uncharacterized. Here, we performed multimodal single-cell sequencing on peripheral blood of patients with acute COVID-19 and healthy volunteers before and after receiving the SARS-CoV-2 BNT162b2 mRNA vaccine to compare the immune responses elicited by the virus and by this vaccine. Phenotypic and transcriptional profiling of immune cells, coupled with reconstruction of the B and T cell antigen receptor rearrangement of individual lymphocytes, enabled us to characterize and compare the host responses to the virus and to defined viral antigens. While both infection and vaccination induced robust innate and adaptive immune responses, our analysis revealed significant qualitative differences between the two types of immune challenges. In COVID-19 patients, immune responses were characterized by a highly augmented interferon response which was largely absent in vaccine recipients. Increased interferon signaling likely contributed to the observed dramatic upregulation of cytotoxic genes in the peripheral T cells and innate-like lymphocytes in patients but not in immunized subjects. Analysis of B and T cell receptor repertoires revealed that while the majority of clonal B and T cells in COVID-19 patients were effector cells, in vaccine recipients clonally expanded cells were primarily circulating memory cells. Importantly, the divergence in immune subsets engaged, the transcriptional differences in key immune populations, and the differences in maturation of adaptive immune cells revealed by our analysis have far-ranging implications for immunity to this novel pathogen.

Tumor mutations can influence the surrounding microenvironment leading to suppression of anti-tumor immune responses and thereby contributing to tumor progression and failure of cancer therapies. Here we use genetically engineered lung cancer mouse models and patient samples to dissect how LKB1 mutations accelerate tumor growth by reshaping the immune microenvironment. Comprehensive immune profiling of LKB1 -mutant vs wildtype tumors revealed dramatic changes in myeloid cells, specifically enrichment of Arg1 ⁺ interstitial macrophages and SiglecF ^Hi neutrophils. We discovered a novel mechanism whereby autocrine LIF signaling in Lkb1 -mutant tumors drives tumorigenesis by reprogramming myeloid cells in the immune microenvironment. Inhibiting LIF signaling in Lkb1 -mutant tumors, via gene targeting or with a neutralizing antibody, resulted in a striking reduction in Arg1 ⁺ interstitial macrophages and SiglecF ^Hi neutrophils, expansion of antigen specific T cells, and inhibition of tumor progression. Thus, targeting LIF signaling provides a new therapeutic approach to reverse the immunosuppressive microenvironment of LKB1 -mutant tumors.