Projects
The cryoEM structure of an antibody Fab/antigen complex.
Antibody/Antigen Interactions
We have studied antibody/antigen recognition for over four decades. Current projects focus on collaboratively characterizing antibodies under development as novel immunotherapies for leukemias, targeting CD33, CD90, and CD123, and Mesothelin, for a range of solid tumors. Our research pipeline is to express antibodies as intact IgG, Fab, scFv, or engineered variants, characterize binding properties by surface plasmon resonance (SPR / Biacore) or boundary layer interferometry (BLI / Octet), and determine three-dimensional structures of antibodies and antigen complexes by x-ray crystallography or cryoEM. These results feed back into ranking antibodies for therapeutic potential and directed engineering of improved biologics, enabled by the flexibility of the Daedalus lentiviral expression platform.
The basic science component of this project is to use antibody reagents as co-crystallization or cryoEM chaperones to obtain previously unknown antigen structures otherwise impossible to determine.
The coordination geometry of actinium determined as a Siderocalin/chelator/nuclide complex.
Scn-Cap Radionuclide Delivery
Siderocalin (Scn / neutrophil gelatinase associated lipocalin (NGAL) / Lipocalin 2 (Lcn2) / 24p3) is released by neutrophils and expressed by epithelial cells in response to pro-inflammatory signals. Scn functions by tightly binding iron as bacterial ferric siderophore complexes, clearing iron earmarked for bacterial use through the urinary tract, thus slowing their growth, allowing adaptive immune system responses to subsequently clear the pathogen. We have previously fully characterized the recognition mechanism of Scn for bacterial ferric siderophores. Scn is also an extremely stable protein and has become a workhorse fusion partner in our laboratory to enable expression via Daedalus of a wide range of wildly engineered proteins.
We have also discovered that, by using synthetic “siderophore” analogs (chelators), wide range of other elements can be efficiently captured, including most lanthanoids and many actinides, under physiological buffer conditions and with near-stoichiometric efficiencies. This has enabled development of the Scn capture (“Scn-Cap”) platform: weaponizing proteins targeting tumors with selected radionuclides by fusions with Scn as novel radio-immunotherapeutics.
The basic science components of this project are 1. using crystal structures of Scn / chelator / actinide complexes to parse actinide coordination chemistry and 2. delineating functional Scn / receptor interactions. The Scn literature is rife with putative functions beyond an antibacterial activity, many requiring functional Scn receptors and/or endogenous siderophore analogs. We have previously identified one receptor (Megalin / LRP2), but refuted other candidate receptors (e.g., SLC22A17 / BOCT) and endogenous “siderophores”. Current efforts focus on elucidating the structural details of the Scn / Megalin interaction and confirming other candidate receptors.
Artemis mapping of HLA class I presented Mesothelin peptides by three alleles.
MHC Class I/II Peptide Processing & Presentation
The immune system monitors cellular proteomes through MHC restriction: peptide fragments from endogenous proteins are presented on the cell surface as complexes with MHC class I proteins (pMHC-Is) for recognition by T cell receptors (TCRs) and NKG2x / CD49 or KIR natural killer cell receptors. Peptides, generated by the proteasome or as defective ribosome products, from self-proteins are presented in the absence of disease; peptides from pathogen or tumor proteins are added during infection or cancer. The peptidome is the peptide repertoire present in the class I loading compartment (ER) of a particular cell; the ligandome is the peptide repertoire presented as pMHCs on a particular cell at one point in time. We have developed a streamlined mass-spec protocol for cataloging ligandomes, using lentiviral transduction of engineered, secreted pMHC-Is and target proteins-of-interest: Artemis. Artemis yields tens-of-thousands of peptides per run across multiple replicates with absolute allele specificity. We are also developing a parallel method for MHC class II proteins. Current efforts focus on:
- Detailing the peptide specificity of classical (HLA-ABC) and non-classical (HLA-EG) MHC-Is and MHC-IIs
- Determining the ligand specificity of non-classical HLA-F
- Determining the peptide specificity of non-human MHC-Is across evolution
- Identifying targetable pHLAs from viral oncoproteins (MCV large T antigen, HPV E6/E7) and tumor antigens
- Exploring immunoevasion mechanisms affecting MHC-I processing and presentation
- Assessing the effects of proteasome variation on ligandomes
- Assessing the effects of altered splicing on ligandomes
- Assessing the effects of AIRE (“autoimmune regulator”) on ligandomes
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