Switchable assembly and function of antibody complexes in vivo using a small molecule.

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.

Human antibody-based chemically induced dimerizers for cell therapeutic applications.

Chemically induced dimerizers (CIDs) have emerged as one of the most powerful tools for artificially regulating signaling pathways in cells; however, currently available CID systems lack the properties desired for use in regulating cellular therapies. Here, we report the development of human antibody-based chemically induced dimerizers (AbCIDs) from known small-molecule-protein complexes by selecting for synthetic antibodies that recognize the chemical epitope created by the bound small molecule. We demonstrate this concept by generating three antibodies that are highly selective for the BCL-xL-ABT-737 complex compared to BCL-xL alone. We show the potential of AbCIDs for application in regulating human cell therapies by using them to induce CRISPRa-mediated gene expression and to regulate CAR T-cell activation. We believe that the AbCIDs generated in this study will find application in regulating cell therapies and that the general method of AbCID development may lead to the creation of many new and orthogonal CIDs.

Engineering Improved Antiphosphotyrosine Antibodies Based on an Immunoconvergent Binding Motif.

Phosphotyrosine (pY) is one of the most highly studied posttranslational modifications that is responsible for tightly regulating many signaling pathways in eukaryotes. Pan-specific pY antibodies have emerged as powerful tools for understanding the role of these modifications. Nevertheless, structures have not been reported for pan-specific pY antibodies, greatly impeding the further development of tools for integrating this ubiquitous posttranslational modification using structure-guided designs. Here, we present the first crystal structures of two widely utilized pan-specific pY antibodies, PY20 and 4G10. The two antibodies, although developed independently from animal immunizations, have surprisingly similar modes of recognition of the phosphate group, implicating a generic binding structure among pan-specific pY antibodies. Sequence alignments revealed that many pY binding residues are predominant in the mouse V germline genes, which consequently led to the convergent antibodies. On the basis of the convergent structure, we designed a phage display library by lengthening the CDR-L3 loop with the aid of computational modeling. Panning with this library resulted in a series of 4G10 variants with 4 to 11-fold improvements in pY binding affinities. The crystal structure of one improved variant showed remarkable superposition to the computational model, where the lengthened CDR-L3 loop creates an additional hydrogen bond indirectly bound to the phosphate group via a water molecule. The engineered variants exhibited superior performance in Western blot and immunofluorescence.

Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers.

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.

Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection.

Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle immunoassays or lateral flow assays, which are either laborious, expensive or lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost, solution-based assay to detect antibodies in serum, plasma, whole blood and to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.

Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection.

Current serology tests for SARS-CoV-2 antibodies mainly take the form of enzyme-linked immunosorbent assays or lateral flow assays, with the former being laborious and the latter being expensive and often lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost solution-based assay to detect antibodies in serum, plasma, whole blood, and saliva, using rationally designed split luciferase antibody biosensors (spLUC). This new assay, which generates quantitative results in as short as 5 minutes, substantially reduces the complexity and improves the scalability of COVID-19 antibody tests for point-of-care and broad population testing.

Theranostic Targeting of CUB Domain Containing Protein 1 (CDCP1) in Pancreatic Cancer.

PURPOSE: The recent emergence of radioligand therapies for cancer treatment has increased enthusiasm for developing new theranostic strategies coupling both imaging and cytotoxicity in the same entity. In this study, we evaluated whether CUB domain containing protein 1 (CDCP1), a single-pass transmembrane protein highly overexpressed in diverse human cancers, might be a target for cancer theranostics. EXPERIMENTAL DESIGN: The ectodomain of CDCP1 was targeted using radiolabeled forms of 4A06, a potent and specific recombinant human antibody that we developed. Imaging and antitumor assessment studies were performed in animal models of pancreatic cancer, including two patient-derived xenograft models we developed for this study. For antitumor assessment studies, the endpoints were death due to tumor volume >3,000 mm3 or ≥20% loss in body weight. Specific tracer binding or antitumor effects were assessed with an unpaired, two-tailed Student t test and survival advantages were assessed with a log rank (Mantel-Cox) test. Differences at the 95% confidence level were interpreted to be significant. RESULTS: 89Zr-4A06 detected a broad dynamic range of full length or cleaved CDCP1 expression on seven human pancreatic cancer tumors (n = 4/tumor). Treating mice with single or fractionated doses of 177Lu-4A06 significantly reduced pancreatic cancer tumor volume compared with mice receiving vehicle or unlabeled 4A06 (n = 8; P < 0.01). A single dose of 225Ac-4A06 also inhibited tumor growth, although the effect was less profound compared with 177Lu-4A06 (n = 8; P < 0.01). A significant survival advantage was imparted by 225Ac-4A06 (HR = 2.56; P < 0.05). CONCLUSIONS: These data establish that CDCP1 can be exploited for theranostics, a finding with widespread implications given its breadth of overexpression in cancer.

A peptide antagonist of the TLR4-MD2 interaction.

Toll-like receptors are an integral part of innate immunity in the central nervous system (CNS); they orchestrate a robust defense in response to both exogenous and endogenous danger signals. Recently, toll-like receptor 4 (TLR4) has emerged as a therapeutic target for the treatment of CNS-related diseases such as sepsis and chronic pain. We herein report a chemical biology approach by using a rationally designed peptide inhibitor to disrupt the TLR4-MD2 association, thereby blocking TLR4 signaling.

Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins.

While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRASG12V, and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell.

Development of ß-amino alcohol derivatives that inhibit Toll-like receptor 4 mediated inflammatory response as potential antiseptics.

Toll-like receptor 4 (TLR4) induced proinflammatory signaling has been directly implicated in severe sepsis and represents an attractive therapeutic target. Herein, we report our investigations into the structure-activity relationship and preliminary drug metabolism/pharmacokinetics study of ß-amino alcohol derivatives that inhibit the TLR4 signaling pathway. Lead compounds were identified from in vitro cellular examination with micromolar potency for their inhibitory effects on TLR4 signaling and subsequently assessed for their ability to suppress the TLR4-induced inflammatory response in an ex vivo whole blood model. In addition, the toxicology, specificity, solubility, brain-blood barrier permeability, and drug metabolism of several compounds were evaluated. Although further optimizations are needed, our findings lay the groundwork for the future drug development of this class of small molecule agents for the treatment of severe sepsis.

Development of agents that modulate protein-protein interactions in membranes.

Membrane proteins account for approximately one third of all proteins in eukaryotic and prokaryotic cells. These proteins are critical in a diverse array of cellular functions. Despite their obvious importance, the effectiveness of research tools to study the structure and function of integral membrane proteins lags behind that of water-soluble proteins. This is due in part to the lack of probing agents that can specifically and selectively recognize these targets. This review focuses on methods developed to overcome the obstacles of studying membrane proteins. We describe TM protein properties as well as biophysical properties of amino acids within the membrane bilayer. We also summarize the known characteristics of membrane regions in their distinctive environments and generate a summary of current research approaches that succeed in probing interactions of TM proteins within their native setting. This allows further insight into protein-protein interactions in a hydrophobic environment as it pertains to drug development.

Selection, Preparation, and Evaluation of Small- Molecule Inhibitors of Toll-Like Receptor 4.

Toll-like receptor 4 (TLR4), a membrane spanning receptor protein that functions in complex with its accessory protein MD-2, is an intriguing target for therapeutic development. Herein we report the identification of a series of novel TLR4 inhibitors and the development of a robust, enantioselective synthesis using an unprecedented Mannich-type reaction to functionalize a pyrazole ring. In silico and cellular assay results demonstrated that compound 1 and its analogues selectively block TLR4 activation in live cells. Animal model tests showed that 1 and its derivatives could potentiate morphine-induced analgesia in vivo, presumably by attenuating the opioid-induced TLR4 activation.