Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

Acknowledgements This work was supported by the Chief Scientist Office, Scottish Government, Grant COV/ABN/20/01 (Elasmogen, Ltd.), a 2018 Prostate Cancer Foundation Challenge Award (AML), a 2013 Prostate Cancer Foundation Young Investigator Award (AML), NCI R01s CA237272, CA233562, and CA245922 (AML). WEM was supported by the NIH T32 HL007741 and JMT by the NIH T32 AI055433. JSM was funded by NIGMS R01 GM088790. HA was funded by NIGMS R35 GM118047 and NCI P01 CA234228. X-ray diffraction data were collected at the Northeastern Collaborative Access Team beamlines, which are funded by the US National Institutes of Health (NIGMS P30 GM124165). The Pilatus 6M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10 RR029205). We thank the Marco Pravetoni lab for providing training and access to the OctetRED96e for BLI experiments.Peer reviewedPublisher PD

The Molecular Imaging of Natural Killer Cells

The recent success of autologous T cell-based therapies in hematological malignancies has spurred interest in applying similar immunotherapy strategies to the treatment of solid tumors. Identified nearly 4 decades ago, natural killer (NK) cells represent an arguably better cell type for immunotherapy development. Natural killer cells are cytotoxic lymphocytes that mediate the direct killing of transformed cells with reduced or absent major histocompatibility complex (MHC) and are the effector cells in antibody-dependent cell-mediated cytotoxicity. Unlike T cells, they do not require human leukocyte antigen (HLA) matching allowing for the adoptive transfer of allogeneic NK cells in the clinic. The development of NK cell-based therapies for solid tumors is complicated by the presence of an immunosuppressive tumor microenvironment that can potentially disarm NK cells rendering them inactive. The molecular imaging of NK cells in vivo will be crucial for the development of new therapies allowing for the immediate assessment of therapeutic response and off-target effects. A number of groups have investigated methods for detecting NK cells by optical, nuclear, and magnetic resonance imaging. In this review, we will provide an overview of the advances made in imaging NK cells in both preclinical and clinical studies

Non-invasive imaging and cellular tracking of pulmonary emboli by near-infrared fluorescence and positron-emission tomography.

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements

Imaging active urokinase plasminogen activator in prostate cancer.

The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU). In the full-length immunoglobulin form, U33 IgG labeled with near-infrared fluorophores or radionuclides allowed us to noninvasively detect active uPA in prostate cancer xenograft models using optical and single-photon emission computed tomography imaging modalities. U33 IgG labeled with (111)In had a remarkable tumor uptake of 43.2% injected dose per gram (%ID/g) 72 hours after tail vein injection of the radiolabeled probe in subcutaneous xenografts. In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissemination prostate cancer model that recapitulated metastatic human cancer. The favorable imaging properties were the direct result of U33 IgG internalization through an uPA receptor-mediated mechanism in which U33 mimicked the function of the endogenous inhibitor of uPA to gain entry into the cancer cell. Overall, our imaging probe targets a prostate cancer-associated protease, through a unique mechanism, allowing for the noninvasive preclinical imaging of prostate cancer lesions

Targeting uPAR with Antagonistic Recombinant Human Antibodies in Aggressive Breast Cancer

Components of the plasminogen activation system (PAS) which are overexpressed in aggressive breast cancer subtypes offer appealing targets for development of new diagnostics and therapeutics. By comparing gene expression data in patient populations and cultured cell lines, we identified elevated levels of the urokinase plasminogen activation receptor (uPAR, PLAUR) in highly aggressive breast cancer subtypes and cell lines. Recombinant human anti-uPAR antagonistic antibodies exhibited potent binding in vitro to the surface of cancer cells expressing uPAR. In vivo these antibodies detected uPAR expression in triple negative breast cancer (TNBC) tumor xenografts using near infrared (NIR) imaging and (111)In single-photon emission computed tomography (SPECT). Antibody-based uPAR imaging probes accurately detected small disseminated lesions in a tumor metastasis model, complementing the current clinical imaging standard (18)F-fluorodeoxyglucose (FDG) at detecting non-glucose-avid metastatic lesions. A monotherapy study using the antagonistic antibodies resulted in a significant decrease in tumor growth in a TNBC xenograft model. Additionally, a radioimmunotherapy (RIT) study, using the anti-uPAR antibodies conjugated to the therapeutic radioisotope (177)Lu, found that they were effective at reducing tumor burden in vivo. Taken together, our results offer a preclinical proof of concept for uPAR targeting as a strategy for breast cancer diagnosis and therapy using this novel human antibody technology

Structural Characterization of a Minimal Antibody against Human APOBEC3B

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes