Following their inoculation by the bite of an infected Anopheles mosquito, the malaria parasite sporozoite forms travel from the bite site in the skin into the bloodstream, which transports them to ...the liver. The thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that is released from secretory organelles and relocalized on the sporozoite plasma membrane. TRAP is required for sporozoite motility and host infection, and its extracellular portion contains adhesive domains that are predicted to engage host receptors. Here, we identified the human platelet-derived growth factor receptor β (hPDGFRβ) as one such protein receptor. Deletion constructs showed that the von Willebrand factor type A and thrombospondin repeat domains of TRAP are both required for optimal binding to hPDGFRβ-expressing cells. We also demonstrate that this interaction is conserved in the human-infective parasite Plasmodium vivax, but not the rodent-infective parasite Plasmodium yoelii. We observed expression of hPDGFRβ mainly in cells associated with the vasculature suggesting that TRAP:hPDGFRβ interaction may play a role in the recognition of blood vessels by invading sporozoites.
The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain ...and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO) cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin) were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.
Vesiviruses, such as San Miguel sea lion virus and feline calicivirus, are typically associated with infection in animal hosts. Following the accidental infection of a laboratory worker with San Miguel sea lion virus, a related virus was isolated in cell culture and named Hom-1. In this study, we found that Hom-1 could be propagated in a number of human cell lines, making it the first calicivirus to replicate efficiently in cultured human cells. Screening of a library of human cell surface membrane proteins showed that the virus could utilize human junctional adhesion molecule 1 as a receptor to enter cells and initiate replication. The Hom-1 virus presents a new system for the study of calicivirus biology and species specificity.
Identifying the cell surface receptors of natural ligands, and deconvoluting the receptor targets of candidate drug leads, presents a challenge in medical research and drug discovery. Traditionally, ...success rates have been low, and screening efforts often generate numerous false-positive hits that require extensive follow-up to either validate or disregard. If successful, receptor identification enables the discovery of previously unknown, disease-relevant targets, provides critical insights into biological pathways and disease processes, and allows for secondary targets to be uncovered. By expressing the majority of the human plasma membrane proteome in human cells on glass slides in situ, human cell microarray technology provides a powerful approach for identifying receptor target interactions. This approach significantly increases the success rates in identifying specific primary receptor targets and off-targets while limiting the number of false-positive hits. Here we describe cell microarray technology, focusing on new advances including the use of whole cells as bait for receptor interactions, and the inclusion of secreted proteins that widens the utility of the technology in off-target screening.
Aggrecan is a critical component of cartilage extracellular matrix. Several members of the 'a disintegrin and metalloproteinase with thrombospondin motifs' (ADAMTS) family have been characterised as ...aggrecanases by their ability to generate fragments containing the NITEGE neoepitope from aggrecan. Increased NITEGE fragments in synovial fluid and articular cartilage are a hallmark of osteoarthritis (OA) and it is hypothesised that the enhanced rate of aggrecan degradation is critical for cartilage destruction in OA. Recently, matrix metalloproteinase 17 (MMP17, also known as MT4-MMP) has been implicated in the activation of one of the key aggrecanases: ADAMTS4. In the present work, the hypothesis that MMP17 mediates the interleukin 1β (IL-1β) induced release of NITEGE neoepitope from human and murine articular cartilage is investigated.
MMP17 was quantified at the protein and RNA level and NITEGE neoepitope generation by immunohistochemistry. Human postmortem articular cartilage explants were treated with recombinant MMP17, or IL-1β in the presence or absence of an MMP17 inhibitor. Glycosaminoglycan (GAG) loss into the media was quantified using the 1,9-dimethylmethylene blue (DMMB) assay. Intra-articular injection (IAI) of IL-1β or meniscotibial ligament transaction was carried out in MMP17 null mice.
The data reveal an association between increased MMP17 protein and NITEGE staining in areas of OA cartilage damage. Ex vivo treatment of normal human cartilage with recombinant MMP17 protein increased NITEGE generation in the cartilage and GAG loss into the media. In addition, IL-1β mediated cartilage GAG loss, and increased NITEGE neoepitope expression, were attenuated with an MMP17 inhibitor. IAI of IL-1β into C57BL6/Jax mice resulted in increased MMP17 expression in articular cartilage and increased GAG content in the synovial fluid. MMP17 null mice were protected against this increase. However, aggrecan loss driven by mechanical stress following medial meniscotibial ligament transection was not dependent on MMP17.
These data further implicate MMP17 in the control of articular cartilage extracellular matrix aggrecan integrity in an inflammatory environment.
Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one ...million deaths every year. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the development of new malaria interventions.
Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like ...orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell–engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.
Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, ...also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs.
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•Generation and validation of a highly diverse naïve rabbit mAb library•>10 billion chimeric rabbit/human Fab clones minable by phage display•Selection of ROR1 and ROR2 mAbs of high diversity, affinity, and specificity•Conversion of ROR1 and ROR2 mAbs to highly potent CAR-Ts
Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for ...drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets.
ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin.
Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model.
Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.
Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy ...targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.
Although the 5-year survival rate of chronic lymphocytic leukemia (CLL) patients has risen to >80%, the only potentially curative treatment is allogeneic hematopoietic stem cell transplantation ...(alloHSCT). To identify possible new monoclonal antibody (mAb) drugs and targets for CLL, we previously developed a phage display-based human mAb platform to mine the antibody repertoire of patients who responded to alloHSCT. We had selected a group of highly homologous post-alloHSCT mAbs that bound to an unknown CLL cell surface antigen. Here, we show through next-generation sequencing of cDNAs encoding variable heavy-chain domains that these mAbs had a relative abundance of ∼0.1% in the post-alloHSCT antibody repertoire and were enriched ∼1,000-fold after three rounds of selection on primary CLL cells. Based on differential RNA-seq and a cell microarray screening technology for discovering human cell surface antigens, we now identify their antigen as Siglec-6. We verified this finding by flow cytometry, ELISA, siRNA knockdown, and surface plasmon resonance. Siglec-6 was broadly expressed in CLL and could be a potential target for antibody-based therapeutic interventions. Our study reaffirms the utility of post-alloHSCT antibody drug and target discovery.
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