RESUMEN
Opioid use disorders (OUD) and overdoses are ever-evolving public health threats that continue to grow in incidence and prevalence in the United States and abroad. Current treatments consist of opioid receptor agonists and antagonists, which are safe and effective but still suffer from some limitations. Murine and humanized monoclonal antibodies (mAb) have emerged as an alternative and complementary strategy to reverse and prevent opioid-induced respiratory depression. To explore antibody applications beyond traditional heavy-light chain mAbs, we identified and biophysically characterized a novel single-domain antibody specific for fentanyl from a camelid variable-heavy-heavy (VHH) domain phage display library. Structural data suggested that VHH binding to fentanyl was facilitated by a unique domain-swapped dimerization mechanism, which accompanied a rearrangement of complementarity-determining region loops leading to the formation of a fentanyl-binding pocket. Structure-guided mutagenesis further identified an amino acid substitution that improved the affinity and relaxed the requirement for dimerization of the VHH in fentanyl binding. Our studies demonstrate VHH engagement of an opioid and inform on how to further engineer a VHH for enhanced stability and efficacy, laying the groundwork for exploring the in vivo applications of VHH-based biologics against OUD and overdose.
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Fentanilo , Anticuerpos de Dominio Único , Fentanilo/química , Fentanilo/inmunología , Animales , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Humanos , Camelidae/inmunología , Camélidos del Nuevo Mundo , Analgésicos Opioides/química , Analgésicos Opioides/farmacología , Analgésicos Opioides/inmunologíaRESUMEN
The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [89Zr]Zr-1E7-Fc to detect MET expression in vivo by PET/CT imaging. [89Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Anticuerpos de Dominio Único , Humanos , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Tomografía de Emisión de Positrones/métodos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Línea Celular TumoralRESUMEN
Here, we document the discovery of a monoclonal antibody that selectively binds to both human and murine fibroblast activation protein alpha (FAP), a serine protease that is overexpressed on cancer-associated fibroblasts (CAFs), making it an attractive therapeutic target for the aiding and abetting tumor microenvironment. The lead antibody, B12, was identified from a naïve murine single-chain variable fragment antibody phage display library screened against recombinant human FAP on magnetic beads. The heavy and light chains of B12 were cloned into full-length human immunoglobulin 1 (IgG) vectors and expressed as a chimeric monoclonal antibody (B12 IgG). We engineered a drug-resistant prostate cancer cell line, CWR-R1-EnzR, to express human FAP for antibody characterization and validation (R1-EnzRFAP). B12 IgG selectively bound to the R1-EnzRFAP cells by flow cytometry and was internalized in vitro by confocal microscopy. B12 IgG was further evaluated as a near-infrared (NIR) optical imaging probe in R1-EnzRFAP and parental xenograft models. High tumor uptake and retention of the NIR probe was observed in the R1-EnzRFAP xenografts, and endogenous expression of murine stromal origin FAP was detected in the parental xenografts. Ex vivo evaluation of these models by immunohistochemistry documented B12 IgG localization to both human and murine FAP-expressing cells.
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Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas , Neoplasias/patología , Células del Estroma/patología , Animales , Técnicas de Visualización de Superficie Celular , Citometría de Flujo , Humanos , Inmunoglobulina G/inmunología , Ratones , Neoplasias/inmunología , Células del Estroma/inmunologíaRESUMEN
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.
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Células Asesinas Naturales/citología , Imagen Molecular , Humanos , Microscopía Intravital , Mediciones Luminiscentes , Imagen por Resonancia MagnéticaRESUMEN
BACKGROUND: The transmembrane glycoprotein CD133 is believed to be a marker of adult prostate stem cells and cancer stem/initiating cells. Investigating the role of CD133 in the normal biology of the prostate and in cancer is complicated by the lack of a sensitive and accurate antibody for its detection. Here, we describe the characterization of a unique antibody identified using human antibody phage display that can recognize CD133 in both formalin-fixed tissues and cell lines. METHODS: A human single-chain variable fragment (scFv) antibody phage display library possessing a diversity of 8 × 109 was screened against fully glycosylated recombinant CD133. A counter screen was performed against deglycosylated CD133 to select for clones that preferentially recognized a glycosylation-independent epitope. The lead scFv was analyzed by flow cytometry and cloned into a rabbit immunoglobulin scaffold for immunohistochemistry (IHC). RESULTS: The antibody designated HA10 was found to bind a glycosylation-independent epitope on the peptide backbone of CD133 with high affinity. As a reagent for flow cytometry, HA10 detected CD133 more accurately than a commonly used commercially available antibody. IHC analysis with HA10 documented the staining of basal cells and luminal cells in healthy prostate sections. Weak staining of luminal cells was observed in adenocarcinoma sections at a very low frequency. Examination of a LuCaP patient-derived xenograft tissue microarray found that only three of the LuCaP models were positive for CD133. The three CD133pos LuCaP models all originated from non-AR driven metastatic prostate cancer with neuroendocrine differentiation. Subsequent interrogation of liver biopsies from a patient who failed second-generation anti-androgen therapy found high levels of CD133 staining. The original transurethral resection of the prostate from that patient was, however, absent of CD133. CONCLUSIONS: We have developed a novel antibody that was able to detect CD133 by both IHC and flow cytometry. Using HA10 as an IHC reagent, we found that CD133 is a marker for a very rare cell type in both healthy prostate and adenocarcinoma sections. Our preliminary investigation also suggests that there may be an association between CD133 and non-AR driven prostate cancer with neuroendocrine differentiation.
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Antígeno AC133/inmunología , Anticuerpos , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Bacteriófagos , Línea Celular Tumoral , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Próstata/patología , Neoplasias de la Próstata/patologíaRESUMEN
Proteases responsible for the increased peritumoral proteolysis associated with cancer represent functional biomarkers for monitoring tumorigenesis. One attractive extracellular biomarker is the transmembrane serine protease matriptase. Found on the surface of epithelial cells, the activity of matriptase is regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Quantitative mass spectrometry allowed us to show that, in selected cancers, HAI-1 expression decreases, leading to active matriptase. A preclinical probe specific for the measurement of emergent active matriptase was developed. Using an active-site-specific, recombinant human antibody for matriptase, we found that the selective targeting of active matriptase can be used to visualize the tumorigenic epithelium. Live-cell fluorescence imaging validated the selectivity of the antibody in vitro by showing that the probe localized only to cancer cell lines with active matriptase on the surface. Immunofluorescence with the antibody documented significant levels of active matriptase in 68% of primary and metastatic colon cancer sections from tissue microarrays. Labeling of the active form of matriptase in vivo was measured in human colon cancer xenografts and in a patient-derived xenograft model using near-infrared and single-photon emission computed tomography imaging. Tumor uptake of the radiolabeled antibody, (111)In-A11, by active matriptase was high in xenografts (28% injected dose per gram) and was blocked in vivo by the addition of a matriptase-specific variant of ecotin. These findings suggest, through a HAI-1-dependent mechanism, that emergent active matriptase is a functional biomarker of the transformed epithelium and that its proteolytic activity can be exploited to noninvasively evaluate tumorigenesis in vivo.
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Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/metabolismo , Células Epiteliales/metabolismo , Imagen Óptica/métodos , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Serina Endopeptidasas/metabolismo , Anticuerpos/inmunología , Humanos , Radioisótopos de Indio/metabolismo , Espectrometría de Masas , Serina Endopeptidasas/inmunologíaRESUMEN
Adeno-associated virus has been remarkably successful in the clinic, but its broad tropism is a practical limitation of precision gene therapy. A promising path to engineer AAV tropism is the addition of binding domains to the AAV capsid that recognize cell surface markers present on a targeted cell type. We have recently identified two previously unexplored capsid regions near the 2-fold valley and 5-fold pore of the AAV capsid that are amenable to insertion of larger protein domains including nanobodies. Here, we demonstrate that these hotspots facilitate AAV tropism switching through simple nanobody replacement without extensive optimization in both VP1 and VP2. We demonstrate highly specific targeting of human cancer cells expressing fibroblast activating protein (FAP). Our data suggest that engineering VP2 is the preferred path for maintaining both virus production yield and infectivity. Our study shows that nanobody swapping at multiple capsid location is a viable strategy for nanobody-directed cell-specific AAV targeting.
RESUMEN
Adeno-associated virus (AAV) has been remarkably successful in the clinic, but its broad tropism is a practical limitation of precision gene therapy. A promising path to engineer AAV tropism is the addition of binding domains to the AAV capsid that recognize cell surface markers present on a targeted cell type. We have recently identified two previously unexplored capsid regions near the 2/5-fold wall and 5-fold pore of the AAV capsid that are amenable to insertion of larger protein domains, including nanobodies. Here, we demonstrate that these hotspots facilitate AAV tropism switching through simple nanobody replacement without extensive optimization in both VP1 and VP2. Our data suggest that engineering VP2 is the preferred path for maintaining both virus production yield and infectivity. We demonstrate highly specific targeting of human cancer cells expressing fibroblast activating protein (FAP). Furthermore, we found that the combination of FAP nanobody insertion plus ablation of the heparin binding domain can reduce off-target infection to a minimum, while maintaining a strong infection of FAP receptor-positive cells. Taken together, our study shows that nanobody swapping at multiple capsid locations is a viable strategy for nanobody-directed cell-specific AAV targeting.
RESUMEN
Cancer-associated fibroblasts (CAF) are a prominent cell type within the tumor microenvironment (TME) where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease fibroblast activation protein (FAP) is expressed on the surface of highly protumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating protumorigenic CAFs will disrupt the cross-talk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell coculture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the proinflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of proinflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies is through effects on the immune microenvironment and antitumor immune response. SIGNIFICANCE: The direct elimination of FAP-expressing CAFs disrupts the cross-talk with cancer cells leading to a proinflammatory response and alterations in the immune microenvironment and antitumor immune response.
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Fibroblastos Asociados al Cáncer , Endopeptidasas , Inmunoconjugados , Microambiente Tumoral , Humanos , Animales , Inmunoconjugados/farmacología , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/patología , Fibroblastos Asociados al Cáncer/inmunología , Ratones , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Endopeptidasas/genética , Endopeptidasas/metabolismo , Línea Celular Tumoral , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Gelatinasas/metabolismo , Gelatinasas/genética , Oligopéptidos/farmacología , FemeninoRESUMEN
Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with beta1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/beta1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular/efectos de los fármacos , Fragmentos Fab de Inmunoglobulinas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Monoclonales , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Unión Proteica/inmunología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/inmunología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Transducción de Señal/inmunología , Spodoptera , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Metastatic castration-resistant prostate cancer (mCRPC) has been largely resistant to immunotherapy. Natural killer (NK) cells are cytotoxic lymphocytes that detect and kill transformed cells without prior sensitization, and their infiltration into prostate tumors corresponds with an increased overall survival among patients with mCRPC. We sought to harness this knowledge to develop an approach to NK-cell based immunotherapy for mCRPC. We engineered an NK cell line (NK-92MI) to express CD64, the sole human high-affinity IgG Fcγ receptor (FcγR1), and bound these cells with antibodies to provide interchangeable tumor-targeting elements. NK-92MICD64 cells were evaluated for cell-activation mechanisms and antibody-dependent cell-mediated cytotoxicity (ADCC). A combination of mAbs was used to target the prostate tumor antigen tumor-associated calcium signal transducer 2 (TROP2) and the cancer-associated fibroblast marker fibroblast activation protein alpha (FAP). We found that CD64, which is normally expressed by myeloid cells and associates with the adaptor molecule FcRγ, can be expressed by NK-92MI cells and mediate ADCC through an association with CD3ζ. Cytotoxicity from the combination approach was two-fold higher compared to treatment with NK-92MICD64 cells and either mAb alone, and seven-fold higher than NK-92MICD64 cells alone at an effector-target cell ratio of 20:1. The cytotoxic effect was lost when using isotype control antibodies, indicating a selective targeting mechanism. The combination approach demonstrated efficacy in vivo as well and significantly reduced tumor growth compared with the saline control. This combination therapy presents a potential approach for treating mCRPC and could improve immunotherapy response.
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Anticuerpos Monoclonales/metabolismo , Células Asesinas Naturales/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores de IgG/metabolismo , Ingeniería de Tejidos/métodos , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Neoplasias de la Próstata Resistentes a la Castración/mortalidad , Análisis de SupervivenciaRESUMEN
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.
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Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Citidina Desaminasa/inmunología , Citidina Desaminasa/metabolismo , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Menor/metabolismo , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos , Cristalización , Células HEK293 , Humanos , Inmunidad Innata , Simulación de Dinámica Molecular , Unión Proteica , Anticuerpos de Cadena Única/metabolismoRESUMEN
Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking viral receptor angiotensin-converting enzyme 2 (ACE2). The lead drug candidate possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD ~3000 times more tightly than ACE2 did and inhibited SARS-CoV-2 pseudovirus ~160 times more efficiently than ACE2 did. Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models. Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented an excellent in vivo stability and a high tissue bioavailability. As effective and inexpensive drug candidates, Nanosota-1 may contribute to the battle against COVID-19.
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Anticuerpos Antivirales/inmunología , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , COVID-19/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Pandemias , Unión Proteica , Conformación Proteica , Receptores Virales/inmunología , Receptores Virales/metabolismo , Anticuerpos de Dominio Único/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Cristalografía por Rayos X , Receptores de Antígenos/química , Receptores de Antígenos/inmunología , Tiburones/inmunología , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Epítopos , Mutación , Filogenia , Unión Proteica , SARS-CoV-2 , Alineación de Secuencia , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The role of prostate-specific antigen (PSA) or kallikrein-related peptidase 3 (KLK3) as a biomarker for prostate cancer is well known; however, the precise physiological role of it's serine protease activity in prostate cancer remains a mystery. PSA is produced at high levels by both androgen-dependent and -independent prostate cancers. Studies have documented high levels of active PSA in the milieu surrounding osseous and soft tissue metastases. This evidence, coupled with growing experimental evidence, suggests that PSA plays an important role in the pathobiology of prostate cancer. These observations support the development of PSA-selective inhibitors as useful tools for the targeted treatment and imaging of prostate cancer. Here, we review the research that has been conducted to date on developing selective inhibitors for PSA. The different approaches used to determine PSA substrate specificity and for creating inhibitors are discussed. In addition, the unique active site characteristics of PSA and how these motifs aided our research in developing PSA targeted agents are highlighted.
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Imagen Molecular/métodos , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Secuencia de Aminoácidos , Animales , Humanos , Masculino , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Antígeno Prostático Específico/antagonistas & inhibidores , Antígeno Prostático Específico/química , Especificidad por SustratoRESUMEN
PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) is a lethal, heterogeneous disease with few therapeutic strategies that significantly prolong survival. Innovative therapies for mCRPC are needed; however, the development of new therapies relies on accurate imaging to assess metastasis and monitor response. Standard imaging modalities for prostate cancer require improvement and there remains a need for selective and sensitive imaging probes that can be widely used in patients with mCRPC. EXPERIMENTAL DESIGN: We evaluated the transmembrane protease fibroblast activation protein alpha (FAP) as a targetable cell surface antigen for mCRPC. Genomic and IHC analyses were performed to investigate FAP expression in prostate cancer. Our FAP-targeted antibody imaging probe, [89Zr]Zr-B12 IgG, was evaluated by PET/CT imaging in preclinical prostate cancer models. RESULTS: Analysis of patient data documented FAP overexpression in metastatic disease across tumor subtypes. PET imaging with [89Zr]Zr-B12 IgG demonstrated high tumor uptake and long-term retention of the probe in the preclinical models examined. FAP-positive stroma tumor uptake of [89Zr]Zr-B12 IgG was 5-fold higher than the isotype control with mean %ID/cc of 34.13 ± 1.99 versus 6.12 ± 2.03 (n = 3/group; P = 0.0006) at 72 hours. Ex vivo biodistribution corroborated these results documenting rapid blood clearance by 24 hours and high tumor uptake of [89Zr]Zr-B12 IgG by 72 hours. CONCLUSIONS: Our study reveals FAP as a target for imaging the tumor microenvironment of prostate cancer. Validation of [89Zr]Zr-B12 IgG as a selective imaging probe for FAP-expressing tumors presents a new approach for noninvasive PET/CT imaging of mCRPC.
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Proteínas de la Membrana/antagonistas & inhibidores , Imagen Molecular/métodos , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Próstata/diagnóstico por imagen , Neoplasias de la Próstata Resistentes a la Castración/diagnóstico , Animales , Línea Celular Tumoral , Endopeptidasas/metabolismo , Células HEK293 , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/farmacocinética , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/patología , RNA-Seq , Radioisótopos/administración & dosificación , Radioisótopos/farmacocinética , Radiofármacos/administración & dosificación , Radiofármacos/farmacocinética , Distribución Tisular , Microambiente Tumoral , Microtomografía por Rayos X , Circonio/administración & dosificación , Circonio/farmacocinéticaRESUMEN
PURPOSE: Aggressive variant prostate cancer (AVPC) is a nonandrogen receptor-driven form of disease that arises in men in whom standard-of-care therapies have failed. Therapeutic options for AVPC are limited, and the development of novel therapeutics is significantly hindered by the inability to accurately quantify patient response to therapy by imaging. Imaging modalities that accurately and sensitively detect the bone and visceral metastases associated with AVPC do not exist. EXPERIMENTAL DESIGN: This study investigated the transmembrane protein CD133 as a targetable cell surface antigen in AVPC. We evaluated the expression of CD133 by microarray and IHC analysis. The imaging potential of the CD133-targeted IgG (HA10 IgG) was evaluated in preclinical prostate cancer models using two different imaging modalities: near-infrared and PET imaging. RESULTS: Evaluation of the patient data demonstrated that CD133 is overexpressed in a specific phenotype of AVPC that is androgen receptor indifferent and neuroendocrine differentiated. In addition, HA10 IgG was selective for CD133-expressing tumors in all preclinical imaging studies. PET imaging with [89Zr]Zr-HA10 IgG revealed a mean %ID/g of 24.30 ± 3.19 in CD133-positive metastatic lesions as compared with 11.82 ± 0.57 in CD133-negative lesions after 72 hours (P = 0.0069). Ex vivo biodistribution showed similar trends as signals were increased by nearly 3-fold in CD133-positive tumors (P < 0.0001). CONCLUSIONS: To our knowledge, this is the first study to define CD133 as a targetable marker of AVPC. Similarly, we have developed a novel imaging agent, which is selective for CD133-expressing tumors, resulting in a noninvasive PET imaging approach to more effectively detect and monitor AVPC.
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Antígeno AC133/metabolismo , Anticuerpos Monoclonales/farmacología , Biomarcadores de Tumor/metabolismo , Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Neoplasias de la Próstata/diagnóstico por imagen , Radioisótopos/farmacocinética , Circonio/farmacocinética , Antígeno AC133/antagonistas & inhibidores , Antígeno AC133/inmunología , Animales , Biomarcadores de Tumor/inmunología , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Radiofármacos/farmacocinética , Receptores Androgénicos/metabolismo , Distribución Tisular , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag ( Nanosota-1C-Fc ) bound to SARS-CoV-2 RBD with a K d of 15.7picomolar (â¼3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND 50 of 0.16microgram/milliliter (â¼6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-F c documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic. IMPACT STATEMENT: Potent and low-cost Nanosota-1 drugs block SARS-CoV-2 infections both in vitro and in vivo and act both preventively and therapeutically.
RESUMEN
Prostate-specific antigen (PSA), a serine protease belonging to the human kallikrein family, is best known as a prostate cancer biomarker. Emerging evidence suggests that PSA may also play a salient role in prostate cancer development and progression. With large amounts of enzymatically active PSA continuously and selectively produced by all stages of prostate cancer, PSA is an attractive target. PSA inhibitors, therefore, may represent a promising class of therapeutics and/or imaging agents. PSA displays chymotrypsin-like specificity, cleaving after hydrophobic residues, in addition to possessing a unique ability to cleave after glutamine in the P1 position. In this study, we investigated the structural motifs of the PSA S1 pocket that give it a distinct architecture and specificity when compared to the S1 pocket of chymotrypsin. Using the previously described PSA substrate Ser-Ser-Lys-Leu-Gln (SSKLQ) as a template, peptide aldehyde based inhibitors containing novel P1 aldehydes were made and tested against both proteases. Glutamine derivative aldehydes were highly specific for PSA while inhibitors with hydrophobic P1 aldehydes were potent inhibitors of both proteases with K(i) values <500 nM. The crystal structure of PSA was used to generate a model that allowed GOLD docking studies to be performed to further understand the critical interactions required for inhibitor binding to the S1 pockets of PSA and chymotrypsin. In conclusion, these results provide experimental and structural evidence that the S1 specificity pocket of PSA is distinctly different from that of chymotrypsin and that the development of highly specific PSA inhibitors is feasible.
Asunto(s)
Quimotripsina/metabolismo , Inhibidores Enzimáticos/química , Péptidos/metabolismo , Antígeno Prostático Específico/metabolismo , Homología Estructural de Proteína , Aldehídos/antagonistas & inhibidores , Aldehídos/metabolismo , Secuencias de Aminoácidos , Animales , Bovinos , Quimotripsina/antagonistas & inhibidores , Quimotripsina/química , Inhibidores Enzimáticos/síntesis química , Glutamina/antagonistas & inhibidores , Glutamina/metabolismo , Humanos , Péptidos/antagonistas & inhibidores , Antígeno Prostático Específico/antagonistas & inhibidores , Antígeno Prostático Específico/química , Especificidad por SustratoRESUMEN
Prostate Specific Antigen's (PSA) role as a biomarker for prostate cancer is well established but the physiological role of its serine protease activity in the pathobiology of normal prostate and prostate carcinogenesis remains largely unknown. In light of recent studies that implicate PSA's enzymatic activity in the initiation and/or progression of prostate cancer, we performed a molecular modeling study of substrate binding at the catalytic site of PSA wherein a PSA-selective substrate (HSSKLQ) was docked in an acyl-enzyme conformation to a three-dimensional homology model of PSA. Additionally, virtual positional scanning studies were conducted to gain mechanistic insights into substrate recognition of PSA. Subsequently, 13 novel peptide substrates of 6-aa length and four peptide substrates with varying length were synthesized and assayed for PSA hydrolysis to evaluate the experimental validity of docking insights. Additionally, six novel aldehyde-containing transition state analog inhibitors were synthesized and tested for their inhibitory potencies. The experimental data on the hydrolysis rates of the newly synthesized substrates and inhibitory potencies of the aldehyde peptides agreed with the docking predictions, providing validation of the docking methodology and demonstrating its utility towards the design of substrate-mimetic inhibitors that can be used to explore PSA's role in the pathobiology of prostate cancer.