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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.
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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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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.
Assuntos
Fibroblastos Associados a Câncer , Endopeptidases , Imunoconjugados , Microambiente Tumoral , Humanos , Animais , Imunoconjugados/farmacologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Fibroblastos Associados a Câncer/imunologia , Camundongos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Endopeptidases/genética , Endopeptidases/metabolismo , Linhagem Celular Tumoral , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Gelatinases/metabolismo , Gelatinases/genética , Oligopeptídeos/farmacologia , FemininoRESUMO
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.
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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 Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Anticorpos de Domínio Único , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Tomografia por Emissão de Pósitrons/métodos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Linhagem Celular TumoralRESUMO
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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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Cristalografia por Raios X , Receptores de Antígenos/química , Receptores de Antígenos/imunologia , Tubarões/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , COVID-19 , Epitopos , Mutação , Filogenia , Ligação Proteica , SARS-CoV-2 , Alinhamento de Sequência , Anticorpos de Domínio Único , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
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.
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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 Membrana/antagonistas & inibidores , Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Próstata/diagnóstico por imagem , Neoplasias de Próstata Resistentes à Castração/diagnóstico , Animais , Linhagem Celular Tumoral , Endopeptidases/metabolismo , Células HEK293 , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacocinética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/patologia , RNA-Seq , Radioisótopos/administração & dosagem , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Microambiente Tumoral , Microtomografia por Raio-X , Zircônio/administração & dosagem , Zircônio/farmacocinéticaRESUMO
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 , Anticorpos Monoclonais/farmacologia , Biomarcadores Tumorais/metabolismo , Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/diagnóstico por imagem , Radioisótopos/farmacocinética , Zircônio/farmacocinética , Antígeno AC133/antagonistas & inibidores , Antígeno AC133/imunologia , Animais , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/farmacocinética , Receptores Androgênicos/metabolismo , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rational drug design creates innovative therapeutics based on knowledge of the biological target to provide more effective and responsible therapeutics. Chagas disease, endemic throughout Latin America, is caused by Trypanosoma cruzi, a protozoan parasite. Current therapeutics are problematic with widespread calls for new approaches. Researchers are using rational drug design for Chagas disease and one target receiving considerable attention is the T. cruzi trans-sialidase protein (TcTS). In T. cruzi, trans-sialidase catalyzes the transfer of sialic acid from a mammalian host to coat the parasite surface membrane and avoid immuno-detection. However, the role of TcTS in pathology variance among and within genetic variants of the parasite is not well understood despite numerous studies. Previous studies reported the crystalline structure of TcTS and the TS protein structure in other trypanosomes where the enzyme is often inactive. However, no study has examined the role of natural selection in genetic variation in TcTS. To understand the role of natural selection in TcTS DNA sequence and protein variation, we examined a 471â¯bp portion of the TcTS gene from 48â¯T. cruzi samples isolated from insect vectors. Because there may be multiple parasite genotypes infecting one insect and there are multiple copies of TcTS per parasite genome, all 48 sequences had multiple polymorphic bases. To resolve these polymorphisms, we examined cloned sequences from two insect vectors. The data are analyzed to understand the role of natural selection in shaping genetic variation in TcTS and interpreted in light of the possible role of TcTS as a drug target. The analysis highlights negative or purifying selection on three amino acids previously shown to be important in TcTS transfer activity. One amino acid in particular, Tyr342, is a strong candidate for a drug target because it is under negative selection and amino acid substitutions inactivate TcTS transfer activity. AUTHOR SUMMARY: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and transmitted to humans and other mammals primarily by Triatomine insects. Being endemic in many South and Central American countries and affecting millions of people the need for new more effective and safe therapies is evident. Here, we examine genetic variation and natural selection on DNA (471â¯bp) and amino acid (157â¯aa) sequence data of the T. cruzi trans-sialdiase (TcTS) protein, often suggested as a candidate for rational drug design. In our surveyed region of the protein there were five amino acid residues that have been shown to be integral to the function of TcTS. We found that three were under strong negative selection making them ideal candidates for drug design; however, one was under balancing selection and should be avoided as a drug target. Our study provides new information into identifying potential targets for a new Chagas drug.