Recombinant intracellular antibodies for molecular gene therapy of HIV-1 infection

Tese de doutoramento em Farmácia (Microbiologia), apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2008The spread of HIV-1 has been dramatic since the early eighties, when the virus was discovered as the causative agent of AIDS. In the absence of an effective vaccine against HIV, a worldwide search has been made in the past two decades to develop small-molecule inhibitors to target essential steps in the viral cycle. Over the recent years, gene therapy has been highly regarded as a new form of molecular medicine in treatment of HIV/AIDS, either asan alternative or as a complement to anti-retroviral chemotherapy. An intrabody consists of an antibody designed to be expressed intracellularly and directed to different subcellular compartments where they can exert their function more effectively. The binding of an intrabody to its molecular target has the potential to block, suppress, alter or even enhance the process mediated by that molecule. Within this context, intracellular antibodies (intrabodies) represent a new class of neutralizing molecules with potential use in gene therapy approaches. The HIV 1 integrase (IN) protein is currently the focus of an intense research effort to develop new anti-HIV-1 drugs. This enzyme catalyses the integration of HIV genome into the chromosome of the host cell, arguably the most insidious step in the infection process. In the first project of this thesis (Chapter 2), we explored the intracellular immunization approach by developing rabbit intrabodies against the HIV-1 IN protein. We immunized rabbits with HIV-1 IN and developed a combinatorial scFv library againstIN. We were able to identify 5 different scFv's antibodies with high binding activity and specificity to IN. These scFv's bound simultaneously to the catalytic and C-terminus domains of IN. In addition, these antibodies have the ability to inhibit the strand transfer processing. Intrabody-expressing cells, either in their cytoplasm or nuclear compartments, were highly resistant to HIV-1 infection. Importantly, when HIV-1 particles where produced in the presence of anti-IN scFv, the expression of intrabodies did not affect virion production significantly. However, it markedly reduced the infectivity of progeny virions due to the incorporation of anti-IN scFv into the viral particles. These findings provide proof-in-principle that rabbit anti- IN intrabodies can be designed to block early and late stages of HIV-1 replication. As a result, our intrabodies might be useful agents for "intracellular immunization"- and used as new tools to study the structure and function of HIV-1 IN due to their epitope binding characteristics. Another potential target for HIV-1 treatment is Vif. This viral protein overcomes the innate antiviral activity of a cytidine deaminase APOBEC3G that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. We previously demonstrated that anti-Vif scFv and camelized VH intrabodies are an effective approach to inhibit this crucial step of the viral replication cycle. In the second project of this thesis (Chapter 3), we showed that the rabbit VL domain can also be very potentially used as an intrabody. Our results demonstrate that the anti- Vif VL single-domain preserve the antigen-binding activity and specificity in the absence of the parent VH domain. In addition, the VL single domain was highly expressed in microbial cell culture and show favourable biophysical properties. The expression of the VL intrabody in eukaryotic cells also showed that the rabbit VL was correctly folded as soluble protein in the reducing environment and could strongly neutralize HIV-1 infectivity. Therefore, the present study suggests that rabbit VL single-domains have also an enormous value as intracellular antigen recognition units. Lentiviral vectors are among the most efficient tools for gene delivery into mammalian cells. A major goal of lentiviral gene delivery systems is to develop vectors that can efficiently target specific cell types. In the last project of this thesis (Chapter 4), we attempted to generate viral particles for targeting gene delivery. To achieve this goal we have used CCR5-positive cells as the target for our strategy. We designed a novel Sindbis pseudotyped vector where the Sindbis E2 receptor binding envelope protein was modified to directly encode a scFv against the CCR5 chemokine receptor. Targeting into specific cells was mediated by the anti-CCR5 scFv display, and viral titers were close to 106 EGFP transduction units/ml. Our data demonstrate that the length of the peptide linker that connects the heavy chain and light chain of anti-CCR5 scFv significantly affects the efficiency of infection. Infection levels obtained with Sindbis envelope displaying a scFv with a longer linker was consistently higher than that with Sindbis envelope displaying a scFv with a short linker. The results presented show that chimeric scFv-Sindbis pseudotyped lentiviral vectors have the potential to become an efficient and broadly applicable approach for targeting gene delivery to specific cells. Furthermore, this strategy has the potential to become a powerful approach for targeting gene delivery in anti- HIV gene therapy due to the important role of CCR5 expression in disease progression.Fundação para a Ciência e a Tecnologia (FCT), (SFH/BD/17039/2004

Chemokine-directed tumor microenvironment modulation in cancer immunotherapy

Research Areas: Biochemistry & Molecular Biology. ChemistryChemokines are a large family of small chemotactic cytokines that coordinates immune cell trafficking. In cancer, they have a pivotal role in the migration pattern of immune cells into the tumor, thereby shaping the tumor microenvironment immune profile, often towards a pro-tumorigenic state. Furthermore, chemokines can directly target non-immune cells in the tumor microenvironment, including cancer, stromal and vascular endothelial cells. As such, chemokines participate in several cancer development processes such as angiogenesis, metastasis, cancer cell proliferation, stemness and invasiveness, and are therefore key determinants of disease progression, with a strong influence in patient prognosis and response to therapy. Due to their multifaceted role in the tumor immune response and tumor biology, the chemokine network has emerged as a potential immunotherapy target. Under the present review, we provide a general overview of chemokine effects on several tumoral processes, as well as a description of the currently available chemokine-directed therapies, highlighting their potential both as monotherapy or in combination with standard chemotherapy or other immunotherapies. Finally, we discuss the most critical challenges and prospects of developing targeted chemokines as therapeutic options.info:eu-repo/semantics/publishedVersio

Liposomes as a nanoplatform to improve the delivery of antibiotics into Staphylococcus aureus biofilms

Research Areas: Pharmacology & PharmacyABSTRACT - Staphylococcus aureus biofilm-associated infections are a major public health concern. Current therapies are hampered by reduced penetration of antibiotics through biofilm and low accumulation levels at infected sites, requiring prolonged usage. To overcome these, repurposing antibiotics in combination with nanotechnological platforms is one of the most appealing fast-track and costeffective approaches. In the present work, we assessed the potential therapeutic benefit of three antibiotics, vancomycin, levofloxacin and rifabutin (RFB), through their incorporation in liposomes. Free RFB displayed the utmost antibacterial effect with MIC and MBIC50 below 0.006 µg/mL towards a methicillin susceptible S. aureus (MSSA). RFB was selected for further in vitro studies and the influence of different lipid compositions on bacterial biofilm interactions was evaluated. Although positively charged RFB liposomes displayed the highest interaction with MSSA biofilms, RFB incorporated in negatively charged liposomes displayed lower MBIC50 values in comparison to the antibiotic in the free form. Preliminary safety assessment on all RFB formulations towards osteoblast and fibroblast cell lines demonstrated that a reduction on cell viability was only observed for the positively charged liposomes. Overall, negatively charged RFB liposomes are a promising approach against biofilm S. aureus infections and further in vivo studies should be performed.info:eu-repo/semantics/publishedVersio

Canine parvovirus : a predicting canine model for sepsis

Research Areas: Veterinary SciencesBackground: Sepsis is a severe condition associated with high prevalence and mortality rates. Parvovirus enteritis is a predisposing factor for sepsis, as it promotes intestinal bacterial translocation and severe immunosuppression. This makes dogs infected by parvovirus a suitable study population as far as sepsis is concerned. The main objective of the present study was to evaluate the differences between two sets of SIRS (Systemic Inflammatory Response Syndrome) criteria in outcome prediction: SIRS 1991 and SIRS 2001. The possibility of stratifying and classifying septic dogs was assessed using a proposed animal adapted PIRO (Predisposition, Infection, Response and Organ dysfunction) scoring system. Results: The 72 dogs enrolled in this study were scored for each of the PIRO elements, except for Infection, as all were considered to have the same infection score, and subjected to two sets of SIRS criteria, in order to measure their correlation with the outcome. Concerning SIRS criteria, it was found that the proposed alterations on SIRS 2001 (capillary refill time or mucous membrane colour alteration) were significantly associated with the outcome (OR = 4.09, p < 0.05), contrasting with the 1991 SIRS criteria (p = 0.352) that did not correlate with the outcome. No significant statistical association was found between Predisposition (p = 1), Response (p = 0.1135), Organ dysfunction (p = 0.1135), total PIRO score (p = 0.093) and outcome. To explore the possibility of using the SIRS criteria as a fast decision-making tool, a Fast-andFrugal tree (FFT) was created with a sensitivity of 92% and a specificity of 29%. Conclusion: These results suggest that increasing the SIRS criteria specificity may improve their prognostic value and their clinical usefulness. In order to improve the proposed PIRO scoring system outcome prediction ability, more specific criteria should be added, mainly inflammatory and organ dysfunction biomarkers.info:eu-repo/semantics/publishedVersio

Highly specific blood-brain barrier transmigrating single-domain antibodies selected by an In Vivo phage display screening

Research Areas: Pharmacology & PharmacyA major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.info:eu-repo/semantics/publishedVersio

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy

Research Areas: Science & TechnologyAnti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identifed six amino acid diferences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived singledomain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.info:eu-repo/semantics/publishedVersio

Novel peptides derived from dengue virus capsid protein translocate reversibly the blood−brain barrier through a receptor-free mechanism

© 2017 American Chemical SocietyThe delivery of therapeutic molecules to the central nervous system is hampered by poor delivery across the blood-brain barrier (BBB). Several strategies have been proposed to enhance transport into the brain, including invasive techniques and receptor-mediated transport (RMT). Both approaches have several drawbacks, such as BBB disruption, receptor saturation, and off-target effects, raising safety issues. Herein, we show that specific domains of Dengue virus type 2 capsid protein (DEN2C) can be used as trans-BBB peptide vectors. Their mechanism of translocation is receptor-independent and consistent with adsorptive-mediated transport (AMT). One peptide in particular, named PepH3, reaches equilibrium distribution concentrations across the BBB in less than 24 h in a cellular in vitro assay. Importantly, in vivo biodistribution data with radiolabeled peptide derivatives show high brain penetration. In addition, there is fast clearance from the brain and high levels of excretion, showing that PepH3 is a very good candidate to be used as a peptide shuttle taking cargo in and out of the brain.The authors thank the Portuguese Funding Agency, Fundação para a Ciência e a Tecnologia, FCT IP, for financial support (grants SFRH/BPD/94466/2013; SFRH/BPD/109010/2015; IF/01010/2013; PTDC/BBBNAN/1578/2014; HIVERA/ 0002/2013) and Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE), call 20-MSCARISE-2014 (grant agreement H20 644167 − INPACT). M.M., L.G., C.F., and J.D.G.C. gratefully acknowledge FCT support through the UID/Multi/04349/2013 project.info:eu-repo/semantics/publishedVersio

Characterization of Plasma Labile Heme in Hemolytic Conditions

The deposited article is the accepted manuscript (post-print version) posted online 7 August 2017 and provided by The Febs Journal. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. The deposited article version contains attached the supplementary materials within the pdf. This publication hasn't any creative commons license associated, although it is in open access.Extracellular hemoglobin (Hb), a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches we demonstrate that labile heme generated during hemolytic conditions is bound to plasma molecules with an affinity higher than 10(-7) M and that 2-8% (~2-5 μM) of the total amount of heme detected in plasma can be internalized by bystander cells, i.e. bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme binding capacity (HBC1/2 ), that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10(-7) M. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme. This article is protected by copyright. All rights reserved.Fundação para a Ciência e Tecnologia grants: (RECI-IMI-IMU-0038-2012, PTDC/SAU-TOX/116627/2010, HMSP-ICT/0018/2011, SFRH/BD/44828/2008, SFRH/BPD/47477/2008, PTDC/SAU-FAR/119173/2010, IF/01010/2013/CP1183/CT0001); ERC grants: (ERC-2011-AdG 294709-DAMAGECONTROL); NHMRC Senior Principal Research Fellowship: (1003484).info:eu-repo/semantics/acceptedVersio

Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugates

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.This work was supported by the Portuguese Funding Agency, Fundação para a Ciência e Tecnologia, FCT IP (SAICT/2017/32085, PTDC/QUI-OUT/3989/2021 and Ph.D. fellowship SFRH/BD/131468/2017 to ASA and SFRH/BD/90514/2012 to JD). CIISA has provided support through Project UIDB/00276/2020, funded by FCT and LA/P/0059/2020-AL4AnimalS. Research Institute for Medicines (iMed.ULisboa) acknowledges the financial support of Fundação para a Ciência e Tecnologia (Projects: PTDC/QUI-OUT/3989/2021; UIDB/04138/2020 and UIDP/04138/2020). The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).info:eu-repo/semantics/publishedVersio

Inibição da replicação do vírus da imunodeficiência humana por anticorpos intracelulares de domínio único VH derivados de coelho

Mestrado em Métodos Biomoleculares AvançadosO Vif (Factor de Infecciosidade Viral) é uma proteína acessória do Vírus da Imunodeficiência Humana (VIH) cuja função principal é bloquear a acção do Apobec3G, uma citidina deaminase que tem a capacidade de tornar o VIH-1 não infeccioso através da indução de hipermutações G para A durante a síntese do ADN viral. Recentemente desenvolvemos anticorpos recombinantes de cadeia única (scFv) específicos contra o Vif, a partir da construção de uma biblioteca genómica de anticorpos resultantes da imunização de coelhos. Os anticorpos foram expressos no citoplasma celular, mantiveram a sua capacidade de ligação ao Vif e inibiram a replicação do VIH-1. Estes resultados sugerem que este tipo de estratégias de terapia génica poderão vir a ter bastante sucesso no tratamento de doenças infecciosas. No entanto, esta nova forma de terapia por imunização intracelular, apresenta ainda alguns obstáculos. A expressão intracelular dos anticorpos no ambiente redutor do citoplasma é normalmente confrontada com problemas de folding, diminuição da solubilidade, níveis de expressão e tempo de meia vida. Neste contexto, o objectivo desta dissertação foi o desenvolvimento de anticorpos de domínio único VH anti-Vif de coelho mais pequenos e robustos. A abordagem efectuada para construir estes anticorpos intracelulares foi baseada nas características dos anticorpos de cadeia pesada dos camelos (VHH). Os domínios VHH apresentam naturalmente solubilidades e estabilidades elevadas em consequência de substituições específicas de aminoácidos (Phe- 37, Glu-44, Arg-45, Gly-47 e Arg103) na região correspondente à interacção com o domínio VL. Desta forma, neste estudo para aumentar a solubilidade e estabilidade do domínio VH anti-Vif, os aminoácidos hidrofóbicos da região de interface foram substituídos por aminoácidos hidrofílicos dos VHH. Os resultados obtidos indicaram que na ausência do domínio parental VL, todos os VHs em estudo apresentaram um reconhecimento específico da proteína Vif . No entanto, somente os VHs mais camelizados é que apresentaram níveis altos de expressão intracelular. O processo de camelização desenvolvido no domínio VH anti-Vif demonstrou ter um papel importante no aumento do tempo de meia vida intracelular. Os resultados apresentados mostram uma excelente correlação entre o aumento da solubilidade e estabilidade do domínio VH, com o aumento da camelização. A actividade biológica de cada um dos domínios VH demonstrou estar fortemente relacionada com a solubilidade e estabilidade da proteína. Os VHs mais camelizados interferem na transcrição reversa e integração do ADN viral, inibindo a replicação. Esta inibição demostrou estar associada com o aumento dos níveis de expressão do Apobec3G. O presente estudo sugere assim que a camelização de domínios VH derivados de coelho poderá ser uma abordagem bastante promissora para o desenvolvimento anticorpos intracelulares cada vez mais pequenos e robustos para futuras aplicações a nível da terapia génica.The Vif Protein (Viral Infectivity Factor) is an accessory protein of Human Immunodeficiency Virus (HIV), whose main function is to block the antiviral activity of Apobec3G; a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. We recently developed a specific single-chain antibody (scFv) from immunized rabbits to HIV-1 Vif protein that was expressed intracellularly and inhibited reverse transcription and viral replication. These results suggest that this kind of gene therapy approaches may have particular promise in treatment of infected diseases. However, in the reducing environment of the cytoplasm, the intracellular expression of antibodies is normally confronted with folding problems, decreased in solubility, expression levels and half-life of protein. Within this context, our goal was to develop small and robust anti-Vif VH single-domains antibodies derived from rabbits. The strategy to develop this intracellular antibodies where based in camelid antibody domains (VHH). VHH single-domains have naturally high solubility and stability due to specific amino acid substitutions in interface region (Phe37, Glu44, Arg45, Gly47 and W103R). In order to improve the anti-Vif VH solubility and stability required for making these molecules potential intrabodies, we mutated the exposed hydrophobic surface area contacting the VL domain. Our results demonstrate that all constructed anti-Vif VH single-domains preserve the antigen-binding activity and specificity in the absence of the parent VL domain. However, only the most highly camelized domains had high levels of intracellular expression. The expression in eukaryotic cells showed that VH single-domains could correctly fold as soluble proteins in the reducing environment. The results demonstrated an excellent correlation between improvements in protein solubility with gradual increasing camelization. Camelized single-domains efficiently bound Vif protein and neutralized its infectivity enhancing function, by reducing late reverse transcripts and proviral integration. Moreover, cell specificity of anti-Vif intrabodies was correlated with an increase of Apobec3G. The present study strongly suggests that camelization of rabbit VH domains is a potentially useful approach for engineering intrabodies for gene therapy