Panobinostat-loaded folate targeted liposomes as a promising drug delivery system for treatment of canine B-cell lymphoma.

Introduction: Cancer is a major public health problem with over 19 million cases reported in 2020. Similarly to humans, dogs are also largely affected by cancer, with non-Hodgkin's lymphoma (NHL) among the most common cancers in both species. Comparative medicine has the potential to accelerate the development of new therapeutic options in oncology by leveraging commonalities between diseases affecting both humans and animals. Within this context, in the present study, we investigated the potential of panobinostat (Pan)-loaded folate-targeted PEGylated liposomes (FA-PEG-Pan-Lip) for the treatment of canine B-cell lymphoma, while contributing to new perspectives in comparative oncology. Methods and results: Two formulations were developed, namely: PEG-Pan-Lip and FA-PEG-Pan-Lip. Firstly, folate receptor expression in the CLBL-1 canine B-cell lymphoma cell line was assessed. After confirming receptor expression, both Pan-loaded formulations (PEG-Pan-Lip, FA-PEG-Pan-Lip) demonstrated dose-dependent inhibitory effects on CLBL-1 cell proliferation. The FA-PEG-Pan-Lip formulation (IC50 = 10.9 ± 0.03 nM) showed higher cytotoxicity than the non-targeted PEG-Pan-Lip formulation (IC50 = 12.9 ± 0.03 nM) and the free panobinostat (Pan) compound (IC50 = 18.32±0.03 nM). Moreover, mechanistically, both Pan-containing formulations induced acetylation of H3 histone and apoptosis. Flow cytometry and immunofluorescence analysis of intracellular uptake of rhodamine-labeled liposome formulations in CLBL-1 cells confirmed cellular internalization of PEG-Lip and FA-PEG-Lip formulations and higher uptake profile for the latter. Biodistribution studies of both radiolabeled formulations in CD1 and SCID mice revealed a rapid clearance from the major organs and a 1.6-fold enhancement of tumor uptake at 24 h for 111In-FA-PEG-Pan-Lip (2.2 ± 0.1 %ID/g of tumor) compared to 111In-PEG-Pan-Lip formulation (1.2±0.2 %ID/g of tumor). Discussion: In summary, our results provide new data validating Pan-loaded folate liposomes as a promising targeted drug delivery system for the treatment of canine B-cell lymphoma and open innovative perspectives for comparative oncology.

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

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.

In Silico Characterization of African Swine Fever Virus Nucleoprotein p10 Interaction with DNA.

African swine fever virus (ASFV) is the etiological agent of a highly contagious, hemorrhagic infectious swine disease, with a tremendous sanitary and economic impact on a global scale. Currently, there are no globally available vaccines or treatments. The p10 protein, a structural nucleoprotein encoded by ASFV, has been previously described as capable of binding double-stranded DNA (dsDNA), which may have implications for viral replication. However, the molecular mechanism that governs this interaction is still unknown, mostly due to the lack of a structural model for this protein. In this work, we have generated an ab initio model of the p10 protein and performed extensive structural characterization, using molecular dynamics simulations to identify the motifs and residues regulating DNA recognition. The helix-turn-helix motif identified at the C-terminal region of the protein was shown to be crucial to the dsDNA-binding efficiency. As with other DNA-binding proteins, two distinct serine and lysine-rich regions found in the two helices were identified as key players in the binding to DNA, whose importance was later validated using experimental binding assays. Altogether, these findings may contribute to a better understanding of the p10 function in ASFV replication.

In vivo Phage Display: A promising selection strategy for the improvement of antibody targeting and drug delivery properties.

The discovery of hybridoma technology, described by Kohler and Milstein in 1975, and the resulting ability to generate monoclonal antibodies (mAbs) initiated a new era in antibody research and clinical development. However, limitations of the hybridoma technology as a routine antibody generation method in conjunction with high immunogenicity responses have led to the development of alternative approaches for the streamlined identification of most effective antibodies. Within this context, display selection technologies such as phage display, ribosome display, yeast display, bacterial display, and mammalian cell surface display have been widely promoted over the past three decades as ideal alternatives to traditional hybridoma methods. The display of antibodies on phages is probably the most widespread and powerful of these methods and, since its invention in late 1980s, significant technological advancements in the design, construction, and selection of antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. With evolving novel disease targets and the emerging of a new generation of therapeutic antibodies, such as bispecific antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cell therapies, it is clear that phage display is expected to continue to play a central role in antibody development. Nevertheless, for non-standard and more demanding cases aiming to generate best-in-class therapeutic antibodies against challenging targets and unmet medical needs, in vivo phage display selections by which phage libraries are directly injected into animals or humans for isolating and identifying the phages bound to specific tissues offer an advantage over conventional in vitro phage display screening procedures. Thus, in the present review, we will first summarize a general overview of the antibody therapeutic market, the different types of antibody fragments, and novel engineered variants that have already been explored. Then, we will discuss the state-of-the-art of in vivo phage display methodologies as a promising emerging selection strategy for improvement antibody targeting and drug delivery properties.

Occurrence of MDR1 1-delta mutation in herding dog breeds in Portugal.

The impact of drug transporters in veterinary medicine has been recognized in recent years. One of the most well-characterized is the product of the MDR1 gene, P-gp. A 4-bp deletion in the MDR1 gene known since 2001 has been described to affect herding dog breeds. Since many used drugs in veterinary medicine are substrates for P-gp, including the macrocyclic lactones, such as avermectins, this 4-bp deletion causes a pathological condition known as "ivermectin toxicosis." For this reason, it is important to determine the animal status concerning this mutation. In Portugal, the information of the occurrence of this mutation in our breeds is limited. The aim of the present study was to determine the occurrence of this mutation and evaluate its association with Portuguese and non-Portuguese dog breeds in Portugal. To achieve this, a total of 105 animals were studied for the presence of the MDR1 4-bp deletion, 23 of which were from Barbado da Terceira, 10 from Cão da Serra d'Aires, 55 belonging to breeds known to carry the mutation (Australian Shepperd, Border Collie and others) and 17 to other breeds (Labrador Retriever, Jack Russel, and others). Despite the small sample size, we observed the presence of the MDR1 1-delta mutation in previously described breeds and identified this mutation in Barbado da Terceira breed for the first time.

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

Anti-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 identified six amino acid differences 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 single-domain 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.

Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening.

A 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.

Immunotherapeutic Strategies for Canine Lymphoma: Changing the Odds Against Non-Hodgkin Lymphoma.

The new era of immune-oncology has brought complexities and challenges that emphasize the need to identify new strategies and models to develop successful and cost-effective therapies. The inclusion of a canine model in the drug development of cancer immunotherapies is being widely recognized as a valid solution to overcome several hurdles associated with conventional preclinical models. Driven by the success of immunotherapies in the treatment of human non-Hodgkin lymphoma (NHL) and by the remarkable similarities of canine NHL to its human counterpart, canine NHL has been one of the main focus of comparative research. Under the present review, we summarize a general overview of the challenges and prospects of today's cancer immunotherapies and the role that comparative medicine might play in solving the limitations brought by this rapidly expanding field. The state of art of both human and canine NHL and the rationale behind the use of the canine model to bridge the translational gap between murine preclinical studies and human clinical trials are addressed. Finally, a review of currently available immunotherapies for canine NHL is described, highlighting the potential of these therapeutic options.

Chemokine-Directed Tumor Microenvironment Modulation in Cancer Immunotherapy.

Chemokines 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.

Liposomes as Antibiotic Delivery Systems: A Promising Nanotechnological Strategy against Antimicrobial Resistance.

Antimicrobial drugs are key tools to prevent and treat bacterial infections. Despite the early success of antibiotics, the current treatment of bacterial infections faces serious challenges due to the emergence and spread of resistant bacteria. Moreover, the decline of research and private investment in new antibiotics further aggravates this antibiotic crisis era. Overcoming the complexity of antimicrobial resistance must go beyond the search of new classes of antibiotics and include the development of alternative solutions. The evolution of nanomedicine has allowed the design of new drug delivery systems with improved therapeutic index for the incorporated compounds. One of the most promising strategies is their association to lipid-based delivery (nano)systems. A drug's encapsulation in liposomes has been demonstrated to increase its accumulation at the infection site, minimizing drug toxicity and protecting the antibiotic from peripheral degradation. In addition, liposomes may be designed to fuse with bacterial cells, holding the potential to overcome antimicrobial resistance and biofilm formation and constituting a promising solution for the treatment of potential fatal multidrug-resistant bacterial infections, such as methicillin resistant Staphylococcus aureus. In this review, we aim to address the applicability of antibiotic encapsulated liposomes as an effective therapeutic strategy for bacterial infections.

Liposomes as a Nanoplatform to Improve the Delivery of Antibiotics into Staphylococcus aureus Biofilms.

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 cost-effective 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.

Loss of α-gal during primate evolution enhanced antibody-effector function and resistance to bacterial sepsis.

Most mammals express a functional GGTA1 gene encoding the N-acetyllactosaminide α-1,3-galactosyltransferase enzyme, which synthesizes Gal-α1-3Gal-ß1-4GlcNAc (α-gal) and are thus tolerant to this self-expressed glycan. Old World primates including humans, however, carry loss-of-function mutations in GGTA1 and lack α-gal. Presumably, fixation of such mutations was propelled by natural selection, favoring the emergence of α-gal-specific immunity, conferring resistance to α-gal-expressing pathogens. Here, we show that loss of Ggta1 function in mice enhances resistance to bacterial sepsis, irrespectively of α-Gal-specific immunity. Rather, the absence of α-gal from IgG-associated glycans increases IgG effector function via a mechanism associated with enhanced IgG-Fc gamma receptor (FcγR) binding. The ensuing survival advantage against sepsis comes alongside a cost of accelerated reproductive senescence in Ggta1-deleted mice. Mathematical modeling of this trade-off suggests that high exposure to virulent pathogens exerts sufficient selective pressure to fix GGTA1 loss-of-function mutations, as likely occurred during the evolution of primates toward humans.

A Prime-Boost Immunization Strategy with Vaccinia Virus Expressing Novel gp120 Envelope Glycoprotein from a CRF02_AG Isolate Elicits Cross-Clade Tier 2 HIV-1 Neutralizing Antibodies.

Development of new immunogens eliciting broadly neutralizing antibodies (bNAbs) is a main priority for the HIV-1 vaccine field. Envelope glycoproteins from non-B-non-C HIV-1clades have not been fully explored as components of a vaccine. We produced Vaccinia viruses expressing a truncated version of gp120 (gp120t) from HIV-1 clades CRF02_AG, H, J, B, and C and examined their immunogenicity in mice and rabbits. Mice primed with the recombinant Vaccinia viruses and boosted with the homologous gp120t or C2V3C3 polypeptides developed antibodies that bind potently to homologous and heterologous envelope glycoproteins. Notably, a subset of mice immunized with the CRF02_AG-based envelope immunogens developed a cross-reactive neutralizing response against tier 2 HIV-1 Env-pseudoviruses and primary isolates. Rabbits vaccinated with the CRF02_AG-based envelope immunogens also generated potent binding antibodies, and one animal elicited antibodies that neutralized almost all (13 of 16, 81.3%) tier 2 HIV-1 isolates tested. Overall, the results suggest that the novel CRF02_AG-based envelope immunogens and prime-boost immunization strategy elicit the type of immune responses required for a preventive HIV-1 vaccine.

Synthetic antibody discovery against native antigens by CRISPR/Cas9-library generation and endoplasmic reticulum screening.

Despite the significant advances of antibodies as therapeutic agents, there is still much room for improvement concerning the discovery of these macromolecules. Here, we present a new synthetic cell-based strategy that takes advantage of eukaryotic cell biology to produce highly diverse antibody libraries and, simultaneously, link them to a high-throughput selection mechanism, replicating B cell diversification mechanisms. The interference of site-specific recognition by CRISPR/Cas9 with error-prone DNA repair mechanisms was explored for the generation of diversity, in a cell population containing a gene for a light chain antibody fragment. We achieved up to 93% of cells containing a mutated antibody gene after diversification mechanisms, specifically inside one of the antigen-binding sites. This targeted variability strategy was then integrated into an intracellular selection mechanism. By fusing the antibody with a KDEL retention signal, the interaction of antibodies and native membrane antigens occurs inside the endoplasmic reticulum during the process of protein secretion, enabling the detection of high-quality leads for expression and affinity by flow cytometry. We successfully obtained antibody lead candidates against CD3 as proof of concept. In summary, we developed a novel antibody discovery platform against native antigens by endoplasmic synthetic library generation using CRISPR/Cas9, which will contribute to a faster discovery of new biotherapeutic molecules, reducing the time-to-market.

Canine multicentric lymphoma exhibits systemic and intratumoral cytokine dysregulation.

Non-Hodgkin Lymphoma (NHL) is among the most common neoplasias in dogs and humans. Owing to remarkable similarities with its human counterpart, the canine lymphoma (cNHL) model has been proposed as a powerful framework for rapid and clinically relevant translation of novel immunotherapies. However, the establishment of cNHL as a predictive preclinical model has been hampered by the limited characterization of the canine immune system. Cytokines are key players of the interaction between tumor and its microenvironment. In human NHL, multiple cytokines have been linked to the development of lymphoma and are relevant biomarkers for treatment response and prognosis. In contrast, few studies have investigated cytokines in cNHL. Within this context, this study aimed to investigate cytokine regulation in cNHL. A multicentric cNHL biobank was successfully constructed. Cytokine mRNA profiles in tumor tissue and circulating PBMC were analyzed by qRT-PCR and compared to a healthy control group. Specific primers were used to evaluate Th1, Th2 and Th17 responses. Systemic cytokine concentrations were measured using a commercial canine multiplex assay which included IL-2, IL6, IL-10 and TNF-α, and compared to a healthy control group. Our results demonstrated a dysregulation of cytokine mRNA expression, representative of the tumor microenvironment and systemic response in cNHL. Intratumoral cytokine response revealed a significant downregulation of humoral and Th1 responses. The systemic response demonstrated a distinct mRNA pattern, however immunosuppression also prevailed. Cytokine serum quantification showed a significant increase of IL-10 concentration in cNHL. Significant differences in hematological parameters were described and a correlation between IL-6 protein serum levels and neutrophil count was shown. Finally, data analysis demonstrated that baseline pretreatment IFN-γ tissue mRNA levels were correlated to survival outcome, predicting a favorable response to chemotherapy. Altogether, these results revealed that cNHL presents a local and systemic dysregulation in cytokine response. By confirming and extending previous research, our work contributed for the evaluation of potential cytokine candidates for diagnostic, prognostic purposes and therapeutic intervention, therefore adding value to comparative oncology.

Establishment of a bioluminescent canine B-cell lymphoma xenograft model for monitoring tumor progression and treatment response in preclinical studies.

Canine diffuse large B-cell lymphoma (DLBCL) is one of the most common cancers in dogs which shares remarkable similarities with its human counterpart, making the dog an excellent model for the investigation of novel therapeutic agents. However, the integration of canine lymphoma in comparative studies has been limited due in part to the lack of suitable xenograft mouse models for preclinical studies. To overcome these limitations, we established and characterized a localized subcutaneous bioluminescent canine DLBCL xenograft mouse model. The canine CLBL-1 cell line stably expressing the luciferase and green fluorescent protein reporters was generated and used to establish the xenograft tumor model. A pilot study was first conducted with three different cell densities (0.1×10(6), 0.5×10(6) and 1×10(6) cells) in SCID mice. All mice presented homogeneous tumor induction within eight days after subcutaneous injection, with a 100% engraftment efficiency and no significant differences were observed among groups. The tumors were highly aggressive and localized at the site of inoculation and reproduced histological features and immunophenotype consistent with canine DLBCL. Importantly, xenograft tumors were detected and quantified by bioluminescent imaging. To assess response to therapy, a therapeutic study with a histone deacetylase inhibitor, panobinostat, was performed. The results demonstrated that panobinostat (20 mg/kg) efficiently inhibited tumor growth and that bioluminescent imaging allowed the monitorization and quantification of tumor response to therapy. In summary, this study provides a bioluminescence canine DLBCL model that offers high engraftment efficiency, preservation of tumor features, and noninvasive monitoring of tumor progression, validating the model as a promising preclinical tool for both veterinary and human medicine.

The histone deacetylase inhibitor panobinostat is a potent antitumor agent in canine diffuse large B-cell lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common causes of cancer-related death in the United States and Europe. Although the outcome of NHL patients has improved over the last years with current therapies, the rate of mortality is still high. A plethora of new drugs is entering clinical development for NHL treatment; however, the approval of new treatments remains low due in part to the paucity of clinically relevant models for validation. Canine lymphoma shares remarkable similarities with its human counterpart, making the dog an excellent animal model to explore novel therapeutic molecules and approaches. Histone deacetylase inhibitors (HDACis) have emerged as a powerful new class of anti-cancer drugs for human therapy. To investigate HDACi antitumor properties on canine diffuse large B-cell lymphoma, a panel of seven HDACi compounds (CI-994, panobinostat, SBHA, SAHA, scriptaid, trichostatin A and tubacin) was screened on CLBL-1 canine B-cell lymphoma cell line. Our results demonstrated that all HDACis tested exhibited dose-dependent inhibitory effects on proliferation of CLBL-1 cells, while promoting increased H3 histone acetylation. Amongst all HDACis studied, panobinostat proved to be the most promising compound and was selected for further in vitro and in vivo evaluation. Panobinostat cytotoxicity was linked to H3 histone and α-tubulin acetylation, and to apoptosis induction. Importantly, panobinostat efficiently inhibited CLBL-1 xenograft tumor growth, and strongly induced acetylation of H3 histone and apoptosis in vivo. In conclusion, these results provide new data validating HDACis and, especially, panobinostat as a novel anti-cancer therapy for veterinary applications, while contributing to comparative oncology.

Characterization of plasma labile heme in hemolytic conditions.

Extracellular hemoglobin, 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 when generated during hemolytic conditions labile heme 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, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, 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.

Albumin-binding domain from Streptococcus zooepidemicus protein Zag as a novel strategy to improve the half-life of therapeutic proteins.

Recombinant antibody fragments belong to the promising class of biopharmaceuticals with high potential for future therapeutic applications. However, due to their small size they are rapidly cleared from circulation. Binding to serum proteins can be an effective approach to improve pharmacokinetic properties of short half-life molecules. Herein, we have investigated the Zag albumin-binding domain (ABD) derived from Streptococcus zooepidemicus as a novel strategy to improve the pharmacokinetic properties of therapeutic molecules. To validate our approach, the Zag ABD was fused with an anti-TNFα single-domain antibody (sdAb). Our results demonstrated that the sdAb-Zag fusion protein was highly expressed and specifically recognizes human, rat and mouse serum albumins with affinities in the nanomolar range. Moreover, data also demonstrated that the sdAb activity against the therapeutic target (TNFα) was not affected when fused with Zag ABD. Importantly, the Zag ABD increased the sdAb half-life ∼39-fold (47min for sdAb versus 31h for sdAb-Zag). These findings demonstrate that the Zag ABD fusion is a promising approach to increase the half-life of small recombinant antibodies molecules without affecting their therapeutic efficacy. Moreover, the present study strongly suggests that the Zag ABD fusion strategy can be potentially used as a universal method to improve the pharmokinetics properties of many others therapeutics proteins and peptides in order to improve their dosing schedule and clinical effects.

Novel Peptides Derived from Dengue Virus Capsid Protein Translocate Reversibly the Blood-Brain Barrier through a Receptor-Free Mechanism.

The 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.