Spray-Dried Inhalable Powder Formulations of Therapeutic Proteins and Peptides.

Respiratory diseases are among the leading causes of morbidity and mortality worldwide. Innovations in biochemical engineering and understanding of the pathophysiology of respiratory diseases resulted in the development of many therapeutic proteins and peptide drugs with high specificity and potency. Currently, protein and peptide drugs are mostly administered by injections due to their large molecular size, poor oral absorption, and labile physicochemical properties. However, parenteral administration has several limitations such as frequent dosing due to the short half-life of protein and peptide in blood, pain on administration, sterility requirement, and poor patient compliance. Among various noninvasive routes of administrations, the pulmonary route has received a great deal of attention and is a better alternative to deliver protein and peptide drugs for treating respiratory diseases and systemic diseases. Among the various aerosol dosage forms, dry powder inhaler (DPI) systems appear to be promising for inhalation delivery of proteins and peptides due to their improved stability in solid state. This review focuses on the development of DPI formulations of protein and peptide drugs using advanced spray drying. An overview of the challenges in maintaining protein stability during the drying process and stabilizing excipients used in spray drying of proteins and peptide drugs is discussed. Finally, a summary of spray-dried DPI formulations of protein and peptide drugs, their characterization, various DPI devices used to deliver protein and peptide drugs, and current clinical status are discussed.

Vaccine formulations in clinical development for the prevention of severe acute respiratory syndrome coronavirus 2 infection.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented effort toward the development of an effective and safe vaccine. Aided by extensive research efforts into characterizing and developing countermeasures towards prior coronavirus epidemics, as well as recent developments of diverse vaccine platform technologies, hundreds of vaccine candidates using dozens of delivery vehicles and routes have been proposed and evaluated preclinically. A high demand coupled with massive effort from researchers has led to the advancement of at least 31 candidate vaccines in clinical trials, many using platforms that have never before been approved for use in humans. This review will address the approach and requirements for a successful vaccine against SARS-CoV-2, the background of the myriad of vaccine platforms currently in clinical trials for COVID-19 prevention, and a summary of the present results of those trials. It concludes with a perspective on formulation problems which remain to be addressed in COVID-19 vaccine development and antigens or adjuvants which may be worth further investigation.

Nanoparticle-based immunotherapy of breast cancer using recombinant Helicobacter pylori proteins.

Breast cancer is the most common cancer in women worldwide and is associated with substantial medical and economic burden. We report the development of a hybrid immunotherapeutic system based on recombinant Nap protein from Helicobacter pylori (HP-Nap) for the treatment of breast tumors. Chitosan nanoparticles with pseudo-spherical morphology and positive zeta potential were synthesized as carriers for HP-Nap. In vitro study was performed on mouse breast cancer cell line (4T1) and human breast cancer cell lines (MCF7). In vivo study was done on 4T1 tomural mice. TUNEL assay and real time PCR test were performed on tumor mice receiving the nanoparticle treatment. The nanoparticle-protein complex induced apoptosis in vitro in cultured breast cancer cells. In-vivo studies on inbred, female BALB/c mice confirmed the shrinkage of tumor mass after administration of the nanoparticle complex containing HP-Nap. The TUNEL assay further confirmed apoptosis in extracted mouse breast cancer cells. A decrease in the expression of VEGF and MMP9 genes was observed in 4T1 cells as shown by real time PCR. Our data suggesting that the therapeutic nanocomplex may have led to decreased tumor growth in mice through changing the production rate of cytokines and increasing tumoricidal activities of the immune system.

Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level.

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.

Recombinant production of a bioactive peptide from spotless smooth-hound (Mustelus griseus) muscle and characterization of its antioxidant activity.

Bioactive peptides are short amino acid sequences with desirable health effects which are derived from animals, plants, and marine sources. In this study, recombinant production of a bioactive peptide (GIISHR) from spotless smooth-hound (Mustelus griseus) muscle and its antioxidant properties is discussed. A gene composed of 12 tandem copies of the peptide sequence was cloned in pET-28a and expressed as a His-tagged polypeptide in Escherichia coli. The recombinant polypeptide was then purified by Ni-NTA affinity chromatography, cleaved by Trypsin and purified by ultrafiltration. DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) and hydroxyl radical scavenging activity assays, ferric reducing antioxidant power (FRAP) assay and ß-carotene bleaching test were used to characterize the antioxidant activity of the GIISHR. Liquid chromatography-mass spectrometry analysis revealed 60% purity for released bioactive peptide. Production yield was estimated as 60-80 mg GIISHR active peptide per 1 L bacterial culture. Antioxidant activity assays indicated that the antioxidant activity was increased with increase in peptide concentration. Though the DPPH radical scavenging activity, FRAP and ß-carotene bleaching power of the peptide were lower than those of the synthetic antioxidant tert-butylhydroquinone (TBHQ), the ABTS and hydroxyl radical scavenging activities of the peptide (at a concentration of 20 mg/mL) were similar to those of TBHQ (at a concentration of 0.1 mg/mL). The findings of the present study may be helpful in development of a process for production of the bioactive antioxidant peptides and its application in food industry.

Immunogenicity of synthetic peptide constructs based on PvMSP9E795-A808, a linear B-cell epitope of the P. vivax Merozoite Surface Protein-9.

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain.

Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2-CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzyme-independent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmoc-solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with Fmoc-SPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the C-terminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified full-length domain.

Synthesis and semisynthesis of selenopeptides and selenoproteins.

The versatile chemistry of the genetically encoded amino acid selenocysteine (Sec) is employed in Nature to expand the reactivity of enzymes. In addition to, its role in biology, Sec is used in protein engineering to modify folding, stability, and reactivity of proteins, to introduce conjugations and to facilitate reactions. However, due to limitations related to Sec's insertion mechanism in Nature, much of the production of Sec containing peptides and proteins relies on synthesis and semisynthesis. Here, we review recent advances that have enabled the assembly of complicated selenoproteins, including novel uses of protecting groups for solid phase peptide synthesis, rapid selenoester driven chemical ligations and versatile expressed protein ligations.

A synthetic peptide derived from domain III envelope glycoprotein of Dengue virus induces neutralizing antibody.

Dengue virus (DENV) is an arthropod-borne human pathogen that represents a severe public health threat in both endemic and non-endemic regions. So far, there is no licensed vaccine or specific drugs available for dengue fever. A fifteen-amino-acid-long peptide that includes the NGR motif was chemically synthesized and conjugated with keyhole limpet hemocyanin. A standard immunization protocol was followed for the production of polyclonal antibodies by immunizing rabbits against the synthetic peptide. The immune response elicited high-titer polyclonal antibodies with the reactivity of the anti-peptide antibody against both synthetic peptide and four serotypes of DENV confirmed by DOT-ELISA. Neutralizing activity of anti-peptide antibody was found to be cross-reactive and effective resulting in 60% reduction of infectivity at 1:200 dilution in all four serotypes of DENV. Our findings have the potential to further improve our understanding of virus-host interactions and provide new insights into neutralizing antibodies and could also be used as a drug target.

Chemoselective Dual Labeling of Native and Recombinant Proteins.

The attachment of two different functionalities in a site-selective fashion represents a great challenge in protein chemistry. We report site specific dual functionalizations of peptides and proteins capitalizing on reactivity differences of cysteines in their free (thiol) and protected, oxidized (disulfide) forms. The dual functionalization of interleukin 2 and EYFP proceeded with no loss of bioactivity in a stepwise fashion applying maleimide and disulfide rebridging allyl-sulfone groups. In order to ensure broader applicability of the functionalization strategy, a novel, short peptide sequence that introduces a disulfide bridge was designed and site-selective dual labeling in the presence of biogenic groups was successfully demonstrated.

An Evaluation of Optimal PEGylation Strategies for Maximizing the Lymphatic Exposure and Antiviral Activity of Interferon after Subcutaneous Administration.

Interferon α2 is an antiviral/antiproliferative protein that is currently used to treat hepatitis C infections and several forms of cancer. Two PEGylated variants of interferon α2 (containing 12 and 40 kDa PEGs) are currently marketed and display longer plasma circulation times than that of unmodified interferon. With increasing realization that the lymphatic system plays an important role in the extrahepatic replication of the hepatitis C virus and in the metastatic dissemination of cancers, this study sought to evaluate PEGylation strategies to optimally enhance the antiviral activity and plasma and lymphatic exposure of interferon after subcutaneous administration in rats. The results showed that conjugation with a linear 20 kDa PEG provided the most ideal balance between activity and plasma and lymph exposure. A linear 5 kDa PEG variant also exhibited excellent plasma and lymph exposure to interferon activity when compared to those of unmodified interferon and the clinically available linear 12 kDa PEGylated construct.

AJIPHASE®: A Highly Efficient Synthetic Method for One-Pot Peptide Elongation in the Solution Phase by an Fmoc Strategy.

We previously reported an efficient peptide synthesis method, AJIPHASE®, that comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long-chain alkyl groups as a protecting group for the C-terminus. To further improve this method, we developed a one-pot synthesis of a peptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. A branched-chain anchor molecule was used in the new process, significantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, which employed precipitation for isolation and a straight-chain aliphatic group. Another prerequisite for this solvent-extraction-based strategy was the use of thiomalic acid and DBU for Fmoc deprotection, which facilitates the removal of byproducts, such as the fulvene adduct.

Expression, Purification, and Characterization of Interleukin-11 Orthologues.

Interleukin-11 (IL-11) is a multifunctional cytokine implicated in several normal and pathological processes. The decoding of IL-11 function and development of IL-11-targeted drugs dictate the use of laboratory animals and need of the better understanding of species specificity of IL-11 signaling. Here, we present a method for the recombinant interleukin-11 (rIL-11) production from the important model animals, mouse and macaque. The purified mouse and macaque rIL-11 interact with extracellular domain of human IL-11 receptor subunit α and activate STAT3 signaling in HEK293 cells co-expressing human IL-11 receptors with efficacies resembling those of human rIL-11. Hence, the evolutionary divergence does not impair IL-11 signaling. Furthermore, compared to human rIL-11 its macaque orthologue is 8-fold more effective STAT3 activator, which favors its use for treatment of thrombocytopenia as a potent substitute for human rIL-11. Compared to IL-6, IL-11 signaling exhibits lower species specificity, likely due to less conserved intrinsic disorder propensity within IL-6 orthologues. The developed express method for preparation of functionally active macaque/mouse rIL-11 samples is suited for exploration of the molecular mechanisms underlying IL-11 action and for development of the drug candidates for therapy of oncologic/hematologic/inflammatory diseases related to IL-11 signaling.

Enzyme-catalyzed expressed protein ligation.

Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.

A potential therapeutic peptide-based neutralizer that potently inhibits Shiga toxin 2 in vitro and in vivo.

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which can cause serious clinical complications in humans, such as hemolytic uremic syndrome (HUS). Recently, we screened and identified two peptide-based Stx2 neutralizers, TF-1 and WA-8, which specifically and directly bind to Stx2. Computer simulations suggested that the majority of TF-1 or WA-8 binds tightly at the receptor-binding site 3 of Stx2. The two peptides also effectively inhibited the cytotoxic activity of Stx2 by blocking the binding of Stx2 to target cells. TF-1 exhibits remarkable therapeutic potency in both mice and rat toxicity models. In mice toxicity models, TF-1 provided full protection when mice were injected with 5 LD50 of Stx2. In rat toxicity models, TF-1 reduced fatal tissue damage and completely protected rats from the lethal challenges of Stx2. In these rats, TF-1 significantly decreased the concentration of Stx2 in blood and diminished tissue distribution levels of Stx2. Furthermore, TF-1 effectively protected rats from the pathological effects caused by Stx2, especially in the kidney, thymus, adrenal gland, and lung. Taken together, these results indicate that TF-1 is a promising therapeutic agent against the pathogenicity of Stx2.

Development and investigation of recombinant immunotoxin protein 4D5scFv-mCherry-PE(40).

Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.

Synthesis of Triphenylethylene Bisphenols as Aromatase Inhibitors That Also Modulate Estrogen Receptors.

A series of triphenylethylene bisphenol analogues of the selective estrogen receptor modulator (SERM) tamoxifen were synthesized and evaluated for their abilities to inhibit aromatase, bind to estrogen receptor α (ER-α) and estrogen receptor ß (ER-ß), and antagonize the activity of ß-estradiol in MCF-7 human breast cancer cells. The long-range goal has been to create dual aromatase inhibitor (AI)/selective estrogen receptor modulators (SERMs). The hypothesis is that in normal tissue the estrogenic SERM activity of a dual AI/SERM could attenuate the undesired effects stemming from global estrogen depletion caused by the AI activity of a dual AI/SERM, while in breast cancer tissue the antiestrogenic SERM activity of a dual AI/SERM could act synergistically with AI activity to enhance the antiproliferative effect. The potent aromatase inhibitory activities and high ER-α and ER-ß binding affinities of several of the resulting analogues, together with the facts that they antagonize ß-estradiol in a functional assay in MCF-7 human breast cancer cells and they have no E/Z isomers, support their further development in order to obtain dual AI/SERM agents for breast cancer treatment.

Synthetic peptides for the immunodiagnosis of hepatitis A virus infection.

VP1, VP2 and VP3 molecules of hepatitis A virus are exposed capsid proteins that have shown to be antigenic and are used for diagnosis in recombinant-antigen commercial kits. In this study, we developed a sequence analysis in order to predict diagnostic peptide epitopes, followed by their spot synthesis on functionalized cellulose paper (Pepscan). This paper with synthetic peptides was tested against a sera pool of hepatitis A patients. Two peptide sequences, that have shown an antigenic recognition, were selected for greater scale synthesis on resin. A dimeric form of one of these peptides (IMT-1996), located in the C-Terminus region of protein VP1, was antigenic with a recognition frequency of 87-100% of anti-IgG antibodies and 100% of anti-IgM antibodies employing the immunological assays MABA and ELISA. We propose peptide IMT-1996, with less than twenty residues, as a cheaper alternative for prevalence studies and diagnosis of hepatitis A infection.

Design Rationale and Development Approach for Pegfilgrastim as a Long-Acting Granulocyte Colony-Stimulating Factor.

Filgrastim, a recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF), is efficacious in stimulating neutrophil production and maturation to prevent febrile neutropenia (FN) in response to chemotherapy. Because of its relatively short circulating half-life, daily filgrastim injections are required to stimulate neutrophil recovery. In an effort to develop a long-acting form of filgrastim that was as safe and efficacious as filgrastim but had a longer in vivo residence time, a number of strategies were considered. Ultimately, fusion of filgrastim to polyethylene glycol (PEG) was selected. Following extensive analysis of conjugation chemistries as well as in vitro and in vivo characterization of a panel of PEGylated proteins, a construct containing a 20 kDa PEG moiety covalently conjugated to the N-terminus of filgrastim was chosen for advancement as pegfilgrastim. Pegfilgrastim is primarily cleared by neutrophils and neutrophil precursors (rather than the kidneys), meaning that clearance from the circulation is self-regulating and pegfilgrastim is eliminated only after neutrophils start to recover. Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim. Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy. This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim. Approaches that may provide advances for therapeutic agonists of G-CSF receptor are also discussed.

Total chemical synthesis of human interferon alpha-2b via native chemical ligation.

Interferon-alpha (IFNα) is a cytokine that orchestrates innate and adaptive immune responses and potently inhibits proliferation of normal and tumor cells. These properties have warranted the use of IFNα in clinical practice for the treatment of several viral infections and malignancies. However, overexpression of IFNα leads to immunopathology observed in the context of chronic viral infections and autoimmune conditions. Thus, it is desirable to develop therapeutic approaches that aim at suppressing excessive IFNα production. To that end, artificial evolution of peptides from phage display libraries represents a strategy that seeks to disrupt the interaction between IFNα and its cell surface receptor and thus inhibit the ensuing biological effects. Mirror-image phage display that screens peptide libraries against the D-enantiomer is particularly attractive because it allows for identification of proteolysis-resistant D-peptide inhibitors. This approach, however, relies on the availability of chemically synthesized D-IFNα composed entirely of D-amino acids. Here, we describe the synthesis and biological properties of IFNα2b of 165 amino acid residues produced by native chemical ligation, which represents an important first step toward the discovery of D-peptide antagonists with potential therapeutic applications.