key: cord-0926079-2qthv38z
authors: Najmeddin, Ali; Shapourabadi, Mina Bahrololoumi; Behdani, Mahdi; Dorkoosh, Farid
title: Nanobodies as powerful pulmonary targeted biotherapeutics against SARS-CoV-2, pharmaceutical point of view
date: 2021-07-31
journal: Biochim Biophys Acta Gen Subj
DOI: 10.1016/j.bbagen.2021.129974
sha: fc92fcbe1d9007856c6f5634f55bc22ad0dfae20
doc_id: 926079
cord_uid: 2qthv38z

Background Since December 2019, the newly emerged SARS-CoV-2 virus continues to infect humans and many people died from severe Covid-19 during the last 2 years worldwide. Different approaches are being used for treatment of this infection and its consequences, but limited results have been achieved and new therapeutics are still needed. One of the most interesting biotherapeutics in this era are Nanobodies which have shown very promising results in recent researches. Scope of review Here, we have reviewed the potentials of Nanobodies in Covid-19 treatment. We have also discussed the properties of these biotherapeutics that make them very suitable for pulmonary drug delivery, which seems to be very important route of administration in this disease. Major conclusion Nanobodies with their special biological and biophysical characteristics and their resistance against harsh manufacturing condition, can be considered as promising, targeted biotherapeutics which can be administered by pulmonary delivery pharmaceutical systems against Covid-19. General significance Covid-19 has become a global problem during the last two years and with emerging mutant strains, prophylactic and therapeutic approaches are still highly needed. Nanobodies with their specific properties can be considered as valuable and promising candidates in Covid-19 therapy.

Based on the SARS-CoV-2 pathogenesis, different strategies for Covid-19 treatment are designed [6] [7] [8] . These approaches mainly include preventing the virus entrance to the target cell particularly by neutralizing antibodies [9, 10] or virus fusion prevention [11] , viral replication inhibition specially by targeting the virus proteases [12] [13] [14] and reducing the severity of immune response [15] which are summarized in Fig. 1 . It is clear that despite all efforts, still therapeutic and preventive interventions against Covid- 19 are an urgent need [16] . A potent target for drug discovery for Covid-19 is RBD-ACE2 interaction that offers a very safe and strong therapeutic target for binding and prevention of infection by antibodies and Nanobodies (Nbs) for researchers who are working on Covid-19( Fig. 2) [17].

Neutralizing antibodies that prevent the virus particles from entering the target cells by inhibiting the RBD-ACE2 interaction, are very interesting anti-viral agents for the treatment seems to be a very effective molecule in inhibiting the RBD-ACE2 interaction and virus entry into the cells [2, 23, 24] . interaction is considered as a therapeutic target for binding and prevention of infection.

Conventional immunoglobulin-γ (IgG) antibodies assembled from two identical heavy (H)chain and two identical light (L)-chain. While sera of camelids contain a unique functional heavy (H)-chain antibody (HCAbs) in addition to conventional antibodies. The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. The smallest intact functional antigen-binding fragment of HCAbs is the single-domain VHH, also known as a Nanobody (Fig. 4C ) [25] . Nanobodies can be used in different formats such as bivalent monospecific, bivalent bispecific, bivalent bispecific, albumin-conjugated, trivalent bispecific and bispecific chimeric antigen receptor (CAR) T cell [23] . Nanobodies (including multi-specific, multivalent and bi-paratopic constructs) are encoded by single genes and are efficiently produced in various prokaryotic and Nanobodies show many interesting characteristics in comparison to conventional antibodies; such as [26] [33, 34] , Chikungunya virus [35] and HCV [36] and also in viral respiratory infections such as Respiratory Syncytial Virus (RSV) [37, 38] and Middle East Respiratory Syndrome Coronavirus(MERS-CoV) [39, 40] .

According to the brilliant experience in previous viral infections, researches incline to study Nanobodies potential in prevention and treatment of Covid-19 caused by SARS-CoV-2. In addition, Nanobodies with their unique biophysical properties (including small size and thermostability) have this potential to be used as the pharmaceutical form of inhalation which can be directly deliver the therapeutic agent to the target organ, lung, conferring high pulmonary drug concentrations with minimal systemic side effects [41] .

Notably, trimeric spike protein is conformationally flexible and allows each of its RBDs to have two different configurations: a "down" conformation that is thought to be less accessible and an "up" conformation that is receptor (ACE2) accessible conformation [2, 42] . One of the best strategies to neutralize SARS-CoV-2 entry to the cells is to develop biotherapeutics with the potential of inducing conformational changes in a way that RBD cannot binds to its receptor anymore. Such as bivalent Nanobodies that induce post-fusion conformation of the SARS-CoV-2 spike to neutralize the virus [2, 43] . Also, researchers have introduced some conserved epitopes for binding to their designed Nanobodies which were inaccessible to antibodies [44] . During the Covid-19 pandemic, several other studies have done to evaluate Nanobodies potentials for treatment of this syndrome, most of them are designed against SARS-CoV-2 spike RBD [45, 46] . Binding kinetics between the SARS-CoV-2 RBD (including association(Ka) and dissociation(Kd) rates) are very important factors that affect the therapeutic outcome of the Nanobody and should be determined accurately [25] which are evaluated in some valuable studies [17, 19, 41, 47] . Some important studies with the aim of development of anti-SARS-CoV-2 nanobodies are summarized in Table 1 . [17]

Local pulmonary delivery of therapeutics may offer benefits for the treatment of lung diseases such as Covid-19. Advantages of pulmonary drug delivery includes the rapid onset of action, reduced systemic side effects, increased therapeutic window and the need for a lower dose to reach the desired therapeutic response, as well as non-invasive administration [59] . Although it is pharmaceutically possible to make different drug delivery systems and drug dosage forms for Nanobodies [26, 60] , if the target tissue of the drug is lung (for example, in diseases such as the Covid-19, which involves the respiratory system), the pulmonary method seems to be the best route of administration. Previous studies have shown that if the drug is administered systemically, only about 0.2% of the drug reaches the lungs, which means that in order to achieve a therapeutic dose in the lungs, much more concentration of the drug (and more side effects and more manufacturing costs as result) is needed in comparison with local pulmonary route. The same result was achieved in another study where it was shown that the inhalation route potentially offers rapid RSV neutralisation, while simultaneously, t 1/2 of about 20 h allows for once daily dosing [37] . (such as resistance against aggregation and shear forces during manufacturing) (Fig. 3) .

Furthermore, being only one-tenth the size of a normal immunoglobulin, a single dose of Nanobody packs in ten times more active molecules compared to the same dose of a classic immunoglobulin [59] . To make a suitable inhaled formulation for a biological medicine such as Nanobody, the formulation method and the delivery device should provide not only the proper droplet size (0.5-5 micrometers aerodynamic diameter for good drug deposition in the lung [59] ) but also in propellants and this results in a dose limitation in addition to changes in protein structure.

DPIs are not easily applicable in critically ill patients, low consciousness or children; and also drying stage which is necessary in DPIs formulations may affect the protein or Nanobody construct which need extensive studies. Most biological products in the market are generally formulated in either solution or suspension or lyophilized powder. Generally, 75% of inhaled proteins in researches are prepared in the form of liquids for nebulization. Pulmonary delivery by nebulizer does not need a drying step as it may affect the drug properties and it can be used easily by all patients. There are different kinds of nebulizer: air-jet, ultrasonic and vibrating mesh. According to previous researches, It seems that the best method for nebulizing Nanobodies is the vibrating mesh method due to minimizing construct changes and multimerization of the molecules (2% vs 40% in air-jet method) [59, 61] . The main advantages of a nebulizer compared to a pMDI or DPI is that oxygen can be administered in combination with the aerosol treatment and higher doses can be administered over a prolonged time. Furthermore, nebulizers can be used without the cooperation of the patient [59] , which is a very important factor in hospitalized patients.

Altogether, it seems that nebulizers are the most appropriate pulmonary dosage form for targeted delivery of Nanobodies to the lung. An example of a Nanobody against a respiratory virus that is formulated in inhalation dosage form is ALX-0171 (manufactured by Ablynx company), a potent trivalent Nanobody with antiviral properties against RSV [37] with good safety results in phase 1 clinical trials [59, 64] . These successful experiences encouraged researches to use their knowledge for the treatment of new emerging corona virus too.

Physical properties of a nebuliser solution may impact drug product stability and device performance. To resist the shear stress during nebulization and to avoid physico-chemical degradation, high solubility, low viscosity, and physical stability in a physiologic buffer is needed (a solubility of at least 100 mg/mL and a viscosity below 2 Centipoise for a nebuliser solution) [59] . As shown in some valuable studies, Nanobodies production should be in a way that pre-and post-nebulization, the stability and function of the Nanobody molecule does not properties. After a defined duration (typically 4 to 6 weeks), the blood is collected and lymphocytes are extracted. A phage display library is made and then the Nanobodies with the most affinity to the RBD of the SARS-CoV-2 spike protein will be selected. After assaying Nanobodies for neutralization of SARS-CoV-2 pseudovirus (by Plaque reduction neutralization test (PRNT) is considered the "gold standard" for detecting and measuring antibodies that can neutralize the viruses [66] ), the next step would be large-scale production in a suitable expression system (bacteria, yeast or mammalian expression system) and

downstream process including purification and pharmaceutical formulation as a pulmonary drug dosage form (Fig. 4) . part of the VHH antibody is called Nanobody. D. The library is screened for Nanobodies against spike SARS-CoV-2 protein (specially the RBD). E. Washing of non-specific phages and elution and amplification of the specific-Nanobodies. F. Virus neutralization assay is performed to select the Nanobody with the most affinity to the SARS-CoV-2 Spike. G. Expression (in E. coli or yeast or any other suitable expression systems) and Largescale production of the selected Nanobody with the highest affinity for the SARS-CoV-2 virus. H. After purification and downstream processes, produced Nanobody will be formulated in a suitable dosage form for pulmonary delivery.

During the last 2 years after appearance of SARS-CoV-2 in China and Covid-19 pandemic, researches and pharmaceutical companies strongly tried to develop therapeutic and prophylactic candidates against this disease. Among different approaches it seems that VHH, Nanobodies with their promising and outstanding properties truly can be a "magic bullet"

against SARS-CoV-2 and Covid-19. It can be suggested that Nanobodies not only can be used as neutralizing agents, but also Nanobodies with anti-inflammatory effects such as anti-IL-6R Nanobody® ALX-0061 which is developed by Ablynx, (Gent, Belgium) primarily for rheumatoid arthritis, probably can be used in Covid-19 with the aim of reducing pulmonary inflammation like Tocilizumab (Actemera ® ) which is an anti-IL-6R monoclonal antibody with extensive use in Covid-19. According to pathogenesis of Covid-19 and its effects on different body tissues like heart and gastrointestinal system, different Nanobodies with different route of administration can be designed and produced to help people against this society annoying virus. It should be noted that computational design can also complement experimental Nanobody development to identify new epitopes according to the Nanobodies structural-conformational information [67] .

SARS-CoV-2: Structure, Biology, and Structure-Based Therapeutics Development

Structureguided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape

Respiratory Virus Infections: Understanding COVID-19

SARS-CoV-2 spike protein: a key target for eliciting persistent neutralizing antibodies

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Comprehensive analysis of drugs to treat SARS-CoV-2 infection: Mechanistic insights

Santosh G Honavar2 , Pooja Khamar SS. Therapeutic opportunities to manage COVID-19/SARS-CoV-2 infection: Present

Neutralizing antibodies for the treatment of COVID-19

Fruitful Neutralizing Antibody Pipeline Brings Hope To Defeat SARS-Cov-2

Broad-spectrum coronavirus fusion inhibitors to combat COVID-19 and other emerging coronavirus diseases

COVID-19: Targeting Proteases in Viral Invasion and Host Immune Response

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARSCoV-2 infection

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

Controlling Cytokine Storm Is Vital in COVID-19

Passive antibody therapy in COVID-19

coronavirus sars-cov-2

Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2

Perspectives on the development of neutralizing antibodies against SARS-CoV-2

Antibodies at work in the time of severe acute respiratory syndrome coronavirus 2

Antibody therapies for the treatment of COVID-19

The Therapeutic Potential of Nanobodies

Immunogenicity Risk Profile of Nanobodies 2021

Natural single-domain antibodies

Nanobody-based delivery systems for diagnosis and targeted tumor therapy

Nanobody approval gives domain antibodies a boost

Single-Domain Antibodies and Their Formatting to Combat Viral Infections

Nanobody; An old concept and new vehicle for immunotargeting

Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity

Nanobodies that neutralize HIV

Intracellular Expression of Camelid Single-Domain Antibodies Specific for Influenza Virus Nucleoprotein Uncovers Distinct Features of Its Nuclear Localization

Potent neutralization of influenza a virus by a single-domain antibody blocking M2 ion channel protein

Selection and characterization of protective anti-chikungunya virus single domain antibodies

Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

Generation and characterization of ALX-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection

Enhanced ability of oligomeric nanobodies targeting MERS coronavirus receptor-binding domain

Yusen Zhoua f a. A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV

multivalent nanobody cocktails efficiently neutralize SARS-CoV-2. Science

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Preparing for the Future -Nanobodies for Covid-19?

Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes

Nanobodies: Prospects of Expanding the Gamut of Neutralizing Antibodies Against the Novel Coronavirus, SARS-CoV-2

Nanobodies, the potent agents to detect and treat the Coronavirus infections: A systematic review

Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain

A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential

Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain

The Development of a Novel Nanobody Therapeutic for SARS-CoV-2

Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

A bispecific monomeric nanobody induces SARS-COV-2 spike trimer dimers

Multimeric nanobodies from camelid engineered mice and llamas potently neutralize SARS-CoV-2 variants

Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2

A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

Nanobodies as inhaled biotherapeutics for lung diseases Gino

Nanobodies as a versatile approach and clinically validated drug platform

Pulmonary delivery of biological drugs

Pulmonary drug delivery: A review on nanocarriers for antibacterial chemotherapy

Drug delivery to the respiratory tract using dry powder inhalers

ALX-0171: Safety and Therapeutic Potential of an Inhaled Anti-RSV Nanobody

A guide to: generation and design of nanobodies

Detection of Neutralizing Antibodies

to Tembusu Virus: Implications for Infection and Immunity

The Answer Lies in the Energy: How Simple Atomistic Molecular Dynamics Simulations May Hold the Key to Epitope Prediction on the Fully Glycosylated SARS-CoV-2 Spike Protein

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: