Monoclonal Antibodies: Usage in the Treatment of COVID-19 Infection

Since the COVID-19 emergence in December 2019, significant efforts are being made in the hunt for appropriate medical interventions. This forces scientists to produce or discover traditional curative medications, preventive vaccinations, or passive immunological techniques as quickly as possible. Therapeutic monoclonal antibodies (mAbs) have drawn a lot of interest throughout this context. COVID-19 approved Emergency Use Authorization (EUA) medications for the outpatient treatment of mild to moderate symptoms for many monoclonal antibodies (mAbs) aimed against the Receptor binding domain of the S protein of the coronavirus 2 (SARS-CoV-2). We investigated the feasibility of monoclonal antibodies for the diagnosis and treatment of COVID-19 infection in this review. Human monoclonal antibodies targeted SARS-CoV-2 viral protein domains, especially the spike protein area, and hyper-immune plasma from recovered COVID-19 patients are also included in this review. In summary, monoclonal antibodies are the promising remedies that could be used to regulate the SARS-CoV-2 (COVID-19 infection causal agent) through immunotherapy, vaccine development, and viral screening.


INTRODUCTION
COVID-19 is a pandemic disease caused by SARS-COV-2 i.e., severe acute respiratory syndrome-2 or coronavirus. The first COVID case is reported at 'Wuhan' which is present in China, in December 2019. Coronavirus has since spread over the globe [1]. More than 279 million

Review Article
illnesses and over 5.3 million fatalities have resulted from the COVID-19 pandemic and several treatments are present based on the stages of infection. SARS-CoV-2 is a positivestranded RNA virus with an envelope and it is a member of the Beta coronavirus family. Coronaviruses have the most complex and massive genome of any RNA virus. Both structural and non-structural proteins are encoded by the viral genome. The SARS-CoV-2 virus's spike (S) protein projects through the viral envelope. One of the most important structures that permits the SARS-CoV-2 virus to enter host cells is the S protein, which is a homotrimeric glycoprotein [2].
Based on our present understanding of COVID-19 pathophysiology, antiviral medicines such as Remdesivir and neutralising antibodies will be most effective in the early stages of the disease i.e., 9 days following the commencement of symptoms at the most and this results in reduced mortality rate. In the later phases, it is the inflammatory response that causes damage, thus medications that reduce inflammation, such as dexamethasone and tocilizumab, are employed [3]. When it comes to immunomodulatory medicines like steroids and tocilizumab, the timing of treatment is crucial. The global spread of COVID-19 necessitates an immediate search for coronavirus illness prevention and therapy [4,5].
Mainly people's attention was concentrated on the development of vaccines, novel antiviral medicines, and plasma infusions during the time, but monoclonal antibodies received less attention, despite the fact that neutralising antibodies are essential components of protective immunity against most viral infections. Neutralizing monoclonal antibodies are used to make vaccines and have a wide range of therapeutic and preventative benefits [6]. Many research organisations have concentrated on monoclonal antibodies since the causal agent of COVID-19 was discovered as SARS-CoV-2.

Definition
Monoclonal antibodies (mAb) are mostly made from convalescent patients' B-cell lymphocytes or humanised mice. These can serve as a means of passive immunotherapy. Monoclonal antibody therapy also called as monoclonal antibody infusion treatment is used to treat COVID-19.
The main aim of this therapy is to prevent or reduce the hospitalizations, reduce the viral loads, reduce the symptom severity. This is an effective therapy but not a replacement for vaccination. So, the people have to get the vaccination to break the virus chain of transmission [7].

Anti-SARS-CoV-2 Therapeutic Monoclonal Antibodies
The spike (S), envelope (E), membrane (M), and nucleocapsid (N) are the 4 major structural proteins, and also non-structural, auxiliary proteins, are all encoded by the SARS-CoV-2 genome. S1 and S2 are two main components of the spike protein that regulates the host cell adhesion and penetration. S1 binds to angiotensin-converting enzyme 2 (ACE2) on the host cell via its receptor-binding domain (RBD), which causes S2 to undergo a shape change which ultimately leads to virus-host cell membrane adhesion and viral penetration [8].

Fig. 1. mAbs bound to SARS-CoV-2 spike protein and neutralises the virus
Anti-SARS-CoV-2 monoclonal antibodies (mAbs) that aim the spike protein have been found to help in the management of SARS-CoV-2 infection clinically. After a probable SARS-CoV-2 contact in a residential environment, some anti-SARS-CoV-2 mAbs have been proven to be successful for post-exposure prophylaxis (PEP) [9] and also in providing specialist care and home care facilities during the SARS-CoV-2 breakouts [10]. When administered before as pre-exposure prophylaxis, several anti-SARS-CoV-2 mAbs were reported to significantly diminish the chances of infection [11].
Many pharma companies, including Celltrion, AstraZeneca, and Regeneron, are in favour for the development of monoclonal antibodies for the diagnosis and treatment of COVID-19. The FDA granted monoclonal antibodies from Regeneron and Eli Lilly during the Delta variant strain of virus, which got a lot of attention. Despite the fact that even more than 70 monoclonal antibodies are in various phases of development, the Food and Drug Administration (FDA) has granted three anti-SARS-CoV-2 monoclonal antibodies Emergency Use Authorization (EUA) for the therapies of mild to moderate COVID-19 infection in non-hospitalized individuals with SARS-CoV-2 infection which is confirmed by research laboratories. These monoclonal antibodies are usually prescribed for the patients who are at a high risk of developing a condition that will require hospitalisation. Bamlanivimab (like a monotherapy), a combination of Bamlanivimab and Etesevimab or Casirivimab and Imdevimab as a combination therapy, and Sotrovimab are the monoclonal antibodies that have received EUA [12]. Some SARS-CoV-2 variants with specific mutations have significantly lowered susceptibility to a majority of the approved anti-SARS-CoV-2 mAbs in preclinical development [13].
There are two monoclonal antibodies that have been proven to be beneficial in lowering Ebola virus disease deaths, especially when treated early in the infection [14], among them one is three-monoclonal antibody mixture, whereas the other one is a single monoclonal antibody.

Bamlanivimab
On November 9, 2020, the FDA granted Bamlanivimab as an Emergency Use Authorization (EUA) for the management of mild to moderate COVID-19 in adults i.e; elderly people and certain paediatric patients. It's the first monoclonal antibody to be officially granted by the FDA for the diagnosis of COVID-19. It should be given in the form of single infusion of 700mg of Bamlanivimab. It is specifically developed to block SARS-CoV-2 spike protein from attaching to and entering host cells via the ACE-2 (Angiotensin converting enzyme-2) receptor. Bamlanivimab monotherapy does not show any significant advantages in terms of viral load reduction. As a result, the FDA terminated the EUA for Bamlanivimab monotherapy on April 16, 2021, stating a growth in the number of SARS-CoV-2 variants resistant to this therapy [15].

Casirivimab and Imdevimab combination
The R10933-10987-COV-2067 research of Phase III trial, includes individuals with mild to moderate COVID-19 infection, provided scientific information for the usage of Casirivimab and Imdevimab [16]. On November 21, 2020, the combo of Casirivimab and Imdevimab monoclonal antibodies gained emergency use authorisation and it gets distributed together as a cocktail under the brand name of REGEN-CoV. These antibodies are recombinant monoclonal antibodies that bind to non -overlapped epitopes of the SARS-CoV-2 spike protein RBD [15]. This combination combines 600 mg of Casirivimab with 600 mg of Imdevimab, which is given as an Intravenous (iv) infusion or injections under the skin. If an IV infusion is not accessible or would create a postponement in the treatment, four subcutaneous injections can be used instead (2.5 ml per injection) [17]. The distribution of Casirivimab with Imdevimab in the US has been suspended due to diminished susceptibility of Omicron strain to Casirivimab and Imdevimab, individuals with Omicron virus infection are unlikely to benefit from this treatment plan [18].
The clinical studies are evaluating the efficacy of REIGN-COV2 in minimizing the symptoms of COVID-19 infection in teenagers (over than 12yrs old), those who are residential connections of SARSCoV-2 positive persons, as well as in lowering SARS-CoV-2 VL in children's (of less than 18 years) [19,20]. Next it compares the efficacy of REIGN-COV2 against placebo in terms of both survival and the need for mechanical ventilation in less-flow oxygen patients in the hospital [20]. People who undergone treatment with the cocktail were not hospitalised as a reason of COVID-19, after the observation of 41 days [21].
Clinical trials demonstrate that Regeneron's combinational monoclonal antibody minimizes COVID-19-related patients' hospitalisation and mortality by roughly 70% in high-risk individuals and when these monoclonal antibodies are given to the persons who came in contact with the infected persons, it reduces the chance of acquiring a symptomatic illness by 80%. People those who had no antibodies to SARS-CoV-2 at beginning of the study showed the greatest clinical benefit [22].

Bamlanivimab and Etesevimab combination
The combination of Bamlanivimab and Etesevimab is used to target various epitopes of the SARS-CoV-2 spike glycoprotein. On February 9, 2021, the FDA granted this combination an EUA for the management of mild to moderate COVID-19 in children and adolescents who are not hospitalised. Bamlanivimab and Etesevimab are kappa neutralization antibodies that work against the SARS-CoV-2 S glycoprotein's receptor binding region to block virus adherence and its entry into human cells [23].
In areas where the cumulative frequency of SARS-CoV-2 mutations is low, a combination dose of Bamlanivimab of 700mg and Etesevimab of 1400mg was given as an Iv drip. COVID-19 vaccination must not be given to these people for up to 3 months following the infusion, according to official recommendations [15]. A phase I randomised, placebo-controlled and double-blind clinical trial was recently conducted to assess the safety, acceptability, pharmacokinetics (ADME parameters) and pharmacodynamics (how drug does to the body) of COVID-19 in 24 hospitalised patients. The obtained results were shown as the reduction of risk by 70% in COVID-19 related hospitalizations who received Bamlanivimab and Etesevimab versus placebo [24]. The distribution of Bamlanivimab with Etesevimab in the US has been suspended due to diminished susceptibility of Omicron strain to Bamlanivimab and Etesevimab in both the Gamma and Beta forms, individuals with Omicron virus infection are unlikely to benefit from this treatment plan [25]. The product's marketing has been reestablished all across United States due to the total frequency of the Gamma and Beta types is less than 5%.

High-risk Conditions
For all patients older than 12 years in each Emergency Use Authorization, high risk is characterized as meeting at least one of these criteria -

Mechanism of Action of Monoclonal Antibodies (mAbs)
SARS-CoV-2 neutralising mAbs are primarily directed against the membrane spike glycoprotein that facilitates entry of pathogens into human host. If virus is unable to enter cells, it will not be able to replicate and spread throughout the body. This protein is the target of almost all monoclonal antibodies. The viral spike makes contact with the angiotensin converting enzyme 2 (ACE 2) receptor, which is located on a various cell causes the viral infection, but neutralising monoclonal antibodies prevent this step. Although knowledge of the viral spike protein's epitopes is expanding, previous experience of other human viral diseases has facilitated quick progress in determining the spike protein's atomic arrangement [40].
In this image broken line indicates cell membrane with ACE2 receptors and TMPRSS2 protease which are essential for virus to enter host cell. A monoclonal antibody attach to the spike protein on virus, hence blocks the virus interaction with host cell.

Fig. 2. It shows interaction of SARS CoV2 virus with the host cell
Majority of monoclonal antibodies discovered so far selectively targets the spike protein's receptor-binding region, which permits corona virus to interact with ACE 2 receptor [41,42]. Neutralizing antibodies are likely to target additional areas of the spike protein as well.
Based on recent SARS-CoV and Middle East respiratory sickness coronavirus information (MERS-CoV), potency is a key characteristic that is typically used to characterise neutralising monoclonal antibodies. It can be used to choose monoclonal antibodies with clinical potential [43].

Production of Monoclonal Antibodies
Monoclonal antibodies are the ones made exclusively by lymphocytes called B cells, such cells are unable to grow for a long period of time in a cultured cell. As a result, numerous techniques were developed for running and producing them in stable cell lines, including protocols based on B cells. When these cells come from mouse lymphocytes, the resulting murine monoclonal antibodies are mostly employed for immunotherapeutic purposes. corona virus patients are treated using human cells, particularly those from recovered patients. Human neutralised monoclonal antibodies produced as a result are considered as effective antibodies against severe acute respiratory syndrome [44].
Traditional process for producing mouse monoclonal antibodies involves fusing splenic cells from immunised mice (for example, immunisation with SARS-CoV-2 proteins) utilizing fusing media like Polyethylene glycol (PEG), particular mouse monoclonal antibodies were produced using mouse myeloma cell lines, followed by cell cloning as well as subcloning. The majority of the mouse monoclonal antibodies produced were indeed employed in immunotherapy [45,46].
The cloning of cDNAs, encoding the numerous sections of the heavy and light chains onto production plasmids carrying the human IgG1 heavy chain and Ig kappa light chain constant regions, is required for creation of potential therapeutic recombinant human monoclonal antibodies. The interleukin-2 signal sequence is included on both plasmids, allowing for effective recombinant antibody secretion. After transfection with sets of IgG1 heavy and light chain expression plasmids, recombinant human antibodies are generated in HEK-293T cells and extracted using protein-A affinity chromatography [47].
In 1975, in order to create significant amounts of homogeneous monoclonal antibodies, the hybridoma technology was introduced. Producing mouse monoclonal antibodies against specific SARS surface epitopes from the spike protein using traditional hybridoma technology; then separating the RNA and conducting humanization and chimeric human monoclonal antibodies using the same hybrid cell line that generates the unique mouse monoclonal antibodies. Human monoclonal antibody 47D11 was created using this approach. In Vero cell culture, the antibody showed neutralisation of shared epitopes from SARS-CoV and SARS-CoV-2 viruses [47].

COVID-19 Prevention Using Monoclonal Antibodies
The epidemic of COVID-19 necessitates the development of an efficient vaccine. Despite the fact that vaccine development often requires years and may even decades, concerted attempts for screening the multiple COVID-19 vaccines at the same time are intended to cut the time to 12 to 18 months [49]. A patient's decision to employ anti-SARS-CoV-2 mAbs should indeed be based on a personalized risk-benefit analysis [17]. For patients who cannot take the vaccine or need more urgent protection before or after exposure, monoclonal antibodies offer an alternative to vaccination in the treatment of corona virus infection. Passive infusion of monoclonal antibodies as a pre-or postexposure prophylactic could provide immediate protection that lasts weeks or months. Even if a vaccination is available, it takes weeks to build an adequate immune response. As a result, monoclonal antibodies against SARS-CoV-2 can be employed simultaneously for prevention and therapy of infection. MAbs should be preserved for the persons who are at a major chance of having a severe COVID-19 illness, such as the obese, getting older or those who have more glucose levels i.e., diabetes, pulmonary disorders, cancer [50]. Although mortality rates may not appear to be consistently higher throughout the studies, the frequency of occurrence of COVID-19 infection is higher in SOT individuals when compared to a people with more chances of respiratory failure [51][52][53][54][55][56].

Adverse Effects
An allergic reaction is one of the possible side effects of monoclonal antibody therapy. These reactions usually occur during or immediately after the infusion, and your healthcare provider will monitor for any signs of an allergic reaction. Patients who have had anti-SARS-CoV-2 monoclonal antibodies are reported to have hypersensitivity, including anaphylaxis and infusion-related events. There have also been reports of hives or itching, rashes, diarrhoea, dizziness and pruritis [17,32,57]. There are also some other adverse effects, such as - Fever and/or chills  Nausea  Headache  Breathing problems  Hypotension  Lips, cheeks, and throat swelling  Wheezing, Muscle pains [35] Clinical study participants those who have injected Casirivimab and Imdevimab combination via Sub cutaneous route experienced injection site reactions such as ecchymosis and erythema.
Headache (6%) and tiredness (4%) have been the most possible side effects of tixagevimab plus cilgavimab. The antibody and placebo treatments had equal rates of overall and severe adverse events. When monoclonal antibodies, like bebtelovimab, are given to hospitally adimitted COVID-19 patients who require highlevel of oxygen or mechanical ventilation, they may have worse clinical results [58].

CONCLUSION
Despite the fact that monoclonal antibodies manufacturing is a time consuming and costly, they have been viewed as a promising therapy option for COVID-19 since the outbreak began. Monoclonal antibodies have proven to be successful and easy to administer in the treatment of a variety of diseases over the last three decades. Monoclonal antibodies are a crucial component of COVID-19 therapies, and they're making their way into therapy guidelines. These compounds have significant advantages for usage against the corona virus infection because they are developed from COVID-19 affected persons, who are in remission. Currently, over than 70 monoclonal antibodies are now being developed in animal or human trials, which could allow for fast production in the event of a pandemic. Bebtelovimab shows action against both the omicron variant and the BA.2 omicron subvariant, according to tests conducted in laboratory. Vaccines are well recognized for providing extended time protective action only after a few days or weeks of use. Monoclonal antibodies might be an excellent addition to vaccinations, particularly in the event of an epidemic, because they provide immediate relief that can remain for weeks or even months after administration. This would also aid with the management of sick patients and the prevention of infection in children's and the elderly people.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.