Monoclonal Antibodies for the treatment of COVID-19

Amid the rush to develop potential treatments for COVID-19, a number of researchers are focusing on monoclonal antibodies that have revolutionized the treatments for cancer, arthritis, autoimmune diseases, etc. Monoclonal antibodies as the name indicates, are the antibodies that are derived from the clones of a single activated B cell that recognizes a particular epitope on an antigen.

In other words, monoclonal antibodies are identical antibodies with the same antigen specificity.

In the case of plasma therapy, a wide range of antibodies, referred to as polyclonal antibodies from the recovered patient are administered into the people who are critically ill with COVID-19. Some plasma antibodies are neutralizing and are specific to epitopes of the SARS-CoV-2, but many other antibodies may have off-target effects, potentially contributing to tissue damage.

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Additionally, the plasma may also contain a lot of other things which may lead to allergic reactions in the recipient’s body. These adverse effects are less likely to occur with the use of single or combinations of monoclonal antibodies that specifically target the SARS-CoV-2.

Currently, the monoclonal antibodies that target spike protein of SARS-CoV-2 are of major interest for researchers. Because spike protein allows the virus to bind to the ACE2 receptors and facilitates its entry into the human cells.

1. The first type of monoclonal antibodies that are in focus are those that target the receptor-binding domain of the S1 unit of spike protein because it docks into the ACE2 receptor on human cells. These types of monoclonal antibodies after binding to the S1 unit can neutralize the virus by blocking its interaction with the ACE2 receptor present on the human cells, which in turn can prevent the entry of coronavirus into the human cells.

2. The second type of monoclonal antibodies are those that recognize the epitopes in the S2 unit of the spike protein of SARS-CoV-2 thus preventing the viral and human cell fusion eventually preventing the entry of the virus into the human cells.

From the studies, researchers have also found that using only one of these two types of monoclonal antibodies is not very effective whereas the combination of these two types of monoclonal antibodies, targeting different epitopes of spike proteins of SARS-CoV-2, is found to be potentially effective against the virus.

These monoclonal antibodies can be mass-produced in the lab, thus unlike plasma therapy, there is no need for plasma donation. These antibodies can be either isolated from recovered patients or can be genetically engineered in the laboratory using hybridoma technology.

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Isolation of monoclonal antibodies directly from recovered COVID-19 patients: Let’s understand how the monoclonal antibodies can be isolated from the recovered patients. The blood of the recovered patient would most likely have multiple unique antibodies that bind to the receptor-binding domain and S2 unit of spike proteins. The researchers can isolate those antibodies from recovering patients that are best in neutralizing the virus and then these antibodies can be mass-produced in the laboratory.

One approach that is being used in isolating the monoclonal antibodies from the recovered patients is the phage display method. In this method, using the recombinant DNA techniques, foreign peptides are fused with a coat protein pIII of bacteriophage M13 in order to display peptides on the bacteriophage surface. A bacteriophage is a type of virus that infects bacteria.

For the isolation of human antibodies, the phage-displayed antibody library is generated. To obtain these antibodies, mRNA from the peripheral blood mononuclear cells (PBMCs) of COVID-19 recovered people is collected and is then reverse transcribed into cDNA. And then the genes encoding the variable regions of heavy and light chains of antibodies are amplified using specific primers. Any antibody molecule consists of two identical light [L] chain polypeptides designated as L chains, and two identical heavy [H] chain polypeptides designated as H chains. And each light chain and heavy chain contains 2 distinct regions: Variable regions [V] and constant regions [C]. The variable regions are selectively amplified because it is the variable region in the light and heavy chains of an antibody molecule which together forms an antigen-binding site.

The amplified variable regions of antibodies are then cloned into a phagemid vector. The Phagemid vector is then electroporated into the E.coli cells. These E. coli cells are then infected with bacteriophage. After infection, the bacteriophage generates a phage-display human antibody library in which the Fv fragments containing variable regions of the light and heavy chains are displayed on the phage coat protein.

After this affinity screening of the phage-displayed antibody, library is done by a process called biopanning. In this, the phages expressing the Fv region of antibodies on the coat protein are added to the antigen immobilized on a solid surface for example on ELISA plates. The antigens coated are receptor binding domain and S2 proteins of spike proteins against which the monoclonal antibodies are required to be raised. The phages that have Fv regions specific for the receptor-binding domain or S2 proteins bind to the coated receptor binding domain and S2 proteins. Non-specific phages are removed by stringent washing. Antigen-bound phages are then eluted and are re-infected into E. coli to produce a subset of phages for the next cycle of biopanning. After several rounds, the specific spike protein binding phages are sufficiently enriched or in other words, the antibody Fv fragments displayed on the phage coat protein that exhibits a strong affinity for the spike protein of SARS-CoV-2 are selected.

These Fv fragments are then isolated from the phage coat and are used to develop intact IgG antibodies. These intact IgG antibodies are then given to the people infected with COVID-19 disease. These administered antibodies bind to the spike protein of SARS-CoV-2, which in turn, prevents the attachment of the virus to the ACE2 receptors present on human cells. Thus the virus can not enter the human cells, and ultimately the viral infection can be controlled. Researchers have obtained a few monoclonal antibodies that have shown excellent results in neutralizing the coronavirus infection. Some of these monoclonal antibodies are now under clinical trials. If trials succeed, then these monoclonal antibodies will prove to be a major advancement in controlling the COVID-19 pandemic.

Fully human monoclonal antibodies against SARS-CoV-2 can also be generated in the laboratory by hybridoma technology: The fully human monoclonal antibodies can be produced by transgenic mice that are created by replacing the entire mouse antibody genes with human antibody genes. Thus, upon immunizing the transgenic humanized mice with the spike protein of SARS-CoV-2, the mice produce the human antibodies. No part of the antibodies produced is mouse-derived.

To derive the monoclonal antibodies, antibody-producing plasma cells are isolated from mice and are then fused with cancerous myeloma cells to generate hybridomas that secrete human monoclonal antibodies. The antibody-producing plasma cells have a definite life span but the myeloma cells are cancerous plasma cells, which are immortal and can divide indefinitely. Thus the hybridoma cells produced

  • Possess the capability to produce antibodies, a property of plasma cells
  • And they also become immortal, a property of myeloma cells

The population of hybridoma cells obtained is heterogeneous, i.e. they produce antibodies with different epitope specificities. But to select the hybridoma that secretes antibodies against the receptor-binding domain of spike protein of SARS-CoV-2, these hybridomas are required to be isolated and grown individually. This is done by a method known as limiting dilution, which dilutes the concentrations of the heterogeneous population such that on an average, each well contains one cell. In practice, some wells may contain no cells, some may contain a single cell, and others may contain multiple cells.

In the next step, each hybridoma cell is screened for the secretion of antibodies with the desired specificity. This screening is done by the ELISA technique and selects only those hybridomas that produce antibodies against the receptor-binding domain of spike protein.

For this, the hybridoma culture supernatant containing monoclonal Abs is added to receptor binding domain coated on microtiter well and monoclonal Abs are then allowed to interact with receptor binding domain. After this, the monoclonal Ab bound to the receptor-binding domain is detected by adding a secondary antibody labeled with an enzyme, which binds with primary monoclonal Ab. Then the chromogenic substrate is added. And upon addition of this substrate if a colored product is obtained it indicates a positive hybridoma.

After this, the hybridoma cell producing antibodies against the receptor-binding domain can be cloned to produce multiple identical daughter clones. These identical daughter cells then mass produce monoclonal antibodies against the receptor-binding domain of spike proteins. These monoclonal antibodies can then be given to the patients who are infected with COVID-19.

How is it different from vaccination? It is very important to understand that monoclonal antibody therapy is not an alternative to vaccines. The concept of vaccination relies on training the immune system’s memory response to detect and respond to specific pathogens when the body encounters them, thus helping prevent sickness from infectious diseases. A component of a pathogen is introduced into the body through a vaccine. While this component does not lead to the disease but it trains the body’s immune system to generate antibodies and memory cells to fight against the actual pathogen that may invade in the future. The antibodies and memory cells built inside the body by the vaccination are long-lasting and stay forever. Therefore, vaccination builds active immunity in the body.

On the contrary, monoclonal antibody therapy provides passive immunity to the patients. It involves administering the antibodies into the people who are sick with COVID-19 to fight the virus early in an infection until the patient’s own immune system generates sufficient antibodies to fight back Covid-19. It is important to note that a dose of antibodies doesn’t directly stimulate a person’s immune system to start creating their own antibodies, but it does offer some protection until their own immune system ramps up. The generation of antibodies isn’t exactly a speedy process. It generally takes 1–3 weeks for the immune system to produce antibodies against SARS-CoV2.

Or in other words, we can say that the administered antibodies provide instant immunity to the infected people but the antibodies remain effective only for a few months, maybe for 3–4 months if they have naturally long half-life or have been modified to extend the half-life.

On the contrary protection from vaccines is long-lasting: from several months to life long. It is because vaccines activate the body’s own immune system to fight against the virus. To summarise, the goal of a vaccine is to generate an immune response that can prevent someone from getting ill with a disease, whereas monoclonal antibody therapy is designed to treat disease.

Therefore, we can say monoclonal antibody therapy is not an alternative to vaccines. Till the time the vaccine does not get materialized, monoclonal antibody therapy can be an important treatment for COVID-19 disease.

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