Designer antibodies: a targeted approach to treat disease

Introduction

Antibodies: we’ve all heard about them and we all need them - but how can we use them to our advantage? Antibodies are the extraordinary proteins that help to protect us from a wide range of infections and have been exploited for their specificity to treat a wide range of diseases. Scientists have estimated that the human body can make as many as a million trillion unique antibodies!

Antibodies in disease

Unfortunately, antibodies are not always the good guys. In fact, they are implicated in a range of inflammatory and autoimmune diseases. In autoimmune diseases, adaptive immune cells fail to differentiate between the antigens on our own cells (also known as ‘self’ cells) and the antigens on invading pathogens (or ‘non-self’ cells). The antibodies produced by these cells then target our body’s own cells for destruction, leading to cell and tissue damage. Antibodies that target our body’s own cells are known as autoantibodies. Autoantibodies can target a wide range of molecules inside, outside, and on the surface of cells, including proteins, lipids and nucleic acids. Autoantibodies have been implicated in a wide range of autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus.  Recent research has shown that patients with COVID-19 have elevated levels of autoantibodies against proteins that regulate the immune response. Researchers also found that these autoantibodies impaired the immune response to the virus.

Using antibodies in research and diagnostics

Each antibody consists of four polypeptide chains: two light and two heavy. Each heavy and light chain is comprised of a constant and a variable region. It is the variable region that determines the antibody’s specificity and allows for the production of trillions of different antibodies. Due to their high specificity, antibodies can form a great basis for research and diagnostic tests aimed at detecting markers of interest. Antibodies can generally be grouped as polyclonal or monoclonal, and each type has its own advantages and disadvantages. Here we focus on monoclonal antibodies, as these the focus of a lot of recently developed treatments.

 Production of monoclonal antibodies (Mabs) involves immunisation of an animal (usually a mouse) to obtain an antibody response. However, what differs in this process is that the B cells are removed from the immunised animal and fused with a cancerous cell line (usually myeloma) to form hybridoma cells which have the potential to continuously divide. Only the hybridoma cells are selected using a selective growth medium. Hybridomas that produce the Mab of interest are then screened for and grown in bulk to obtain large amounts of the antibody (see figure 1).

Figure 1. Monoclonal antibody production. An animal is immunised with the antigen of interest (A). Antibody- producing B cells from the animal are extracted and fused with myeloma cells to (B) form hybridoma cells (C). The hybridoma cells are selected for and grown in culture, then screened for production of the antibody of interest (D). Bulk amounts of the antibody of interest are produced (E). Figure created with BioRender.com.

Mabs have numerous clinical and research uses. Mabs targeting the human chorionic gonadotropin hormone form the basis of pregnancy tests. Moreover, Mabs can also be used for diagnostic tests, to detect the presence of a disease marker in patient samples, as well has having several uses in research to detect markers of interest. One of the main uses of Mabs is creating targeted therapies for diseases, particularly in cancer, which I will go on to discuss.

Antibody therapy

Mabs have been used particularly in diseases where current treatments are not as effective or have severe side effects. Due to their targeted approach, Mab-based therapies tend to have fewer side effects than other conventional drugs.

Initially, murine Mabs were used as therapies, but problems with efficacy created a need to make more ‘humanised’ antibodies. Researchers used techniques to alter the structure of these murine antibodies to do just that, making them more suitable for binding human targets and hence allowing for more efficient, long-term treatments in disease.

Mabs are most commonly used in the treatment of oncological diseases, by targeting proteins known to play a role in disease progression. A widely recognised example of this is Mabs targeting the HER2 protein in HER2-positive breast cancer. Around 20% of people with breast cancer have overexpression of the HER2 growth hormone on the surface of the cancer cells. As a result of this, these cancer cells tend to grow more rapidly, and the cancer itself can be more aggressive.  Herceptin is a Mab used in the treatment of HER2-positive breast cancer. It binds to HER2 to block cancer cell growth. More information on Herceptin can be found on the NHS website.

In addition to having several uses in cancer, Mabs are also commonly used in the treatment of inflammatory diseases such as RA. A prime example of this is Adalimumab, a Mab  which targets Tumour Necrosis Factor-alpha (TNF-α). TNF-α is a signalling protein that has been found to be overproduced in the blood and joints of people with RA and has been linked with the uncontrolled inflammation seen in this disease. Anti-TNF-α Mabs have been very effective in the treatment of RA, particularly for people for which conventional drugs have not worked. You can read more about the different types of anti-TNF-α antibodies used in RA treatment on the Versus Arthritis website. 

Limitations of antibody therapy

Although they are a fantastic scientific creation, Mabs do not come without their flaws. They tend to be better tolerated than other treatments such as chemotherapy, however, in some people they can result in several unwanted side effects such as high blood pressure and bleeding. Therapeutic efficacy is also less than optimal in the treatment of some diseases such as certain tumours, where there is poor penetration of the tumour tissue by Mabs. Production of Mabs can be both extremely time consuming and expensive, with the cost of the therapy a major limiting factor in the production of some treatments.

Research is currently ongoing to address some of these limitations, particularly the high production costs and poor tumour penetration in cancers. Nanobodies are the ‘smallest known antibody fragment’ and have proved effective in several studies. They were identified after heavy-chain-only antibodies (HcAbs) were discovered in camelids. HcAbs contain two heavy chains and a one variable domain. The single variable chain was isolated from these HcAbs and termed the nanobody (see figure 2). Nanobodies retain all their antigen binding potential, despite their size and do not require a complex process to produce. They also tend to be more stable than conventional antibodies.

In the field of cancer, nanobodies have the properties to allow them to deeply penetrate various tumours. Nanobodies have been widely investigated for the treatment of cancers and have proved successful at targeting proteins on the surface of tumours and blocking components involved in angiogenesis.

More recently, investigations have been made into the role of nanobodies in the treatment of COVID-19. The benefit of using these nanobodies for COVID-19 treatment is that they can recognise epitopes that standard antibodies often cannot. Recent research showed that nanobodies were able to neutralise different variants of the SARS-CoV-2 virus successfully. Groups of neutralising antibodies in this study were unable to neutralise these variants. This study suggests that nanobodies could be instrumental in the treatment of COVID-19, particularly as new variants emerge.

 

Figure 2. Conventional antibody, HcAb and nanobody structure. Conventional antibodies consist of two heavy and two light chains (each with one constant and variable region). HcAbs contain two heavy chains which each have one variable region, and nanobodies are comprised of a single variable region. Figure created with BioRender.com.

What is new in the world of antibody therapeutics?

mAbs published a list of ‘antibodies to watch’ in January 2021, outlining which antibodies currently in development might be rolled out in 2021. Disease targets for these antibodies range from osteoarthritis to Alzheimer’s disease, and even COVID-19. You can read more about these novel therapeutic targets in the extensive article published in the journal, but for the purposes of this article, let’s focus on progress in COVID-19 therapeutics.

With COVID-19-associated inflammatory cytokines taking the world by storm, many companies have been focused on producing antibodies to help target the key triggers in this inflammatory process. The virus-induced ‘cytokine storm’ has been frequently linked with increased mortality in COVID-19 patients. A cytokine storm involves overproduction of pro-inflammatory cytokines, leading to widespread inflammation and tissue damage in the lungs, and eventually multiple organs throughout the body. You can read more about this here.

Many of the current COVID-19 therapy approaches have involved repurposing antibodies. This involves taking antibodies that have been implicated in the treatment of other diseases with similar pathology and trialling them in COVID-19. Antibody repurposing has the key advantage of skipping various time-consuming initial dosage and safety steps in the clinical trials process, as these drugs are already deemed safe to use. Towards the end of 2020, antibodies of this nature were already in late-stage clinical trials, with the hope that they would be granted emergency use. Included in these antibodies is Levilimab, an antibody against the interleukin (IL)-6 receptor, to supress the effects of IL-6, a pro-inflammatory cytokine implicated in the COVID-19 cytokine storm.

Other companies such as Regeneron have produced antibodies which target specific parts of the COVID-19 virus, in particular the spike protein that enables the virus to enter and infect our cells. Regeneron have produced an antibody mixture, or ‘cocktail’ called ‘REGEN-COV’, containing two Mabs which bind ‘tightly and non-competitively to non-overlapping parts of the spike protein’. This makes REGEN-COV effective at binding to COVID-19 variants, which tend to have spike proteins with altered structures at certain parts of the protein. REGEN-COV reduced the viral load in non-hospitalised patients with COVID-19 and the need for medically-attended visits. This antibody cocktail is aimed at those with mild to moderate COVID-19, with the aim of supressing the virus in its early stages, as opposed to the antibodies above (targeting parts of the cytokine storm) which are typically used in those with more severe disease. Having multiple Mabs that can potentially treat a range of disease severities is great, especially early-stage disease where there is a potential to block disease progression and reduce tissue damage. This is in contrast to other COVID-19 treatments such as steroids (immune modulators) which are only recommended in patients with severe COVID-19, where there is already significant tissue damage.

The world of COVID-19 antibody therapeutics is still very much developing, and research is revealing new targets and pathways involved in the pathogenesis of COVID-19. Whilst this helps us to understand the disease better, it also provides a basis for researchers all over the world to work to create better, more targeted treatments for this hugely detrimental disease.

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