Comparison

In vivo antibodies

In vitro vs. in vivo

At the beginning a central question arises: What does "in vitro" and "in vivo" actually mean?

Both terms originate from Latin. In vitro means 'within the glass' (lat. vitrum). In biology and chemistry one thinks directly of a test tube and this is exactly what is meant.

An in vitro experiment is carried out outside a living organism.

The term "in vivo", however, is the exact opposite. It means that something happens inside a living being (lat. vivum = life). Accordingly, in vivo experiments are carried out within a living organism.

In most cases, in vivo experiments are carried out following newly gained in vitro knowledge. This is due to the complexity of living organisms, which puts the in vitro observations obtained under controlled, well-defined circumstances to the test.

The best known applications for in vitro applications are in vitro fertilization (IVF), in which an artificial fertilization of an egg cell with sperm is performed, and in vitro diagnostics (IVD). The latter involves the examination of human samples within a laboratory for the purpose of diagnosing abnormalities or even diseases using products known as diagnostic agents.

 

In vivo antibody production

For the in vivo production of monoclonal antibodies, an adjuvant is first injected into the abdominal cavity of the mouse. Such an injection leads to an irritation of the peritoneum. The resulting accumulation of fluid in the abdominal cavity is called ascites.

In addition, additional fluid is secreted into the animal's peritoneal cavity, which promotes the growth of ascites.

Once this first step is completed, another injection into the abdominal cavity follows. Here, a concentrated hybridoma cell suspension corresponding to the desired monoclonal antibody is used.

The hybridoma cells now adapt to the new environment, start to grow and there is a continuous secretion of monoclonal antibodies into the ascites fluid within the peritoneal cavity.

Finally, the ascite fluid can be removed. In most cases, this is followed by purification of the antibodies on Protein A or G columns.

An advantage of this method is the low cost compared to common in vitro methods.

A disadvantage, however, are the components of the ascites fluid. The ascites fluid may contain proteolytic enzymes, which can damage the monoclonal antibodies produced, and endogenous antibodies, which lead to a decrease in antibody specificity after the purification step.

However, the production of monoclonal antibodies that can be used in vivo can also take place free from the use of animals in so-called bioreactors.

This allows the cultivation of the corresponding hybridoma cells over a longer period of time. This is due to the ability of the bioreactor to administer fresh cell culture medium and to remove used medium at the same time. The harvesting of the final antibody and its purification can also be done automatically by a bioreactor.

However, the creation and maintenance of a suitable environment within a bioreactor is linked to numerous parameters, which must be set with high precision.

On the one hand, temperature, gas throughput and stirring speed are the three most important physical parameters. The chemical parameters include dissolved oxygen and carbon dioxide levels, pH, osmolality, redox potential and numerous metabolite levels.

On the other hand the biological parameters are of great importance. They include the concentration of living cells and the general viability of the cells. Additionally, intracellular - and extracellular parameters are measured.

 

In vivo antibody purity

Low endotoxin level

Perhaps the most important question before using an antibody in vivo is its purity or endotoxin content.

The outer cell membrane of certain bacteria consists of chemical compounds called endotoxins. The origin of the word is in ancient Greek and roughly means "toxic substance inside".

Endotoxins are lipopolysaccharides (LPS) which can be divided into a lipophilic lipid and a hydrophilic polysaccharide part. They gain their significance for the in vivo use of antibodies through their activation of cellular signaling pathways which, in addition to inflammatory reactions, can even have an apoptotic effect on cells. Endotoxins can also have a negative influence on the correct performance and evaluation of cell-based assays.

The detection of endotoxins is performed by means of the amebocyte lysate of horseshoe crabs, the so-called LAL test. The measured endotoxin units are subsequently stated per milliliter (EU/mL).

Azide-free

In addition to endotoxins, azides also play an important role in in vivo experiments. Azides, which belong to the pseudohalides, have drastic effects on cellular processes. Their irreversible inhibition of the respiratory chain enzyme cytochrome C oxidase, the terminal electron acceptor within the respiratory chain, is of particular importance.

Carrier-free

The absence of carrier proteins, transmembrane proteins with important function in the passive transport of substrates, is also ensured prior to the sale of antibodies for in vivo use.

 

In vivo antibody application

T-cell depletion using in vivo antibodies

T-cell depletion describes the removal or lowering of specific T-cells and can reduce the risk of a graft-versus-host response.

Neutralization using in vivo antibodies

Especially in view of the SARS-CoV-2 pandemic, the search for neutralizing antibodies is a central part of research. Neutralization is achieved by binding the antibody to a specific antigen. This antigen may, for example, be located on the virus surface and be responsible for virus entry into the cell or its binding to cellular receptors. The antibody-antigen binding thus creates a physical barrier and prevents the virus from interacting with the cell.

Frequently asked questions about in vivo antibody neutralization

What is neutralization in immunology?

The binding of an antibody to a pathogen and the resulting (physically) prevented infection of a cell is called neutralization in immunology.

Do antibodies carry out neutralization?

Only few antibodies are able to neutralize invading pathogens. Neutralization depends on the specific binding site of the antibody.

How do antibodies neutralize antigens?

Antibodies can neutralize antigens by binding to sites that are particularly important for pathogen infection.

What is a virus neutralization test?

A virus neutralization test demonstrates the ability of a specific antibody to physically prevent the interaction between a viral surface protein and a cellular receptor by binding to it.

 

In vivo imaging using antibodies

In addition to their therapeutic use, in vivo antibodies are becoming more and more popular for their use in non-invasive in vivo imaging and thus for in vivo diagnostics.

This allows research on diseases to be conducted in the in vivo model, enabling even better analysis and investigation of biomarkers on cell surfaces, therapy-influenced signaling pathways, metastatic cancer or general immune responses.

Non-invasive in vivo imaging using specific antibodies can thus enable the visualization of specific biomarkers within an entire organism.

In the long term, it can be further complemented by X-ray computed tomography (CT), magnetic resonance imaging (MRI) and ultrasound, as the latter is limited to the patient's anatomy and physiology. Furthermore, this new method offers great hope of further improving targeted therapies through molecular observations.

 

Therapeutic antibodies

If a monoclonal antibody is used to fight a disease, it is called a therapeutic antibody. Especially in the fight against cancer, antibodies that bind to the immune checkpoints of T-cells help. In this process, also known as immune checkpoint therapy, the antibody blocks the interaction of cancer cells with the immune checkpoints on the surface of the T-cell membrane.

As a result, the cancer cell cannot escape the immune response and attack by the T-cell (immune evasion). In this case, the cancer cell would normally use the activation of these immune checkpoints, which are originally intended to cause an overfunction of immune cells by suppressing the immune system.

The naming of such therapeutic antibodies follows a fixed scheme: antibodies generated in the mouse always end in -omab. The first of its kind was Muromonab-CD3 in 1986. Since the current WHO nomenclature was not yet active at the time of its development, its name does not follow the now valid guidelines. However, the use of monoclonal antibodies of murine origin has a major disadvantage, as they are often recognized as "foreign" by the patient's own immune system, which initiates a defense reaction. Therefore, the newer therapeutic antibodies are antibodies of exclusively human origin (ending in -umab).

 

Two further examples of the mode of action of therapeutic antibodies

Blocking of certain overexpressed receptors

For a cancer cell to grow indefinitely, it must receive appropriate growth signals. In order for this to happen, it produces, for example, a particularly high proportion of HER2 receptors compared to other cells. This receptor is responsible for such a growth and division signal. However, the binding of an anti-HER2 antibody blocks its action and thus prevents further growth of the cancer cell.

Blocking angiogenesis

The formation of new blood vessels to supply oxygen to tissues is called angiogenesis. When a tumor reaches a certain size, the lack of oxygen makes it necessary to stimulate the formation of new blood vessels by means of messenger substances. If this process is blocked, the oxygen supply to the tumor is cut off.

 

Therapeutic antibodies by gene transfer

One of the latest approaches to make the production of monoclonal antibodies for therapeutic purposes even more efficient is their production within the patient himself. This method is called in vivo gene transfer. Such an introduction of the genetic sequence for the production of the antibody can be done by means of special viruses (adenoviruses). This method uses the ability of the viruses to integrate their own genetic material into the host cell.

Nevertheless, this new approach is affected by side effects such as germ line transfer, the development of cancer or unwanted reactions of the immune system to the virus infection.

A non-viral approach involves the use of plasmids that can be transfected into muscle tissue. However, their disadvantage is the low expression level of the encoded protein.

The process of transfer is one of the most central approaches of modern science. Especially the support of non-viral gene transfer by electroporation seems to be very promising. Parts of the tissue membrane are made permeable to the plasmids used by means of electrical voltage pulses, which can lead to a 10-100 times higher efficiency of the gene transfer.

 

Biosimilars

Biopharmaceuticals are biological drugs. Their active ingredients are produced within living cells. This is in contrast to chemical drugs, whose active ingredients are produced synthetically.

If the patent of a biopharmaceutical expires, other manufacturers can copy this product. However, the original active ingredient is not used. As a result, the approval of a biosimilar is a complex process and its monitoring measures are critical.

Although biosimilars do not differ from the original biologics in their clinical effect, the former show significant differences such as a changed glycosylation pattern. However, the amino acid sequence of the biosimilars is identical to their original.

Four characteristics distinguish a biosimilar:

  1. Marketing begins after the original patent expires
  2. Significantly lower selling price than the original
  3. Despite biological variation especially similar to the origina
  4. The effect, quality and safety are almost identical to the original

The imitation products of a therapeutic protein, known as biosimilars, despite their proximity to the original drug, require their own investigations and tests regarding their efficacy and safety.

In the field of therapeutic monoclonal antibodies, numerous biosimilars for adalimumab, bevacizumab, infliximab, rituximab and trastuzumab have been successfully launched in recent years.