What if therapeutic antibodies were a key for curing cancer? Selecting and optimizing our natural defense mechanisms to develop treatments against rare or incurable diseases; these are the challenges that are trying to overcome researchers focusing on therapeutic antibody development.

Therapeutic antibodies an innovative form of immunotherapy. Credit: Pixabay
Therapeutic antibodies an innovative form of immunotherapy. Credit: Pixabay

Antibodies are proteins produced by the immune system, targeting one or several antigens, typically pathogens or foreign bodies. The recent major advances in biotechnology allow antibody generation and development for therapeutic purposes, offering several advantages over traditionally used small molecules.

Thanks to their high specificity and selectivity and their ability to recruit the immune system, their use as therapeutics knows a growing success in many fields, such as oncology, inflammatory diseases, transplantation, infectious and cardiovascular diseases (Figure 1).

This success leads to major repercussions in the biopharma market, which is reflected by the increase of antibody generation for therapeutic use and the number of them reaching the clinic: in 2018, 520 monoclonal antibodies were in preclinical and clinical development against 350 in 2013, and 88 were already approved – 39 in 2013.

Figure 1. Use of therapeutics in medicine (2018)
Figure 1 Use of therapeutics in medicine (2018)

The growing number of therapeutic mAbs (monoclonal antibodies) currently in development and the progresses made in antibody production and engineering suggest that the quantity of approved therapeutics should probably increase significantly during the next years.

Therapeutic antibodies can have different mechanisms of action, such as:

  • Binding to a pathogen to prevent its penetration in the cells
  • Activation or inhibition of different cell-mediated mechanisms (immune checkpoints…)
  • Leading to an antigen-cells clumping, making them more likely to be phagocyted (g. ingested by dedicated immune cells)
  • Activating the complement system, to induce cellular lysis (g. cellular breakdown) or phagocytosis
  • Allowing the delivery of drugs to a specific locus, thanks to Antibody Drug Conjugate (ADC) technology

The growth of therapeutic antibody use is however limited by several factors, such as a low efficiency due to the size of the molecule (tissular penetration issues, blood brain barrier penetration), immunogenicity leading to potential side effects, or high production costs. The whole industry is currently striking to find solutions to improve these key parameters.

Lowering immunogenicity and side effects

The immunogenicity is an issue that concerns every therapeutic antibody, and must always be taken into account for the risk/benefits ratio calculation.

It occurs when the antibody is detected as a foreign body, triggering an immune response. Several methods can be developed to reduce this immunogenicity. Between them:

  • Increasing the sequence identity between the antibody and a human germline sequence
    • Chimerization: This antibody engineering technique consists in associating the constant portions of a human antibody with murine variable domains.
    • Humanization: In the case of humanized antibodies, the CDR sections (the parts responsible for linking the antigen) of a murine antibody are replaced by their human version.
    • Fully human antibodies: Fully human antibodies can be developed using transgenic mice or thanks to phage display technology by panning human antibody libraries. They are mostly used to treat cancers and immunologic issues.
  • Using antibody fragments: An antibody is typically composed by 2 single chains, a light (LC) and a heavy one (HC). The variable regions of each chain (respectively VL and VH) is responsible for binding the antigen, and the Fc region also called “effector” plays a major role in recruiting the immune system. A possible method to reduce immunogenicity is to exploit the small size of antibody fragments as immunogenicity is often related on the size of the antibody.
  • Building synthetic libraries: In silico studies and engineering of the antibody framework sequences allow to identify potential problematic regions of the antibody that may trigger immune responses. This allows developing optimized monoframework synthetic libraries allowing to reduce immunogenicity response potential.

Improving antibody efficiency

Several methods can be used to enhance the antibody properties, to improve their efficiency. Here are some examples:

  • Selecting the best targets: Two main strategies can be deployed to develop new high-potency therapeutics for a same pathology: on one side, it is possible to enhance existing antibodies’ action modes, or to find out and develop antibodies targeting other epitopes of the same antigen when possible. These are called second (2G) or third generation antibodies (3G). It is also possible to improve the properties of existing antibodies to make them “me-better” antibodies. A second strategy consists in targeting new or less studied antigens. This strategy appears to be much more risky, but offers a greater potential.
  • Improving the affinity for the antigen: To proceed, it is possible to use phage display libraries to select the antibodies presenting the best affinities for the antigen. Affinity can also be improved thanks to affinity maturation process. This method consists in inducing punctual mutations on the CDRs, in order to replace non-polarized amino acids by charged ones, enhancing by this way electrostatic interactions. In certain cases, it can be desirable to obtain less affinity to allow a better tumor penetration and to prevent a “Binding site barrier effect”.
  • Improving antibody action efficiency: The interaction between the Fc domain of the antibodies and the Fcγ receptors displayed on the surfaces of macrophages, neutrophils, dendritic cells and natural killers (NK), is responsible for recruiting the immune system against the antigen. Therefore, an enhancement of the efficiency of the Fc domains thanks to mutations and glycan modifications can lead to an increased ADCC (antibody-dependent cell-mediated cytotoxicity).
  • Improving access to the target: A solution is to enhance the antibody penetration through the tissues and cellular membranes. This major issue is conditioning antibody efficiency - particularly in the case of solid tumors or blood-brain barrier penetration.
    • The use of antibody fragments is a possible solution: thanks to their small size, fragments penetrate more quickly and efficiently than full-size mAbs, but as a counterpart they have a low serum half-life, leading to a lower antigen adherence.
    • Bispecific antibodies are a very promising tool in immunotherapy. They combine two antibody parts, and that makes them able to bind two epitopes at the same time. They offer the possibility to bind antigens, membrane cell receptors, immune cells. As an example, they can be used to retarget effector cells of the immune system, while stimulating their activity by activating a receptor allowing an efficient tumor cell lysis.
    • Intrabodies are conceived to be expressed in the intracellular locus. They can, therefore, target antigens located in the cytoplasm, the nucleus, the RES (reticuloendothelial system), mitochondria, peroxisomes or the plasma membrane. Intrabodies interact specifically with a target antigen to block or modify the interactions. This can lead to modifications in the biological activity of the protein of interest.
    • ADC (antibody drug conjugates) development is a very promising technique consisting in linking an antibody with a drug. This allows the direct delivery of small molecules to a specific locus. This method combines the high specificity and affinity offered by antibodies, with some interesting small molecules properties (for example toxicity).
    • Optimizing the affinity: The “Binding site barrier effect” is a phenomenon able to lower antibody efficiency; too much affinity to the antigen can lead to a saturation of antibodies on the membrane surface, resulting into a low penetration inside the tumor. It is therefore necessary to find out an optimal balance between a sufficient affinity to allow an efficient targeting of the tumor, and an optimal retention rate allowing diffusion inside the cell.
  • Improving the antibodies’ pharmaco-kinetics (e.g. their absorption, distribution, metabolism, and excretion): the FC domain can be modified thanks to mutations within the amino acids, in order to improve the antibody circulation time. Another way to increase antibodies’ half-life is by enhancing their affinity for the neonatal Fc receptors (FcRn). This parameter has to be modulated carefully, as a too elevated half-life can induce instability or aggregation, possibly leading to a higher immunogenicity.

Reducing the economic costs

An important drawback for therapeutic antibodies’ use is their high cost, due to their low production yields. As an example, Canakinumab is a therapeutic used to fight some rare inflammatory diseases. Its cost can reach 12 k€ for a 150mL dose. This explains why efforts are increasingly conducted to reach more affordable prices.

A simpler and better productivity and reduced production times can be obtained by improving the cell lines, the purification methods or by generating small fragments’ sizes which allow a cheaper and faster production in microbial systems. Increasing the serum half-life is another method which can lead to a significant cost-reduction, as it can result in decreasing the number of injections. For example, PEGylation (covalent attachment of PEG) increases the size of a fragment and by this way, it offers a better stability resulting in a serum half-life gain, possibly improving the anti-tumoral activity, and reducing immunogenicity. As a counterpart, this method can sometimes conduct to the inactivation or to a lower affinity of the fragment.

Not only the use of therapeutic antibodies by itself has pushed back the frontiers of medicine, bringing very promising results in fields such as oncology or immunology, but the recent advances in antibody engineering techniques should provide even more efficient and affordable treatments in the next years.

The development of mAbs from human sequences thanks to the new coming phage display and transgenic mouse technologies have led to a better efficiency in addressing novel therapeutic targets, and a significant immunogenicity decrease. Other techniques of recombinant antibody production such as ribosome, yeast and mammalian display are emerging providing an alternative to accelerate even more the drug discovery process.

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