Antibody Drug conjugates (ADCs) are the connection between the best of the new innovative therapeutic technologies. They are composed of an antibody (mAb) linked to a highly potent cytotoxic anti-cancer molecule (toxin). By combining the unique tumor-targeting capabilities of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, ADCs are intended to target and kill only the cancer cells and spare healthy cells.

The covalent link between the mAb and the toxin is also critical for the efficiency of the molecule since it must be:
  • stable in the bloodstream in order to prevent systemic toxicity due to the premature release of the toxin,
  • able to release the toxin after internalization of the ADC into the cancer cells.

Monoclonal antibody

Antibodies are large biomolecules secreted by plasmocytes and are involved in the immune system of superior living organisms. Their structure allows them to bind very specifically to another macromolecule called epitope via strong interactions.
Each plasmocyte always secrete the same antibody structure. But through a complex gene recombination system within plasmocytes populations during immune response, the possibilities of refolding are extremely numerous. Antibodies can thus virtually target a very wide range of molecules, not only proteins but smaller molecules such as haptens as well.
For the past 30 years, monoclonal antibodies have been developed and produced in large scale by the means of antibody engineering. Monoclonal antibodies stem from one single plasmocyte which has been selected and immortalized to become a hybridoma. Hybridomas grow indefinitely and secrete large amounts of antibodies which all have the same specific target.
Characterizing and isolating the specific tumor markers of cancer cells is thus of great importance to make an efficient ADC. Monoclonal antibodies raised against those markers will have the ability to bind them specifically once in the vicinity of the tumor cells. Cytotoxic payloads can therefore be released only where they are expected to achieve their role, without interfering with healthy tissues.

Cytotoxic payload

The cytotoxic payload is the part of the ADC which has the real inhibiting action over cancer cells. It must be selected in accordance with the desired purpose of the final ADC, but must in all cases meet some requirements :

High Cytotoxicity

Only few molecules succeed in reaching their intracellular target, and need a highly effective action despite their low quantity. In order to be interesting for the development of ADCs, their IC50 should be lass than the nanomolar scale. At the same time, however, it should have a very limited activity while present in the bloodstream to ultimately prevent from adverse effects.

Appropriate attachment to linker

The structure of the payload should both make it easy to attach to the linker and not interfere with its activity. The chemical modification of the payload due to its attachment to the linker should not disrupt its natural cytotoxic activity once it is released into the lysozyme.

Characterized mechanism of action

Before being involved in the development of an ADC, the payload should first have been described for its action over its target. Several types of actions have been described in the literature so far :

Disruption of cytoskeleton

These are inhibitors of microtubule network assembly, which then cause apoptosis by cell cycle stop. They are therefore very effective against fast-dividing cells (such as cancer cells) by drastically lowering their expansion. However, their impact over differentiated or quiescent cells will be very low.

DNA alteration

Some cytotoxic molecules have been described for their altering action over DNA strands. Such actions can be chemical modifications on the double helix structure, breaks in the backbone or folding constraints

RNA alteration

Other class of payloads have an action over RNA transcription through an inhibition of RNA polymerase. Cells enter in senescence and die by subsequent apoptosis.

Linker

The linker is the spacer that connects the monoclonal antibody and the cytotoxic payload. Its main role is to ensure the stability of the whole molecule while being transported within the bloodstream to the antibody’s target. The choice of the linker will also define the global pharmacokinetics of the ADCs. If it is too weak, the payload might be released off-target which increases ADC’s toxicity. If it is too strong, the payload will not be released within the cancer cell which decreases ADC’s effectiveness. Two categories of linkers have been described so-far:

Non-cleavable linkers

Even if the linker is too strong to release the payload from the antibody, lysosomal elements are still able to degrade the whole antibody. The payload is then free to exert its role within the cancer cells’ cytoplasm. The main advantage is an increased stability in the plasma and a decreased off-target activity, since the payload will remain bound to the antibody.

Cleavable linkers

This type of linker will be sensitive to local physicochemical conditions (such as pH) or the presence of proteases. This confers a good stability of the assembly in the plasma, but is easily destabilized once in a protease-rich environment. Cleavable linkers can be separated into two subcategories:

Chemical cleavage

Chemically cleavable linkers include acid-cleavable ones such as hydrazine, which remains stable at neutral pH, but is easily hydrolyzed in the acidic pH of cells compartments. They also include reducible linkers, which rely on higher reducing potential of cancer cells’ cytoplasm. However, this category of linkers shows non-specific release of the payload.

Enzyme-mediated cleavage

On the contrary, selective release of the payload can be achieved using enzyme-sensitive linkers. Lysosomes are full of specific degradation enzymes whose activity can be used to release the payload only once the ADC has undergone endocytose. Such linkers have a very good stability within the bloodstream as well, since such enzymes are absent from plasma.

Attachment site

Most coupling reactions are made through the free amine group of lysines residues or reduced cysteine disulfide bonds. The use of natural amino acids do not require pretreatment and allow efficient reactions. But the variable distribution of these non-specific attachment sites among antibody structure can lead to a great heterogeneity of the conjugates.

Site-specific conjugation methods include hydrazine (using formylglycine-generating enzymes) or isopeptide (using transglutaminase) bonds. Engineered cysteine have also been developed by inserting specific groups into disulfide bonds.

Most of these molecules are very efficient and are currently used in multiple cancer treatment protocols. For the last few years though, results have revealed that we are still far from what we expected, and that ADCs still have a great potential for improvement. We are confident that our technology will be a very important contribution to the way of designing ADCs and to cancer therapy in the next few years.