Advancements in research have shifted the way we think of combating cancer, and traditional treatment options are on the path to being replaced by safer and more effective methods.
Antibody-drug conjugates, or ADCs, are at the forefront of this change. They and are being developed to meet the challenges that cancer treatment has continually faced.
The chemistry that is used to attach an antibody, via a linker, to an cytotoxic agent affects the performance and efficacy of an ADC. That’s why linker chemistries have become an important target for scientists involved in the development of novel antibody-drug conjugates.
There are several requirements for the successful developments of antibody-drug conjugates, much of which has to do with the use of chemical linkers that hold an antibody and cytotoxin together. Linker research is aiming to produce linkers that suit the particular antibody and cytotoxin being used, provide stability before entering the cell, and provide efficient payload release once inside the target cel.
With traditional chemotherapy drugs, cancer cells are only killed slightly faster than normal cells, making treatment harmful and often ineffective. This lack of specificity was addressed by the development of mAbs that aimed to target antigens present on the surface of tumors, which allowed drugs to be targeted to specific regions and avoid contact with healthy cells. However, the use of mAbs alone has generally been unsuccessful due to their large size and poor penetrability.  
Better understanding of the biology of cancer
Now, as technology and our understanding of the biology of cancer and other diseases has advanced, targeted therapies like antibody-drug conjugates (ADCs) are paving the way for next generation cancer treatment. ADCs are able to take potent cytotoxic agents – up to a thousand times more powerful that traditional chemotherapeutic drugs – and conjugate them to a monoclonal antibody that can specifically target a tumor antigen. This conjugation is done through the use of a chemical linker, which can undergo endosomal or lysosomal degradation and release the cytotoxin once inside the cell.
Because the cytotoxic agent is released only inside the targeted cancer cell, there is possibility for highly tumor-targeted specificity, which enables the use of more powerful drugs while reducing off target toxicity. However, in order to prevent the cytotoxic agent from being released before reaching the cell, as well as to ensure that the payload is efficiently released, the development of linkers must be optimized. There are a variety of linkers that have been developed for ADCs, all of which fall into the two general categories of cleavable and non-cleavable linkers. 
Cleavable vs. Non-Cleavable
Non-cleavable linkers are broken down once inside in order to release the active cytotoxic agent. After being internalized, they generate metabolites containing the cytotoxin and may or may not contain a portion of the linker as well.
One advantage of non-cleavable linkers is that they possess a greater degree of plasma stability. Additionally, they can potentially provide a greater therapeutic window due to the fact that they may carry a more potent cytotoxin, since internalization is required for any payload release.
Cleavable linkers rely on the physiological environment and release drug payload by either hydrolyzation or proteolysis. There are various types of cleavable linkers that have been developed for ADCs, which either fall into the category of chemically labile or enzyme cleavable linkers. 
In the past, several in vivo studies have shown non-cleavable linkers to outperform their cleavable counterparts. In fact, in a study of huC242-SMCC-DM1 (Immunogen), which uses the non-cleavable thioether linker N-succinimidly-4-(N-maleimidomethyl) cylcohexane-1-carboxylate (SMCC), has shown that the ADC was only effective for tumors in which all proliferating cells expressed the target antigen. This was not the case for huC242-DM1 which contained a cleavable disulfide linker, which displayed higher in vivo activity in multiple xenograph tumor models as compared to its non-cleavable counterpart.  
Chemically Labile Linkers
Chemically labile linkers include acid cleavable and reducible linkers, and they have been extensively applied in the manufacturing of ADCs.
Acid-cleavable linkers such as hydrazones and silyl ethers at the forefront of ADC development. Acid cleavable linkers are cleavable under acidic conditions in the cell, but they are designed to remain stable at the pH of the blood. However, non-specific drug release has remained a challenge for acid cleavable linkers, since they have often been associated with releasing cytotoxin in other acidic parts of the body.
Reducible linkers, on the other hand, take advantage of the cellular reducing environment. Reduced glutathione in tumor cells cytoplasm is very high compared to that of normal cells, and this is able to act on the disulfide bonds that hold these linkers together.
Chemically labile linkers have been used since the first approved ADC gemtuzumab ozogamicin (Mylotarg®; Pfizer/Wyeth). Gemtuzumab ozogamicin was withdrawn from the market in 2010 for toxicities related to poor plasma stability, leading to further research for this linker variety. That being said, inotuzumab ozogamicin (CMC-544; Pfizer), which has a structure closely related to gemtuzumab ozogamicin, has recently shown good stability in human plasma and serum with the use of acid labile 4-(4’-acetylphenoxy) butanoic acid linker for targeting CD22 expressing B-lymphoid malignancies.  
Currently, Immunomedic’s IMMU-110, which uses an acid-labile linker to release doxorubicin (DOX), has shown high activity against multiple myeloma (MM; also called Kahler disease), a cancer that begins in the blood’s plasma cells, and appeared to be safe in monkey models, and is in now in phase I and II studies.
Milatuzumab (IMMU-115), as a doxorubicin conjugate currently in a Phase I/II clinical trial for the treatment of patients with relapsed multiple myeloma and also DOX based, uses a hydrazone linker and providing a basis for novel therapeutics against B-cell malignancies.  
For disulfide linker based conjugates, which include the cytotoxic maytansinoids, progress is being seen through the development of the novel antibody-drug conjugate, IMGN242, which has shown excellent efficacy and clearance rates, about four times faster, when compared to its antibody component alone. 
Enzyme Cleavable Linkers
Enzyme cleavable linkers are different from chemically-cleavable linkers in that they rely on the presence of hydrolytic enzymes in the cell. These linkers can be peptide based or include a beta-glucuronide linker.
Peptide-based linkers, including valine-citrulline (Val-Cit) dipeptide linkers and phenylalanine-lysine (Phe-Lys) dipeptide linkers have been used in many antibody-drug conjugates. Designed to keep antibody-drug conjugates intact in systemic circulation, cleavage by specific intracellular proteases provide an excellent balance between plasma stability and intracellular protease cleavage.
Currently, AGS-5ME (Astellas Pharma) is an ADC based on a valine-citrulline (Val-Cit) depeptide linker, a substrate of cathepsin B, that is in Phase I clinical trials for the treatment of pancreatic and prostate cancer.
Beta-glucuronide linkers rely on cleavage by the enzyme β-glucuromidase, which is over expressed in the lysosome of many tumor cell types. These linkers have hydrophilic properties, which allow them to promote solubility of the ADC when compared to their dipeptide-based counterparts.  
New Approaches: Quaternary Ammonium Linker
Despite recent progression, linker development remains limited in scope. Drugs that are conjugated as part of an antibody-drug conjugate must have certain reactive functional groups present in order to fit the drug linker chemistries that already exist. Functional groups, including primary and secondary amines, phenols, and sulfhydryls, are commonly used for linker attachment and have been developed in recent years.
However, the scope of ADC payloads has recently been expanded by development of quaternary ammonium linkers. This novel linker can expand ADC payloads to include tertiary amines, a functional group commonly present in biologically active compounds, but that had not been used as a linker element thus far.
The conjugate for which this novel strategy is being used is a monomethyl auristatin E (MMAE) construct which uses a β-glucuronidase–cleavable linker which is showing impressive plasma stability thus far. Anti-CD30 conjugates comprised of the glucuronide-MMAE linker were potent and immunologically specific in vitro and in vivo, and the pharmacologic properties were similar to the carbamate-linked glucuronide-MMAE construct that was used as a comparison.
The novel linker was then used for a tubulysin antimitotic drug that contained an N-terminal tertiary amine. When the glucuronide-tubulysin quaternary ammonium linker was synthesized and evaluated as a payload, the resulting conjugates displayed a high level of activity in a Hodgkin lymphoma xenograft. This conjugate was potent and immunologically specific in vitro as well. These results were superior to those obtained with a related tubulysin derivative that contained a secondary amine N-terminus for conjugation, and which was developed using previous linker technologies.
The development of quaternary ammonium linker is currently being expanded to include new tertiary amine containing compounds. Researchers are hoping that this new strategy will allow for antibodies to combine with tertiary amine containing payloads and provide highly plasma stable, potent and better targeted ADCs that can contribute to the rapidly expanding ADC effort for cancer treatment.