Antibody-Drug Conjugates > Linker Chemistry

The complexity associated with linking an antibody to a cytotoxic agent (used interchangeably with “drug” in this entry) was greatly under-appreciated in early days of ADC research, but has become a core development focus for companies that are developing proprietary ADC platforms.  As ADC research has matured, it has also become clear that choosing the right linker / cytotoxic agent pair can have a profound effect on the toxic agent’s ability to kill cancer cells.  If not properly optimized, ADCs can aggregate while in circulation, linkers can get cleaved before cellular entry, and the potency of the toxic agent can be compromised.

Crosslinking the antibody to the linker is essential for proper drug delivery to the intended cellular compartment.  ADCs are constructed through the reaction of drugs or chemical crosslinking reagents with solvent accessible reactive amino acids such as lyseine and cysteine on the antibody.  Linkers generally fall into one of two categories: cleavable (peptide, hydrazone, or disulfide) or non-cleavable (thioether).    

Choosing the right linker to use in an ADC is more of an art than a science.  For instance, linkers inherently have shorter half-lives than their antibody counterparts, and therefore typically need to be modified to improve their solubility.  Usually this is done by adding PEG, or PEG-like derivative, to the linker.  Each antibody-linker-drug pairing requires tweaking and optimization.  Unfortunately, there is no real off the shelf solution to creating an ADC. 

Cleavable linkers

Cleavable linkers have reasonable stability during systemic circulation but can be cleaved under certain intracellular conditions, such as in an acidic environment.  In the case of the lysosome, the entire ADC is processed in the lysosome by exposing the cytotoxic agent to a strong acidic environment and lots of digestive enzymes.  Just because the drug is freed in the lysosome does not mean that it is incapable of leaving the cell.  Therefore, with cleavable linkers there is some risk of unwanted side effects as the drug can leak back into the vasculature.

I thought it might be interesting to do a mini chemistry lesson on the various linker chemistries that are currently being used:

Disulfide-based: The use of disulfide linkers exploits the observation that the intracellular concentration of thiols, such as glutathione and cysteine are much higher than those in plasma.  Disulfide linkers are selectively cleaved in the cytosol due to a more reductive intracellular environment and were originally designed to be used with the cytotoxin, maytansinoid (ex: thiol containing DM1 or DM4). 

Hydrazone linkers: Have been designed to be selectively cleaved within the intracellular compartment of lysosomes (lower pH compared to the systemic blood circulation).  Hydrazones have typically been linked to antibody thiol groups generated through interchain disulfide bone reduction.

Peptide linkers:  Have the potential to be selectively cleaved by lysosomal proteases (ex: cathepsin-B) and have demonstrated increased serum stability and improved anti-tumor effects compared to hydrazone linkers.  Valine-citruline (Val-Cit) pairs are the most commonly used peptide linkers and are ideally suited to work with the auristatin family of drugs such as monomethyl auristatin E (MMAE).  MMAE is totally synthetic, quite stable, very potent, and is ideally suited for chemical modification.

Non-cleavable Linkers

Original ADC research overlooked non-cleavable linkers as researchers were convinced the cleaving of the linker was the most reasonable way to free the drug.  ImmunoGen has shown that upon binding to the transmembrane target some ADCs get rapidly internalized and once internalized, the antibody can be degraded to the point where the active drug is exposed.  To say it another way, the antibody is degraded amino acid by amino acid through the linker and up to the drug, which then becomes activated.    

Chemistry - Thioethers: Non-reducible (“non- cleavable”) thioether bond using the SMCC (N-succinimidyl-4-(N-maleimidomethyl)- cyclohexane-1-carboxylate) linker.

ImmunoGen has had quite a bit of success linking Herceptin to DM1 via its SMCC linker (T-DM1 program).  Originally, ImmuoGen worked with peptide linkers such as Val-Cit and linked them to DM1 or DM4, their scientists demonstrated that a thioester could replace Val-Cit in some cases.  Drug activity was retained while increasing serum half-life and decreasing the likelihood of the drug being cleaved before it entered the cell, thus overcoming many of the side effect concerns that plague the cleavable linkers.

Antibody-Drug Conjugates

Over the next few entries I will dive into the world of antibody-drug conjugates or ADCs.  An ADC is an antibody that has been conjugated (bound) to a cell-killing drug by a chemical linker.  Most ADCs in development are directed against solid tumors, and to a lesser extent to hematological cancers.  This is an area of personal interest as well as being highly relevant to my work at Osage as some of the hottest ADC startups – Ambrx, Allozyne, Redwood Biosciences – are all university spinouts.  My first entry will be a rough overview on ADCs followed by more specific blog posts about startup activity in the space, drug development challenges, promising ADCs in late stage development, and maybe even an entry on some ADCs that have failed in the clinic.

The concept of ADCs has been around for the last 30 years, but it was really within the last five or so years that drug development interest in the space has really accelerated.  The reasons for the increased enthusiasm for ADCs are multifold.  First, healthcare reform granted biologics 12 years of exclusivity, even if the patent has expired.  This incentivizes pharma companies to develop antibodies and also encourages them to develop ADCs and other strategies to extend the brand life of marketed biologics.  Second, many first generation antibodies are starting to see higher rates of relapse than previously anticipated.  By linking first generation antibodies with cell-killing agents, pharma companies could keep relapsed patients within their brand family.  Finally, scientists have uncovered many new and exciting targets that provide ideal candidates for ADCs.

Antibodies bind to ligands with incredible specificity and it is this functionality that researchers seek to exploit in developing ADCs.  When antibodies bind to specific membrane-bound targets they often get rapidly internalized into the cell.  In developing ADCs, scientists try to link various payloads to antibodies in hopes that the antibodies will more efficiently traffic those payloads into specific cells.  Conceptually this seems pretty simple, but in practice, it is quite hard to efficiently get a conjugated antibody into a cell in high enough quantities to produce a therapeutic effect.

Linking an antibody to another molecule, large or small, is quite a feat of chemical engineering.  Because antibodies are large molecules that provide numerous locations to attach a linker, choosing the right location for the linker can have profound effects on the antibody’s ability to stably carry the toxic agent, bind to its ligand, and get internalized.

Once the linker location is locked down, researchers can then go about choosing the optimal cell-killing agent to conjugate to the antibody.  Most cell-killing agents are chemotherapies that were originally designed for systemic administration and not selective insertion into cells.  Standard assumptions about the properties of chemotherapies often break down in the context of ADCs which forces researchers to tinker with various antibody-chemotherapy combinations until they find a suitable combination.  Such tinkering takes considerable time and money, with little assurance of clinical predictability (ADC could be way more or less toxic than predicted) after all of the hard work. 

Antibodies are great because they are large molecules that persist in the blood stream for a much longer time than their smaller peptide counterparts.  This strength becomes a weakness in the context of ADCs.  As the ADCs travel around the body the chemical linker must keep the antibody bound to the cell-killing agent.  As the ADC gets knocked around the body, the chances of the cell-killing agent breaking off increases greatly making off target cell-killing a significant risk factor.    

While there are significant drug development challenges associated with ADCs, the ability to selectively kill cancer cells continues to intrigue big pharma and encourage the development of novel ADCs.

FierceBiotech's 2010 Fierce 15

Every year FierceBiotech publishes its list of the top 15 up and coming biotech companies.  Anyone can vote for his or her favorite company making the list rather subjective, but for the purposes of this blog entry that does not really matter.  The main point I want to stress is the fact that 11 of the 15 companies (listed below) are university spinouts (according to my count).  The remaining 4 companies were either created de novo or were corporate spinouts. 

• Accerlon: Columbia, Harvard, MSKCC
• Adimab: Dartmouth, MIT
• Anchor Therapeutics: Tufts
• Catabasis: Harvard
• Dicerna: City of Hope
• Eleven Therapeutics: Harvard, UAB, Stanford, MIT
• iPieran: Harvard, Children’s Boston
• Kymab: Sanger Institute
• Plexxikon: NYU
• Tetraphase: Princeton
• Gelesis: MIT 

I looked at a number of these companies as investment opportunities over the past year and believe that the above list has a number of fantastic companies.  But, there are tons of other companies out there that could have made equally strong cases to be included in the Fierce 15.  So what separates these 11 university spinouts from companies not included in the Fierce 15? 

Scientific Talent:  Many of these companies were founded by leading academics that are both thought leaders in their respective fields and have also had prior entrepreneurial success.  For instance, Joseph Schlessinger, the founding scientist of Plexxikon, is one of the world’s top kinase researchers and previously founded another kinase-focused startup, Sugen (sold to Pfizer).  VCs tend to be attracted to such scientists because they understand the challenges associated with creating a startup, are experts in their respective fields, and are able to attract top researcher scientists to their startups. 

Business Strategy: Of the 11 companies, almost all of them are real companies, not virtual.  VCs are on the hunt for startups that have multiple shots on goal and/or are developing a platform technology.  Supporting the development of multiple products is way more expensive than for a single asset, but diversification provides a safety net for investors.  Also, products can be partnered or licensed which results in non-dilutive capital generation for the company.  

Management Talent:  VCs like to invest in repeat entrepreneurs with significant domain expertise.  To use Adimab as an example, the CEO, Tillman Gerngross, is a former Dartmouth Professor and a co-founder of GlycoFi (sold to Merck).    

Hot Technology Area:  There aren’t a lot of me-too or reformulation companies on the Fierce 15 this year.  VCs are looking for creative ideas in the areas of antibody discovery, iPSC, kinases, and protein-protein interactions.