ADCs-Magic Bullets, Failed Drugs, and Heterobifunctional Linkers

Guest Author: Brian Rivera, Product Manager: Bio Separations

Brian Rivera Product manager Bio Separations PhenomenexOver a hundred years ago, Nobel Prize laureate, Paul Ehrlich, envisioned a drug which could kill microbes but not harm the body, a “magic bullet” he called it. From mustine to monoclonal antibodies, researchers have implemented Ehrlich’s side chain theory in attempts at discovering this so-called “magic bullet”, specifically targeting treatments for cancer1. Many cancer cell-killing, cytotoxic compounds, have been isolated from natural sources, which showed promise for cancer therapy. Some, like taxols and camptothecin’s, saw great success. Others like calicheamicins, maytanisoids, auristatins and duocarmycins, not so much. However, on their own, these all failed clinically because they were just too potent for the human body to process safely.

Enter the world of Antibody Drug Conjugates, or ADCs. These highly potent drugs just might be the magic bullet Ehlrich always imagined existed. ADCs are monoclonal antibodies (mAbs) coupled with a small molecule, which is commonly one of the failed cytotoxic drugs mentioned before. The combined binding specificity that only a well-characterized monoclonal antibody has, along with the ultra-potent cytotoxic agent, ADCs can be up to a thousand times more potent than chemotherapy2.

As much promise as an ADC has as a therapeutic, there are inherent challenges in the analytical characterization of them. One being that mAbs are difficult to characterize – ~1300 amino acid, 150 kD proteins with a host of post-translational modifications. Now randomly attach a cytotoxic agent to them and try to monitor all the different species of ADC and see if they become deconjugated.

Difficult stuff!

But let’s not get ahead of ourselves. We need to first understand how an ADC is made. As the first part of this article series, we’ll simply discuss all the different parts of an ADC, and this will provide better context as to why they are so difficult to analyze.

The Anatomy of an ADC

An ADC is made up of four components—the monoclonal antibody, a protein specific to an antigen, linker (which can be used to covalently bond a small molecule), and the cytotoxic agent, or “payload.”

Figure 1. A cartoon of an antibody-drug conjugate and its components-monoclonal antibody, linker, space, and “payload”.
antibody drug conjugates

As a review, mAbs are Immunoglobulin G’s, which have a molecular weight of approximately 150 kDa, and are composed of two different polypeptide chains, which are made up of 1320 amino acids. mAbs are produced from a single, isolated B-cell, making them specific to only one antigen. Each IgG has two heavy two light chains that are linked to each other by disulfide bonds. There are 4 isotypes of IgG’s, the most common in ADCs being an IgG1, which has 4 interchain disulfide bridges. These disulfides will become critical when we start talking about cysteine conjugates.

In addition to heavy and light chains, there are other fragments of a mAb, such as the fragment crystallizable (Fc) and the “Fab” fragment (fragment—antigen binding). The Fab is critical in an ADC, as it is the portion of the antibody that is antigen specific. For example, a mAb that binds to CD30, a known cell membrane protein that is expressed by Hodgkin’s Lymphoma. Typical chemotherapy for Hodgkin’s Lymphoma involves administering a cytotoxic drug. With ADCs, the specificity of the monoclonal antibody is used as the drug delivery. Seattle Genetics’ brentuximab vedotin is an anti-CD30 mAb conjugated to the very cytotoxic auristatin. This way, we can selectively only target the cancerous cell, lymphoma, which is expressing the CD30 antigen.

Of course, the ADC can only work if it just “deconjugates” before delivery. The mAb must reach the target cell before the payload is released. Otherwise, this is no longer a magic bullet, it becomes the shotgun approach typically done with cancer treatment.

This is where the “linker” comes in.

The linker is used to attach a small molecule to a protein, or to “conjugate” the protein to a small molecule. The linker needs to be stable during delivery, and then be conditionally released. Since ADCs will be engulfed by the target cell (receptor-mediated endocytosis), taking advantage of the intracellular environment is one way to selectively cleave the linker to release the cytotoxic agent.

One approach is using an acid labile linker, such as hydrazones. Since the inside of a cell is relatively acidic, the thought is the drug will be released upon entry in the cell3.

Another common linker type that is used is the “cysteine-linker-auristatin” motif. This uses a construct of a maleimide (which reacts with free thiols), valine and citrulline (which will cleave by the intracellular protease Cathepsin B), and a spacer—PABC—which allows room for the protease to cleave4. This motif is used by various companies and is common with cysteine conjugates.

Speaking of which, there are many different conjugation chemistries that can be used. However, let’s look at cysteine and lysine conjugates, since the two FDA-approved drugs Adcetris and Kadcyla use these chemistries respectively.

Cysteine conjugates are produced by mildly reducing the mAb using dithiothreitol (DTT) or tris-carboxyethyl phosphine (TCEP). Recall that an IgG1’s has 4 disulfide bonds. These will get reduced to thiols, which then react to a linker with a maleimide group; the classic “metal-free thiol-maleimide click” reaction. This is of course random or “stochastic” as many chemists call it, and this leads to heterogenous mixtures of ADC with various numbers of drug loads attached to them. With these so-called cysteine conjugates, drug loads are typically evenly numbered (i.e. 2, 4,8) since hydrophobic interactions of the hydrophobic payload will hold the antibody together. Figure 2 shows a nice example of the heterogeneity of the drug.

Figure 2. A “cysteine conjugate” and the multiple isomers formed from a stochastic conjugation.
cysteine conjugates

The other conjugation method, Lysine, is a two-step process which uses a “heterobifunctional linker” (NHS-ester SMCC) to react with the lysine, then a subsequent reaction to a sulfhydryl on a cytotoxic agent. This is the chemistry behind ado-trastuzumab emtansine- or KADCyla, which is also stochastic, as most IgG1’s have 80-90 lysine residues.

Finally, the last component of the conjugate to discuss is the payload or warhead. First of all, how is awesome is the terminology here? Way more fun than just saying cytotoxic drug.

As previously alluded to, the payload is a super potent, cytotoxic drug. In fact, take any chemotherapy that failed in clinical trials because of cytotoxicity and use it for an ADC. A common cytotoxic agent is the previously mentioned auristatin used with Adcetris, specifically, the monoethyl auristatin E (MMAE) and monoethyl auristatin F(MMAF) are used. These are analogs of dolastatin 10, an antimitotic cytotoxic agent which was initially investigated for treatment of drug-sensitive mammary and colon tumors5. Dolastatin 10 and its analogs were found to be too toxic; in fact, about 100-1000 times more potent than doxorubicin, another form of chemotherapy. However, conjugation to mAbs as the delivery agent makes a failed drug into a potential blockbuster, just like Adcetris (which uses the MMAE payload). ADCs with higher potency are preferred as they enhance tumor penetration as well as prevent multi-drug resistance6. There are of course other payloads, maytansinoids (i.e. DM1 and DM4) and new novel warheads like pyrrolobenzodiazepines (PBDs).

Putting it All Together

As you can tell, there’s a lot of moving parts that go into an ADC. The mAb itself must be well-characterized and have a known target. The linker must be stable and not deconjugate before the package has been delivered. The warhead needs to be ultra-potent. These components come together, often in a very random manner, and all this makes for a large molecule that becomes difficult to characterize. Chromatographically, this creates several challenges, especially as approaches are considered for the difference conjugate types and the specific chemistry that is used.

In the next part of this article series, we’ll get into current chromatographic solutions and challenges, for the most critical quality attribute for an ADC- the drug-to antibody ratio, or DAR.

Craving more information? See our Pharmaceutical Technical Resources and our Pharmaceutical/Biopharmaceutical industry page.


1Strebhardt K, Ullrich A. Paul Ehrlich’s magic bullet concept: 100 years of progress. Nat Rev Cancer. 2008;8(6):473-80.

2Sievers EL, Senter PD, et al. Antibody– Drug Conjugates in Cancer Therapy. Annu. Rev. Med. 64, 2013: 15–29.

3Finniss, M. C., Chu, K. S., Bowerman, C. J., Luft, J. C., Haroon, Z. A., & Desimone, J. M. (2014). A versatile acid-labile linker for antibody-drug conjugates. MedChemComm, 5(9), 1355-1358

4Jain N, Smith SW, Ghone S, Tomczuk B. Current ADC Linker Chemistry. Pharmaceutical Research. 2015;32(11):3526-3540. doi:10.1007/s11095-015-1657-7.

5Luesch H, Moore RE, Paul VJ, Mooberry SL, Corbett TH. Isolation of dolastatin 10 from the marine cyanobacterium Symploca species VP642 and total stereochemistry and biological evaluation of its analogue symplostatin 1. J Nat Prod. 2001;64(7):907-10.

6Beck A, Goetsch L, Dumontet C, Corvaïa N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov. 2017;

ADCs-Magic Bullets, Failed Drugs, and Heterobifunctional Linkers
Article Name
ADCs-Magic Bullets, Failed Drugs, and Heterobifunctional Linkers
Enter the world of Antibody Drug Conjugates, or ADCs, which are monoclonal antibodies (mAbs) coupled with a small molecule.

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