Antibody-Drug Conjugates - BiopharmaDirect

Antibody-Drug Conjugates

Chemotherapy is currently one of the most widely used therapies in the field of tumor treatment. However, due to the huge side effects of chemotherapy, some patients are often unable to receive chemotherapy because of their low tolerance. Antibody-drug Conjugate (ADC) is a new type of therapy that combines the high efficacy of small molecule drugs and the targeting abilities of monoclonal antibodies. With its high specificity of antibodies, normal cells in the body can be avoided from killing, thereby adverse reactions are reduced during treatment.

ADC consists of the following three parts:

• Antibody: its role is to specifically recognize target cells.
• Toxins (drug): play a role in killing cells.
• Linker: connect antibody and toxin.

Figure 1 Structure of an antibody–drug conjugate (ADC) (source: ResearchGate)

As we mentioned above, ADC is a combination therapy of monoclonal antibodies and chemotherapy, which has both the selectivity of monoclonal antibodies and the lethality of chemotherapy. Although the principle of ADC drugs is simple, there are many factors that need to be considered in the actual industrial production process. Below are short explanations to the three components of ADC.

When designing ADC drugs, the first thing to consider is which target should be selected. The targets selected by the existing ADC drugs are often those that have been verified by the efficacy of commercial antibodies, or directly develop targets on the basis of the monoclonal antibodies that have been marketed. For instance, the already marketed Roche T- DM1 was developed on the basis of its blockbuster drug Herceptin (trastuzumab, which targets the HER2 protein). The antibody targets can be generally divided into the following categories:

Table 1 Target antigens in solid tumours under research (Source: British Journal of Cancer)

Internalization efficiency: Another factor that needs to be considered is the ability of the antibody to enter the tumor cell. If the antibody has a good specificity but cannot enter the tumor after being bound to the surface of the tumor cell, then such an antibody is not suitable. Some studies believe that the higher the affinity of the antibody to the target, the better. A 2012 study measured the internalization efficiency of HER2 antibodies with different affinities (as shown in the figure below, PMCID: PMC3077882), and found antibody H3B1 with the highest affinity behaves slightely poorer in entering tumor cells than antibodies with slightly lower affinity. Therefore, for ADC, it is not that the higher the affinity of the antibody to the target, the better. Instead, the affinity, internalization efficiency and other indicators should be comprehensively considered in the antibody design process.

Figure 2 Affinity limits the tumor uptake of antibodies. (PMCID: PMC3077882)

Antibody type: In choosing different types of antibody, people generally prefer IgG, especially IgG1. Compared with other types of IgG, IgG1 can induce multiple immune responses after binding to target cells, such as ADCC and CDC. This is undoubtedly an icing on the cake for ADC's killing of tumor cells. Take T-DM1 as an example: the antibody trastuzumab used can not only target the conjugated emtansine to tumor cells, but also continue to kill tumor cells with its ADCC effect, while IgG2 and IgG4 is far inferior to IgG1 in these aspects. Although IgG3 can also induce ADCC and CDC, its half-life is short and therefore unstable, so it is not often used.

What factors need to be considered when designing linkers? First, the linker needs to connect the monoclonal antibody to the toxin molecule. This connection must be stable enough to ensure that the ADC will not be broken when circulating in the blood vessel. Second, when ADC enters the tumor cell through internalization, the linker must ensure that toxin molecules are released. A clinical trial of the world's first ADC drug Mylotarg showed that the Mylotarg treatment group can cause severe fatal liver damage, and the death rate of the combined drug group is higher than that of the chemotherapy group alone, which directly led to the withdrawal of Mylotarg. The failure of Mylotarg was caused by the poor connection stability of the linker used. Linkers currently used by ADC drugs can be divided into two types: degradable linkers (Cleavable Linkers, see figure a) and non-degradable linkers (Non-Cleavable Linkers, see figure b).

Figure 3 cleavable linkers and non-cleavable linkers (source: Pharmaceuticals)

Furthermore, the degradable linker can be divided into the following categories according to different technical routes.

Acid-sensitive linkers: This type of linker contains a hydrazone group, which is sensitive to acid. It is relatively stable and difficult to decompose in the blood where pH is about 7.5. After ADC enters the tumor cells, where the pH value in the tumor cells is generally low (about 4-5), it begins to release toxins. The world’s first ADC drug, Pfizer’s Mylotarg, used this linker. However, as we mentioned above, Mylotarg often caused the linker to decompose prematurely, resulting in greater side effects, which led to the drug’s withdrawal from the market in 2010.

Glutathione-sensitive disulfide linkers: Glutathione (GSH) is a small molecule compound whose concentration in normal cytoplasmic matrix is ​​about 0.5-10mmol/L, and its concentration in plasma is about 2-20μmol/L. However, the concentration of GSH in tumor cells is often several times higher than that in normal cells. According to the difference of GSH concentration, the corresponding small molecule linker can be designed. In addition to GSH, disulfide isomerase (PDI) can also reduce disulfide bonds.

Lysosomal protease-sensitive peptide linkers: Many lysosomal-related proteases, such as cathepsin B, are often overexpressed in tumor cells. Based on this, cathepsin B-sensitive linkers can be designed. Due to the high plasma pH and the acidic environment for protease catalysis, a small amount of protease in the plasma will not affect the linker. The ADC drug Adecetris of Seattle Genetics, approved by the FDA in 2018, uses a valine-citrulline linker that is sensitive to cathepsin B.

β-glucuronide linker: β-glucuronide is a substrate of β-glucuronidase. The lysosome in tumor cells often contains a large amount of β-glucuronidase, so it is also feasible to use derivatives of β-glucuronide as linkers. At the same time, due to the hydrophilic properties of β-glucuronide, connection with some hydrophobic drugs can enhance the hydrophilicity of the drug, thereby making ADC more stable in the blood.

Compared with the degradable linker, the non-degradable linker is obviously more stable, at least there is no need to worry about ADC being decomposed in plasma in advance and causing toxicity to normal cells. When the ADC containing the non-degradable linker enters the tumor cell through internalization, the lysosome of the tumor cell will partially degrade the ADC antibody, and the linker-drug complex cannot be degraded, so the non-degradable linker must ensure that the linker-drug complex still has the ability to kill tumors. Take T-DM1 as an example: it uses a linker called SMCC (N-succinimidyl-4-(maleimidomethyl) cyclohexane-1-carboxylate) to connect to DM-1 (toxin), and the metabolite lysine of T-DM1—MCC-DM1, although cannot continue to be degraded by lysosomes, still has the same toxicity as DM-1, making it possible to still kill tumor cells.

The toxins used by ADCs currently on the market or under development are basically chemotherapeutics that have been on the market. Generally, the underlying mechanisms of action are listed below:

Cause DNA damage: such as calicheamicins, duocarmycins, pyrrolobenzodiazepines, SYD983, etc.
Inhibit tubulin polymerization: such as auristatins, MMAE, MMAF, maytansinoids, etc.

The connection of linker and antibody needs to be carefully considered in ADC design. The widely used method is to connect the linker to the cysteine ​​or lysine residues on the antibody, but this method has serious disadvantages: an antibody often contains multiple cysteine ​​or lysine residues. A variable number of linkers and toxins are often connected to each antibody, which will lead to uneven physical and chemical properties of ADC drugs. For example, random chemical connection will cause too many toxins to be attached to some of the antibodies, and less or no toxins on the other part of the antibodies, and antibodies without toxins can still bind to the target protein. This binding in turn will inhibit other toxin-loaded antibody to bind with the target protein.

Subsequent research will focus on site-specific conjugation, that is, random chemical binding is no longer used to connect linker and antibody. Some technical routes of site-specific conjugation are summarized as follows:

Table 2 Comparison between different site-specific conjugation technologies. (source: Pharmaceuticals)

Judging from academic publications, the research enthusiasm in ADC is increasing day by day in recent years. There are currently more than 50 ADC drugs in clinical trials (either alone or in combination with chemotherapy) worldwide, but most of them focus on lymphoma, leukemia, and solid tumors such as breast cancer:

Figure 4 (a) Status of clinical trials on ADCs; (b) Different ADC payloads in clinical trials; (c) Different ADC linkers in clinical trials; (d) Clinical trials of ADCs for different type of oncologic indications based on clinicaltrials.gov database search. (source: Pharmaceuticals)

In recent years, with the success of Adcetris (2011), Kadcyla (2013), and Besponse (2017), ADC drugs have set off a small upsurge in clinical trials. With the establishment of more startups focusing on ADC drugs, more ADC drugs will be successfully listed in the future, which is definitely good news for cancer patients.

Reference

• Diamantis N, Banerji U. Antibody-drug conjugates—an emerging class of cancer treatment[J]. British journal of cancer, 2016, 114(4): 362-367.
• Dan N, Setua S, Kashyap V K, et al. Antibody-drug conjugates for cancer therapy: chemistry to clinical implications[J]. Pharmaceuticals, 2018, 11(2): 32.
• Rudnick S I, Lou J, Shaller C C, et al. Influence of affinity and antigen internalization on the uptake and penetration of Anti-HER2 antibodies in solid tumors[J]. Cancer research, 2011, 71(6): 2250-2259.