Antibody-drug conjugates (ADCs) are a novel class of highly targeted biopharmaceutical drugs that conjugate a cytotoxic drug with a monoclonal antibody (mAb) through an applicable chemical linker (Fig. 1). Drug molecules are transported to target tissues via the specific targeting effect of antibodies, which can reduce the systemic side effects of drugs, expand the scope of drug treatment, and enhance the therapeutic potential of antibodies.

Fig. 1. Schematic structure of an antibody drug conjugate (ADC) (Int J Mol Sci. 2016, 17(4): 561).

Antibody Drug Conjugate Review

The concept of ADC drugs was proposed as early as 1900, but due to technical limitations, it remained at the theoretical level for a long time. From 1990 to 2000, monoclonal antibody drugs gained widespread clinical application, lowering the barriers to developing ADC drugs. After more than 90 years of research and development, ADC has developed from early humanized/fully human monoclonal antibodies to second-generation fully human monoclonal antibodies, and currently the third generation of ADC fully human/engineered monoclonal antibodies. In the third stage of development, the continuous emergence of new technologies and new conjugation forms has brought new power to ADC drugs. ADC drugs have begun to truly enter clinical practice, and ADC drugs targeting different targets have obtained corresponding indications in the fields of hematological tumors and solid tumors.

• ADC Target

The successful development of ADCs relies on the specific binding of antibodies to target antigens. The ideal ADC target is highly expressed on the surface of tumor cells, low or not expressed in normal tissues. Targets expressed in normal tissues will ingest ADC drugs, which will not only lead to "off-target" toxic effects but also reduce the dose of ADC enriched in cancer tissues and shorten the ADC drug treatment time window.

At present, leukocyte surface differentiation antigen is the first widely used ADC target. Among the 20 ADC drugs currently in clinical development, 10 ADC drug targets (CD33, CD30, CD79b, CD22, CD19, CD56, CD138, and CD74) are leukocyte surface antigens. In addition, some solid tumor surface receptor molecules have gradually been found to be suitable clinical ADC targets. For example, ADC drugs for PSMA on prostate cancer cells, epidermal growth factor receptor EGFR, and nectin 4 in ovarian cancer tissue have all entered the clinic Phase II.

• ADC Antibody

The high specificity of antibody molecules is the basic requirement for achieving ADC drug efficacy and concentrating cytotoxic agents on tumor sites. To prevent the detection and binding of the antigen from being affected, cytotoxic drugs are usually attached to the Fc part or constant region of mAb. Currently, all ADC antibodies are IgG molecules, because of their high affinity to target antigens and longer half-life in the blood, which helps to increase the accumulation of drug molecules at the tumor sites. In addition, the immunogenicity of ADCs is one of the main determinants of circulating half-life. Early ADCs used mouse monoclonal antibodies to cause a strong acute immune response (HAMA) in humans. Currently, most ADCs use humanized antibodies or fully humanized antibodies. The final construction of the ADC requires conjugation of linkers and drugs to native antibodies. Since drug conjugation can occur with natural amino acid residues, complex antibody engineering is required. Currently, there are two conjugation methods in FDA-approved ADC products: non-specific conjugation and site-specific conjugation.

• Non-specific Conjugation

Random conjugation does not require modification of the antibody, but directly utilizes alkylation and acylation of lysine residues on its surface or releases cysteine residues through reduction of disulfide bonds to connect to the linker. There are large differences in the number and position of randomly coupled cytotoxic drugs. The resulting conjugate is a mixture with uneven distribution of DAR and different pharmacokinetics, activity, and safety, resulting in reduced efficacy. The cysteine (8) and lysine (80) residues on the antibody are easier to undergo chemical reactions and be modified, so they are often used as binding sites for effector molecules.

Lysine Conjugation: In the early development of ADC, lysine on the antibody was usually selected as the binding site, and its synthesis efficiency was high. However, since each antibody has up to 80 lysine residues, it leads to great heterogeneity. The average DAR is 3.5-4, distributed between 0-7, which affects the in vivo distribution and cytotoxicity of ADC drugs.

Cysteine Conjugation: There are 8 free cysteines on each antibody that can be connected to the linker through disulfide bonds. Due to the limited number of binding sites and the unique reactivity of the thiol group, cysteine conjugation is used. Amino acids serve as attachment sites to help reduce ADC heterogeneity. The DAR values are 2, 4, 6, and 8, which are relatively better than lysine.

• Site-Specific Conjugation

Due to the shortcomings of randomly coupled ADC drugs such as DAR and uneven conjugation positions, companies have gradually begun to favor site-specific conjugation. Compared with random conjugation, ADC drugs obtained by site-specific conjugation have better uniformity and pharmacokinetic properties. It reduces non-therapeutic toxic side effects caused by drug shedding and has a wider therapeutic window. Site-specific conjugation technology transforms the antibody itself to connect the drug to specific sites on the antibody, which greatly improves the uniformity of the ADC. Site-specific conjugation technologies mainly include Thiomab, ThioBridge, introduction of unnatural amino acids, introduction of sequence tags or glycans, etc.

• Cysteine conjugation technology
• Engineered mutation unnatural amino acid conjugation technology
• Glycosyl conjugation technology
• Amino-site conjugation technology
• Enzymatic conjugation technology

• ADC Payload

Payload is a key factor in the successful development of ADC drugs. Only a small part of the antibodies injected into the body are accumulated in the solid tumor tissue. Therefore, the toxic molecules with sub-nanomolar (IC50 value of 0.01-0.1nM) are suitable payloads. In addition, toxic molecules must have suitable functional groups that can be coupled, and the functional group should have strong cytotoxicity and hydrophobicity, and be very stable under physiological conditions. The toxic molecules currently used for ADC drug development can be divided into two categories: microtubule inhibitors and DNA damaging agents. Other small molecules such as α-amanitin (selective RNA polymerase II inhibitors) are also under investigation.

• Microtubule Inhibitors

Microtubules are hollow fibrous filaments composed of α-tubulin and β-tubulin. Along with actin polymers and intermediate fibers, microtubules are an integral component of the cytoskeleton. It is critical for a variety of cellular functions, including maintenance of cell shape, cell motility, and intracellular transport of vesicles, mitochondria, and other components. Therefore, drugs that interfere with microtubule dynamics can have significant effects on spindle formation and ultimately the process of cell division. Currently, the most important tubulin inhibitors used as ADC payloads are Auristatin and Eribulin derived from marine species and Maytansinoids derived from plants.

• DNA Damaging Agents

DNA damaging agents are divided into three categories due to different mechanisms of action: DNA double-strand destroying agents, DNA inserting agents, and DNA alkylating agents. DNA is crucial in the growth and proliferation process of cells. Its destruction can effectively kill tumor cells and inhibit their rapid proliferation. Some DNA damaging agents, such as PBD dimer and Duocarmycins, may produce delayed toxicity when administered at high doses in animals, so careful consideration should be given to the design of the dosing cycle.

• ADC Linker

Choosing a suitable linker to constrain antibodies and payloads is the key to successful ADC construction. The linker needs to exist stably in the blood circulatory system and can release active payloads quickly when positioned in or near tumor cells. Based on their release mechanisms, linkers can be divided into two categories: cleavable and non-cleavable. Non-cleavable linkers rely on degradation of the antibody after endocytosis into lysosomes, thereby releasing the drug while the linker remains covalently attached, such as Kadcyla. In contrast, cleavable linkers are more common, and their drug release mechanisms include acidic pH hydrolysis, thiol reduction, and protease cleavage. Among them, specific recognition of dipeptides (such as valine-citrulline) by enzymes in lysosomes is the most common cleavage mechanism, leading to the release of drugs. An ideal linker must meet the following conditions:

• The instability of the linker will cause the premature release of the payloads and cause damage to normal tissue cells.
• Once the ADC is internalized into the target tumor tissue, the linker needs to have the ability to quickly dissolve and release toxic molecules.
• Hydrophobicity is also an important feature when considering linker. Hydrophobic connection groups and hydrophobic payloads usually promote the aggregation of ADC small molecules, thereby causing immunogenicity.

What is the Mechanism of ADC Toxicity?

There are two main ways for ADC to exert anti-tumor activity. First, the specific mAb binds to the targeted cell surface antigen, is internalized by the tumor cells and processed by the endolysosomal system, and the payload is released into the cytoplasm, ultimately inducing cell apoptosis through the cytotoxic pathway. The second is to induce tumor cell death through the bystander killing effect.

Fig. 2. Mechanism of action and biological activity of ADCs (Bioorganic & Medicinal Chemistry Letters. 2014, 24(23): 5357-5363).

Specifically, ADCs rely on highly targeted tumor antigen recognition capabilities to recognize and bind to cell surface-specific tumor antigens, followed by internalization of the ADC via endocytosis. Upon entry into tumor cells, ADCs are transferred to endosomes or lysosomes, which digest potential linkers or antibodies and actively release cytotoxic drugs into the cytoplasm. The released cytotoxic drugs then flow into the cytoplasm and induce apoptosis through DNA insertion or inhibition of microtubule synthesis. Therefore, the correct target, antibody, linker and cytotoxic payload become the four key factors affecting ADC drugs. An ideal ADC should have the following characteristics:

• A monoclonal antibody (mAb) that targets a specific cancer antigen while sparing healthy cells.
• A potent cytotoxic small molecule drug with high systemic toxicity designed to induce target cell death after being internalized and excreted by tumor cells.
• Stable linkers in circulation to release drug agents in tumors.
• Monoclonal antibodies are covalently linked to small molecule cytotoxic agents that focus on specific cancer cells to reduce overall systemic toxicity.
• Enhance the cytotoxic potential of monoclonal antibodies.
• Inducing higher tumor selectivity while improving drug tolerability.
• In contrast to standard chemotherapeutic biologic agents or drugs, systemic exposure is limited.

ADC Antibody Drug

As a new type of biological agent, ADCs take advantage of the highly specific selectivity of antibodies against cancer cells, allowing cytotoxins to target cancer cells more precisely, thereby improving the therapeutic effect and reducing the side effects of drugs. Since the first approval of Mylotarg (gemtuzumab ozogamicin) in 2000, numerous advances have been made in the discovery, development and manufacturing of ADC technology. Below are some representative antibody drug conjugates.

• Enfortumab Vedotin

Enfortumab vedotin (Padcev®) is an ADC targeting nectin-4, consisting of a human monoclonal antibody targeting nectin-4, a protease-cleavable linker, and MMAE. Nectin-4 is a cell adhesion molecule that is overexpressed in 97% of urothelial carcinomas and is associated with tumor growth and proliferation. Nectin-4 is expressed at very low levels in adult healthy tissues, but is highly expressed in a variety of tumor cells, such as urothelial cancer, bladder cancer, breast cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, and pancreatic cancer. The specific high expression of Nectin-4 is closely related to tumor occurrence and metastasis, and can promote tumor cell proliferation, differentiation, migration, and invasion by activating the PI3K/AKT pathway. Therefore, nectin-4 has become an important target for the diagnosis and treatment of many solid tumors. Currently, Padcev® is the first ADC approved to treat urothelial cancer and the first approved drug targeting nectin-4.

• Sacituzumab Govitecan

Sacituzumab govitecan is a monoclonal antibody targeting Trop-2, which is conjugated to SN-38 through an acid cleavable hydrazone linkers. SN-38 is a topoisomerase-1 inhibitor and the active metabolite of the chemotherapy drug irinotecan. Trop-2 is a transmembrane glycoprotein. It is overexpressed in various tumor tissues such as breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, colon cancer, and pancreatic cancer. Especially in triple-negative breast cancer, the expression rate is as high as more than 90%. After administration, sacituzumab govitecan binds to Trop-2 on tumor cells and promotes the release of SN-38, causing DNA damage and subsequent cell cycle arrest. Due to the membrane permeability of SN-38, it can stimulate the anti-tumor effect of nearby cells without being internalized, exerting a bystander effect. In April 2020, Trodelvy® received accelerated approval from the FDA for the treatment of metastatic triple-negative breast cancer in adults who have received two or more prior treatments for metastatic disease.

• Trastuzumab Deruxtecan

Trastuzumab deruxtecan (Enhertu®) is a representative of the third generation ADC, consisting of Her2-targeting trastuzumab, a cleavable tetrapeptide linker and a cytotoxic topoisomerase I inhibitor. Studies have shown that T-DXd is effective against tumor cells with high or low expression of Her2 or tumor cells with Her2 mutations. The reasons may include: 1) T-DXd has a DAR value as high as 8, so it has an efficient payload; 2) the released payload has high membrane permeability, allowing it to enter adjacent tumor cells and produce a bystander effect; 3) the novel tetrapeptide-based linker has high stability in plasma. In December 2019, Enhertu® received accelerated approval from the FDA for the treatment of adult patients with unresectable or metastatic Her2 positive breast cancer who have previously received Her2 therapy.

• Brentuximab Vedotin

Brentuximab vedotin (Adcetris®) is a CD30-targeting ADC composed of the chimeric lgG1 antibody cAC10, MMAE, and a protease-cleavable linker. Brentuximab vedotin is a representative drug of the second generation ADC. It uses human-mouse chimeric antibodies or humanized monoclonal antibodies instead of mouse-derived monoclonal antibodies, which not only reduces immunogenicity but also enhances tumor targeting. Brentuximab carries an average of 4 MMAE molecules, has a DAR of 4, and a molecular weight of approximately 153 kDa. After internalization, proteolysis releases MMAE, which induces cell cycle arrest and apoptosis by disrupting the microtubule system. Because MMAE has a bystander effect, brentuximab vedotin is also effective in histologically heterogeneous tumors that express CD30. Adcetris® was approved by the FDA in August 2011 for the treatment of Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (sALCL).

With more than a decade of experience in ADC development and manufacturing, BOC Sciences has become a trusted partner for customers seeking to advance novel ADC therapies from concept to commercialization. We assist customers at every stage of the ADC development process, from early-stage development to late-stage clinical manufacturing, BOC Sciences offers comprehensive services to accelerate the development of novel ADC therapeutics. Our team of process development scientists has extensive experience in optimizing conjugation chemistries, reaction conditions, and purification methods to maximize the purity and stability of ADCs. We utilize multiple analytical techniques, including mass spectrometry, high-performance liquid chromatography, and bioassays, to characterize and evaluate the quality of ADC products throughout the manufacturing process. If you are interested in our ADC services, please contact us for more information.