Antibody-drug Conjugates: A Comprehensive Guide

1. What Are Antibody-drug Conjugates?

Antibody Drug Conjugate (ADC) is an innovative class of powerful biological drugs that integrate a highly effective cytotoxic drug with a specially designed monoclonal antibody through an appropriate linker. They enable targeted delivery of their conjugated compounds to cancerous tissues while minimizing toxicity to healthy tissues and enhancing the therapeutic benefit for patients.

An ADC mainly comprises three components: a monoclonal antibody (mAb) connected to a payload through a chemical linker. The ADC is like a missile with the ability to accurately strike the target. The mAb acts like a missile guidance system. It is engineered to target specific antigens expressed on the surface of cancer cells. The payload is typically a cytotoxic drug with highly anti-tumor properties, similar to a high-explosive warhead of great killing power. The linker is the critical structure that combines mAb with cytotoxic drug, akin to the missile’s connection device.

2. Critical Elements in the Design of Effective Antibody-drug Conjugates

Designing a perfect ADC needs to optimize multiple parameters, including target antigen, antibody, linker, payload, and conjugation methods.

ADC Development Solutions

2.1 Target Antigen Selection

A key aspect of developing a successful ADC for cancer is the selection of the specific antigenic target of the mAb component. The selected target antigen should have four characteristics.

First, the target antigen should be highly and homogeneously expressed on the surface of the tumor cells as compared to normal tissues, which can minimize the toxicity of on-target and off-tumor ^[2]. ADC-targeted antigens are tumor-associated antigens (TAAs) usually overexpressed in cancer cells but not in normal cells.

Secondly, the binding site of the targeted antigen should be directed toward the outer surface of the tumor cell rather than the internal area, allowing the circulated ADC to attach to the target antigen before internalization occurs ^[3].

Thirdly, the target antigen should have internalization capability to enable the ADC to enter the cell, which will subsequently improve the efficacy of the cytotoxic drug.

Fourthly, the target antigen should not be shed into the systemic circulation to prevent unintended binding of the ADC outside the tumor.

Figure 2. Target Antigens for ADCs in development and in the clinic and developed

2.2 Antibody Selection

The antibody of an ADC determines its duration in plasma circulation, immunogenicity, immune functions, and target specificity. The antibody should have a high specificity and high binding affinity for the target antigen and low immunogenicity. It also should possess the capability to induce receptor-mediated internalization. In addition, it should maintain a long plasma half-life and facilitate rapid internalization ^[4].

The human IgG isotypes are the most common antibodies used in ADCs because of their most abundant antibody class and potency to initiate an immune effector. ADCs mostly choose IgG1 considering the half-life, structural stability, Fc-mediated effector functions (antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP)) and coupling convenience of different IgG types antibodies.

According to the immunogenicity of antibodies, they can be divided into fully humanized antibodies, humanized antibodies, and chimeric antibodies. Completely humanized or humanized antibodies are more selected to reduce immune response.

2.3 Linker Selection

The linker is the bridge between the mAb and the cytotoxic drug. The design, structure, and chemistry of the linker directly affect the efficacy and safety of the ADC. The ideal linker guarantees adequate stability of the cytotoxic drug in the bloodstream, effectively prevents its premature release, and promotes the targeted release of the drug into tumor cells, thereby improving the overall efficiency and tolerability of an ADC ^[5]. Linkers can be classified into two categories: cleavable and non-cleavable, based on their release mechanism.

Cleavable linkers include chemically cleavable and enzyme cleavable linkers, which are cleaved after exposure to acidic or reducing environment or proteolytic enzymes, resulting in the release of the cytotoxic payload of the ADC. Cleavable linkers release a payload that may have membrane permeability, which can cause a bystander effect. Approximately two-thirds of ADCs in the current clinical trials use cleavable linkers ^[6], which are predominantly dipeptide and disulfide linkers.

Non-cleavable linkers do not contain any biologically or chemically labile bond. The drug release depends on the mAb’s complete degradation within the lysosome following ADC internalization. If this degradation is not insufficient, the released payload will contain the polar amino acids that connect the linker-payload, which are membrane-impermeable and may reduce the bystander effect.

The two most commonly used non-cleavable linkers are maleimidocaproyl (Mc) and succinimidyl trans-4-(maleimidylmethyl) cyclohexane-1-carboxylate (SMCC). A non-cleavable linker is better tolerated but less efficacious.

The important parameters to consider in conjugation include the conjugation site on the antibody component, a well-defined Drug to Antibody Ratio (DAR), homogeneity, and linkage stability.

2.4 Payload Selection

The criteria for choosing the ADC payload are solubility, availability to conjugation, stability, and drug potency ^[7]. ADC payloads should meet several key criteria ^[8]. They possess a high cytotoxic capacity linked to the pronounced lipophilic nature. The payload needs to remain stable during preparation, storage, and circulation. They must target components located within the cells. The active cytotoxic payload should be small, non-immunogenic, and sufficiently soluble in aqueous buffer solutions to ensure optimal conditions for conjugation.

Payloads are typically microtubule-disrupting agents and DNA-damaging agents (topoisomerase inhibitors or inter travelers DNA), which are designed to disrupt key cellular processes such as microtubule function or DNA replication, respectively. Commonly used microtubule-disrupting agents are auristatins and maytansinoids. DNA-damaging drugs include duocarmycin, pyrrolobenzodiazepine, calicheamicins, and doxorubicin.

The drug-antibody ratio (DAR), the number of drug molecules coupled to each antibody molecule, directly affects the efficacy, pharmacokinetics, and safety of an ADC. It is usually determined by the conjugation method used during the development stage. Reasonable optimization of DAR can not only improve the cytotoxicity and biological activity of ADC but also enhance its therapeutic effect.

Additionally, conjugation techniques used to connect mAb and payload determine the fast payload unleash. There are two conjugation methods: stochastic ADC conjugation and site-specific ADC conjugation.

ADCs generated by stochastic conjugation are usually heterogeneous. The number and position of cytotoxic drug molecules attached to the antibody vary, forming mixtures with different DARs such as combinations of DARs of 0, 2, 4, 6, and 8. Due to the uneven distribution of drugs on the antibody, the therapeutic effect may be unstable and the response to patients may vary greatly.

Compared with stochastic conjugation, ADCs generated by specific site conjugation have narrower heterogeneity because the drug is only attached at a specific position of the antibody, ensuring that each molecule has a consistent drug load and a defined structure. This consistency helps improve the expected efficacy and safety of the drug and reduces the risk of adverse reactions.

3. How Do Antibody-drug Conjugates Work?

The tumor-suppressive function of ADCs is primarily mediated by the potency of tumor markers in promoting ADC internalization, rather than by inhibiting cell growth. The mechanism of action of ADCs can be broken down into a series of steps that ensure selective delivery of the cytotoxic drug to the tumor site.

3.1 ADC Targeting the Tumor Cell

ADCs are intravenously injected into the bloodstream to avoid gastric acid and proteolytic enzyme degradation of mAb. The antibody of ADC recognizes the target antigen on tumor cells and binds to it with high affinity, forming an ADC-antigen complex.

3.2 Internalization of ADC-antigen Complex into Tumor Cells

After binding to the tumor cell, the formed ADC-antigen complex is internalized via receptor-mediated endocytosis. The cell engulfs the complex, bringing it into the cell in an endosome.

3.3 Linker Cleavage and Drug Release

After entry into the cell, the ADC-antigen complex in the endosome fuses with the lysosome which leads to low pH. The acidic environment and lysosomes abundant in proteases such as cathepsin-B and plasmin cleave the ADC, resulting in the efficient release of the free cytotoxic drugs into the cytoplasm.

3.4 Drug Cytotoxicity

The drug is released into the cytoplasm where it interferes with the cellular mechanisms, induces apoptosis, and eventual cell death ^[1]. The pathway of cell death depends on the type of used drug. For instance, auristatins and maytansinoids disrupt microtubule formation, preventing proper cell division and causing cell death. Calicheamicins and duocarmycins trigger cell death through DNA intercalation.

4. Advantages and Challenges of Antibody-drug Conjugates

ADCs are an emerging cancer treatment strategy, which can achieve targeted therapy by binding cytotoxic drugs to specific antibodies. Although ADCs have demonstrated significant efficacy and safety in the clinic, they still face many challenges in their development and application.

4.1 Advantages

● Targeted Therapy

ADCs combine the specificity of mAbs with the potent cytotoxic effects of chemotherapy drugs. This allows for precise targeting of cancer cells while sparing healthy tissues, reducing off-target toxicity.

● Enhance the Bioavailability of Drugs

The design of ADCs can improve the bioavailability of drugs in the body, ensuring that more therapeutic ingredients can reach the tumor site, thus improving the therapeutic effect.

● Improve Therapeutic Index

The targeting of ADCs improves the therapeutic index of cell-killing agents. This means less frequent dosing can achieve greater effectiveness and produce fewer side effects during treatment.

● Broad Applicability

ADCs can be designed to target a wide range of cancer types, including those difficult to treat with conventional therapies. They have shown remarkable success in treating certain cancers, such as breast cancer, lymphoma, and leukemia.

4.2 Challenges

Despite their promise, ADCs face several challenges in development and clinical application.

● Short Blood Residency Time

Early ADCs faced a major problem in that they have a short retention time in the blood. This may cause the drug to be quickly cleared before reaching the tumor, thus affecting the efficacy ^[9].

● Low Tumor Microenvironment Permeability

The ability of ADCs to penetrate the tumor microenvironment limits their range of action. Even if ADCs can target tumor cells, they may not be able to fully cover the entire tumor due to insufficient permeability, thus affecting the efficacy ^[9,10].

● Insufficient Payload Potency

Although ADCs are designed to enhance drug efficacy, some ADCs are not more potent than cytotoxic drugs used alone. This shows the need to optimize the selection and design of drug payloads during development ^[10].

● Immunogenicity Issues

The development of ADCs is also accompanied by the problem of immunogenicity. The antibody component of ADCs can elicit an immune response in some patients, leading to the generation of anti-drug antibodies (ADAs) that can neutralize the ADC or cause adverse effects. Therefore, how to reduce immunogenicity is an important direction of current research ^[10].

● Pharmacokinetics and Safety

The pharmacokinetic properties and safety of ADCs still need further study. Although some ADCs have achieved clinical success, improving their pharmacokinetic properties and safety remains a challenge ^[11].

5. FDA-Approved Antibody-drug Conjugates

Among the 14 ADCs approved for marketing, six of them are for the treatment of hematological tumors, and the others are for solid tumors (Table 1). More than 100 ADCs are currently undergoing active clinical trials, most of which are in Phase I and Phase I/II. More than 80% of clinical trials are studying the safety and effectiveness of ADCs in various solid tumors, while the rest involve hematological malignant tumors. This indicates that following the success of T-DM1 early and the recent approval of sacituzumab govitecan and Loncastuximab tesirine, research on ADCs has gradually turned into the solid tumor in recent years.

Table1 FDA-approved ADCs ^[12]

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