The Road to ADC Optimization: Long and Challenging, Yet Will Reach Its Destination

ADC is a powerful therapeutic drug class in cancer treatment, with more than 100 ADCs in preclinical stages. When designing ADCs, there are five key considerations: (1) target; (2) antibody; (3) conjugation strategy; (4) linker; (5) cytotoxic payload. This article mainly provides a detailed analysis of considerations regarding the target, antibody, chemical conjugation, and intracellular payload. As for the ‘linker,’ it has been discussed in depth in the previous article ‘More Than Conjugation: In-Depth Interpretation of the ADC ‘Covalent Bridge’ – Linker,’ which interested readers can access and read on this public account.

1、 Target

Currently, there is a wide range of target antigens or receptors that can be utilized in ADCs. Most of the targets are molecules that are internalized through receptor-mediated endocytosis (RME), but a few targets are located on the cell surface or in the tumor vasculature. For these internalizing targets, the optimal condition for designing ADCs is to ensure they are highly and evenly expressed on tumor cells, with no or low expression in healthy tissues and organs. Then, after ADC binding, they should quickly internalize and be transported to the lysosome.
Table 1: Summary of Clinical ADCs.

 

Figure 1: ADC Structure. The main considerations in designing ADCs include molecular targets, antibodies, chemical conjugation, cytotoxic payloads, and the linker between the antibody and the cytotoxic payload.

CD30 is a perfect example of this standard. CD30 is highly and evenly expressed on tumor cells, with low expression in normal tissues. After binding with the antibody, it is rapidly internalized. The FDA-approved Adcetris® is an ADC targeting CD33. However, not all ADCs perfectly meet these criteria.

For example, the target HER2 in Kadcyla®, which is highly expressed in breast cancer tissue and rapidly internalized after antibody binding, is also expressed in other tissues such as the skin, heart, and gastrointestinal (GI) tract. However, HER2 expression in these organs is still lower than in tumor cells, which is why Kadcyla® has good tolerability. When comparing ADCs in clinical development, most of the differences are due to the target, as the antibody design changes depending on the target. The range of targets is broad, including:

1. Tumor cell surface antigens (e.g., HER2 in Kadcyla®),
2. Surface receptors, such as epidermal growth factor receptor (EGFR; IMGN-289) and folate receptor (IMGN-853),
3. Cell adhesion molecules, such as Nectin-4 (ASG-22ME),
4. Glycoproteins, such as mucin 1 (SAR566658) and GPNMB (CDX-011).

A key issue hindering target development is tumor antigen density and heterogeneity, as well as the acceptable expression levels in normal tissues and organs. Studies have shown that the efficacy of ADCs is related to antigen density, but recent research suggests that expression levels may not be the determining factor for ADC efficacy. A study on anti-CD79b ADC (DCDS4501a) found that in cell lines where CD79b expression levels were above a certain threshold, the expression level had a nonlinear relationship with in vitro activity. Therefore, there may be a minimum threshold, and the threshold for different targets may vary.

For off-target effects in healthy tissues, it is important to consider whether the target is expressed in vital organs or regenerating tissues, as well as the accessibility of the ADC. For example, expression in bone marrow means that toxicity can be controlled, as B cells and myeloid cells are generally regenerable.

PMSA is another typical example. While it is expressed in both cancerous and healthy prostate tissue, the toxicity of the ADC on the prostate is not particularly severe, as most prostate cancer patients opt for prostatectomy. PMSA is also expressed on the apical membranes of the kidneys and small intestines, but in reality, ADCs that are restricted to the basolateral side have difficulty accessing the apical membranes, so the adverse effects are not significant.

Thus, a deeper understanding of the relationship between molecular target expression, specificity, efficacy, and side effects will aid in the future development of ADCs.

2、 Antibody Design

The antibody component is a crucial determinant of the targeting efficiency and pharmacokinetics (PK) of ADCs. Factors such as the size of the antibody, its transport mechanism, effector functions, and ability to bind to Fc receptors are all considered in ADC design. ADCs generally exhibit low clearance and long half-lives, similar to the parent antibody. However, due to the drug-to-antibody ratio (DAR) and additional metabolic pathways for drug cleavage, the half-life and clearance of the ADC may slightly differ from the parent antibody.

One of the considerations in antibody design is its subtype (IgG1, IgG2, IgG3, and IgG4), as differences in the Fc region may affect the effector functions. In antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), IgG1 and IgG3 subtypes are typically more active, while IgG4 generally lacks effector functions. Although these secondary immune functions contribute to anti-tumor activity, they may affect tumor localization.

IgG1 is the most common subtype used in ADCs, such as Kadcyla® and Adcetris®, which exhibit strong ADCC effects. Mylotarg®, which uses the IgG4 subtype, has no ADCC or CDC effects.

A potential approach to improving the antibody component of ADCs is to use smaller antibody fragments, such as nanobodies, which have higher tumor penetration. Another method for enhancing antibody functionality is through engineered strategies that modify specific sites.

3、 Target

The conjugation sites and DAR (drug-to-antibody ratio) both impact PK/PD, binding affinity, and aggregation state, making them essential in ADC development. Most clinical ADCs utilize lysine’s ε-amino group or cysteine’s thiol group for conjugation. When lysine is involved in modification, the drug molecules distribute across many lysine residues on the antibody, resulting in a variable DAR.

Cysteine residue modifications are related to the reduction of disulfide bonds in the antibody’s hinge region and lead to even-numbered DARs. Kadcyla® (with 3.5 DM1 molecules per antibody) and Adcetris® (with about 4 MMAE molecules per antibody) are generated by lysine modification and cysteine conjugation, respectively.

Although more drug molecules bound to the antibody implies a higher payload, it also leads to higher aggregation of the final product, reducing PK and binding affinity compared to the parent antibody. In the case of drug conjugation, the ADC’s half-life is often shorter than that of the unmodified antibody. Therefore, to maintain a longer half-life and higher antibody binding efficiency, the average DAR is typically kept around 3-4.

A significant drawback of current chemical conjugation strategies is the generation of heterogeneous final products with varying DARs at different positions. Computational models show that modifying lysine residues can distribute the drug molecules across approximately 40 different positions, generating over 10^6 different ADC species. While conjugation with cysteine residues reduces heterogeneity, it still produces more than 100 types of ADCs.

To overcome this issue, several companies are attempting to modify specific sites to generate homogeneous ADCs. The main techniques involve engineering cysteine residues, introducing unnatural amino acids, and enzyme conjugation, as described below.

01. Engineered Cysteine Residues

One of the strategies for site-specific modification is to introduce cysteine residues into the antibody sequence through single-point mutations. This technology, developed by a genetic technology company, is known as ThioMAb.

Firstly, phage display technology is used to select suitable conjugation sites within the antibody sequence. Then, through single-point mutations, cysteine residues (usually n=2) are introduced into the antibody’s amino acid sequence. Finally, a maleimide linker is used to connect to the engineered cysteine residues.

02· Incorporating Non-Natural Amino Acids

Sutro Biopharma and Ambryx are using non-natural amino acid-based methods for site-specific modification of ADCs. In this approach, unique functional groups, not found in natural amino acids, are incorporated into the antibody sequence, allowing for specific modification via bioorthogonal chemical methods. These technologies overcome the limitations of maleimide and disulfide exchange chemistries used in cysteine modifications, without requiring additional enzymatic processing steps.

Ambryx introduced a p-acetylphenylalanine (PAcPhe) group, which contains a ketone functional group. This ketone can selectively conjugate with alkoxyamine-based drug molecules, forming a stable oxime linkage. Sutro Biopharma, on the other hand, introduced a p-azidomethyl-l-phenylalanine (PAMF) group, which can undergo strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry with drug molecules, facilitated by microbial strains.

4、 Conjugation

Innate Pharma is utilizing enzymatic post-translational modification to achieve precise site-specific conjugation. This technology requires the removal of N-linked glycans from the antibody or the use of mutated glycosylated variants produced through site-specific mutation. The bacterial transglutaminase (BTGase) is then used to covalently attach the drug substrate, which lacks cadaverine, to the antibody for site-specific conjugation. Other site-specific technologies, such as GlycoConnect™ from SynAffix and Medtope-Enabling technology from Medtope Biosciences, are also being developed.

5、 Cytotoxic Payloads

The cytotoxic payloads used in ADCs primarily fall into two categories:

1. Anti-mitotic agents that disrupt microtubule assembly and play a critical role in mitosis.
2. Drugs that bind to DNA minor grooves and cause DNA double strand breaks (as shown in Figure 2).

Figure 2: Advances in ADC Internalization and Toxicity Mechanisms
After binding to the cell surface receptor, ADCs are internalized via receptor-mediated endocytosis. Once inside the cell, the cytotoxic drug is released through cleavage of the linker or degradation of the antibody, subsequently exerting its cytotoxic effects.

The anti-mitotic drugs MMAE and DM1/DM4 are among the most commonly used payloads (as shown in Table 1 and Figure 3). These drugs preferentially disrupt highly proliferative cells and increase malignant cell sensitivity to mitosis, providing additional specificity. Compared to traditional chemotherapy drugs like doxorubicin and paclitaxel, these ADC payloads are significantly more potent (100-1000 times more effective).

Figure 3: Comparison of Clinical ADC Applications
Although ADCs primarily target tumor sites, statistics show that only 1-2% of the administered dose accumulates at the target. Therefore, improving the efficacy of ADCs is a key focus in their development.

Despite significant variability in the antigen/antibody and linker components of ADCs, the types of drugs used remain limited. Developing new drugs suitable for ADCs is a challenging task. First, the drug needs to be highly cytotoxic. Secondly, the drug target should be intracellular. Additionally, the drug must be small enough to reduce the risk of immunogenicity and water solubility issues. It must remain stable in circulation and have a linker that can effectively conjugate with the antibody. Therefore, there is still considerable room for development in identifying drugs suitable for ADCs.

Conclusion

After decades of trials and research, ADCs have emerged as the latest class of cancer therapies. With two approved drugs showing good clinical performance, and over 30 drugs in various stages of clinical research, the prospects for ADC drug discovery and development are very promising. Optimizing payloads, targeted antibodies, and linker technologies will help generate next-generation ADCs with higher drug delivery efficiency, fewer side effects, and reduced resistance and cancer metastasis potential.