September 24, 2021

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Experts comprehensively analyze challenges and prospects of ADC drugs

DExperts comprehensively analyze the challenges and prospects of ADC drugs

Experts comprehensively analyze the challenges and prospects of ADC drugs

Antibody-conjugated drugs (ADCs) are currently undergoing rapid development, which use antibodies to selectively deliver cytotoxic drugs to tumor sites. As of May 2021, the U.S. Food and Drug Administration (FDA) has approved 10 ADCs. In addition, there are currently more than 80 ADCs under investigation in approximately 150 active clinical trials.

Although ADC is getting more and more popular, it is still a challenge to expand its therapeutic index (better efficacy and less toxicity). However, the development of some new technologies, such as fixed-point coupling, linkers, payloads, new biological platforms and advanced analysis technologies, are helping to shape the future development of ADCs.

The mechanism and key elements of ADC

Most ADCs follow a similar mode of action, including antibody-mediated receptor binding, ADC internalization, and subsequent payload release and cytotoxicity (below), and of course bystander effects. The success of ADC depends on several key factors.

1) Target antigen (for example, CD30, HER2, CD22. CD33, CD79b, Nectin 4, Trop2, BCMA, CD19)

2) The type of antibody (eg IgG1, IgG2, IgG4, Nanobody, bispecific antibody)

3) The type of payload (for example, (MMAE, DM4, calicheamicin, DM1, MMAF))

4) The type of linker (such as valine-citrulline, Sulfo-SPDB, hydrazone linker)

5) Linker platform (such as lysine, cysteine ​​and site-directed coupling)

6) Target indications (for example, breast cancer, lymphoma, leukemia, uremia, lung cancer, ovarian cancer, etc.).

Experts comprehensively analyze challenges and prospects of ADC drugs
The mechanism of ADC

ADC drugs in markets

Among the 10 ADC drugs approved for marketing, 6 are for the treatment of hematoma (Table 1 and Figure A below), namely Adcetris (Brentuximab vedotin), Besponsa (inotuzumab ozogamicin), Mylotarg (gemtuzumab ozogamicin), Polivy (polatuzumab vedotin), Blenrep (belantamab mafodotin) and Zynlonta (loncastuximab tesirine).

The other 4 treatments for solid tumors (Table 1 and Figure B below) are Kadcyla (Ado-trastuzumab emtansine), Enhertu (trastuzumab deruxtecan), Trodelvy (sacituzumab govitecan) and Padcev (enfortumab vedotin)

Experts comprehensively analyze challenges and prospects of ADC drugs Experts comprehensively analyze challenges and prospects of ADC drugs

The structure and antibody subtypes of 6 ADC drugs for the treatment of hematoma

Experts comprehensively analyze challenges and prospects of ADC drugs
4 subtypes and structures of ADC drugs for the treatment of solid tumors

ADC drugs in the clinic

There are currently more than 80 ADCs in active clinical trials, most of which are in phase I and phase I/II (Figure a below and the table at the end of the text). More than 80% of clinical trials are studying the safety and effectiveness of ADC in various solid tumors, while the remaining trials involve hematological malignancies (Figure b below).

This shows that following the early success of T-DM1 and the recent approval of T-Dxd, sacituzumab govitecan and enfortumab vedotin, ADCs that have turned to research on solid tumors have gradually increased in recent years. The ADCs in more than 80 clinical trials targeted about 40 different targets (Figure c below).

Currently, HER2 is one of the most attractive targets in ADC drug development. There are currently three anti-HER2 ADCs in phase III trials. Among them, RC48 is produced by RemeGen, which connects the anti-HER2 IgG1 antibody hertuzumab through a valine-citrulline linker that can be cleaved by protease and about four MMAE molecules through cysteine. In preclinical studies, lower doses of RC48 showed anti-tumor activity in sensitive and resistant xenograft models of trastuzumab and lapatinib. Compared with T-DM1, it has a better anti-tumor effect.

Studies have shown that in multiple phase I trials for HER2-positive malignant tumors, its safety can be effectively controlled, and in a pivotal phase II trial (NCT03507166), it has achieved encouraging results: including In pretreated HER-2 positive locally advanced or metastatic urothelial carcinoma, the overall response rate (ORR) was 51.2%.

In terms of small molecule drugs, most use drugs to induce the disturbance of tubulin and arrest mitosis, while a few cause DNA damage (Figure 3d). Of course, some new small molecule drugs are gradually being used in ADC drugs, such as TRL7/8 (Toll-like receptor agonists), RNA II polymerase inhibitors, and target BCL-xL anti-apoptotic proteins.

Coupling method: the coupling method can directly affect the quality of ADC. The quality of ADC affects the safety and efficacy of the product. There are currently three main coupling methods: cysteine ​​through reduction of interchain disulfide bonds, lysine exposed on the surface of antibodies, and site-directed coupling technology.

Currently, most of the ADC drugs in clinical trials are through traditional cysteine ​​coupling or site-specific coupling technology approved by the manufacturer. Only a small percentage of them use the traditional lysine coupling method, which may be due to the large heterogeneity of ADC drugs that this method may cause (Figure e below).

Experts comprehensively analyze challenges and prospects of ADC drugs
ADC drugs for the treatment of tumors in the clinic

Challenges in ADC development

There are many challenges to be faced in the development of ADC drugs. The first is to prove the effectiveness of the drug. Many drugs have been forced to terminate because they cannot be proven clinically, such as MM-302 and Rova-T.

In addition, the toxicity associated with ADC drugs is also a challenge. For example, the use of calikamycin as a load is associated with an increased incidence of liver damage and liver toxicity. For example, the incidence of venous occlusive disease (also known as sinus obstruction syndrome) has increased and the incidence of drug-induced liver injury has increased. During clinical trials and post-approval use of gemtuzumab, an increase in venous occlusive disease (also known as sinus obstruction syndrome) and drug-induced liver damage was observed. At the same time, there are also reports of side effects related to peripheral neuropathy and neutropenia caused by MMAE, ocular side effects caused by MMAF, and neutropenia caused by topoisomerase I inhibitors.

The emergence of drug resistance: Over time, tumors can develop some mechanisms to overcome the efficacy of the drug, resulting in weaker or disappearance of the drug. Since ADC is a drug that is effective in multiple ways, drug resistance can occur in any part of the drug’s effect (the figure below).

One mechanism of drug resistance may come from the regulation of antigen recognition by antibodies. This may be due to the down-regulation of target expression on the cell surface. In some preclinical studies, cells continuously treated with ADC eventually developed a model of acquired resistance over time, and these cells showed reduced expression of target antigen proteins. In response to this type of resistance mechanism, some bispecific antibody ADC drugs targeting bi-epitope have been in clinical development, such as ZW49 (targeting different epitopes of Her2) and M1231 (targeting EGFR and UC1).

Another common mechanism of resistance is to clear the payload through ATP binding cassette transporters. Many cytotoxic warheads used for ADCs may be the substrate of these pumps, which may cause the drug to flow out of the target cell and reduce the efficacy of the drug. Clinical data shows that the efflux pump is one of the reasons for the reduced efficacy of gemtuzumab.

Of course, drug resistance may occur at any step in the process of ADC drugs: 1) defects in antibody internalization, transport and recovery, 2) lysosomal degradation leading to drug release barriers, and 3) cell death pathways. Change (picture below)

ADC resistance mechanism

Important considerations in ADC design

Target antigen

Improving the safety and effectiveness of ADCs depends to a large extent on the choice of target antigen and its interaction. Two key parameters are involved in the selection of target antigen: tumor specificity and expression level. Ideally, the selected target will exhibit a high degree of tumor-specific or disease-specific expression, and the expression of little or no specificity of the target in normal tissues is essential to reduce the toxicity of ADC. The overall success of the ADC plays an important role. The specificity of the target is critical to reducing the toxicity of ADCs and therefore plays an important role in its overall success.

From the perspective of oncology, antigens can be expressed as surface receptors in tumor cells, tumor stem cells, or in tumor blood vessels and microenvironment. In the best case, the antigen is evenly expressed at similar levels throughout the tumor-associated cells. In this way, ADCs drugs can make full use of the bystander effect and overcome tumor heterogeneity.

Monoclonal antibodies

After selecting the target antigen, it is necessary to screen antibodies and antibody subtypes according to the ability of the antibody to penetrate the tumor. The ADCs being developed and approved all belong to the IgG1, IgG2, or IgG4 subclass. These subclasses differ in cross-linking ability and biological activity, including ADCC and complement-dependent cytotoxicity (CDC). Compared with IgG2 and IgG4, IgG1 is used the most because of its stronger delivery capability and more effector functions. However, when considering the target characteristics or different mechanisms, in some cases, the ADCC and CDC effects of IgG1 need to be avoided. At this time, IgG2 and IgG4 antibodies become the first choice. At the same time, the choice of subtype has a great influence on coupling, especially when considering the use of cysteine ​​for coupling.

The size of the monoclonal antibody

After the selection of antigen and antibody subtypes, the size of the antibody needs to be considered. In the past, full-length IgG antibodies were mostly selected, but such antibodies have certain limitations in terms of endocytosis and tumor penetration. In order to solve this problem, some new antibody forms have emerged, such as Fab-ADC, ScFv-ADC, etc. These antibodies are relatively small and therefore have good permeability, but because they do not contain Fc, they have a relatively short half-life.

Antibody modification

Antibodies are similar to other proteins and will be modified (PTMs) during the production or storage process, such as deamination, sialylation, or cleavage of the C-terminal lysine. These modifications will affect the stability of the antibody, so It will also affect the stability of the ADC, thereby affecting the effect of the ADC.

Internalization of ADC

Most ADCs are designed for target antigens, and these antigens show efficient internalization through receptor-mediated endocytosis to promote the ADC’s entry into the cell after recognition. For a long time, in order to make the release of cytotoxic load have less impact on healthy cells, receptor internalization has been a requirement for effective ADC design. In order to design a successful internalized ADC, the ADC’s target accessibility, density, internalization rate, and intracellular transport must be evaluated.

Generally speaking, compared with hematomas, ADCs targeting targets expressed on solid tumors have more physical barriers that need to be overcome before they can reach the antigen after administration. In hematological malignancies, the target is easily exposed to ADCs in the circulation. In addition, the target will sometimes “fall off” from the surface and be released into the blood, which will cause the loss of ADC in the circulation and reduce the efficacy of the drug. Measuring receptor expression (receptor copies/cell), internalization rate, and recovery rate can all directly affect ADC entry into target cells.

Considerations in ADC design

Cytotoxic load (warhead)

Antibodies are the most important component of ADC drugs, and the cytotoxic load is the final component that kills tumor cells. Cytotoxic payload (sometimes called “warhead”) is usually a small molecule drug whose purpose is to induce cell killing of target tumor cells/tissues. The first generation of ADCs used small molecule poisons including doxorubicin, but their clinical activity was low. The second-generation ADCs use more potent small molecule drugs. These drugs are too toxic as independent treatments, but when selectively delivered to target cells, IC50s are in the range of 0.01-0.1 nM, which shows very good Efficacy.

Even so, due to biodistribution, absorption, and loss of conjugated drugs in circulation, it is estimated that only 1-2% of the ADC payload reaches the intracellular target. Therefore, the effectiveness of the payload must be high (preferably in the sub-nanomolar range) so that ADC can eradicate target cells even at low cumulative concentrations. In order to achieve this goal, most of the current ADCs contain powerful molecules that either disrupt tubulin polymerization or induce DNA damage.

It is essential to understand the mechanism of ADC payload and its applicability to the target. Although many ADCs currently under development use anti-mitotic tubulin destroyers to selectively destroy rapidly proliferating cells, these payloads may not be effective against tumor cells that are not highly proliferating. It is worth noting that a variety of new small molecule toxicants have been studied in clinical trials. For example, less toxic topoisomerase inhibitors have attracted more and more attention. Such small molecule poisons make ADCs with higher DAR possible.

For example: Compared with T-DM1, the recently approved trastuzumab deruxtecan has a higher DAR value, but its toxicity is lower, its stability is better, and it has a better therapeutic effect. Other payloads, such as PBD dimers that exhibit high potency, also appear in ADC designs. The recently approved Lonca-T utilizes PBD dimer, which can exert its efficacy at a lower concentration, and has a highly effective bystander effect, and because of its short half-life, the probability of systemic toxicity is lower. . In addition, some other types of “warheads” such as immunostimulants, RNA polymerase II inhibitors, and BCL-xL inhibitors that promote apoptosis are also under development.

Insufficient folding of the antibody and the exposed hydrophobic amino acids will cause the antibody to aggregate, and some hydrophobic small molecule poisons as payloads will increase the risk of antibody aggregation. The aggregation of antibodies will not only reduce the efficacy of ADC drugs, but may also cause the drugs to enter non-target cells and cause side effects. Studies have shown that the use of hydrophilic linkers, spacer sequences, and hydrophilic small molecule poisons can effectively avoid antibody aggregation.

Linker and coupling method

Linker is an important part of ADC, it connects mAb and cytotoxic load. This linker helps stability in the circulation until the ADC reaches the target cell and releases the payload. At present, linkers are mainly divided into two categories: cleavable and non-cleavable. The cleavable linker can be lysed under the action of certain environmental factors, thereby releasing the free drug into the cell sap. This includes hydrazine linkers that are cleaved in the acidic environment of endosomes and lysosomes, such as those used for gemtuzumab ozogamicin.

In the presence of proteases or reducing agents, cleavable linkers can also be cleaved, such as type B proteases or high concentrations of glutathione. For non-internalized ADCs, drug release depends on extracellular cleavage, such as glutathione and protease cleavage of linkers to release drugs to kill tumor cells.

The non-cleavable linker can resist degradation by proteases and rely on complete degradation of the antibody to release the linker-load complex. This requires the cytotoxic load to remain active when the linker is attached. Because of this, non-detachable linkers have been proposed as a strategy to overcome the linker-load complex no longer being a substrate of MDR1. Therefore, ADCs of different MOAs may be the decisive factor.

For some ADCs, chemical linkers can also balance the hydrophobicity between the antibody and the payload, thereby reducing potential aggregation. In this regard, the bioanalytical significance of all components analyzed is very important for evaluating the safety and effectiveness of ADC. Hydrophilic linkers and spacers, including cyclodextrin, polyethylene glycol and other polymers, may play a role in improving the stability of ADC drugs in the circulation, their efficacy on target cells, and overall pharmacokinetics.

In addition to the selective choice of linkers, the method of linking the payload with the antibody is crucial in regulating the uniformity and efficacy of the ADC. Traditional methods rely on lysine and interchain cysteine ​​to link cytotoxic molecules to antibodies. The heterogeneity of ADC drugs conjugated via lysine is inevitable because of the large number of lysines available for coupling in the antibody. At the same time, since the coupling site and quantity cannot be controlled, the coupling of lysine related to Fc receptor binding may affect the binding of antibody Fc to receptor, which may reduce the efficacy of ADC drugs.

At present, most ADCs in use or development rely on interchain disulfide cysteine ​​for coupling, and 4 of them (IgG1 and IgG4) or 6 (IgG2) interchain disulfide bonds are used by excessive reducing agents such as Reduction of phosphine or dithiothreitol. This method avoids the destruction of the intrachain disulfide bonds, and at the same time releases the sulfhydryl group from the cysteine ​​residues participating in the interchain disulfide bond.

The resulting product is a mixed ADCs. Each ADC of IgG1 or IgG4 type contains 0-8 drugs, and each ADC drug of IgG2 type contains 0-12 drugs. The DAR values ​​in these mixtures are (0, 2, 4, 6, 8, 10, 12). Therefore, compared with the lysine coupling method, the uniformity of ADCs is improved because the cysteine ​​available for coupling after reduction is greatly reduced.

However, even with a more uniform coupling method, the control of DAR value and drug load distribution (DLD) still needs to be strengthened. Because optimizing the DAR value and DLD is very important for the pharmacokinetics of ADC. Ensuring the homogeneity of all ADCs produced is a key aspect of quality control for developers and manufacturers to ensure product safety. Early studies have shown that it is ideal for each antibody to have DAR values ​​of 2-4 drug molecules, which can ensure the stability and efficacy of ADC in the circulation. A low DAR value may affect the efficacy of ADC, while a high DAR value may increase the off-target toxicity of the drug and accelerate the rate of drug clearance.

However, the recently approved trastuzumab deruxtecan and sacituzumab govitecan challenge the previous limit of 4 DAR. In both cases, each antibody carries nearly eight drug molecules. In addition, the ADC drugs currently in clinical evaluation have a wide range of DAR values. The smallest DAR value is 1, such as BDC-1001, and the highest DAR value is 15, such as ASN004.

In addition, DAR may also affect the dose, the concentration of the antibody administered, and the subsequent absorption of the drug by the tumor. According to the effectiveness of small molecule toxicants, ADCs with low DAR values ​​can be administered at higher doses to increase the concentration of antibodies to promote the penetration of ADC drugs into solid tumors. ADCs with high DAR can be administered at lower doses, which may result in lower antibody concentrations and poor tumor uptake. This view is supported by in vitro studies.

At present, some new site-directed coupling technologies have emerged. These technologies can use special linkers to produce ADC drugs with a single DAR value. The position and coupling through the thiol.

THIOMAB™ Site-specific Coupling Platform

There are some methods that introduce unnatural amino acids for site-specific coupling, such as the introduction of p-acetylphenylalanine and p-azidomethyl-L-phenylalanine. In addition, the glycosyl in the antibody can also be used for coupling. This method can not only solve the heterogeneity, but also has consistency;

Another site-specific coupling technology, SMARTag™, uses a compound enzyme reaction to install an aldehyde tag to achieve coupling at a specific location. As mentioned above, this method uses a chemical enzymatic reaction to install an aldehyde tag for coupling at a specific location. The coupling site is a formylglycine (aldehyde) residue, which is based on a specific pentapeptide consensus in the antibody. Cysteine ​​is produced by enzymatic oxidation.

SMARTag™ platform

Another site-specific coupling method has received more and more attention-disulfide bridges, which can control DAR and DLD without the need for redesign. This technology uses the traditional cysteine ​​coupling method to couple bifunctional drug molecules. Therefore, a drug molecule is coupled to the disulfide between each chain. Using this method, developers can get ADC drugs with DARs values ​​of 4 and 6 (the DAR value depends on the immunoglobulin subtype).

Site-specific coupling of disulfide bridges

ADC product quality evaluation

ADCs are complex molecules with unique critical quality attributes (CQAs). In addition to the quality requirements for naked antibodies, it also includes DAR, DLD, the number of unbound payloads or unbound antibodies, antigen binding, and cell viability. In order to ensure product quality and production consistency, each CQA must be fully evaluated. In addition to conventional antibody analysis methods, some advanced techniques have been developed to evaluate ADC-specific CQA. Such as high-resolution mass spectrometry (MS). Native ion mobility (IM) mass spectrometry, and two-dimensional high performance liquid chromatography (2D-HP), etc. (the table below).

Analysis of DAR value and DLD

For cysteine-linked ADCs, hydrophobic interaction chromatography is usually used to determine the average value of DAR, DLD, and unconjugated antibody. Emerging technologies include high-resolution MS and IM-MS. The average value of DAR, DLD, and unconjugated antibody can be determined using MS-compatible ammonium acetate buffer under neutral pH conditions. ADCs coupled via lysine are highly heterogeneous, so their quality analysis is challenging.

For this type of ADC drugs, the DAR value is usually determined by measuring the specific absorbance of the drug and the absorbance of the antibody at 280 nm. Antibody coupling sites and PTMs (post-translational modifications) can be determined by peptide maps. For ADC drugs coupled by cysteine, a single tryptic peptide map can be generated for analysis. Lysine-conjugated ADC drugs may need to be analyzed by peptide maps combined with trypsin, Asp-N and Glu-C; in addition, unsheathed capillary electrophoresis (CE) combined with MS/MS can also be used to evaluate these properties.

Impurities related to processes and products

Impurities related to the specific process of ADCs include unbound payloads, free linkers, or other chemicals used in the manufacturing process. Reversed phase (RP)-HPLC is used to evaluate these potential impurities in the final product. Removal of protein-containing species (such as intact ADC, unconjugated antibody) can improve the detection ability of protein precipitation, size exclusion chromatography (SEC) or SEC×RP 2D-LC for process-related magazines. In addition, two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) can improve the sensitivity of detection and can detect a small amount of free payload. Product-related impurities, such as polymers, fragments, charge variants and other PTMs on antibodies, can be evaluated by SEC, analytical ultracentrifugation, CE, capillary isoelectric focusing, ion exchange chromatography and peptide maps.

Pharmacodynamic determination

Pharmacodynamic testing is an important part of the quality control strategy of complex pharmaceutical products. It is combined with physical and chemical property testing to ensure the consistency of production during the product life cycle. Generally speaking, the efficacy determination should reflect the MOA of the product. For multifunctional products, more than one efficacy test is required to fully grasp its biological activity. An ADC can retain its MOA associated with the antibody, such as signal blocking, ADCC, or CDC.

ADC may also cause bystander effects, so when small molecule poisons are released into the tumor microenvironment, both antigen-positive cells and antigen-negative cells will be affected. Therefore, ADCs should be evaluated by two methods: antigen binding test and cell-based function test.


The advent of ADCs provides a promising treatment for many types of cancer. As more and more ADCs enter clinical trials, the industry is gradually shifting from traditional technologies to newer and more powerful technologies to develop this complex product. This includes exploring new tumor antigens, new antibody structures, new payloads, new linkers, and advanced coupling methods to improve the therapeutic window of ADC.

In the new antibody grid structure, scFv has better solid tumor permeability and uptake; bispecific and bi-epitope ADCs may be able to overcome the obstacles of tumor heterogeneity. Probodies and other conditionally activated technologies (CABs) may be able to reduce the impact of off-target effects.

A variety of payload classes other than microtubule disruptors, including PBD dimers, topoisomerase inhibitors, anthracyclines, and protein-specific modulators, are being introduced into a new generation of ADCs. In addition, some fixed-point coupling technology platforms are used to improve the stability of the ADC in the circulation while maintaining the effective release of the payload.

The complexity of ADC brings unprecedented challenges to the analysis of drugs, especially when adding hydrophobic payloads. ADC analysis requires advanced analysis techniques, and these techniques need to be continuously updated with the rapid development of ADC. Apply appropriate analytical techniques for product consistency.

ADC treatment is shifting from hematoma (lymphoma and leukemia) to solid tumors (such as breast, urethral cancer, lung cancer and ovarian cancer). The expansion of these clinical indications also highlights the therapeutic potential of ADC.

Many ADCs in the clinical pipeline are being evaluated in conjunction with other treatments (such as immune checkpoint inhibitors and mAbs for different antigens, etc.). The accumulated clinical data, combined with the product quality information described here, is helping to shape the future development of ADC.

Table 2: ADC drugs in the clinic

(source:internet, reference only)

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