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Antibody-drug conjugates (ADCs) optimization and future development

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Antibody-drug conjugates (ADCs) consist of recombinant monoclonal antibodies (mAbs) that are covalently bound to cytotoxic chemicals (called warheads) through synthetic linkers. ADCs not only have the anti-tumor efficacy of highly cytotoxic small molecule drugs, but also combine the high selectivity, stability and favorable pharmacokinetic characteristics of the mAb. In this article, the selection of antigen targets in ADC development, the warheads used in ADCs in clinical stages, the design and optimization of linkers, antibody selection and optimization, site-specific and alternative conjugate chemistry, and strategies to enhance potency are discussed. Non-oncology ADCs are also included.


  1. Antigen target selection

A major problem in the development of cancer ADCs is the identification and validation of sufficient antigenic targets for the mAb. Several factors need to be considered in antigen selection. First, an ideal antigen of interest has a high level of expression in a tumor, but little or no expression in normal tissues, or at least expression is limited to a given tissue type to reduce ADC target toxicity and result in an acceptable therapeutic index. Second, the target antigen should be present on the cell surface so that circulating mAbs can enter. Third, the target antigen should be an intrinsic antigen, so that after binding, the ADC is transported into the cell, and the cytotoxic agent can exert its function.

However, non-internal ADCs may show significant toxicity in some cases and often cause a strong "bystander effect." Future designs should consider the relative role of cytotoxic drugs and antibodies in the antitumor activity and toxicity profile of the entire ADC.

  1. Warhead for clinical ADC

ADCs currently undergoing clinical trials, most targeting DNA or microtubules, have been efficiently optimized. Since the number of antigens on the surface of tumor cells is limited (about 5,000-106 antigens per cell) and the average drug-to-antibody ratio (DAR) of ADC is limited to 3.5-4, that is, the amount of ADC transferred to tumor cells is very low, and the cells that are combined with cell toxicity may cause clinical failure.

Auristatin and maytansin, which act by inhibiting tubulin assembly, are the most commonly used warheads for ADCs today. Other warheads used are based on pyrrolobenzoquinazoline (PBD), dihydroindolothiazepine, Tubulysins, calicheamicin, irinotecan derivatives, docamomycin, camptothecin analog, adrenaline and doxorubicin and so on. In fact, due to fierce competition, more and more ADCs in early clinical trial studies did not disclose the chemical structure of antigen targets and/or warheads and linkers.

  1. Linker design and optimization

Premature release of the drug in the circulation can lead to systemic toxicity and lower therapeutic indicators. An effective linker design must balance good stability over a few days in the cycle as well as efficient lysis when delivered to target cells. To enhance the solubility and DAR of ADCs and overcome the resistance induced by extracellular proteins (such as MDR1) delivered by chemotherapeutic agents, several strategies are being investigated: conditional release of drugs in the cytoplasm of target cells (removable and non-removable cleavage of the linker); the enhancement and limitation of the bypass effect is achieved by the ability to cross the biofilm-linker-drug metabolite; the polar linker increases solubility and reduces MDR.

  1. Antibody selection and optimization

Both antibody and ADC are forced to increase the homogeneity and developability of the antibody to reduce the rate of drug loss. Analytical techniques such as liquid chromatography, electrophoresis and mass spectrometry not only help to select clones of the best antibodies with suitable glycosylation characteristics, but also for the comprehensive structural identification of research fields and potential clinical candidates, as well as for identification "Hot spots" on antibodies that are detrimental to stability and pharmacokinetic and pharmacological properties. Mass spectrometry also helps to optimize the structure of next-generation mAbs from a pharmaceutical perspective, allowing the development of drug candidates (OptimAbs) and ADCs (OptimADCs) with reduced CMC loading and better drug properties. Selection of antibodies includes chimeric, humanization and selection of human antibodies as well as isotype selection.

  1. New coupling strategy

The second-generation of ADCs are controlled mixtures of different drug loading materials with a typical average DAR of 3.5 or 4. Species with DAR greater than 4 show lower tolerance, higher plasma clearance and reduced in vivo efficacy. Most ADCs today have a common structural feature, such as a thiosuccinimide linkage formed by a maleimide reaction of a thiol and an alkyl group. However, most ADCs cause measurable removal of maleimide during long-term circulation, which can be solved by site-specific coupling and alternative conjugate chemistry: for example, engineering cysteine, unnatural amino acid engineering, enzymes auxiliary ligation, sugar recombination and sugar binding, amino-terminal engineered serine, ligation to the Fab nucleotide binding site, re-bridging of native cysteine, avoiding retro-Michael disintegration and high load of ADC.

  1. Improve the performance of the ADC

Additional strategies to improve ADC performance can be designed to avoid potential resistance to warheads, by using smaller protein scaffolds to enhance tumor penetration, or by combining ADCs with recently approved mAb-based immune checkpoint inhibitors.

  1. ADC for non-oncological indications

There are few studies on ADCs for non-cytotoxic drugs. For example, an ADC directed against C-X-C chemokine receptor type 4 is an antigen that is selectively expressed on serum cells; antibody-antibiotic conjugates (AAC) against S. aureus in cells.

  1. Conclusions and future development

The development of ADCs has benefited from general improvements in the design of therapeutic mAbs, as well as specific improvements in conjugate synthesis methods that enhance homogeneity. The linker strategy and the diversity of warheads provide new opportunities to improve drug delivery in tumors, while also reducing the chances of drug exposure to normal tissues. In fact, it is important to better understand the toxicity determinants of ADC, whether it is used as a single drug or in combination with other therapies. To increase the therapeutic index, the ADC needs to reduce the minimum effective dose under the potency of the cytotoxic agent, or increase the maximum tolerated dose in terms of tumor selectivity. The next generation of ADCs relies on the synthesis and characterization of more homogenous and stable ADCs with macromolecular structures with medicinal chemical-like control. Recent ADC developments have revived interest in cytotoxic natural products, and in the future, the breakthrough in ADC performance may involve warheads with new combat mechanisms. In addition, alternative forms of new mAbs have emerged, but must be compared to full mAb in terms of therapeutic indicators such as toxicity, efficacy, and pharmacokinetics.

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