What are ADC Linkers?

Antibody-drug conjugates (ADCs) are the most popular chemoimmunotherapy agents in recent years. ADCs can enhance the ability to target and kill tumors, while preserving normal tissues from damage, so as to maximize the efficacy and reduce the drug systemic effect. Toxicity has always been a hot area of ​​drug research and development, but the complex structure of ADC drugs brings certain difficulties to drug design and development. More or less drugs that have been marketed have low internalization speed and off-target toxicity. Its safety and effectiveness are related to the design of the linker of ADC drugs.

Structure and mechanism of action of ADCs

The ADC drug structure is divided into three parts, the antibody part (Antibody) that specifically recognizes the tumor target, the drug with cytotoxicity (payload/Cytotoxic drug), and the linker (Link) that connects the payload to the antibody. After the ADC drug binds to the corresponding antigen on the tumor cell surface, the ADC/antigen complex is internalized, and then the payload is released, leading to cytotoxicity and cell death.

ADC Linker Standard

ADC Linker design is the most important part of ADC drugs, and all aspects of ADC pharmacology may be affected by the specific design of the linker, such as drug stability in circulation, tumor cell permeability, drug-to-antibody ratio (DAR) – i.e. the number and range of payload molecules carried by each antibody, bystander effects, etc. Several criteria must be met to successfully build an ADC.

(1) The linker needs to have sufficient stability in plasma (circulating stability) so that ADC molecules can circulate in the blood and localize to the tumor site without premature cleavage. Linker instability leads to premature release of toxic payloads and undesired damage to untargeted healthy cells, which can lead to systemic toxicity and adverse effects.

(2) The linker needs to have the ability to be rapidly cleaved and release free and toxic payloads after ADC internalization into target tumor cells.

(3) Another property to consider in Linker design is hydrophobicity. The hydrophobic linker, combined with the hydrophobic payload, usually promotes the aggregation of ADC molecules.

Several factors need to be considered in the actual design of ADCS linkers, including conjugation site, conjugation method, linker length, linker chemistry, cleavable/non-cleavable ligation, and steric hindrance of the proximal junction.

Structure of ADC Linker

The way the ADC drug releases its payload determines its stability in blood circulation. According to this division, the linker can be divided into cleavable linker and non-cleavable linker. Cleavable linkers are cleaved in response to extracellular and intracellular environments (pH, redox potential, etc.) or by specific lysosomal enzymes, which has the benefit of allowing researchers to estimate conjugation based on known pharmacological parameters of the payload The cytotoxic potency of the payload.

The cleavable linker can be released when the ADC is not internalized, which can produce a bystander effect in the vicinity of the tumor target and expand the killing area. However, most of the cleavable linkers are less stable than non-cleavable linkers, and are prone to premature breakage in the process of blood circulation, resulting in off-target toxicity. Therefore, it is necessary to continuously optimize the structure and strike a balance between ADC specificity and normal tissue damage. Cleavable linkers can be divided into two broad categories: enzymatic and chemical linkers.

Cleavable Linker – Chemical Linker

linker with disulfide bond

Disulfide linkers are glutathione-sensitive linkers whose cleavage relies on a higher concentration of reducing molecules in the cytoplasm of glutathione (GSH), a thiol-containing tripeptide that readily acts as an S-nucleophile reaction. For the stability of the linker in the blood circulation, a methyl group is often placed next to the disulfide bond, which resists reductive cleavage in the circulation. Upon internalization, abundant intracellular GSH reductively cleaves disulfide bonds, releasing free payload molecules.

Disulfide-containing linkers are primarily linked to maytansinoid payload classes, and the reactivity of disulfide bonds can be regulated by steric hindrance: alpha methyl substitution significantly affects reduction rate and resistance to thiol-disulfide exchange force.

Hydrazone

Two of the earliest approved ADCs, Mylotarg and Besponsa, used hydrazone linkers. The hydrazone is an acid labile group, and the ADC of the hydrazone linker is transported to acidic endosomes (pH 5.0-6.0) and lysosomes (pH about 4.8), where the free drug is released by hydrolysis. However, hydrazone linkers are less stable in the circulatory process (blood circulation), and ADCs with hydrazones as linkers are slowly hydrolyzed under physiological conditions (pH 7.4, 37°C), resulting in the slow release of toxic payloads.

non-cutting linker

ADCs with non-cleavable linkers must be internalized, and the antibody moiety needs to be degraded by lysosomal proteases to release the active molecule, thus having absolute cycling stability. The most representative is SMCC (N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate). The representative drug using SMCC as a linker is TD-M1, and its most important tumor metabolite is Lys-SMC-DM1.

Cleavable linkers – enzymatic linkers

Cathepsin B substrate linker

The mechanism of such linker cleavage is that after the ADC is internalized by endocytosis and transport to the lysosome, cathepsin B selectively cleaves the linker, and the payload is released from the ADC in a traceless manner, consisting mainly of dipeptides Linkers and Tetrapeptide Linkers.

Dipeptide linkers began with the study of cleavable dipeptides as cathepsin B substrates for doxorubicin prodrugs. The P1 position can bind hydrophilic residues, the P2 position can bind lipophilic residues to increase the stability of plasma tension, and the PABC moiety acts as a spacer between the Val-Cit moiety and the payload, limiting the steric position of the payload blocking, allowing cathepsin B to exhibit its full protease activity towards linkers attached to bulky payload molecules such as doxorubicin.

The most used dipeptide linkers include the Val-Cit dipeptide (25 clinicals) and the Val-Ala dipeptide (7 clinicals). Buffer stability, cathepsin B release efficiency, cell viability and histopathological characteristics were comparable between the two linkers. However, it is difficult for Val-Cit to achieve high DAR due to precipitation and aggregation, in contrast, Val-Ala linker can achieve DAR as high as 7.4 with limited aggregation (<10%). Compared to Val-Cit, Val-Ala has less (insignificant) hydrophobic behavior, so this linker is more advantageous in the context of lipophilic payloads, such as PBD-dimers. If a hydrophobic spacer (acetyl) is used, then Val-Cit has a higher localized DAR than Val-Ala, and if a small amount of linear PEG12 is added, the difference will not be significant.

The tetrapeptide Gly-Gly-Phe-Gly shows all the characteristics of a stable and efficient cleavable ADC linker and was successfully applied to Daiichi Sankyo’s Enhertu (DS-8201a), the linker spacer of DS-8201a was A compact hemiamine.

Pyrophosphodiester linker

The pyrophosphodiester linker is an anionic linker with higher water solubility and excellent cycle stability than traditional linkers. Upon internalization, pyrophosphodiester passes through the endosome-lysosome pathway, which undergoes a two-step enzymatic linker cleavage, releasing first the payload-monophosphate molecule, followed by the free payload.

Studies have shown that the conjugates of anti-human CD70 and various glucocorticoids constructed by this linker can maintain stability in human plasma for 7 days, and can be rapidly cleaved to release the payload after internalization.

The rate of payload release following internalization of such linkers varies, mainly related to substituents proximal to the pyrophosphate moiety, suggesting that the release rate can be controlled by designing different structures.

β-glucuronidase substrate

β-glucuronidases are a class of glycosidases that catalyze the hydrolysis of β-glucuronic acid residues. This linker exhibits low levels of aggregation, high plasma stability, an in vitro validated cleavage process, and robust in vivo efficacy. The linker is also applied to other amine-containing payloads such as CBI microgrooved adhesives, camptothecin analogs and hydroxyl-containing molecules SN38, bisgamycin, via an additional dimethylethylenediamine (DMED) self-ignition spacer and Linghai protein.

The better hydrophilicity of this linker makes it easier to prepare ADCs of DAR 8 compared to cathepsin-sensitive linkers.

β-galactosidase substrate

A β-galactosidase-cleavable linker has been reported for ADCs incorporating a PEG10 spacer. The spacer is replaced with a nitro group to increase the rate of self-immolation. By analogy with the β-glucuronidase linker, the cleavage mechanism involves the hydrolysis of the β-galactosidase moiety, which confers hydrophilicity to the chemical precursor. Another advantage is that β-galactosidase is only present in lysosomes, whereas β-glucuronidase is expressed in lysosomes and is also present in the microenvironment of solid tumors. The study demonstrated that β-galactosidase-containing ADCs were more potent than the gold standard Traztuzumab emtansine (T-DM1) both in vitro and in vivo in the context of anti-HER2 ADCs releasing MMAE.

Sulfatase substrate

The sulfatase-cleaved linkers act as glycosidase and (pyro)phosphatase-based linkers, which exhibit good hydrophilicity due to the nature of the target substrate (permanently charged sulfate), while mainly in lysosomes Express. Compared to the classical cleavable Val-Cit and Val-Ala linkers, the sulfatase linker showed similar cellular potency against the Her2+ cell line.

Conjugation method of linker

The site of linker conjugation and the method of conjugation determine the DAR (drug to antibody ratio) of the drug, and the conjugation site has a significant impact on ADC stability and its pharmacokinetic-pharmacodynamic profile, high drug load Usually results in fast plasma clearance, while low DAR (drug-to-antibody ratio) ADCs show less activity.

Chemical conjugation and enzymatic conjugation are currently the two most used methods of attachment of antibody and payload components.

chemical coupling

With accessible amino acid residues on the surface of the antibody, chemical conjugation is the coupling of the handle portion of the linker to the amino acid residue portion of the antibody, avoiding the complexity of identifying suitable mutation sites and the potential challenges of scaling up and optimizing cell cultures. The DAR and the coupling site of the early chemical coupling method are very random, and the resulting ADC is very different and the quality is not uniform.

Lysine amide coupling

Amide conjugation is a major ADC conjugation method using linkers containing activated carboxylate esters to attach payload and solvent-accessible lysine residues to antibodies.

The activated carboxylic acid moiety reacts with the lysine residue, resulting in an amide bond bond between the mAb and the payload. The optimized conjugation conditions resulted in an average drug-to-antibody ratio (DAR) of 3.5–4 with a distribution between 0–7.

Cysteine ​​coupling

Specific reaction between cysteine ​​residues of antibodies and thiol-reactive functional groups installed on the payload. Antibodies have no free thiols, all cysteine ​​residues form disulfide bonds. The advantage is that the coupling site is limited and the thiol group has obvious reactivity, so it is superior to lysine conjugation in DAR controllability and heterogeneity.

Enzyme coupling

Attachment of the payload can be achieved in a very selective manner by using genetically encoded amino acid tags inserted into the antibody sequence. These tags are specifically chosen to be recognized by enzymes capable of performing site-specific conjugation, such as formylglycine-generating enzymes (FGEs), microbial transglutaminases (MTGs), sorting enzymes, or tyrosinase.

Sortase transduction

NBE Therapeutics has developed a further enzymatic conjugation method based on S. aureus Sortase A-mediated transpeptidation. Their strategy utilizes the enzyme Sortase A (Srt A), which cleaves the amide bond between threonine and glycine residues in the LPXTG (X = any amino acid) pentapeptide motif. It then catalyzes the attachment of the glycine-derived payload to the newly generated C-terminus, creating a peptide bond at physiological temperature and pH. This approach was applied to different antibodies, such as anti-CD30 and anti-Her2, with pentylglycine-tagged linkers containing maytansine and MMAE.

Microbial glutamine transduction

Microbial transglutaminase (MTGase) strategies are also frequently exploited to specifically bind different payloads. The MTGase enzyme catalyzes the formation of a peptide bond between the glutamine side chain at position 295 of the deglycosylated antibody and the primary amine of the substrate. In contrast to other enzymatic strategies, MTG is a flexible technique that does not require a peptide donor for conjugation. Acyl acceptors have no structural restrictions as long as they contain primary amines

Binding to glycans

Since human IgG is a glycoprotein, it contains an N-glycan at position N297 in each heavy chain of the CH2 domain of the Fc fragment. This glycosylation can be exploited as a point of attachment for the linker payload. Distant localization of glycans from the Fab region reduces the risk of impairing the antigen-binding capacity of the antibody after conjugation. Furthermore, their different chemical composition compared to the peptide chains of antibodies allows for site-specific modification, making them suitable sites for conjugation.

Glycan bioconjugation can be differentiated according to the techniques used to target carbohydrates: glycan metabolic engineering, glycotransferase treatment followed by glycan oxidation, endoglycosidase and transferase treatment, and ketone or azide labeling.

Prospect

Although most of the ADCs currently in clinical practice are based on the modification of natural amino acid sites, due to the difficulty of obtaining a stable DAR ratio at natural amino acid sites, many companies have put their sights into the field of engineering modified amino acid residues with the intention of obtaining A more mean stable ADC. For example, tiimumab technology, which incorporates non-canonical amino acids (ncAA), provides another possibility for site-specific conjugation. ncAA technology allows the incorporation of amino acids with unique chemical structures, enabling the introduction of linker-payload conjugates in a chemoselective manner.

AxisPharm develops a broad range of Linkers and provide custom linker synthesis. Our current product catalog covers click chemistry tools such as DBCO, Tetrazine, TCO, BCN and Cycloprpene etc., biotin linkers, PEG linkers, peptide linkers, glucuronide linkers, photo cleavable linkers, Fluorescent Dye probe linkers and folic acid peg linkers.

As a customer-oriented contract research company, we offer flexible ADC Linker product kits and Bioconjugation services with competitive price.

Reference source:
[1] The Chemistry Behind ADCs
[2] Antibody-drug conjugates: recent advances in conjugation and linker chemistries
[3] The Analysis of Key Factors Related to ADCs Structural Design
[4] Summary of ADC linker technology – Target Society