During the early development of Antibody-Drug Conjugates (ADCs), lysine is often selected as a well binding site with antibodies. As a leading service provider in ADC research and discovery, BOC Sciences can perform advanced lysine conjugation technologies to develop antibody-drug conjugates. Based on a thorough understanding of customers' project requirements, our scientists are capable of inventing ADCs suitable for large-scale production to support commercial needs at a low cost.
Lysine is an amino acid naturally found in proteins. It has a reactive amino group that can react chemically with drugs. By engineering the antibody to introduce lysine at specific positions on the antibody, a special catalyst is used to couple the payload to the native lysine to form an ADC. Currently, lysine coupling has been used in the production of three FDA-approved ADCs. A typical immunoglobulin G1 (IgG1) antibody has approximately 90 lysine residues. When targeting DAR values of 2 to 4, the non-selective conjugation strategy has the potential to provide statistically up to 106 different isomers. In industry, process conditions are optimized and coupling occurs at 8-10 kinetically preferred sites of 90 lysines. This method has the following advantages:
ADCs prepared using native lysine site-directed coupling technology have high stability and efficiency. Due to the stable carbon-carbon bonds formed between the antibody and the drug, ADC will not dissociate in the blood, ensuring the delivery and release of the drug. At the same time, because the number and position of the drug on the antibody are controllable, the ADC can achieve optimal structure and function, improving the selectivity and effectiveness of the drug. In addition, since native lysine is an amino acid naturally found in proteins, using it as a connection point will not cause rejection by the immune system, nor will it affect the structure and activity of the antibody itself.
Lysine-directed conjugation technology is an innovative ADC preparation method that utilizes the Michael addition reaction between lysine and drugs. In this reaction, the NH2 group on lysine acts as a nucleophile and attacks the electrophilic group containing carbonyl or thioether on the payload, forming a new carbon-carbon bond and releasing a proton. This reaction is very fast and efficient and can be completed under mild conditions. When using lysine site-directed coupling technology to prepare ADC, the antibody needs to be engineered first to introduce native lysine at specific positions on the antibody. Based on this, BOC Sciences can be achieved in two ways: one is to replace the original amino acid on the antibody with native lysine by mutation technology; the other is to use insertion technology to insert native lysine into a certain position of the antibody. Both methods can ensure that the structure and activity of the antibody are not affected, while increasing the sites available for coupling on the antibody. This method can achieve high efficiency and high selectivity coupling reaction, avoiding the interference of other reactions and the generation of by-products.
Individual lysine residues in proteins can have significantly different reactivity toward reagents due to differences in solvent accessibility, protein higher-order structure, and their surrounding environment. Therefore, site-directed modifications can be achieved by exploiting the microenvironment surrounding target lysine residues in proteins. Significant benefits of this approach are that the commercially available reagents are efficient and the bioconjugation procedure itself is simple. Among them, N-hydroxysuccinimide (NHS) ester is widely used as a coupling linker. The nucleophilic NH2 group of lysine reacts with the electrophilic NHS on the linker payload. Due to differences in lysine position and microenvironment, the pKa of the protonated amino group is different, thus affecting the reaction rate. Taking advantage of this difference in properties, BOC Sciences offers a variety of conjugation strategies for lysine residues and linkers.
Enzymes provide important chemical biology tools for site-selective modification of ADCs because enzymes have very good site selectivity, high conversion efficiency, and can achieve one-pot multiple modifications. Currently, enzymes widely used in ADC lysine modification include transpeptidase A (SrtA) and microbial transglutaminase (MTG). SrtA from Staphylococcus aureus is a lysine-targeted enzyme that catalyzes the covalent linkage of two amino acids through an isopeptide bond. SrtA recognizes the amino acid sequence LPXTG. It first cleaves the amide bond between threonine (T) and glycine (G), and then thioesterifies its reactive cysteine sulfhydryl group with the threonine carboxyl group in the sequence. This thioester can be substituted with an amino group to form a stable amide bond. Another enzyme used in the enzymatic synthesis of ADC is microbial transglutaminase (MTG), which enables the formation of an amide bond between the γ-amide of glutamine and the ε-amino group of lysine. At the beginning of this process, the cysteine in the active site of MTG attacks the carbonyl group of the γ-amide and replaces the amino group to undergo thioesterification. The amide is then formed by reaction with the primary ammonia of lysine. MTG is a cheap and easily available enzyme that works under different temperatures, buffer salt concentrations and pH values.
Lysine enzymatic modification services provided by BOC Sciences are performed by a team of experienced scientists who are experts in antibody chemistry and enzymology. Our scientists work closely with customers to customize enzymatic modification strategies to meet their specific requirements, such as type of modification required, modification sites and project size. This personalized approach ensures clients receive customized solutions that meet their unique needs and goals.
Enzymes can achieve precise selectivity through proximity control, ensuring that reactions with substrates only occur at the active site. Inspired by nature, researchers have also developed the use of structural units and functional groups close to lysine to guide selective modification. There are two main strategies to achieve proximity-guided modification. One is to use affinity tags to use peptides or small molecule binding agents to guide the reaction of reagents with predetermined sites; the other is to form a temporary covalent binding site through covalent linkage before modifying the target residue. BOC Sciences is a leading provider of proximity-guided lysine modification services, including affinity tags and covalent conjugation. By targeting specific lysine residues, researchers can introduce affinity tags or covalently bind small molecules to proteins for a variety of applications such as protein purification, protein-protein interaction studies, and drug discovery.
Lysine conjugation is a post-translational modification process involving the covalent attachment of the amino acid lysine to another molecule (usually a protein, peptide, or small molecule). Lysine conjugation can occur through a variety of mechanisms, including acetylation, ubiquitination, sumoylation, and neddylation.
Cysteine conjugation involves linking the molecule to the thiol group (-SH) of the cysteine residue in the antibody. Lysine conjugation involves attaching a molecule to the epsilon amino group (-NH2) of a lysine residue in an antibody. The main difference between these two methods is the reactivity of the amino acid residues involved. Due to the presence of thiol groups, cysteine is highly reactive and highly specific. Cysteine therefore becomes a preferred target for bioconjugation when high reactivity is required. In contrast, lysine conjugation is less specific because the amino group of lysine can react with a wider range of functional groups. This lack of specificity can be either an advantage or a disadvantage, depending on the desired application.
Taking Kadcyla as an example, the DM1 toxin molecule is connected to the lysine residue in the trastuzumab sequence through the SMCC linker. After forming a drug-linker, it will cause a mass difference of +956.36 Da (MCC-DM1). When analyzing peptide map data through mass spectrometry data processing software, set MCC-DM1 as a variable modification, perform automatic search by the software, and perform manual data confirmation through other differences caused by coupling reactions.
a. Chromatographic behavior: After coupling hydrophobic drugs, in RPLC chromatography mode, the retention time of the coupled peptides increased compared with the uncoupled peptides. At the same time, there are two types of MCC-DM1 connected through maleimide. The three-dimensional configuration causes the coupled peptides to emit peaks in pairs.
b. Coupling DM1 will prevent Trypsin enzymatic cleavage of the corresponding Lys site, so the coupled peptide will contain a missed cleavage site (except for the subsequent C-terminal Pro).
c. DM1 will produce characteristic fragments during the secondary dissociation process. The secondary mass spectrum of the coupled peptide will contain DM1 characteristic fragment ions.
Based on the software search and combined with the above manual confirmation strategy, the coupled peptides can be accurately identified. In addition, due to differences in ADC coupling methods and the diversity of linkers and optional toxic small molecules, different ADC drug molecules will have their own unique situations, and a suitable coupling peptide screening program needs to be developed based on the actual ADC molecular structure.
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