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Antibody-drug conjugates (ADCs) are composed of antibodies and cytotoxic drugs linked through a linker. This type of drug accurately delivers drugs to the lesion by targeting antigens specifically expressed on the surface of tumor cells, releasing cytotoxic drugs with highly effective therapeutic effects inside the tumor, and ultimately specifically killing tumor cells. Therefore, ADC drugs have great clinical therapeutic value.
Site-specific conjugation technology can achieve fixed-point and quantitative coupling of antibodies and small molecule toxins. The ADC obtained through this technology has a suitable drug-antibody ratio (DAR), high uniformity, good stability, and reproducibility between batches. It is highly reactive, has better activity and pharmacokinetic properties, and is also more suitable for large-scale production of ADC. The choice of conjugation technology is crucial to the homogeneity and stability, efficacy and safety of ADC drugs. The conjugation of cytotoxin-linker to antibodies is a key step in producing ADC and affecting the quality attributes of ADC, and plays an important role in ADC pharmacokinetics and therapeutic index. Currently, more than 95% of ADCs are based on two chemical conjugation technologies: cysteine alkylation technology or lysine acylation technology. In addition to the traditional two methods, new technologies are rapidly emerging, including not only innovative methods for conjugation cysteine (alkylation and cross-linking) and lysine (acylation or alkylation) to produce a more homogeneous ADC and improve the therapeutic index, but also extended to new conjugation techniques, such as conjugation techniques based on carbonyl, amide, and azide groups. Drugs with precise control over conjugation sites to DARs are easier to optimize, manufacture and develop, and have fewer regulatory hurdles. Therefore, site-specific conjugation technology has become the focus of new generation ADC drug research.
| Conjugation Methods | Advantages | Shortcomings |
| Lysine Conjugation | Naturally high abundance, easy to couple; little structural disturbance. | Heterogeneity; modification sites are often highly restrictive. |
| Cysteine Conjugation | Fast conjugation reaction; little structural disturbance. | May affect antibody stability; lack of easily accessible residues complicates technology; engineered cysteine may confer immunogenicity. |
| Enzymatic Conjugation | The DAR and conjugation sites are basically determined; the operation is simple and the off-target rate is low. | Enzyme conversion efficiency depends on the site; optimization possibility of DAR is small; tag sequence insertion may bring immunogenicity. |
| Glycan Conjugation | Natural glycosyl sites facilitate technology; various bioorthogonal ligation reactions may be realized. | Modification sites are immutable; DAR tends to be lower; glycoengineering may bring immunogenicity. |
| Unnatural Amino Acids Conjugation | Structural disturbance is small; conjugation efficiency is high: DAR and conjugation sites are basically determined; various bioorthogonal connection reactions may be realized. | The operation is quite complicated; the antibody expression rate is low and the stability is affected: the incorporation of unnatural amino acids may cause immunogenicity. |
Relying on BOC Sciences' small molecule business capabilities, we have completed the construction of one-stop ADC CDMO capabilities covering multiple aspects such as antibody modification, toxin-linker production, and antibody conjugation. BOC Sciences has built a diverse and complete coupling technology process platform and has the ability to support various types of conjugated drug process development platforms, including the preparation of pre-clinical candidates (PCC) molecules, early process development and optimization, process amplification, and process characterization, process transfer and commercial production, etc.
| Catalog | Product Name | CAS Number | Category | Price |
| BADC-00031 | Brentuximab vedotin | 914088-09-8 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-01595 | Datopotamab deruxtecan | 2238831-60-0 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-01593 | Cantuzumab mertansine | 400010-39-1 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-00023 | Trastuzumab emtansine | 1018448-65-1 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-01592 | Gemtuzumab ozogamicin | 220578-59-6 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-01599 | Anetumab ravtansine | 1375258-01-7 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-01600 | Sirtratumab vedotin | 1824663-83-3 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-01601 | Tusamitamab ravtansine | 2254086-60-5 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-01594 | Labetuzumab govitecan | 1469876-18-3 | Antibody-Drug Conjugates (ADCs) | Inquiry |
| BADC-01596 | Enfortumab vedotin-ejfv | 1346452-25-2 | Antibody-Drug Conjugates (ADCs) | Inquiry |
As one of the most commonly used conjugation technologies for early ADC products, highly abundant lysine is the most common and easiest target to modify. Lysine conjugation uses the nucleophilic -NH2 in lysine on the antibody surface to connect to the linker. Although the technical route is simple, due to the excessive number of lysine sites that can be coupled on the antibody surface, the product heterogeneity is large and requires strict process control. In order to solve this problem, scientists have proposed more lysine-directed conjugation technologies, such as selective modification of the most active lysine, and site-directed modification through enzymes.
pClick technology is a convenient method that does not require antibody engineering or complex reaction conditions. This affinity peptide scheme was pioneered by Han's team, who used the B domain of protein A (FB protein) from Staphylococcus aureus to synthesize peptides containing unnatural amino acids. Favorable positioning and proximity are achieved by FB protein, and selective non-natural amino acids react with specific lysine. When trastuzumab (Tras) and the FB-E25FPheK (4-fluorophenylcarbamate lysine) mutant were selected as model substrates, the researchers demonstrated that excess FB-E25FPheK mutant was used for 48 h. The coupling reaction can achieve a coupling yield of Tras-FB conjugates >95% with a single modification site K337. This technology can also be used to prepare multifunctional ADCs and bispecific antibodies with imaging and drug delivery functions, both of which have achieved good results. Recently it has also been used in bone targeting (BonTarg) technology to generate first-class bone targeting antibodies.
AJICAP technology is a method of site-specific modification of natural antibodies using Fc (antibody crystallizable fragment) affinity reagents. The first-generation AJICAP utilizes Fc affinity peptide reagents to achieve coupling, and then reduces it with tris (2-chloroethyl) phosphate (TCEP) to install a thiol group on a specific lysine. Then, dehydroascorbic acid was used to oxidatively reconstruct the disulfide bond between the antibody chains that was cleaved due to reduction, thereby successfully modifying site K248. However, the re-oxidation step often leads to disruption of disulfide bonds and aggregation problems, so the second generation AJICAP avoids redox treatment and directly uses the linker cleavage reaction. In addition to successful conjugation to K248, ADCs conjugated to K288 can also be synthesized using appropriate Fc affinity peptide reagents with appropriate spacers. The researchers used two different analytical methods, time-of-flight tandem mass spectrometry and hydrophobic interaction high-performance liquid chromatography, to determine the drug-to-antibody ratios of multiple antibody subtypes and toxin conjugates. Both were maintained at 1.7~2.0; the aggregation percentages were all<3.0. %, not significantly different from naked antibodies. In the efficacy evaluation, it was found that AJICAP-ADC showed significant tumor regression, sufficient stability and excellent tolerability.
The Rajagopalan team found that there is a highly conserved nucleotide binding domain (NBD) between the light chain and the heavy chain of the antibody, which is present in all immunoglobulin Fab (antigen binding fragment) groups. Through modeling analysis and computer screening, the Bilgicer team determined that IBA has a high affinity for this NBD, with a Kd (dissociation constant) value ranging from 1 to 8 μmol·L−1, depending on the antibody. So they designed a UV cross-linking method that relies on IBA coupling to the nucleotide binding site of the antibody, and found that up to 1.41 couplings can be achieved without UV light damaging the activity of the antibody.
The linchpin-directed modification was pioneered by the Rai team. Through reversible intermolecular reactions, they place the linchpin point on all accessible specific amino acid residues, and then make the other functional group irreversibly selectively modify the adjacent specific group, and finally dissociate the key modification to complete the specific modification of the protein. Among them, they used spacer adjustments to precisely match the relative orientations of neighboring modifications. The researchers envisioned achieving specific modification of low-abundance amino acids by key modification of high-abundance lysine, and successfully obtained a DAR 4 ADC with good selectivity and good inhibitory effect on tumor cells.
Cysteine coupling is a widely used technology in the field of ADC at this stage. It partially reduces the disulfide bond on the antibody and connects it to the maleimide and other groups on the linker. Cysteine coupling is a convenient method to solve the problem of heterogeneity, and the unique nucleophilicity and low natural abundance of cysteine provide the possibility of regioselectivity. Although most proteins do not have solvent-accessible cysteine residues, they can be generated by protein engineering or reduction of native disulfide bonds.
The interchain disulfide reduction and alkylation technology was originally proposed by Willner et al. As a convenient alternative to avoid the heterogeneity issues associated with conjugation to lysine, maleimide is often used as the conjugate. However, this connection often undergoes a reverse Michael reaction, causing drug instability. Hydrolyzed maleimides and other conjugates with clever structures have been used for optimization, such as sulfonamides, vinyl/alkynypyridines, etc. Recently, Schumacher's team proposed a novel and modular coupling method. Azide-containing proteins are coupled to ethynylphosphonates via the Staudinger-phosphite reaction, which reduces the electron-rich nature of the triple bond and thus facilitates thiol addition reactions. Then it is added to the reduced interchain disulfide bonds to obtain functionalized antibodies with excellent stability. By further enhancing the hydrophilicity with diethylene glycol, an ADC with stably linked DAR 4 can be generated.
Interchain disulfide bond bridging technology is the first method pioneered by Brocchini's team to reduce DAR. They found that chemical modification of natural disulfide bonds in proteins does not affect the protein's tertiary structure and thus its biological activity, which provides the possibility for this technology. Afterwards, their team successfully coupled Fab fragments using PEG-disulfone reagent, and Godwin's team connected MMAE to this disulfone reagent, thus obtaining a homogeneous and stable ADC with a DAR of 2.8. It retains antigen-binding properties, is stable in serum, and exhibits potent targeted cell killing in in vitro and in vivo cancer models. The next generation of maleimide, divinylpyrimidine, divinyltriazine, etc. have also been proven to be well used in interchain disulfide bond bridging technology to generate homogeneous and stable ADCs.
THIOMAB technology is a classic method for conjugation with cysteine. It has proven the feasibility and benefits of site-directed coupling of drug antibodies and has derived a series of site-directed coupling technologies realized by engineered cysteine. The THIOMAB technology pioneered by Junutula's team uses phage display methods to predict suitable coupling sites and perform cysteine substitutions, because the substituted residues usually form disulfides with cysteine or glutathione, so the reactive cysteine thiol needs to be obtained through a reduction and oxidation process, and then coupled with a specific linker and payload through a maleimide group. Currently, a series of studies are being conducted to optimize THIOMAB technology. For example, site adjustment can prevent the generation of redundant disulfide bonds between antibody Fabs; choosing a positively charged environment can promote hydrolysis, thereby improving the stability of the drug. Another example is that optimizing the maleimide group can reduce the generation of the reverse Michael reaction, thereby preventing the premature release of cytotoxic drugs from the antibody.
Metal-mediated techniques offer high regioselectivity, which makes them attractive for bioconjugation. Myers' team has proposed a method of labeling proteins in aqueous media using arylpalladium(II) reagents, but the need for functional linkers may cause off-target or purification problems. To achieve optimization, the Pentelute team proposed to directly transfer aryl groups to cysteine residues in proteins using palladium complexes produced by oxidative addition of aryl halides or triflates. The palladium reagent is easy to store and reacts quickly, and can successfully synthesize the ADC trastuzumab-vandetanib with a DAR of 4.4 that retains antibody affinity.
In the early stages of antibody design, specific tags can be introduced into the antibody through genetic engineering to achieve site-specific coupling of enzymes. Enzyme-mediated site-specific reactions can provide almost certain DAR and coupling sites and are a promising method for synthesizing homogeneous ADCs. This approach takes advantage of the enzyme's precise localization capabilities and low off-target rate, showing great potential in next-generation antibody-drug conjugation. Currently, enzymes widely used for enzyme-mediated ADC conjugation include:
| Types | Description |
| Transglutaminase | Transglutaminase can catalyze the formation of a covalent bond between the glutamine side chain and the primary amine, and cannot recognize the naturally occurring glutamine in the constant region of the glycosylated antibody. This feature provides an opportunity to design a specific glutamine tag, which has now been widely used in ADC technology. |
| Formylglycine-generating enzyme | The formylglycine synthase is an enzyme used to convert cysteine into an aldehyde-containing amino acid formylglycine. The minimum consensus sequence is CXPXR ( X is any amino acid ), but the recognition sequence of human formylglycine synthase is LCXPXR. After introducing the LCXPXR sequence using standard molecular biology techniques, it has been recognized by formylglycine synthase in the presence of copper cofactors to convert the sulfhydryl group on cysteine into an aldehyde group. |
| Sortase (SrtA) | SrtA was originally discovered in Staphylococcus aureus, where it anchors many surface proteins to the cell wall of Gram-positive bacteria. SrtA can recognize the C-terminal modified sequence LPXTG and cleave the amide bond between threonine (T) and glycine (G) to form LPXT-SrtA, and then enter the amino group of the glycine (Gly) chain as a nucleophile. Reconstruct intermediates. In this way, a new peptide bond is formed between threonine and the incoming glycine, and SrtA is released into the next catalytic cycle. |
| Phosphatidylinositol transferase (PPTase) | PPTase is a catalytic enzyme that catalyzes the synthesis of 4'-phosphotidinyl cofactor (P-pant) from coenzyme A (CoA) and acyl carrier protein (ACP) or peptidyl carrier protein (PCP). Enzymes covalently linked to highly conserved serine residues have an unusually broad substrate tolerance, which provides the possibility for site-selective modification of proteins. |
| Tubulin-tyrosine ligase(TTL) | TTL is an enzyme that plays an important role in microtubule homeostasis and can be repurposed to add tyrosine derivatives to any protein carrying a Tub-tag (VDSVEGEGEEEG EE) at the C-terminus. TTL has also been shown to have broad substrate tolerance, not only tyrosine derivatives, but also some amino acids unrelated to the tyrosine structure, which provides more possibilities for TTL-mediated labeling. |
Lysine and cysteine are widely used targets and have significant advantages in chemical reactions. However, heterogeneity issues negatively impact their pharmacodynamic properties, which in turn affects toxicity manifestations. To deal with this problem, Reddy's team reported a chemical replacement method based on endogenous amino acids. Among them, because the aromatic side chains of tyrosine, tryptophan, and histidine show unique reactivity and selectivity, a variety of chemical technologies based on aromatic side chain modification have emerged.
For example, there is also a suitable chemical modification method for methionine, which contains the same sulfur as cysteine but has relatively weak nucleophilicity, called the ReACT method. The researchers used oxazodine compounds to convert methionine into the corresponding imine sulfide coupling product, and then used bioorthogonal chemical reactions to install the payload at specific locations on a given protein. The researchers used liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis to identify probe labeling sites throughout the proteome, observing labeling of 235 methionine residues and 1 lysine residue. No modifications were detected on the cysteine side chain or other nucleophilic amino acids, demonstrating the high selectivity of the ReACT strategy for methionine residues under mild biocompatibility conditions. They also found that the linkage exhibited reasonable stability under acidic and alkaline conditions, even in the presence of certain reducing reagents. When designed into drugs, high tumor targeting efficiency can be observed.
There is a conserved glycosylation site on the antibody. Glycosyl chemical structures differ from amino acids and therefore do not interact with peptides. Furthermore, the glycosylation site is far from the variable region, so conjugation to the glycosyl moiety is unlikely to affect binding to the antigen. These make glycosyl groups convenient targets for specific conjugation. Currently, there are two main methods for site-directed coupling by modifying sugar groups. One is to oxidize the adjacent diol in the sugar chain to produce an aldehyde group capable of aldehyde-amine or aldehyde-hydrazine coupling. The other is to use specific glycosyltransferases transfer monosaccharides containing bioorthogonal groups. These unique functional groups are then used to perform specific chemical connection reactions with drugs or small molecule targets. At present, there are very few experiments on the targeted synthesis of ADCs from sugars. This is related to factors such as the harsh reaction conditions required, the toxicity of the required Cu catalyst, and interference with the reaction. In addition, direct glycoconjugation lacks flexibility due to the fixed coupling site, and glycoengineering may make ADC immunogenic.
Incorporating non-natural amino acid ribosomes with orthogonal chemical reactivity into antibodies provides a new idea for the synthesis of homogeneous ADCs. This method is expected to precisely control the coupling site and DAR. In the expressed antibodies, alkoxy-amine derivatized drugs can be specifically coupled to the ketone group of p-acetylphenylalanine to obtain a homogeneous ADC. This coupling method proved to be highly productive and stable. The constructed ADC (anti-HER2-oristatin conjugate) has strong targeting efficacy and excellent serum half-life against Her2 tumors in vivo. In addition, methods based on non-natural amino acids are most suitable for precise localization and regulation of reactive residues, but often require special techniques and biological agents for rather complex genetic engineering, and the incorporation of non-natural amino acid residues may lead to instability of antibodies or trigger unwanted immune responses. Only by properly addressing these issues can the method be truly universal and practical.
ADC is one of the research hotspots in tumor treatment. In the past 10 years, many ADCs have been launched on the market, driving the rapid development of ADCs. Currently, many ADCs are in the clinical research stage. ADCs have also begun to appear in more forms of monoclonal antibodies, such as nanobodies, antibody Fab fragments, single-chain variable peptides, etc. The design of linkers is also constantly improving, and more and more small molecule drugs will be used for conjugation antibody. With the development of coupling technology and the improvement of processes, ADC will develop in the direction of high uniformity, high stability, and high efficacy.
