ADC Conjugation Technologies

This article introduces conjugation technologies based on carbonyl, amide, azido, and other amino acid functional groups.

Carbonyl (-C(O)R)

Carbonyl groups (aldehydes and ketones) are not naturally present in proteins; they undergo various bioorthogonal conjugation reactions with natural protein functional groups. Carbonyl groups can be easily introduced into monoclonal antibodies via genetic encoding of non-natural amino acids (NNAA) or enzymatic steps. Below, we describe two major bioconjugation reactions ( bioconjugation service ) involving aldehydes and ketones used in ADCs.

Oxime Linkage

O-alkylhydroxylamines (aminooxy) can specifically react with aldehyde or ketone functional groups. The reaction between aminooxy and either group forms an oxime bond. This bond is relatively stable when formed with ketones but less stable with aldehydes.

Aminooxy related products:

Aminooxy-PEG2-BCN | CAS:2253965-14-7

Aminooxy-PEG8-acid, CAS 2055013-68-6

Aminooxy-PEG2-alcohol, CAS 185022-12-2

Natural antibodies do not possess aldehyde or ketone functional groups. Schultz et al. developed genetic encoding of NNAA featuring ketone groups (p-acetylphenylalanine, p-AcF), a method applied by Ambrx.

p-AcF can be engineered at any given position in monoclonal antibodies, with subsequent oxime linkage producing ADCs (Figure 1). Two ADC products, ARX788 and AGS62P1/ASP1235, generated using this technology, have been approved for clinical use.

Figure 1. ADC Generation by Genetically Encoded p-ACF and Oxime Ligation
Figure 1. ADC Generation by Genetically Encoded p-ACF and Oxime Ligation

Another method for installing ketones into antibodies is LegoChem, which also involves antibody engineering. After the initial introduction of a C-terminal CAAX fusion on the antibody light chain, subsequent alkylation with a synthetic oxo-isoprenoid derivative can be performed under the influence of farnesyltransferase, a process commonly referred to as isoprenylation (Figure 2).

Similarly, the final generation of ADCs occurs through the connection of ketones with appropriately O-alkylated hydroxylamines. ADCs utilizing LegoChem technology, such as FS-1502 (LCB14-0110), have also progressed into clinical trials.

Figure 2. LegoChem Method
Figure 2. LegoChem Method

Hydrazino-Pictet–Spengler Connection

One drawback of oxime coupling chemistry is the requirement for large excess of reagents and acidic conditions (pH 4.5) to drive the reaction at a reasonably fast pace.

As an alternative, Agarwalet et al. developed a variant of the Pictet-Spengler reaction, which reacts with aldehydes ( Aldehyde PEG – ADC Linkers ) under neutral pH conditions to generate iminium intermediates. These intermediates rearrange to form cyclic products containing highly stable C-C bonds.

Based on this Pictet-Spengler reaction, a strategy was devised involving the enzymatic oxidation of engineered cysteines (part of the C-terminal LCTPSR) to aldehydes by methionine gamma-lyase (FGE).

Subsequent incubation with N,N-dimethyl hydrazine-indole reagents functionalized with cytotoxic payloads leads to the spontaneous formation of stable cyclic conjugates (Figure 3). This method has been applied to the clinical drug TRPH-222/CAT-02-106.

Figure 3. Hydrazino-Pictet–Spengler Connection Method
Figure 3. Hydrazino-Pictet–Spengler Connection Method

Explore more about Carbonyl related products:

Carbonyl reactive Linkers

Benzyloxy carbonyl-PEG3-acid | CAS: 2100306-73-6

Azidohexanyl-amino-carbonyl-amino-tris tri-acid

Benzyloxy carbonyl-PEG4-NHS ester | CAS:2639395-44-9

Acylamine Group (-C(O)NHR)

So far, the acylamine group ( Acrylamide-PEG-Azide) is the most common functional group found in proteins, as all amino acids in the protein backbone are linked in this manner. Additionally, glutamine and asparagine have side chains containing acylamine groups.

Therefore, the acylamine group appears to be an illogical candidate for site-specific coupling, further reinforced by its low reactivity.

However, this unique class of acylamine-based bioconjugates can be cleverly utilized by natural enzymes to install a functional group onto acylamine groups containing natural amino acids (primarily glutamine).

Amino acids related products:

Amino-PEG10-acid, CAS 196936-04-6

Amino-PEG1-acid, CAS 144942-89-2,Formula C5H11NO3



Amino-PEG32-acid, CAS 196936-04-6

Transglutaminase-mediated Glutamine Acylation

Transglutaminases (TGases) are widely present in vertebrates, invertebrates, plants, and microorganisms. TGases catalyze the formation of isopeptide bonds between glutamine and lysine residues, primarily by catalyzing the formation of an isopeptide bond between the glutamine-carbonyl group and the lysine-amino group, resulting in inter- and intramolecular protein cross-linking complexes.

Under appropriate conditions, TGases can also be used to selectively attach lysine-functionalized LD or other LDs with terminal amino groups to the side chain carboxamide groups of glutamine residues in antibodies (Figure 4).

Figure 4. Enzymatic Binding of Lysine-Functionalized Antibodies Under Transglutaminase Action
Figure 4. Enzymatic Binding of Lysine-Functionalized Antibodies Under Transglutaminase Action

It is worth noting that natural glutamines in monoclonal antibodies are typically not substrates for native TGases. Therefore, various strategies have been employed to facilitate the formation of isopeptide bonds: (A) through TGase mutations; (B) by inserting or fusing glutamines containing C-terminal peptide tags; (C) by enzymatically removing antibody glycans to expose glutamines. For example, Dophen has developed an engineered form of TGase and is developing DP303c, targeting HER2, for the treatment of solid tumors. Researchers at Rinat/Pfizer, on the other hand, have used native TGase, combined with antibody engineering, to introduce TGase recognition sequences (Q-Tag) at different positions. Pfizer has advanced the clinical development of the TROP-2-targeting ADC drug PF-06664178/RN927C, fused with the C-terminus LLQGA, which has since been discontinued. The third strategy, explored by Innate Pharma, is based on a technology initially developed by Schibli et al., which involves enzymatically deglycosylating antibodies to release glutamine HC-Q295. The Innate ADC (IPH43) utilizing this technology is currently in preclinical stages.

Acylation Reaction of Sortase-Mediated Glutamines

The primary function of sortases is to assist in attaching proteins to the bacterial cell wall and assembling pili. Sortases act on secreted proteins containing C-terminal cell wall sorting signals, which consist of a five-residue recognition motif, such as LPXTG, capable of undergoing acylation reactions with specific oligoglycine receptor substrates, thereby replacing the terminal glycine of LPXTG with a receptor’s oligoglycine fragment. This method has been used to produce ADCs (Figure 5).

For example, NBE Therapeutics utilizes this method by fusing an LPETG tag to the heavy chain C-terminus, then connecting it with a five-glycine-modified cytotoxic payload PNU-159,682, resulting in the development of the clinically advanced ADC drug NBE-002. Similarly, GeneQuantum has developed the GQ-1001 drug based on the heavy chain C-terminus LPGTG.

Figure 5. Sortase-Mediated Coupling of Gly5-Linker Payload with C-terminal LPXTG-Tagged Antibodies
Figure 5. Sortase-Mediated Coupling of Gly5-Linker Payload with C-terminal LPXTG-Tagged Antibodies

Azido Group (-N3)

The azido group is most commonly known for its application in the “click reaction” with alkynes. The click reaction, also known as copper-catalyzed azide-alkyne cycloaddition (CuAAC), is now considered the preferred method for any bond-forming reaction.

However, copper can also lead to undesirable side reactions, such as histidine or tyrosine oxidation, and due to its toxic nature, it is rarely used in pharmaceutical manufacturing. SPAAC ( What is SPAAC? ) has emerged as a powerful alternative, and various cyclooctyne reagents have been developed.

Metal-Free Click Chemistry of Genetically Encoded Amino Acids

Genetic encoding of non-natural amino acids (NNAAs) is an effective method for installing unique chemical tags at desired positions in monoclonal antibodies. Sutro Biophma has developed a cell-free protein expression system by incorporating p-azidomethyl-L-phenylalanine (pAMF) at specific positions, suitable for subsequent LD-click chemistry coupling to prepare ADCs (Figure 6).

Due to the efficiency of cell-free protein expression, site scanning has been performed to determine the optimal coupling positions, and it has been used in various ADC projects. For example, STRO-001 (DAR2 ADC, coupling at HC-F404) and STRO-002 (DAR4 ADC, coupling at HC-Y180 and HC-F404).

Figure 6. Genetic Encoding of p-AMF Enables Specific Coupling via Metal-Free Click Chemistry
Figure 6. Genetic Encoding of p-AMF Enables Specific Coupling via Metal-Free Click Chemistry

Enzyme-Modified Glycans for Metal-Free Click Chemistry

Bacterial strain-promoted cyclooctyne-azide cycloaddition has also been utilized for GlycoConnect, a technology developed by Synaffix. Its core involves enzymatically introducing azido sugars ( Azido-PEG-sugar – ADC Linkers ) onto the glycans of natural antibodies. Initially, antibodies undergo enzymatic actions by both endoglycosidases and galactosyltransferases (GalNAc-T) in the presence of UDP-6-azido GalNAc to convert natural sugars into homogeneous, truncated, and azido-labeled trisaccharides (Figure 7).

Subsequently, LD coupling of azido-labeled antibodies is carried out to generate homogeneous ADCs. The GlycoConnect technology is currently employed in three clinical ADC drugs: ADCT-601 (ADC Therapeutics), XMT-1592 (Mersana Therapeutics), and MRG004A (Miracogen).

Figure 7. Modeling Polysaccharides with Endoglycosidase and GalNAc-T in the Presence of UDP 6-Azido-GalNAc and Achieving Uniform ADCs via Metal-Free Click Conjugation of Payloads
Figure 7. Modeling Polysaccharides with Endoglycosidase and GalNAc-T in the Presence of UDP 6-Azido-GalNAc and Achieving Uniform ADCs via Metal-Free Click Conjugation of Payloads

Other Functional Groups for Antibody Conjugation

In addition to the conjugation technologies currently used in marketed and clinical ADCs, many next-generation conjugation methods are also being developed.

Phenol (Tyrosine Side Chain)

Extensive bioorthogonal labeling strategies are based on the selective chemistry of the phenol side chain of tyrosine (Tyr). Tyrosine appears at a moderate frequency in protein sequences (natural abundance of 3.3%), providing an opportunity for site-selective labeling of its unique phenol side chain.

Despite tyrosine often being partially buried in the protein surface, it can participate in hydrogen bonding and, due to its redox potential, form tyrosyl radicals facilitating electron transfer. Therefore, tyrosine residues undergo highly diverse biological modifications, such as nitration, oxidation, cross-linking, AMPylation, halogenation, or glycosylation.

Tyrosine related products:

Mal-PEG2-BocNH Tyrosine Methyl Ester

Amino-PEG2-BocNH Tyrosine Methyl Ester

Propargyl-PEG1-BocNH Tyrosine Methyl Ester

Azide-PEG1-BocNH Tyrosine Methyl Ester | CAS

HO-PEG1-BocNH Tyrosine Methyl Ester | CAS

Bruins et al. reported a recent method based on generating 1,2-benzoquinone derivatives from specially designed tyrosine moieties, followed by forming stable cyclooctene bonds through metal-free click cycloaddition with bicyclononyne (BCN). By using mushroom tyrosinase to selectively oxidize solvent-exposed tyrosines, 1,2-benzoquinone groups are generated on the antibody surface.

Thioether (Methionine Side Chain)

Methionine is an amino acid primarily responsible for protecting the body from oxidative stress. Thus, its functionalization is less likely to impair protein function compared to other amino acid residues. Another advantage is the low abundance of methionine (about 1.8%), reducing the likelihood of non-specific labeling.

In 2017, Lin et al. reported highly selective, rapid, and robust methionine labeling using redox chemistry to selectively label aziridine-based reagents under biocompatible conditions, followed by click chemistry to attach the payload. This technique is applicable for identifying highly active methionines across the proteome and for generating ADCs, such as selectively binding to the C-terminal methionine tag on engineered trastuzumab Fab fragments.

In 2018, Taylor et al. reported another selective reaction for C-terminal engineered methionine, based on the electrophilicity of hypervalent iodine reagents and the complete chemical selectivity of the methionine side chain’s SMe group in click chemistry. Although conceptually elegant, the resulting methionine conjugates exhibited generally moderate stability, requiring further optimization for successful application in the ADC field.

Imidazole (Histidine Side Chain)

Like methionine, histidine is another amino acid with relatively low natural abundance (2.9%) and low intrinsic nucleophilicity, making it a potentially suitable amino acid for site-specific antibody conjugation.

In 2017, Sijbrandi introduced a new antibody conjugation method using cationic organoplatinum-based linkers to randomly connect to histidine residues on the antibody, stably attaching the LD. The binding of the platinum-based linker resulted in ADCs with unaffected antibody binding activity, DARs in the range of 2.5-2.7, and approximately 85% of the payloads attached to the Fc region.

Know more about ADCs:

What is ADC(Antibody-drug Conjugates)?

What are ADC Linkers?

ADC Linker Design and ADC Empowerment

Cleavable Linkers Play a Pivotal Role in the Success of Antibody-Drug Conjugates (ADCs)

Antibody-drug conjugates(ADCs) list Approved by FDA(2000-2023)


1. Chemical Linkers in Antibody–Drug Conjugates (ADCs).

2. Characterization of disulfide bond rebridged Fab-drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine cytotoxic payload.

3. Site-specific conjugation of native antibodies using engineered microbial transglutaminase.

4. Structure and dynamics of a site-specific labeled Fc fragment with altered effector functions.