Cutting-edge bioconjugation technology for ADCs

preface

The design of clinically successful ADCs depends not only on the efficacy of the payload and its attachment point, linker stability, and effective drug release, but also on the choice of
antibody and bioconjugation techniques.
Over the past 10 years, all FDA-approved ADCs have come in the form of heterogeneous mixtures of ADCs, with different numbers of drugs
attached to different locations of monoclonal antibodies.
The coupling site has a significant effect on ADC stability and its pharmacokinetics, with high DAR (drug-antibody ratio) often leading to rapid plasma clearance, while ADCs with low DAR exhibit weak
activity.
Among drugs of ADC, the presence of namoclonalab is a potent competition inhibitor
.
As a result, over the past decade, a host of new conjugation strategies have been developed with the aim of controlling the location and quantity of small molecule drugs while maintaining structural integrity and homogeneity
.

Chemically based specific in situ antibody modifications

The natural structure of monoclonal antibodies offers a variety of possibilities for biological conjugation, and chemical-based, specific natural (non-engineered) antibody conjugation offers some advantages
.
It avoids the complexity of antibody-specific site mutations and the challenges
that can be faced in scaling up and optimizing cell cultures.

Depending on the antibody sequence, the junction sites between disulfide bonds of endogenous amino acids such as lysine, histidine, tyrosine, and cysteine are very attractive
.
All FDA-approved ADCs, until 2021, utilize these endogenous amino acids for conjugation
.
However, antibody scaffolds also contain glycans, which result from
post-translational modification of the FC region during monoclonal antibody production.
Some studies have reported new strategies for sugar engineering, which appears to be an interesting alternative to bioconjugation
.

Conjugation with endogenous amino acids

One of the most common coupling methods is the use of lysine residues of antibodies, amino acid nucleophilic NH2 groups, to react with the electrophilic N-hydroxysuccinimide (NHS) group on the Lick payload.

Despite the simplicity of the reaction, the high abundance of available lysine residues has led to the formation
of inhomogeneous mixtures of many ADCs under random distributions.
DAR is controlled by the drug/antibody stoichiometric ratio, which is widely used, including approved ADCs such as Besponsa, Mylotarg, and Kadcyla
.

More recently, specific modifications
to lysine sites and residues have also been reported.
Through computer-aided design, sulfonyl acrylates are used as intermediate reagents for modifying
individual lysine residues on native protein sequences.

The regional selectivity of the reaction is attributed to the design of sulfonyl acrylate and the unique local microenvironment
around each lysine.
By computational prediction, lysine with the lowest pKa tends to react
preferentially at weakly alkaline pH in a site-specific manner.
This reaction
was observed even in the presence of other nucleophilic residues such as cysteine.
This technique has been applied to 5 different proteins and trastuzumab, all of which retain the original secondary structure and protein function
after coupling.

In 2018, Rai et al.
reported another site-specific modification
utilizing reversible intermolecular reactions of "chemically key proteins".
This reagent carries a variety of functional groups that reversibly form imine moieties
on all available lysine residues.
The key protein then reacts
with the proximal histidine residue via the epoxide in the reagent.
Thus, under physiological conditions, the key protein is separated from lysine and the aldehyde is regenerated to enable the antibody
to be labeled by oxime binding.

This targeted modification technique for key proteins was later developed into monolysine residue labeling technology, which has unquestionable selectivity
even in the presence of N-terminal amines.
The success of the method relies on the Fk1-spacer-Fk2 reagent
.

The^Fk1 functional group reacts reversibly with lysine to regulate the microenvironment
of the proximal lysine moiety of_Fk2.
The conjugation is then performed at the lysine residues (K169 and K395) at Fk2 by amide bonding, and the location of
the coupling is adjusted by the design of the spacers.
This method has been successfully applied to the synthesis of ADC (trastuzumab-emtansine), demonstrating that its cell activity is comparable
to the approved Kadcyla.

Merlul et al.
recently reported a different binding strategy that effectively targets histidine residues
on native antibodies.
They introduced a cationic organometallic platinum(II)-based linker, [ethylenediamine platinum(II)]2+, represented in the figure as Lx
.

This technique is based on two steps
: complexation and conjugation.
Azetidine ligands such as piperidine coordinate with Lx to form complex precursors, and stable intermediates contain a payload and a chloride ion
on the ligand.
The complex contains a positively charged Pt(II) center, which improves the water solubility of the linker and payload complex and minimizes antibody aggregation, and the method is also extended to similar iodine complexes
.
In a recent report, the use of sodium iodide was shown to significantly improve the coupling yield and selectivity
of this technique.
The exchange of residual chlorine ligands on the Cl-Lx-drug loading complex with iodide results in a more active I-Lx-drug load, resulting in higher coupling yields
.
This technology has been applied to the large-scale production
of ADC drugs.

Disulfide heavy bridging strategy

IgG antibodies contain four interchain disulfide bonds, two linking light and heavy chains, and two located in the hinge region connecting the two heavy chains, which maintain the integrity
of monoclonal antibodies.
Another classical bioconjugation pathway explores the role
of these cysteines as payload attachment points.
The reduction of the four disulfide bonds typically results in eight sulfhydryl groups that are able to react with maleimide linkers to produce an ADC
with a DAR of 8.

Doronina and colleagues report examples of ADCs conjugated with a chimeric anti-CD30 monoclonal antibody conjugated MMAE with DAR=8
.
This payload loading method is better controlled
compared to classic lysine coupling.
However, it has been reported that a higher drug load increases the risk of aggregation, which leads to high plasma clearance and reduces efficacy
in vivo.

Badescu and al reported a novel site-specific rebridging conjugation strategy in 2014, and they were the first to demonstrate that the new bis-sulfone is able to alkylate two sulfhydryl groups from antibody and antibody fragments that reduce disulfide bonds with minimal
effect on antigen binding.
Later, Wang and al described a new water-soluble allyl sulfone that enhances reactivity without in situ activation
.
It exhibits high stability, high water solubility, and site specificity
.

In addition, there is a rebridging technique for biocoupling of mercaptoalkynes with terminal alkynes and cyclooctyne, which further develops a new generation of maleimides, such as dibromo- (DBM) and dithiomaleimide (DTM), for site-specific conjugation
.
These maleimide analogues contain good release groups at positions 3 and 4, enabling fast, efficient, and high-yield coupling
.
Hybrid thiobromide maleimide (TBM) that binds the properties of dibromine and dithiomaleimide has recently been reported, and this TBM reagent binds faster and shows a higher percentage of DAR=4, possibly due to the reduced steric hindrance
of bromine.

In 2015, Chudasama et al.
introduced a new class of heavy bridging reagents, dibromopyrididazinediones
.
They demonstrated that it can be efficiently inserted into disulfide bonds, and the resulting structure exhibits excellent hydrolytic stability
even at high temperatures.
However, heterogeneity is also observed as the temperature on the reduction step increases, and this structure also allows the selective introduction of different functional groups
.

Divinylpyrimidine is another effective re-bridging reagent capable of producing a stable ADC
with DAR=4.
Spring et al.
studied the effect of vinyl heteroaryl scaffolds on cysteine rebridging, and they believed that replacing pyridine with pyrimidine could make the heteroaryl ring a better electron acceptor, thereby improving crosslinking efficiency
.
Their work extended to divinyltriazine, where heavy bridging showed greater efficiency
at high temperatures.

To avoid the drawbacks of in vivo instability associated with classical maleimide conjugation, Barbas et al.
investigated methylsulfonylphenyloxadiazole, an agent that responds
specifically to cysteine.
They are more stable than cysteine-maleimide conjugates in plasma
.
Inspired by this, Zeglis designed the DiPODS reagent, which contains two oxadiazolyl methyl sulfone moieties linked by phenyl groups, and DiPODS form covalent bonds
with two sulfates in a heavily bridged manner.
Compared to maleimide conjugation, conjugation in this way has superior in vitro stability and in vivo performance
.

Glycan conjugation

Since IgG is a glycoprotein, it contains an N-glycan at the CH2 domain N297 location of each heavy chain of the Fc fragment, and this glycosylation can serve as an attachment point
to the connecting payload.
Long-distance localization of polysaccharides with Fab regions reduces the risk of impairing the antigen-binding capacity of antibodies after conjugation, and furthermore, their chemical composition differs compared to the peptide chains of antibodies, allowing site-specific modifications that make them suitable conjugation sites
.

Glycan bioconjugation can be distinguished according to the technique used to target carbohydrates: including glycan metabolism engineering, glycostransferase treatment after glycan oxidation, and ketone or azide labeling
after endoglycosidase and transferase treatment.

Neri et al.
reported site-specific modification
of fucose at the N-glycosylation site of IgG antibodies.
This sugar contains a cis-glycol fraction that is suitable for selective oxidation
.
They oxidized fucose residues with sodium metaperiodate to create an aldehyde group that reacts with hydrazine-containing linkers, so that the antibody is linked
to the drug by hydrazine bonds.

Senter and colleagues added sulfur-based analogues to cell culture media, which metabolically brought 6-thiofucose into antibody modification
.
They believe that substitution is accomplished by hijacking the fucosylation pathway, which introduces chemical sites to achieve site-specific binding
.
Compared to classical cysteine conjugates, this approach significantly reduces the level of heterogeneity and produces conjugates
with more predictable pharmacokinetic and pharmacodynamic properties.

Recombinant IgG rarely contains sialic acid, however, glycine
has been shown to be enzymatically engineered using galactosyl and sialyltransferases.
Galactose is added by an enzymatic reaction to obtain G2 glycans, followed by the addition of terminal sialic acid
.
This modification is oxidized by periodic acid to form aldehyde groups, which can be coupled to a linker-payload
with a hydroxylamine group.
The conjugate has high targeting selectivity and good
antitumor activity in vivo.
Periodic acid can also oxidize sensitive amino acids such as methionine, affecting the binding
to FcRn.

In addition to these coupling strategies, galactose residues can also serve as modification sites
.
Several studies have reported the replacement of galactose with a galactose containing ketone or azide functional group galactose through the use of mutated β-1,4-galactosetransferase, a galactose derivative with biorthogonal functional groups that opens the way
for efficient coupling.
These techniques have been developed for imaging and anti-cancer applications
.

The endoglycosidases EndoS and EndoS2 found in Streptococcus pyogenes, enzymes are capable of hydrolyzing N-glycans of IgG, thus making the hydrolyzed residues an effective site for bioconjugation
.
This method helps to homogenize the glycan structure of the monoclonal antibody, and it is also suitable for any IgG subtype
.
Such methods were applied to trastuzumab-maytansine to prepare sugar-coupled ADCs
with good in vitro and in vivo efficacy.

Site-specific bioconjugation of engineered antibodies

Advances in bioorthogonal chemistry and protein engineering have contributed to the generation of more homogeneous ADCs
.
Although there are many attachment methods available on native monoclonal antibodies, site-specific bioconjugation on engineered antibodies enables more efficient control of DAR and avoids changing affinity
for antigen binding.
In this way, natural or unnatural amino acids are added at certain locations to obtain a homogeneous product with excellent pharmacokinetic and pharmacodynamic characteristics
.

Enzymatic method

Attachment of the payload can be achieved
in a very selective manner by inserting specific amino acid tags into the antibody sequence.
These tags are recognized by specific enzymes, such as formylglycine-producing enzyme (FGE), microbial transglutaminase (MTG), transpeptidase, or tyrosinase, enabling site-specific coupling
.

Aaron et al.
explored a novel site-specific conjugation
that utilizes aldehyde to label proteins.
This technique utilizes a genetically encoded pentapeptide sequence (Cys-X-Pro-X-Arg) in which cysteine residues are recognized by FGE and co-translated oxidized to formylglycine
during protein expression in cells.
In this way, engineered antibodies are selectively coupled to aldehyde-specific linkers by HIPS (hydrazino-Pictet–Spengler) chemical methods.

Microbial transglutaminase (MTGase) strategies are also often developed to localize-specific conjugations
.
MTGase catalyzes the formation of peptide bonds
between the glutamine side chain at the deglycemic antibody 295 position and the primary amine of the substrate.
Compared to other enzyme strategies, MTG is a flexible technique that does not require a peptide donor to achieve conjugation
.
As long as the acyl receptor contains a primary amine, there is no structural limitation
.

Glutamine residues are naturally present in the Fc region
of each heavy chain of the monoclonal antibody.
After deglycosylation at position 295, glutamine residues are coupled by MTGase-mediated reactions to produce a homogeneous DAR=2 ADC.

To improve efficiency, linkers with branched chains can be conjugated, thus doubling DAR, and mutations of asparagine at position 297 to glutamine can also increase DAR.

NBE Therapeutics developed Staphylococcus aureus transpeptidase A-mediated conjugation
.
Their strategy utilizes transpeptidase A (SrtA) to cleave the amide bond
between threonine and glycine residues in the motif of LPXTG (X = any amino acid) pentapeptide.
It then catalyzes the coupling of glycine-related payloads to the newly generated C-terminus to generate peptide bonds
at physiological temperature and pH.

The method was applied to different antibodies, such as anti-CD30 and anti-Her2, and conjugated maytansine and MMAE using a linker containing 5 glycine, both ADCs showing in vitro cell killing activity
similar to classical conjugation.
Enzymatically produced trastuzumab-maytansine perfectly matched Kadcyla
in in vivo tests.

In another example, an ADC
for the highly potent anthracycline toxin derivative PNU-159682 was generated by transpeptidase.
Interestingly, with this technique, the coupling efficiency is even higher than that of Adcetris and Kadcyla analogues
.
In addition, the prepared PNU-159682 ADC has high in vitro and in vivo stability and shows more potency than ADCs
containing tubulin-targeted payloads.

Another emerging new method is site-specific antibody labeling through a tyrosine tag, which is fused
with the C-terminal gene of the monoclonal antibody light chain.
Given site accessibility, Bruins and colleagues used an engineered tetraglycyltyrosine residue as a label, which provides an easily accessible site for conjugation
.
Tyrosinase oxidizes tyrosine to 1,2-quinone, allowing cycloaddition reactions with various bicyclic [6.
1.
0]nonyne (BCN) derivatives.

This method can be efficiently conjugated
to MMAE containing BCN linkers.

Cysteine engineering: thimonoclonal antibody technology

Random cysteine conjugation and rebridging are techniques
that utilize naturally occurring cysteine residues within the antibody structure.
However, the heterogeneity of the random cysteine method and the fragmentation of monoclonal antibodies in the rebridging strategy need to be considered in ADC synthesis, especially when
hydrophobic drugs are conjugated.

Unlike them, thimonoclonalab technology achieves selective and homogeneous modification
of desired sites on the antibody by utilizing engineered reactive cysteine that does not involve structural disulfide bonds.
In general, cysteine mutations are designed to facilitate cytotoxic payload conjugation while maintaining the stability, affinity, and ADC aggregation
of monoclonal antibodies.
To determine the optimal location of mutations, several techniques are commonly employed, including computational modeling, model system screening, and high-throughput scanning
.

Junudula et al.
first reported a thiobizumab strategy that replaces alanine (HC-A114) at position 114 of the heavy chain of anti-MUC16 antibody with engineered cysteine residues, and the reactive thiol within the engineered position is able to react
with maleimide-loaded linkers.
The synthetic anti-MUC16 ADC demonstrated potency in xenograft mouse models and high dose tolerance in rats and cynomolgus monkeys, a finding that established a general approach
to thiomoclonalab conjugation strategies.

In addition, succinimide ligation can undergo two parallel reactions within the cytoplasm: the reverse Michael reaction leading to a loss of linker-payload, and the hydrolysis of succinimide, both of which have significant effects
on ADC activity in vivo.
To improve stability, Lyon and collaborators designed a linker
integrated with a basic amino group adjacent to maleimide.
The addition of diaminopropionic acid (DPR) to the linker promotes rapid quantitative hydrolysis of thiosuccinimide at neutral pH and room temperature, so that non-specific decoupling is blocked, thereby improving stability in vivo
.
In addition to the commonly used maleimide, different cysteine reactants such as iodoacetamide, bromoformamide, carbonyl acrylate, N-alkyl vinylpyridine salts
have been explored.

Biocoupling with engineered unnatural amino acids

In addition to thiomomonoclonal antibody technology, the addition of non-standard amino acids (ncAAs) provides another possibility
for site-specific conjugation.
The technique uses amino acids with unique chemical structures, enabling the introduction of linker-payload complexes
in a chemically selective manner.
This technique requires recombinant antibody sequences utilizing tRNA and aminoacyl tRNA synthetase (aaRS) orthogonal to all endogenous tRNAs and synthetases within the host cell to bring ncAA into proteins
in response to unassigned codons.
Typically, ncAA is added to the
medium during fermentation.
It is important to choose unnatural amino acids because they may stimulate immunogenicity
.
Commonly used ncAAs are analogues of natural amino acids with unique groups, such as ketones, azides, cyclopropene, or dienes
.

Acetylphenylalanine (pAcF) has been successfully integrated into anti-CXCR4 antibodies
.
The payload Auristin is effectively coupled to the antibody by oxime ligation, resulting in a chemically homogeneous ADC.

This ADC showed good in vitro activity and complete removal of lung tumors in mice
.

Due to the acidic conditions required for oxime ligation and the kinetics of slow ADC release, another option is to add ncAA-containing azides
.
The widely used p-azidoperianiline (pAzF) enables rapid CuAAC or SPAAC responses under physiological conditions, using this strategy to successfully conjugate glucocorticoid payloads on anti-CD74 antibodies
.
In addition to pAcF technology, azide-containing lysine analogues (AzK) have been successfully introduced into antibodies to generate site-specific ADCs
with Auristin, PBD dimer or tubulin payloads.

In addition, cyclopropene derivatives (CypK) of lysine as well as naturally occurring atypical amino acids such as selenocysteine (Sec) have been successfully integrated into the antibodies
.
The resulting ADC exhibits good stability, selectivity, and in vitro and in vivo activity
.

brief summary

Over the past few years, many advances
have been made in the optimization of the structure and mechanism expansion of ADCs.
New conjugation techniques have been developed to achieve higher selectivity
for tumors.
These coupling techniques result in better stability, selectivity, and in vitro and in vivo activity
of the ADC.
Preliminary data on these new conjugation techniques are encouraging and will greatly facilitate the rapid development
of ADC drugs in the future.

References:

1.
The Chemistry Behind ADCs.
 Pharmaceuticals (Basel).
 2021 May; 14(5): 442.

： ，
。   ， ，