Antibody-Drug Conjugates (ADCs) are designed to bind to cell surface receptors and get internalized thorugh receptor medaited endocytosis, then trafficked into endosomes and ultimately lysosomes. Following internalization, ADC antigen complexes are fused with endosomes, which break them up for receptor recyling and transport ADs to lysosomes. Finally, an ADC molecule undergoes lysosomal degradation through a number of pathways to release its cytotoxin, which then binds to its target. (Chamow “Manufacturing challenges of therapeutic antibody -drug conugates, BioProcess Internaitonal, September 2023).

Preparation of Antibody-Cytotoxin Conjugates

In general, antibody cytotoxin conjugates are prepared by a multi-step process. First, antibody is covalently attached to a linker in a modification reaction and unreacted components and reaction products separated from antibody linker conjugate by desalting. Next, purified antibody-linker conjugate is reacted with modified cytotoxin to form the antibody-cytotoxin conjugate. The conjugate is purified from unreacted components, solvent and reaction products as by, for example, size exclusion chromatograph. For more information on purification of antibodies see antibody purification. Mazzola (US 2005/0031627)

The majority of ADCs are built on IgG1 scafolds. These complex about 150 kDa biomolecuels contain mltiple native sites for connjugation and can be modified to include additional reactive sites. (Own “Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry” AAPS Jouranl, 17(2), 2015). 

Dai (US 2011/0166319) discloses a prcoess for preapring an atnibody chemically coupled to a drug which cindlues the steps of frist covalently attaching a linker ot the antibody, an optional purificaiton step, conjugated a drug to the antibody-linker and a subsequent purificaiton step, and optional holding steps. 

Kaiser (US Patent Application No: 16/631,759, published as US 2020/0164338) discloses a method of prdoucing an antibody drug conjugation which includes the steps of pumpinng an antibody solution and a linker solution into a mixing device and then into a residence time device, followed along with a connection to the next unit operation, followed by conjugation of the drug. 

Chemical Modifications & Linking Sites on Antibodies:

Chemical modificiations for the functionalization of antibodies include modification with an ezyme such as alkaline phosphatase or peroxidase (HRP), iodation or addition of a chelating compound for radioisotopes and modification with a low molecular compound such as biotin. These modification are typically performed via lysine amino group, a cystein thiol group and an activated carboxyl group. These modificaitons are specific for the functional groups, but are not site-specific. Thus, the problems include reduction in the activity of antibodies due to the modification or the like of the antigen-bnding sties of the antibodies, and difficult control of the number of compounds to be bound. For ADCs, anticancer agents are bound to antibodies in a site-nonspecific manner. In order to overcome these problems, antibody modificaiton has been practiced using antibodies haivng a particular site specificaly introduced functional group. For example, a particular non natural amino acid or free cysten can be introduced by genetic manipulation. Also, a glutamine can be intorduced using transglutaminase (TG). (Ito US 2018/0141976). 

Antibody-drug conjugates (ADCs) are a subclass of biotherapeutics designed to faicilitate the targeted delivery of potent cytotoxic drugs to cancer cells. Depending on the conjugation chemistry, different types of ADCs can be constructed such as cystein-conjugated, lysine conjugated or site-specific ADCs. At a molecular level, all ADC molecuels bear complex chemical structures, combining the molecular characteristics of small molecule drugs with those of large molecule mAbs. In addition, the conjugation reaciton employed increases the complexity of ADC samples by producing a mixture of ADC molecules that are heterogeneous with respect to different drug to antibody ratios (DARs) and also the fact that conjugation is typically a random process becasue there are many surface accessible lysine residues. (Chen, “in-depth stuctural characterization of Kadcyla (ado-trastuzumab emtansine) and its biosimilar candidate” MABS 2016, 8(7), 1210-1223).

Although many drugs are amenable to direct conjugation to an antibody scaffold, heterobifunctional linkers often facilitate ADC bioconjugation. Linkers provide a functional handle for efficient conjugation to antibodies. Reactive handles often fetured in linker chemistry are grouped based on teh site of conjugation. N-hydroxysuccinimide esters are the most common choice for functionalizing amines, especially when coupling to E-lysine residues. For conjugation to cysteines, thiol-reactive maleimide is the most applied handle, alhtough it is also possible to create a disulfide bridge by oxidaiton with a linker bearing a sulfhydrl group. Aldehyde or keto functional groups such as oxidized sugar groups or pAcPhe unnatural amino acids can be reacted with hydrazides and alkoxyamines to yield acid-labile hydrozones or oxime bonds. In addition, hydrozine can be couple with an aldehyde via HIPS ligation to generate a stable C-C linkage. The mechanism of drug release is an important consideration in linker selection. Non-cleavable linkers rely on degradation of the scaffold within the lysosome after internalization. Alternatively, cleavage linkers respond to physicologic stimuli such as low pH, high clutathione concentrations and proteolytic clevage. Several non-clevable alkyl and polymeric linkers have been explored in ADC development. Non-clevable linkers require mAb degradation within the lysosome after ADC internalization to release active drug. Clevage linkers are popular in the ADC clinical pipeline with acid sensitive linkers such as hydrazones and silyl ethers. Hydrozones are easily synthesized and have a plasma half-life of 183 h at pH 7 and 4.4 h at pH 5, suggesting that they are selectively clevable under acidic conditions such as those found in the lysosome. Enzymatically clevage linkers are gaining significant attention due to superior plasma stability and release mechanism. The most popular enzymatic clevage sequence is the dipeptide valine-citrulline, combined with a self immolative linker p-aminobenzyl alcohol. Clevage of an amide linked PAB triggers a 1,6-elimination of carbon dioxide and concomitant rlease of the free drug in parent amine form. (Owen, “Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry” AAPS Journal, 17(2), 2015). 

At N-terminal Amino Acid Residue:

Kim (EP 3015116) discloses an antibody-drug conjugate having a drug conjugated to an N-temrinal amino acid residue of a H or L chain of an antibody. Examples of the N-temrinus include not only aino acid residues at the distal end of th N-temrinus but also residues near the N-temrinus. Exampels of the reactive gorup capable of crosslinking with the alpha-amine group include any reactive groups known in the arm which can crosslink with the N-temrinal alpha-amine group of the H or L chain and include isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride. 

At cystein/Lysine/Glutamine residues:

In theory, bioconjugation is possible via most amino acids. Nevertheless, the nucleophilic primary amine of lysine and reactive thiol of cysteine are the most commonly used amino acids inthe bioconjugation of antibodies. Lysine is one of the most commonly used amino acid residues for linking substrates to antibodies, becasue they are usually exposed on the surface of the antibody and thus easily accessible. Alkylation and acylation are the most important reaction with the nucleophilic E-amine. Antibodies can ontain up to 80 lysine residues and as a result, conjugaiton wa lysines inevitably leads to different number of substrates per antibody and antibodies with teh same number of substrates attached at idfferent sites. Furthemore, modifed lysines in proximity to the antibody-bnding site may influence the interaction of the IgG with the corresponding antigen. (In contrast to lysines, the number of cysteine residues within the sequence of IgGs is much lower. Tehre are only 4 interchain disulfide bonds that can be targeted as potential conjugation sites and thus the heterogeneity of immunoconjugates can be dramatically reduced. Moreover, these cysteines form covalent disulfide bonds to stabilize the tertiary sturcture of teh antibody and thus under non-reducign conditions are not reactive. Schibli “Antibody Conjugates: form heterogeneous populations to defined reagents” Antibodies 2015, 4, 197-224). 

Cysteine based conjugation methods rely on a specific reaction between cysteine residues of the antibody and a thiol reactive functional group intalled on the payload. Amide coupling is a major ADC conjugation ethod connecting a payload and solvent accessible lysine residues on the antibody using linkers containing activated carboxylic acid esters. Amide coupling of an amine and an activated carboxylic acid is one of the most reliable high yielding chemical conversions in oranic synthesis. However, there are about 80 lysine residues on a typical antiboy and about 10 reisdues are checmially accessible. Thus, this conjugaiton modality often gives multiple ADC species with variable DARs and conjugation sites. (Tsuchikama, Protein Cell, 2018, 9(1)): 33-46.)

Employing the thiols of interchain cysteine residues in monoconal antibodies as attachment sites for drug molecules if one of the most used conjugation methods. In a human IgG1, there are four interchain disulfide bonds that can be used as potential conjugation sites. The four interchain disulfide bonds can be reduced by tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DT) which results in eight thiol gorups that are available for conjugating drug molecules. Through this method, different drug antibody ratio (DAR conjugates will be obtained when targeting typical DARs of 2-04. Amines of the antibodies can also react with the carboxyls that dervied form the drugs in the effect of the NHS to give antibdoy drug conjugates. Amines of lysines are commonly used for linking drugs to antibodies becasue lysines are usually exposed on the surface of the antibodies and thus easily accessible. Antibodies contain up to 80 lysines and as a resutl, conjugation through lysine residues inevitably leads to heterogenicity such as different number of drugs per antibody and antibodies with the same number of drugs attached at different sites. (Yao “Methods to design and synthesize antibody-drug conjugates (ADCs) International J. of Molecular Sciences, 17, 194, 2016). 

The traditional linking sites in the preparation of protein conjugates are primary amines on lysines. The epsilon amino group in lysine reside can be easily acylated. However, depending on the number and relative accessibility of lysine residues on the protein surface, the random acylation process would generally result in a heterogeneous mixture of conjugates. These challenges have led to the use of thiols for mAb conjugation, as the number of thios is limited and fixed in the different subtypes of IgGs. The commercial antibody products are primarily IgG1s, which possess 4 inter and 12 intra chain disulfide bonds. Drugs can be linked to inter chain disulfides following reduction. Nonethelss, the use of disulfides for conjugation may still yield a hterogeous populaiton of conjugated products that contains positional isomers. Goswami (Antibodies 2031, 2, 452-500)

Cytotoxic drugs are generally conjugated to antibodies either thorugh lysine side-chain amines or through cysteine sulfydryl groups activated by reducing interachain disulfide bonds. Both of these procedures yield heterogenous products containing a mixture of species with different molar ratios of drug to antibody, linked at different sites, each with distinct in vivo pharmaokinetic, efficacy and safety profiles. To limit the potential liabilities associated with such conjugation methods, Junutula engineered reactive cysteine residues at specific sites in anitobodies, termed THIOMABs”, to allow drugs to be conjugated with defined stoichiometry without disruption of interchain disulfide bonds. For conjugation to covalently attach thiol reactive probes to engineered cysteines, first, the cysteine and glutathione adducts were removed from the THIOMABs by partial reduction followed by diafiltration. This partial reduciton also disrupts interchain disulfide bonds but not intrachain disulfide bonds. The interhain disulfide bonds were allowed to reform by air oxidation or by accelerated oxdiation using CuSO4 of dehydro-ascorbic acid. After this treamtnet, THIOMAB variants were then conjugated with biotin-PPO-maleimide. (Junutul “Site-specific conjugaiton of a cytotoxic drug to an antibody improved the therpaeutic index” Nature biotechnology, 24(8), 2008).

Hu (WO 2015/1091883) discloses an Fc cotnaining polypetpide such as an antibody which includes an N-glycosylated Fc region that includes an acceptor gluamine residue flanked by an N-glycosylation site and where the conjugate moiety is conjugated to the Fc containing plypeptide via the acceptor glutamine residue. The term “acceptor glutamine residue” refers to an amino acid that under suitable conditions is recognized by a transglutaminase (TGase) and can be crosslinked to a conjugate moeity that includes a donor amino group by a TGase through a reaction between the glutamine and donor amino groups (such as lysine or a structurally related primary amine such as amino pentyl group). The conjugation between the conjugation moeity and the acceptor glutamine residue is carried out by conjugating the amine donor group of the conjugation moeity or the small molecule handle to the acceptor glutamine residue. Thus, any conjugate moeity containing an amine donor group can be directly conjugated to the Fc bontianing polypetpide. Any conjugate moeity not containing an amine donor group can be indirectly conjugated to the Fc containing polypeptide via a small molecule handle which cotnains an amine donor group. For example, the conjugate moeity can include an amine donor group (e.g., primary amine-NH2), an optional linker, and an active moeity (e.g., a small molecule) The conjugate moeity can also be a polypeptide or a biocompatible polymer containing a reactive Lys. The amine donor group such as a primary amine (-NH2) provides a substrate for transglutaminase to allow conjugation of the agent moetiy ot the Fc contiaining polyeptide via the acceptor glutamine. Accordingly, the linkage between the donor glutamine and the amine donor group can be of –CH2-CH2–CO–NH–. In some embodiments, the Fc containing polypetpide and the conjugate moeity are linked throguh a cleavable linker. In some embodiments, the conjugate moeity includes an active moeity that is a cytotoxic agent. In some embodimetns, the N-glycosylation site and the acceptor glutamine resiude are 5 or less amino acid residues apart.

Shen (Nature biotechnology, 30(2), 2012) discloses generation of antibodies with cystein residues engineered into the IgG H chain that provide reactive thiols for conjugation to the auristatin and maytansine classes of cytotoxic drugs. 

Thiol maleimide linkage has been widely used in the synthesis of ADCs because of its high slectivity, rapid reaciton kinetics and compatibility with aqueous reaciton conditions. For example, Bentuximab vedotin is prepared by reducing the endogenous antibody cystines, followed by conjugation of cysteine thiols with a maleimide-funcitonalized monomethyl auristatin D derviative. (pone, “understanding how the stbiliyt of the thiol-maleimide linkage impacts the pharmacokinetics of Lysine-linked antibody-maytansinoid conjugates). Biconjugate Chemistry, 2016). 

Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics) are produced. by conjugation to surface exposed lysines or partial disulfide reduction and conjugaiton to free cysteines, respectively. These stochastic modies of conjugaiton lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. (Zimmerman, “Production of stie-specific antiboy-drug conjugates using optimized non-natural amino acids in a cell free expresison system). 2014). 

Matsuda (US 2021/0139541) discloses compounds having an affinity substance to an antibody and a bioorthogonal functional group of the formula A-L-E-B where B is a bioothogonal functional gruop, E is a divalent group that includes an electrophilic group coupled with a leaving group and having ability to reactive with a nucleophilic group in the antibody, L is a divalent group that incldues a leaving rup and A is an affinity substance to the antibody. 

–Ezyme-directed Modification:

Chilkoti “A noncanonical function of sortase enables site-specific conjugation of small molecules to Lysine residues in proteins” Angew, Chem. Int. Ed. 2015, 54, 441-445) disclsoes that the enzyem sortase A can be used to conjugate small molecules to a mAb with an ezquisite level of control over the site of conjugation. Sortase A recognizes the primary sequence LPXTG (where X is any amino acid) in a protein and cleaves the peptide bond between threonine and glycine, forming a stable intermedaite that joins the catalytic thiol in Sortase A to teh carboxyl group of threonine in a thioster bond. Chilkoti cloned the 4D5 mAb against human epidermal growth factor receptor 2 (Her2) and genetically modifed it to contain a pilin domain at the carboxy termini of its H chain. The recombinant antibody was incubated with SortageA-ELP and biotin-LPETGRAGG peptide overnight. Only the anti-Her2 H chain modifed to contain the pilin domain was biotinylated by SortageA-ELP. 

–Affinity Guided Conjugation:

Affinity guided conjugation strategies invovle the incorporation of a scaffold that enables non-covalent interactions to occur between the affinity reagent and the target protein. This, in turn, increased local concetnraiton ofa.reactive moeity to a particular site, allowing for site-specific modificaitons to take place. Chilkoti “A noncanonical function of sortase enables site-specific conjugation of small molecules to Lysine residues in proteins” Angew, Chem. Int. Ed. 2015, 54, 441-445)

Small domains that bind with high affinity to a conserved sequence in teh Fc domain of antibodies are used to form non-covalent antibody conjugates. This approach does not require any modificaiton of the antibody. Among numerous Fc binding domains, teh ZZ domain –a dimer of the modified immunoglobulin binding site of protein A is the most widely used. Its high affintiy interaciton with the Fc part of antibodies has been exploited to by genetically fusing the ZZ domain to Pseudomonas exotoxin A. Incubaton with a antibody yielded hihgly toxic non-coavlently coupled ADCs. Schibli “Antibody Conjugates: form heterogeneous populations to defined reagents” Antibodies 2015, 4, 197-224).

Ito (US 2018/0141976 and EP 3299383) dicloses an IgG binding peptide having an amino acid sequence of 13-17 amino acid residues having the formula (X1-3)-C-(X2)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3). Xaa1 is a protein constituting amino acid such as a lysine residue, a cysteine residue, an aspartic acid or a glutamic acid residue or a non-protein constituting amino acid such as diaminopropionic acid or 2-aminosuberic acid. It is preferred that Xaa1 is modified with a cross-linking agent. The cross-linking agent is a chemical substance for linking the IgG binding peptide to IgG Fc via a covalent bonds. This can be a compound having at least two sites capable of binding to the desired amino acids (such as Lysine, cysteme). Examples include cross-linking aagents containing two or more succinimidyl grups such as DSG and DSS, cross-linking agents containing two or more imidic acid moieties such as DMA. It is also preferred that the peptide has no or little the same residue as Xaa1 (e.g., has only one or two same residues as Xaa1) in the sequence. Two cycteins in the peptide can form a disulfide bond to form a cyclic peptide. Alternatively, sulfide groups in the two cysteine residues on the other sides of the peptide may be linked via linker. The peptide can then be mixed with IgG causing a cross-linking reaction of the petide with the cross-linking agent with the IgG. This eliminates the need of altering the sequence of the antibody molecule. The IgG binding petpide can be modified with additional functional substances such as a labelling agent or an additional drug. The cross linking to IgG can occur site specifically between the amino acid residues Xaa1 of the IgG bidning peptide and Lys248 or Lys246. 

Yamada (US Patent Applicaiton No: 16/663,791, published as US 2020/0190165) discloses an antibody which includes lysine residue(s) in a target region and 5 or more lysine residues in a non-target region and at least one functional substance which is a drug, a label or stabilizer which binds to the lysine residue(s) in the target reigon with 30% or more regioselectivity through a linker which does not include a peptide portion. The antibody is prepared by preparing a compound haivng the formula A-L-B-R where A is an affinity substance to a soluble protein, L is a cleavable linker which is a divalent group comprising a cleavable porition, B is a divalent group that includes a bioorthogonal functional group or a divalent group with no bioorthogonal functional group and R is a reactive group to a soluble protein such as an antibody. The reactive group can be specific to a side chain of a lysine residue. The bioorthogonal functional groups can bind to the soluble protein via a side chain of a lysine, tyrosine or tryptophan residue. First a soluble protein (T) such as an antibody assocaites with the compound through an affinity substance (A) to the soluble protein. Next, the compound reacts with a side chain of a specific amino acid residue (e.g., a side chain of a lysine resiude) in a target region present near an association site of the affinity substance and the coluble protein through a reactive gorup (R) (e.g., an activated ester) to form a conjugate between the compound and the soluble protein. The cleavable portion enables relase of the affinity substance (polypeptide) to an antibody.