The number of cytotoxic drugs connected to each antibody is the drug-to-antibody ratio (DAR). When the DAR increases, the drug metabolism rate of ADC drugs increases, the half-life decreases, and the systemic toxicity increases. Ideally, when the DAR is 4, the drug has the highest efficacy. Therefore, in actual production, the drugs with DAR less than 2 or DAR greater than 4 are removed through quality control and purification processes to ensure the uniformity of the drugs.
|The key factor||Potential adverse effects||Improvement direction|
|Drug-antibody ratio||Toxicity, pharmacokinetics, safety.||DAR directly affects the safety and efficacy of drugs; DAR is directly controlled during the production process, and the variation range of DAR should be reduced.|
|DAR species composition||Toxicity, pharmacokinetics, safety.||Drugs may be highly heterogeneous, and production needs to maintain the batch-to-batch consistency of DAR.|
|Free drug||Safety.||Free drugs may increase systemic toxicity and need to be removed.|
|Polymer or fragment||Immunogenicity, pharmacokinetics, toxicity.||The direction of process improvement is to prevent the formation of polymer or remove polymer, its formation may be due to the hydrophobic aggregation of the linker drug, reduction/oxidation reaction, and the solvent changes the antibody.|
|Remaining solvent||Safety.||The solvent is used to dissolve small molecule drugs when coupling antibodies and removed after the reaction.|
ADC has experienced three generations of technological changes, and the treatment window has been greatly improved. The small molecules of the first-generation ADCs were not toxic enough, and the ADCs were not stable enough, and most of them ended in failure. The second generation of drugs used more toxic small molecules to overcome the weakness of the first generation of insufficient potency and optimized the antibody but still used traditional chemical coupling. The antibody coupling ratio (DAR) has poor uniformity (0-8 or even higher) and poor linker stability, which is easy to lyse in the blood and cause serious side effects. There are currently two second-generation ADC drugs approved for marketing (Adcetris and Kadcyla).
The birth of the third-generation ADC drugs mainly benefited from the development of site-specific coupling technologies, such as ThioBridge technology, unnatural amino acid coupling, and enzymatic coupling. More precise conjugation technology can control the position and number of highly active drug molecules conjugated on the antibody, and the heterogeneity of antibody-conjugated drugs will directly affect their distribution and metabolism in the body. Therefore, the higher homogeneity of antibody-conjugated drugs will improve drug purity and quality control. In addition, if the size of the antibody part is smaller and the binding force is higher, the drug molecules can penetrate into the solid tumor better, such as a new class of DARPin (designed ankyrin repeat proteins)-toxin-conjugated drugs. DARPins are a new generation of target-binding proteins, with smaller size and higher binding force. So DARPins are ideal targeted drugs, which can be used to deliver toxic small molecule drugs to tumors to achieve efficient targeted killing of cancer cells.
Table 1: Comparison of Cysteine conjugation and Lysine conjugation.
|Cysteine residues||Maleimides, haloacetyls, other Michael acceptors||Simple and reproducible method|
Used in FDA approved Adcetris widely employed in pipeline candidates, DAR ~0-8.
Comparatively less heterogeneous by products than lysine conjugation.
Easier to characterize pharmacokinetically.
|Lysine residues||Activated ester functional groups like N-hydroxysuccinimide esters||Though highly heterogeneous, this method is employed in FDA approved Kadcyla, Mylotarg, DAR ~3.5 (Kadcyla), ~2.5 (Mylotarg).|
Mostly used to crosslink via non-reducible linkers.
A certain amino acid residue in the antibody molecule is mutated to cysteine, and then it is specifically coupled with the drug to synthesize ADC, eliminating the influence of the destruction of the interchain disulfide bond. Insert a cysteine residue at a specific position of the antibody through genetic engineering technology, and then couple the sulfhydryl group on the cysteine to the drug molecule to synthesize a site-specific antibody-drug conjugate. This method will neither interfere with the folding and assembly of immunoglobulins nor change the binding mode of antibodies and antigens.
Through the use of extended genetic code to achieve breakthrough protein therapy, using a tyrosyl-tRNA/aminoacyl-tRNA synthetase that can specifically recognize unnatural amino acids, transfected by Chinese hamster ovary cells (CHO) to replace the 21st amber amino acid codon, the resulting cells can be used to synthesize various antibodies with gene-decoded para-acetyl phenylalanine residues, and then undergo an oximation reaction with hydroxylamine to obtain products with a DAR of 2.
Enzyme catalysis uses genetic engineering technology to produce related amino acid sequences in antibodies that can be recognized by certain enzymes and then uses enzyme specificity for substrates to modify specific amino acid residues to achieve site-directed coupling. Currently, transglutaminase, glycosyltransferase, and sortase A are mainly used. Sortase A (Srt A) is an enzyme with membrane-bound sulfhydryl transpeptidase catalytic function. It can recognize the main sequence LPETG in the protein and cleave the peptide bond between threonine and glycine to form a stable sulfhydryl group in Srt A connected to the carboxyl group of threonine through a thioester bond.
The principle of disulfide bond reduction transformation is to reduce the disulfide bond of the monoclonal antibody itself and use a dibromo (or disulfonate) reagent to react with the reduced interchain disulfide to provide a re-bridged mAb to obtain products with a DAR of 4.
|Technology||Connection between linker and antibody||Blood stability||Toxin molecular load|
|Introduction of cysteine residues||Sulfur bond||Relatively stable||2-4|
|Disulfide bond reduction||Sulfur bond||Relatively stable||4-8|
|Introducing unnatural amino acids||Sulfur bond||Stable||2|
|Sorting enzyme catalysis||Relatively stable||3.2|
In terms of the above four quantitative coupling principles and techniques, the characteristics of disulfide bond reduction and the introduction of unnatural amino acids are more obvious. The disulfide bond reduction theory can connect 8 small toxin molecules, which have a greater advantage of the number of toxin connections. The introduction of unnatural amino acids has very stable chemical bonds between linkers and small molecules, and it is not easy to dissociate in the blood. Therefore, good blood stability is its advantage.
|Drugs||Target||Payloads||Linker||Monoclonal Antibody (mAb)||Indications||Developer|
|Adcetris||TNFRSF8 (KI-1, CD30)||Monomethyl Auristatin E (MMAE)||Valine-citrulline||IgG1||Hodgkin Lymphoma (HL); Anaplastic Large Cell Lymphoma (ALCL).||Seattle genetics, Inc./Takeda Pharmaceutical Co. Ltd.|
|Kadcyla||HER2||Maytansine DM1||Noncleavable succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker||Trastuzumab||HER2+ metastatic breast cancer (MBC)||Genentech Roche in collaboration with ImmunoGen|
|Mylotarg||CD33||Calicheamicin (N-acetyl-γ calicheamicin 1,2-dimethyl hydrazine dichloride)||Covalent linkage (condensation) of a bifunctional linker, 4-(4-acetylphenoxy)butanoic acid (AcBut linker)||IgG4||Relapsed AML (Acute myeloid leukemia) | CD33 positive||Pfizer (Wyeth)|