The Complete Guide to Site-directed Conjugation in Antibody-drug Conjugates

Antibody drugs have become the mainstay of modern biopharmaceutical development with high specificity, high stability and low toxicity. In recent years, antibody-drug conjugates (adc), the combination of antibodies and small molecule drugs, have made a breakthrough, and it is very likely to grow into a new generation of heavy drugs in antibody tumor therapy. The ADC drug connects the cytotoxic drug to the monoclonal antibody through a small molecule linker, and relies on the unique site-directed conjugation of the monoclonal antibody to bind the drug and kill the cancer cell, making up for the side effects of the cytotoxic drug and the weak effect of the monoclonal antibody. There are four different site-conjugation methods below.

1 Introduction of reactive cysteine The study found that by mutating an amino acid residue in an antibody molecule into cysteine, and then using it to specifically conjugate with a drug to synthesize an ADC, the effect of interchain disulfide bond destruction can be eliminated. However, if the design of the mutation site is inappropriate, a wrong intrachain or interchain disulfide bond will be formed. Junutula et al. developed the PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) technique, which first selects some amino acids that are completely or partially exposed to the surface of the antibody such as Val, Ala, Ser, etc., which are mutated to Cys; and then the Fab fragment mutant is displayed on the surface of the phage by phage display technology and reacts with Biotin. Then, a mutant can be screened, which can stably bind to the biotin and conjugate with streptavidin by the introduced Cys residue by an ELISA method. At the same time, the research team screened three mutation sites on the heavy chain (HC) or light chain (LC) that met the above conditions based on the Fab fragment of trastuzumab: HC-A114, HC -A175 and LC-V110, and used this technique to obtain a reactive IgG mutant THIOMAB, the conjugation drug of which is called TDC (THIOMAB-drug conjugates).2 Introduction of non-natural amino acids Some researchers have used the genetic code extension technology to synthesize a tRNA that recognizes the termination codon and designed an aminoacyl tRNA synthetase that catalyzes the attachment of unnatural amino acids to the tRNA to form an amino acid tRNA synthetase/tRNA orthogonal pair. Then, an amino acid codon in the DNA sequence of the antibody is mutated to a termination codon, and the orthogonal pair is used to synthesize an antibody containing an unnatural amino acid in or out of the cell. Since the introduced non-natural amino acid contains some special functional groups such as an azide group, a ketone group, an alkynyl group and the like, the drug and the antibody can more easily achieve site-directed conjugation. The key to this technology is to obtain strictly orthogonal molecular pairs, using their high reactivity specificity to introduce non-natural amino acids into antibody molecules.3 Through the enzymatic method The method refers to artificially causing the related amino acid sequence which can be recognized by some enzymes in the antibody by genetic engineering technology, and then utilizing the specificity of the enzyme to the substrate to modify the specific amino acid residue, thereby achieving site-conjugation. Formylglycine-generating enzyme (FGE) recognizes a pentapeptide sequence CXPXR, and oxidize Cys to formylglycine (fGly), and the resulting formyl group can form a stable CC bond with the drug through the HIPS reaction. Transglutaminase (TG) recognizes the LLQGA pentapeptide sequence and can catalyze Gln’s?-amide group forms a heteropeptide chain with a compound containing a primary amine.4 Through the disulfide bond modification When the interchain disulfide bond of the antibody is opened, the reduced Cys can be rejoined by reacting with the bireactive reagent to replace the traditional interchain disulfide bond with the bireactive reagent. Accordingly, the drug can be positioned at the disulfide bond site of the antibody to form an ADC using a drug-conjugated bireactive reagent. Moreover, the molecular weight of the dual reactive reagent is small enough to affect the spatial structure of the antibody molecule. The ADCs prepared by the traditional methods have large differences between batches, and it is difficult to develop an evaluation standard for drug analysis and in vitro and in vivo activity investigation. In addition, small molecule drugs have strong hydrophobicity and ADCs with a large number of drug (DAR of 4 or higher) are prone to cohesion with a reduced stability. In vivo experimental data show that the ADC produced by site-directed conjugation has excellent pharmacokinetic properties compared to conventional ADCs, greatly reducing the non-therapeutic side effects caused by drug shedding, and thus has a wider therapeutic window.

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