Unnatural Amino Acids for Antibody-Drug Conjugates (ADCs)

Unnatural amino acids (uAAs) are a class of amino acids that do not exist in nature and are synthesized through chemical or bioengineering methods. They can provide unique chemical reaction sites, making the coupling process of ADC more controllable and efficient. For example, by integrating uAAs, researchers can introduce specific reactive groups, such as alkyne groups, azide groups, etc., to achieve click chemistry reactions between antibodies and drugs. This method not only improves the efficiency of coupling, but also makes the distribution of drug molecules on antibodies more uniform, thereby reducing side effects and improving the therapeutic index of drugs. In this way, the use of uAAs in ADCs not only improves the stability and safety of the conjugates, but also significantly enhances their therapeutic effects. This technological advancement provides important support for the research and development and clinical application of anticancer drugs, and opens up new horizons for precision medicine and personalized treatment.

Antibody Drug Conjugate

Antibody-Drug Conjugates (ADCs) are composed of three parts: monoclonal antibodies, highly effective cytotoxic substances, and linkers. It effectively reduces the adverse reactions of cytotoxic antitumor drugs and improves the selectivity of tumor treatment by organically combining the targeting of antibodies with the anti-tumor effects of cytotoxic drugs. For the development of ADC drugs, the key lies in the selection of targets, antibodies, cytotoxic payloads, linkers, and conjugation methods. Among them, the conjugation method of ADC drugs will directly determine the properties such as drug-to-antibody ratio (DAR), conjugation site distribution, and conjugation stability. This is also the most difficult barrier to the development of ADC drug technology. At present, ADC has become a new hot spot and important trend in the research and development of anti-tumor antibody drugs due to its excellent targeting and anti-cancer activity, attracting more and more attention.

Fig. 1. Antibody-drug conjugates (ADCs) (Int J Mol Sci. 2016, 17(4): 561).

ADC Drug

Since the first ADC drug Mylotarg® (gemtuzumab ozogamicin) was approved by the FDA in 2000, a total of 15 ADC drugs have been approved for hematological malignancies and solid tumors worldwide as of December 2023. In addition, there are currently more than 100 ADC candidates in different stages of clinical trials.

Antibody Conjugation

Antibody drug conjugation refers to the connection method of connecting the various components of ADC, which determines the DAR and uniformity, and affects the activity, tolerance and stability of the drug. It is generally divided into two categories: random conjugation and site-specific conjugation. Common methods of non-site-specific conjugation are Lys residue conjugation and Cys residue conjugation. Site-specific conjugation includes the introduction of reactive cysteine, disulfide bond heavy bridge, non-natural amino acid technology, enzyme catalysis technology, glycosyl conjugation technology and proximity-induced antibody conjugation (pClick) technology. Site-specific conjugation has become a research and development trend of conjugation methods because its DAR value is easier to control and the ADC drugs produced have good uniformity.

• Random Conjugation

The traditional random chemical conjugation method (natural amino acids) is based on non-selective modification using naturally occurring amino acid residues in antibodies, such as native lysine and reduced interchain native cysteine.

Natural Lysine Conjugation

Lysine residues have a high natural abundance (7.2%) and surface accessibility, and immunoglobulin G1 (IgG1) contains about 90 lysine residues, of which more than 30 can be chemically modified. Therefore, this conjugation method is rapid and convenient, but its selectivity is relatively poor, the uniformity is insufficient, and it usually produces multiple ADC drugs with variable DARs and conjugation sites, which has an impact on the PK/PD of ADCs. Kadcyla, Mylotarg, and Besponsa on the market currently use lysine residue conjugation.

Fig. 2. Lysine and cysteine conjugation (Cancers (Basel). 2024, 16(2): 447).

Natural Cysteine Conjugation

Cysteine-based reactions provide another coupling method: conjugation with cysteine residues, that is, disulfide bonds are reduced and converted to cysteine residues, which can be conjugated. Typically, IgG1 antibodies have interchain disulfide bonds and intrachain disulfide bonds. The interchain disulfide bonds are exposed on the outside of the antibody and can be easily reduced to expose free cysteine residues, providing available sites for coupling the linker payload to the antibody. Due to the limited number of binding sites and the unique reactivity of thiol groups, the use of cysteine as a linking site helps reduce heterogeneous ADCs. After reduction, up to 8 cysteine residue sites can be exposed for interchain disulfide bonds, which can generate drugs with DARs of 2, 4, 6, and 8. Cysteine conjugation can significantly reduce the heterogeneity of ADCs and obtain higher uniformity than lysine-based coupling ADCs. Polivy, Padcev, and Adcetris on the market use this method for conjugation.

* Natural amino acid products:

• Site-Specific Conjugation

Site-specific conjugation is an important technology in ADC development. For ADC, the antibody part is responsible for targeting specific antigens, while the drug part is used to kill cancer cells. The main advantage of site-specific conjugation is to control the binding position of the drug to the antibody, thereby optimizing the efficacy and safety of the drug. Traditional random conjugation methods easily lead to the inability to accurately control the binding position of the drug on the antibody, resulting in a mixture, which affects the uniformity and pharmacokinetic properties of the ADC. In contrast, site-specific conjugation can be achieved through a variety of methods, including enzymatic conjugation, site-directed mutagenesis, and the introduction of non-natural amino acids. These methods accurately control the drug connection point, ensure the uniformity and consistency of ADC, and improve its therapeutic effect and safety.

Introduction of Reactive Cysteine

The ThioMab technology developed by Genentech is a representative of this technology. It is a method of inserting cysteine residues into different positions of the heavy chain (HC) or light chain (LC) of an antibody by means of genetic engineering, and connecting drugs. The percentage of ADCs with a DAR of 2 is as high as 92.1%. In addition, ThioMab technology does not affect the folding and assembly of immunoglobulins or the binding of antibodies to antigens. A major disadvantage of ThioMab technology is that the thiol introduction step may lead to the formation of incorrect disulfide bonds between two fabs in the antibody, which remains a problem to be solved.

Disulfide Conjugation

Disulfide re-bridging has also attracted attention, despite its low coupling efficiency and the presence of intrachain staggered bridges. Similar to traditional cysteine coupling, the coupling sites are also obtained by reducing interchain disulfide bonds. Unlike random coupling, disulfide re-bridging involves reaction with cysteine-selective cross-linking reagents, such as new generation maleimides (NGMs), pyridazinediones (PDs), etc.

Unnatural Amino Acid Conjugation

UAAs provide a new technical means for the development of ADC. Antibody drug site-specific coupling can be achieved through uAAs. The synthesis of proteins on ribosomes is carried out through the recognition of tRNA anticodon and mRNA codon. tRNA introduced that can specifically recognize uAAs and the corresponding aminoacyl tRNA synthetase. Under the action of aminoacyl tRNA synthetase, tRNA and the corresponding uAAs combine to form aminoacyl tRNA, and then complement the codon on mRNA through its anticodon, so that the uAAs are integrated into the polypeptide chain to synthesize a recombinant antibody containing uAAs. The uAAs introduced are usually acetylphenylalanine, azido methyl-l-phenylalanine and azido lysine. The ketone and azido functional groups on the uAAs can react with the drug linker to obtain an ADC with uniform drug to antibody ratio. Keto group can form oxime bond with hydroxylamine group, azide group can form cycloaddition reaction of 1,2,3-triazole with alkynyl group under the catalysis of copper, and azide group can also combine with cyclooctyne without the catalysis of copper to generate azide octyne cycloaddition reaction.

* UAAs for antibody conjugation:

By artificially adding uAAs to the original sequence of the antibody, it presents a specific site on the antibody surface that can be easily coupled, thereby obtaining an ADC with a fixed site and uniform DAR value. UAA coupling can mutate uAAs at will and obtain ADCs with arbitrary DAR values. The ADCs generated by introducing uAA technology show longer circulation half-life, better efficacy and safety.

Enzymatic Modification

Through genetic engineering, specific amino acid sequences are artificially induced to be expressed in antibodies. These sequences can be recognized by specific enzymes, and then specific amino acid residues are modified by enzymes to achieve site-specific binding. Currently, formylglycine generating enzyme (FGE) and transglutaminase (TG) are commonly used.

Glycosyl Conjugation

Glycosyl conjugation technology uses endoglycosidase to modify different sugars in natural proteins to expose N-acetylglucosamine. Then, N-acetylgalactosamine modified by azide chemistry is connected to the N-acetylglucosamine of the antibody using glycosyltransferase, and finally a click chemistry reaction is performed to obtain a site-specific conjugated ADC. The advantage of this technology is that the drug linker is coupled to the sugar chain without changing the amino acid sequence, and they are connected far away from the amino acid residue.

Genetically incorporated uAAs allow for unique orthogonal conjugation strategies compared to those used for the 20 natural amino acids. Therefore, uAAs offer a new paradigm for creating next-generation ADCs. Furthermore, uAA-based site-specific conjugation can also be used to create other multifunctional conjugates that are important for biopharmaceuticals, diagnostics, or reagents.

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