Since 2000, the research and development of precision therapy represented by small molecule targeted drugs and antibodies has made great progress and become the main targeted therapy for tumors. However, since small molecules and antibody drugs can specifically target tumor signaling pathways or antigens on the surface of tumor cells, and the heterogeneity between tumor cells is strong, precision therapy is very important for precision therapy. Antibody drug conjugates (ADCs) combine the dual advantages of small molecule and antibody drugs compared to antibody or small molecule drugs alone. Many ADCs have demonstrated impressive activity against treatment-refractory cancers, resulting in their approval for both hematologic malignancies and solid tumorindications.

What is Antibody Drug Conjugate?

ADCs are hybrid therapies that deliver cytotoxic payloads directly to the desired site of action to enhance efficacy and minimize off-target effects. It consists of three basic components, namely an antibody that recognizes the target antigen, a small molecule cytotoxic drug, and a chemical linker that connects the two. Each factor affects the ultimate efficacy and safety of the ADC. In general, ADC development needs to consider all of these key factors, including the selection of target antigen, antibody, cytotoxic payload, linker, and conjugation method. After ADC drugs enter the blood, they work through the following main steps: 1) The monoclonal antibody first binds to the target antigen highly expressed on the surface of tumor cells; 2) The tumor cells internalize the ADC-antigen conjugate into the cells; 3 ) The linker is digested by lysosomes in the cell, and the linker is cleaved, releasing small molecule cytotoxic drugs; 4) Cytotoxicity plays a role in destroying DNA or preventing tumor cells from dividing, killing cells; 5) The synergistic effect of monoclonal antibodies, because they bind the target antigen, will also play the role of targeted drugs, such as HER-2 antibodies.

Fig. 1. The modular components of an antibody-drug conjugate (Front Pharmacol. 2023, 14: 1274088).

FDA Approved Antibody Drug Conjugates (ADCs) by 2024

After decades of research and troubleshooting, significant technological advancements and an improved mechanistic understanding of ADC activity culminated in the FDA approval of 15 ADCs, each providing demonstrable therapeutic benefit to cancer patients. Of these, belantamab mafodotin-blmf (Blenrep) was withdrawn from the FDA on November 22, 2022.

Table 1. FDA approved ADCs.

ADC Formulation Analysis

A typical antibody formulation contains ADC, excipients (sugars or salts), buffers, surfactants (Tween) and other metal ion chelators. Table 2 lists the formulations of ADCs that have been marketed, which show that even with the same linker-payload, the final formulation buffer used by different antibodies will be different.

Table 2. ADCs formulation analysis.

Screening Process for ADC Preparations

• pH and Buffer Screening

Among the 14 ADCs on the market, most ADCs have pH values between 6.0 and 6.9. Among them, histidine salt was the most important buffer type. Among the 14, 5 were histidine, 3 phosphate, 2 citrate, 2 succinate, 1 tromethamine and 1 MES. Generally, the selection of ADC buffer needs to follow the following principles: according to the buffer range of different buffer pairs, the pH gradient was set from 4.0-8.0 to prepare the ADC concentration for clinical use. Then, they were placed at 25°C and 40°C for 7 days, observed and sampled on day 0, day 1, day 3 and day 7, respectively, including observation of clarity, BCA detection concentration, SEC-HPLC detection of purity, DSC detection of thermal stability, etc.

When the suitable pH range is determined, two buffers at the pH can be selected. For example, when the suitable pH is 6.0, 30 mM histidine or 20 mM citric acid can be selected as the buffering agent of the preparation, and then the next step is screened.

Table 3. ADCs pH and buffer analysis.

• Surfactant Screening

Surfactants reduce aggregate production primarily by reducing surface tension. Of the 14 ADCs, 7 used 0.01-0.02% (W/V) Tween 80, 3 used 0.01-0.02% (W/V) Tween 20, and 1 used 0.03% Tween 80, 1 used 0.1% Tween 20, and the other 2 did not add surfactant. Generally, there are two screening principles for surfactants.

• Common processing method: stir the sample horizontally at 250-300 rpm on an orbital shaker at 25°C for 8 hours or longer. After stirring, some samples will aggregate and the solution will become cloudy.
• Reference detection methods: visual observation of solution clarity, concentration determination, SEC-HPLC detection of aggregate content, turbidity meter detection of turbidity, HIAC automatic liquid particle counter detection of particle number.

• Excipient Screening

Among the 14 ADC formulations, various sugars or salts were added as excipients mainly. When setting different sugars and salts, there are many combinations and types. 10% trehalose, 9% sucrose, 5% sorbitol, etc. can be used as the basic formula for preliminary screening. When it is necessary to combine, the content of the two is halved. After 14 days of storage at 4°C, 25°C, and 40°C, characterizations were performed for transparency observation, BCA concentration detection, SEC-HPLC purity detection, DSC thermal stability detection, CIEF pH detection, etc.

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