ADC Drug R&D Solutions: Key Elements, Preparation, and Core Products

1. Introduction to Antibody-Drug Conjugates (ADC)

Antibody-Drug Conjugate (ADC) is a novel and highly effective biological drug that connects antibodies with biologically active small-molecule cytotoxic payloads through linkers (Figure 1).

The three core components of ADC drugs include:

1.       Specific targeting antibody;

2.       Cytotoxic payload (toxin);

3.       Cleavable or non-cleavable linker.

Specific target recognition and effective toxin action make ADC a veritable "biological missile". The development of ADC drugs has high technical barriers, requiring comprehensive consideration of multiple factors such as the rational selection of targets; the rational combination of antibodies, linkers, small-molecule drugs and their interactions; antibody conjugation technology, and the drug resistance of ADC drugs. Among them, target selection, linker selection, conjugation technology, and preclinical efficacy evaluation are the focus of attention in the ADC R&D process.

2. Preparation of ADC

The preparation of ADC starts with the selection of target antigens. The safety and efficacy of ADC largely depend on the selection of target antigens and their interactions. Then, highly specific antibodies are first developed against the target antigens, and then cytotoxic payloads are connected to the antibodies through lysine conjugation, cysteine conjugation, site-specific conjugation and other methods using specific cleavable or non-cleavable linker fragments.

There are two common approaches for ADC preparation: one-step method and two-step method. The one-step method involves first connecting the linker and the cytotoxic payload, then performing antibody conjugation. The two-step method involves first conjugating the linker to the antibody, then connecting the cytotoxic payload. One of the biggest challenges in ADC development is selecting an appropriate linker and conjugation mode for the antibody and cytotoxic payload. Because in addition to effectively delivering the cytotoxic payload, the stability of the antibody-payload connection system is a key factor determining the efficacy and toxicity of ADC. It is necessary to ensure that it will not break in advance in the blood circulation before reaching tumor cells, leading to off-target effects, and that the toxicity can efficiently release the cytotoxic payload after reaching tumor cells. This is a key factor determining the therapeutic potential of ADC. The cytotoxic payload is the most important core component of ADC, which is the active ingredient that ultimately executes the killing of tumor cells (direct killing or bystander effect). A highly toxic payload is the key to enabling ADC to exert the expected efficacy.

3. Key Elements of ADC

ADC is composed of antibodies, linkers, and cytotoxic small-molecule drugs. It achieves precise killing of tumor cells by high-activity cytotoxic drugs through the binding of antibodies to specific antigens on the surface of tumor cells. In addition, the selection of target antigens and conjugation methods are also extremely important for whether the designed ADC has safe and effective clinical therapeutic effects.

3.1 Starting Point of ADC Design: Antigen Target

The selection of antigen targets needs to consider the following aspects:

1.       The target antigen is highly expressed in tumors and not expressed or lowly expressed in normal cells;

2.       Whether the target antigen is distributed on the cell membrane surface of tumor cells, so that it can be recognized by specific antibodies;

3.       Whether the antigen is not easy to shed to prevent antibodies from binding to antigens in the circulation;

4.       Whether the target antigen has internalization characteristics, which will help carry the toxin molecules conjugated by ADC into tumor cells to play a role, and the target antigen should not be down-regulated after ADC treatment.

At present, more than 50 antigens have been used as target antigens recognized by ADC. Antigens overexpressed in solid tumor cells include HER2, EGFR, CD56, Trop2, CD70, Tissue factor, Collagen IV, etc. Currently, the most commonly used target antigens for ADC include Her2, CD19, CD33, CD22, MSLN (mesothelin), etc.

3.2 Antibodies in ADC

Antibodies are another important component in ADC design. They should be able to specifically recognize the target antigen of tumor cells and have high affinity with the target antigen. The lack of high specificity or cross-reaction with other antigens will lead to unpredictable side effects. At the same time, antibodies should also have the characteristics of weak immunogenicity, long half-life, and good stability in blood circulation. According to the sequence of their heavy chain constant regions, antibodies can be divided into five categories: IgG, IgA, IgD, IgE, and IgM. All currently approved ADCs adopt IgG antibodies. Among them, IgG1 is the most widely used in ADC antibody R&D due to its moderate molecular weight, high affinity, long half-life, simple preparation, and strong Fc effector function.

ADCs generally use human-mouse chimeric antibodies (mouse-derived light and heavy chain variable regions and human-derived constant regions, such as Adcetris) and humanized antibodies (mouse-derived CDR fragments, other sequences are human-derived, such as Mylotarg and Kadcyla). This reduces immunogenicity to a certain extent and reduces the production of human anti-mouse antibodies. However, early ADCs still had problems such as a certain degree of off-target toxicity, heterogeneous products, easy aggregation or rapid clearance, and a narrow therapeutic window.

The specificity, affinity, and internalization rate of antibodies are also factors that need to be considered. High specificity helps to concentrate toxin small molecules to tumor sites, thereby achieving targeted pharmacological effects. ADCs with low specificity are more likely to be toxic to normal tissues. ADC antibodies should have high binding affinity, and the binding affinity of most ADCs is between 0.1-1.0nM. Compared with small molecules, the speed of antibodies entering tissues from plasma is slower, and faster internalization efficiency can improve the efficacy of ADC.

The antibody part of ADC can adopt forms such as bispecific antibodies or single-domain antibodies. Bispecific antibodies can target two different epitopes of the same target antigen, or two different target antigens. Single-domain antibodies have also been used to conjugate killer radioactive elements or toxic molecules to develop new ADCs.

3.3 Linkers in ADC

Linkers connect antibodies with payloads. The stability of linkers in the blood is very important. They need to remain stable in the blood to keep the cytotoxic payload attached to the antibody. However, once the ADC enters the tumor cell or is transported to the lysosome, the linker should decompose rapidly to release the payload. Linkers affect many important properties of ADC, such as drug-antibody ratio (DAR), payload release time, therapeutic index (Therapeutic Index, TI), and pharmacokinetics.

According to the cleavage characteristics of linkers in tumor cells, linkers can be divided into cleavable and non-cleavable types. Cleavable linkers can be cleaved through various mechanisms, including acid-labile cleavage of hydrazone bonds, reductive cleavage of disulfide bonds, and enzymatic hydrolysis of peptide bonds. For example, acid-labile linkages are usually stable in the blood but are rapidly cleaved in the low-pH lysosomal environment (such as Besponsa and Mylotarg), releasing small-molecule toxins to exert cell-killing effects. Disulfide bond types can release cytotoxic payloads to kill tumor cells through intracellular glutathione (GSH) reduction reactions, and their steric hindrance can limit the immature cleavage of ADC before entering cells. If the released cytotoxic payload can cross the tumor cell membrane, they can kill nearby cancer cells, which is the so-called "bystander effect". However, cleavable linkers do not necessarily produce a bystander effect, which mainly depends on the membrane permeability and charge characteristics of the released payload. It should be noted that the bystander effect also has some defects, such as killing normal cells or immune cells near the target tumor cells. Another defect of cleavable linkers is that there may be a certain degree of metabolic degradation during in vivo circulation, leading to off-target toxicity.

Non-cleavable linkers are composed of structures that are stable in the blood. ADCs containing such linkers can only release the payload after being degraded by proteases in the lysosomes of tumor cells. Stable linkers greatly reduce off-target toxicity caused by extracellular release, but have low release efficiency and require a good internalization process. After non-cleavable linkers are endocytosed into lysosomes, the linkers will not be degraded, and the connected antibodies will be degraded into amino acids, forming amino acid-linker-small molecule cytotoxic complexes. Due to the charge of the "linker-amino acid residue", its membrane penetration and diffusion are limited, so it usually does not produce a bystander effect.

3.4 Cytotoxic Payloads in ADC

The cytotoxic payload drugs carried by ADC are its most important effector components, also known as payloads. Currently commonly used cytotoxic molecules include microtubule formation inhibitors, DNA-damaging agents, and DNA transcription inhibitors. Microtubule formation inhibitors prevent microtubule polymerization by binding to microtubules, thereby blocking the cell cycle, producing cytotoxicity, and exerting anti-tumor effects. Examples include Monomethyl auristatin E (abs810174), Maytansine (abs817384), and their analogs. DNA-damaging agents bind to the minor groove of DNA and promote DNA strand alkylation, breakage, or cross-linking, such as Calicheamicin (abs819016). DNA transcription inhibitors include Amatoxin and Quinoline Alkaloid (SN-38), such as CL2A-SN-38 (abs819783), a drug-linker conjugate connected by linker CL2A and toxic molecule SN-38, which can be used to prepare ADC.

3.5 Conjugation Methods of ADC

Conjugation methods are mainly divided into non-site-specific conjugation and site-specific conjugation. The non-site-specific conjugation method was used in the early stage, mainly through lysine or cysteine conjugation. The site-specific conjugation method is to perform specific conjugation through genetic engineering sites to achieve more homogeneous ADCs, enabling the connection of cytotoxins at specific sites.

ANT BIO PTE. LTD.'s ADC Toxin-Antibody Site-Specific Conjugation Kit (abs580253) is easy to operate, does not require complex operations such as antibody engineering, and can quickly achieve site-specific conjugation of monoclonal antibodies. The conjugated product is homogeneous and stable. It is suitable for site-specific conjugation in the conjugation stage and early ADC conjugation research.

Conjugation Principle:

1)    Antibody Azidation Modification

First, glycosidase (EndoS) is used to expose the N-acetylglucosamine (blue square) on the conserved N-glycan of the monoclonal antibody constant region, and then the bovine galactosyltransferase mutant (b.GalTY298L) is used to connect N-acetylgalactosamine with an azide functional group (yellow square-N3) to N-acetylglucosamine.

2) Toxin Molecule Connection

    Subsequently, copper-free catalyzed alkyne-azide cycloaddition reaction (such as SPAAC) can be used to connect biotin, fluorescein, or toxin molecules to the monoclonal antibody.

ANT BIO PTE. LTD. can provide researchers with reagents such as ADC cytotoxins, ADC linkers, ADC linkers with payloads, ADC site-specific conjugation kits, ADC popular target proteins, ADC positive reference antibodies, ADC overexpressed drug target cell lines, as well as antibody customization and mIHC services. It can realize from antibody preparation, screening, conjugation to later mIHC pathological detection, helping to accelerate ADC drug R&D.

    (Absin Antibody and Protein Customization, Free Analysis Plan Provided! Service Advantages: Mature antibody preparation technology (supported by high-scoring literatures), free analysis plan provided. Ultra-low price, step-by-step payment to avoid the risk of high investment costs. Polyclonal Antibodies, Mouse Monoclonal Antibodies, Rabbit Monoclonal Antibodies. Antibody Preparation: For monoclonal antibodies, high-titer animals are selected for cell fusion, subclonal screening to establish stable monoclonal positive cell lines, and ascites preparation; for polyclonal antibodies, multiple hosts such as rabbits, mice, and rats are available. Purification: ProteinA/G is used to purify antibodies/proteins; Limulus amebocyte lysate method is used to determine endotoxin. Labeling: Antibody/protein labeling, multiple dyes available (Alexa Fluo series, Cy series, biotin labeling, HRP labeling, etc.). Protein Expression, Purification Services, Labeling Services)

 (Absin Panoramic Multi-Target Staining and Imaging Analysis Platform. Sample Coverage: Paraffin sections, frozen sections, tissue microarrays (TMA), organoids, etc. Provide multi-positive cell counting, colocalization analysis, average fluorescence intensity analysis, optional spatial distance analysis, infiltration analysis, etc. Standardized Services: For existing Absin indicators, only samples need to be provided, and results can be obtained in 1-2 weeks. Customized Services: IHC + TSA single staining + pre-multiple staining + formal experiment, multi-step verification is more reliable. Automated Staining Machine: Compared with traditional manual staining, the operation time is shorter, the effect is more stable and uniform; the proprietary Covertile technology can better maintain the morphology and integrity of tissues. Multi-Platform Scanner: Meet different imaging needs, TG in situ multi-label single-cell spatial quantitative, qualitative, and positional in-depth analysis platform, which can achieve up to nine labels and ten colors. HALO Digital Pathology Image Analysis Platform: High-throughput, full-section tissue analysis image analysis, based on morphology and multiple data expressions, analyze full-section images cell by cell, with accurate and detailed results. Equipment: 3Dhistech, Leica Bond RX, TissueFAXS Spectra, HALO Digital Pathology Image Analysis Platform)

References

[1] Fu Z, Li S, Han S, Shi C, Zhang Y. Antibody drug conjugate: the "biological missile" for targeted cancer therapy. Signal Transduct Target Ther[J]. 2022 Mar 22;7(1):93.

[2] Wu G, Fu Z H, Xu G L, et al. Research Progress in Antibody-Drug Conjugate R&D[J]. Biomedical Translation, 2021, 2(4):11.

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