Antibody-drug conjugates (ADCs) have emerged as an innovative therapeutic approach in cancer treatment in recent years. The basic principle behind ADCs is to link antibodies with cytotoxic drugs through a linker, allowing the drug to be selectively delivered to tumor cells, thus enhancing therapeutic efficacy and reducing toxicity to healthy cells. In the design of ADCs, the linker plays a critical role, determining the drug's selectivity, stability, and efficacy. The valine-citrulline (Val-Cit) linker has become a key focus in ADC research due to its advantages in stability and selective degradation.

What is an ADC linker?

An ADC linker is a chemical bridge between the antibody and the cytotoxic drug (payload). The design intention is to keep the drug stable when away from the target cell, and to allow the drug to be released inside the target cell (most often tumour cells) by chemical synthesis. The linker's role is to ensure the specificity of drug delivery—maintaining the stability of the drug in the body and delivering it at the target site. Hence the linker has to take into account stability of the drug, a manageable release process, and high affinity between the antibody and the drug. ADC linkers can be separated into cleavable and non-cleavable linkers. The key distinction between the two is that the linker breaks or dissociates in a particular context inside the target cell, thus releasing the cytotoxic agent.

Fig. 1. Antibody-drug conjugates (Cancers (Basel). 2024, 16(2): 447).

Cleavable vs Non-Cleavable Linkers

• Cleavable Linkers

Cleavable linkers are those that will cleave in certain environments (like the tumor microenvironment, which can be acidic or enzymatic) to liberate the drug. Such linkers are normally made to be cleaved within the cell or tumour environment by enzymatic cleavage or other processes. The benefit of cleavable linkers is that they can trigger precisely controlled release of drug within the tumour cells, maximizing the effectiveness of the drug and minimizing damage to healthy cells. There are various typical cleavable linkers:

• Enzyme-sensitive linkers: Linkers that are cut by specific enzymes present in tumor cells, such as proteases, peptidases, or other intracellular enzymes.
• Acid-triggered linkers: These linkers break down under low pH conditions, such as the acidic microenvironment of tumor cells.

• Non-Cleavable Linkers

Non-cleavable linkers are stable during the treatment course and cannot be cut off until the antibody reaches the target cell and is cleaved through receptor-mediated endocytosis to release the drug. These linkers are generally chemically stable enough to keep the drugs from spilling out prematurely. Non-cleavable linkers may be attached to the antibody and drug through stable chemical ties like disulfide ties. The major benefit of non-cleavable linkers is that they are stable in the bloodstream, meaning that they do not release the drug early during transport, which makes them less toxic to normal tissue. However, these linkers require appropriate mechanisms inside the target cell (such as enzymatic cleavage) for drug release.

ADC Linker Synthesis

The synthesis of ADC linkers typically involves multiple steps of chemical reactions, including linking the antibody, drug, and linker. The most common synthesis method is through the chemical modification of specific amino acid residues in the antibody (such as cysteine or lysine) to react with functional groups in the linker, forming stable covalent bonds. The synthesis steps of the linker can be briefly summarized as follows:

• Linker Design: The structure of the linker is designed based on the therapeutic target and drug properties, ensuring that the linker can function effectively in the tumor microenvironment while ensuring stable and efficient binding of the drug to the antibody.
• Linker Synthesis: The linker is synthesized using chemical synthesis methods, typically through solid-phase synthesis, solution-phase synthesis, or enzyme-catalyzed synthesis.
• Linking Antibody and Drug: The linker is connected to the antibody and drug through appropriate chemical modification reactions (such as coupling reactions). These reactions are often achieved using crosslinking agents or specific chemical groups in the linker.

Val-Cit Linker

The Val-Cit linker is a dipeptide sequence formed by linking valine (Val) and citrulline (Cit) through a peptide bond. In this sequence, citrulline serves as an electronic analog of arginine, and its weaker basicity makes it more stable than arginine under environmental conditions. This structural design allows the Val-Cit linker to be selectively degraded in vivo by specific proteases, such as cathepsin B, to release the cytotoxin. Cathepsin B is an endogenous enzyme widely distributed in lysosomes that can recognize and cleave the citrulline-valine bond in the Val-Cit linker, thereby releasing its cytotoxic payload. This characteristic gives the Val-Cit linker strong targeting effects in ADCs, enabling selective release of toxic drugs within tumor cells while minimizing toxicity to normal tissues.

Val-Cit Linkers from BOC Sciences

Advantages of Val-Cit Linker

One of the notable advantages of the Val-Cit linker is its high stability and low non-specific degradation. Compared to other conventional linkers, the degradation of Val-Cit depends on the enzymatic cleavage by cathepsin B, an enzyme that is more abundantly expressed in tumor tissues and relatively sparse in normal tissues. As a result, the Val-Cit linker can precisely release cytotoxic drugs within tumor cells, enhancing the cytotoxic effects on tumor cells while minimizing the drug's toxicity to healthy cells. In addition, to overcome the impact of bulky payloads on protease cleavage activity, researchers have introduced the amino-benzyloxycarbonyl (PABC) spacer. This spacer effectively prevents large drug payloads from interfering with the degradation of the linker, expanding the applicability of the Val-Cit linker in various drug payloads and enhancing its versatility in different ADC formulations.

Val-Cit Linker in ADC

The valine-citrulline linker plays an important role in ADC development, especially in scenarios where rapid drug release is required. Its structure and properties make it an ideal choice for targeted drug therapy. Below are key applications of the Val-Cit linker in ADC development:

• Improving Drug Targeting and Efficacy: The Val-Cit linker allows ADCs to release the drug within tumor cells through a specific enzymatic cleavage system, thereby enhancing therapeutic efficacy. This linker ensures precise drug release in the tumor microenvironment without causing excessive toxicity to healthy tissues.
• Reducing Side Effects: Since the Val-Cit linker can be cleaved by enzymes within tumor cells, the drug remains stable in the blood, reducing toxicity to normal cells. This results in better tolerability and side-effect control for ADCs using the Val-Cit linker in clinical applications.
• Applicable to Various Cytotoxic Drugs: The Val-Cit linker can be combined with various cytotoxic drugs, including microtubule inhibitors and DNA-damaging agents, thus expanding its application scope in ADCs. This allows developers to design more targeted and diverse ADC products.
• Enhancing Drug Stability: The Val-Cit linker maintains chemical stability until the drug reaches the target cells, preventing premature release in the bloodstream and ensuring long-lasting, precise drug delivery.

Val-Cit Linker in Clinical Trials

The Val-Cit linker has shown significant potential in various clinical studies and drug developments. A typical example is the use of Monomethyl auristatin E (MMAE) as a cytotoxic drug conjugate. MMAE is a derivative of the marine toxin dolastatin 10, which inhibits microtubule polymerization and prevents cancer cell mitosis, exhibiting strong anti-proliferative effects. The C-terminal of MMAE is modified with a methylamino group (MMAE), allowing it to bind with the PABC spacer and form a degradable drug-linker combination with the Val-Cit linker. Many currently available ADC drugs, such as RemeGen's Aidixi (for HER2+ gastric cancer/UC treatment), Seagen's Padcev (for urothelial cancer treatment), and Tivdak (for cervical cancer treatment) co-developed by Seagen and Genmab, use the Val-Cit-MMAE combination as the linker-payload structure. These drugs utilize the Val-Cit linker to release cytotoxic drugs within tumor cells, achieving precise targeted therapy, showcasing the widespread application and significant advantages of the Val-Cit linker in ADC drugs.

• Aidixi

Aidixi is an ADC targeting the HER2 receptor, used for the treatment of HER2-positive gastric cancer and urothelial carcinoma (UC). The drug consists of an anti-HER2 monoclonal antibody conjugated to the cytotoxic drug MMAE through a Val-Cit linker. The anti-HER2 antibody part specifically recognizes and binds to HER2-positive tumor cells, while MMAE targets the microtubules to inhibit mitosis in cancer cells. The Val-Cit linker has a protease B-sensitive degradable property, selectively releasing the toxic drug in the tumor microenvironment and minimizing toxicity to normal cells. This ADC has shown high efficacy, particularly for patients with HER2-overexpressing gastric cancer and UC, significantly improving patient survival rates.

• Padcev

Padcev (Enfortumab vedotin) is an ADC used for the treatment of metastatic urothelial carcinoma (mUC). Its structure consists of a monoclonal antibody targeting the Nectin-4 receptor, conjugated to the cytotoxic drug MMAE via a PABC spacer. The anti-Nectin-4 antibody specifically binds to the Nectin-4 receptor, delivering MMAE directly into the cancer cells. MMAE inhibits microtubule polymerization, preventing mitosis in cancer cells and exerting an anti-cancer effect. Padcev has high targeting specificity and selectivity, effectively reducing toxicity to normal cells. It has been approved for the treatment of metastatic or locally recurrent urothelial carcinoma, providing a new treatment option.

• Tivdak

Tivdak (Tisotumab vedotin) is an ADC used for the treatment of advanced or metastatic cervical cancer. Tivdak is composed of an anti-Trop-2 monoclonal antibody conjugated to MMAE via a degradable Val-Cit linker. Trop-2 is a cell surface antigen widely expressed in various cancer cells, particularly in cervical cancer. The anti-Trop-2 antibody portion binds to Trop-2, directing MMAE into cancer cells, while MMAE inhibits microtubule polymerization and blocks mitosis in cancer cells. This ADC demonstrates strong anti-tumor effects, especially in cervical cancer patients, significantly improving treatment outcomes and survival rates.

• Adcetris

Adcetris (Brentuximab vedotin) is an ADC used for the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL). The drug consists of an anti-CD30 monoclonal antibody conjugated to the cytotoxic drug MMAE via a Val-Cit linker. The anti-CD30 antibody targets CD30-positive tumor cells, while MMAE disrupts microtubule polymerization and inhibits cancer cell proliferation. The Val-Cit linker has selective degradation properties, allowing MMAE to be released within tumor cells, minimizing toxicity to normal cells. Adcetris has been widely used for the treatment of relapsed or refractory HL and sALCL patients, significantly improving survival rates and quality of life.

• Polivy

Polivy (Polatuzumab vedotin) is an ADC used for the treatment of diffuse large B-cell lymphoma (DLBCL). Polivy is composed of an anti-CD79b monoclonal antibody conjugated to the cytotoxic drug MMAE via a degradable PABC linker. The anti-CD79b antibody targets the CD79b antigen on the surface of DLBCL cells, precisely delivering MMAE into the tumor cells. Inside the cells, MMAE inhibits microtubule polymerization, preventing mitosis and achieving an anti-tumor effect. Polivy has been approved for the treatment of relapsed or refractory DLBCL patients, significantly improving survival rates, and is a highly effective therapeutic option.

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