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Antibody–drug conjugates (ADCs) have attracted considerable attention in recent years. HER2 and TROP2 are already the focus of numerous development programs, while less extensively explored targets often offer opportunities for differentiation. CD44 variant isoforms (CD44v) represent one such area of interest.

Initially recognized as markers of cancer stem cells (CSCs), CD44v isoforms are now being explored as ADC targets. Development has faced challenges, however: clinical development of one CD44v6-targeted ADC was discontinued because of skin toxicity. This experience highlights the importance of both target selection and precise targeting.

At Reqbio, we have developed two overexpression cell models targeting CD44v6 and CD44v9. Rather than presenting a product specification sheet, this article discusses our perspective on this target and the research questions these two tools can help address.

I.What Is CD44v and Why Does It Matter

CD44 is a widely expressed transmembrane glycoprotein involved in cell adhesion and signal transduction. Its gene contains 20 exons, and alternative splicing generates numerous isoforms. The standard isoform, CD44s, is broadly distributed in normal tissues, whereas variant isoforms, collectively termed CD44v, acquire distinct functions through the inclusion of different variable exons, such as v3, v6, and v9.

CD44v expression is frequently dysregulated in malignant tumors. This change extends beyond structural variation: it contributes to malignant characteristics at multiple levels, including metabolism and phenotype, and is closely associated with the acquisition of cancer stem cell properties.

CD44v therefore serves as more than a cell surface marker; it also participates in tumor progression. This functional role makes it an attractive ADC target: targeting CD44v means targeting a molecule important to tumor survival, rather than simply a marker.

II. CD44v9 and xCT in Drug Resistance and ADC Targeting

By integrating signals from multiple growth factors, CD44v activates key pathways, including MAPK/ERK and PI3K/AKT, promoting proliferation, resistance to apoptosis, invasion, and metastasis. CD44v9 is of particular interest in this context.

CD44v9 helps tumor cells survive chemotherapy by regulating defenses against oxidative stress and upregulating antiapoptotic signaling. Specifically, CD44v9 binds to the cystine/glutamate antiporter (xCT), enhancing glutathione (GSH) synthesis and antioxidant defenses. This constitutes an important mechanism of tumor drug resistance.

The CD44v9–xCT interaction thus provides cancer cells with a defense against treatment. A CD44v9-targeted ADC uses this same protective component as a target, bypassing its protective function to kill cancer cells directly.

This rationale is particularly compelling for patients whose tumors have become resistant to existing therapies. It also explains why CD44v9 is considered an important direction for the next stage of ADC development.

III. Lessons from CD44v6 ADC Development and Skin Toxicity

Bivatuzumab mertansine, a CD44v6-targeted ADC comprising an anti-CD44v6 antibody and a DM1 payload, initially generated considerable interest. Its development was subsequently discontinued because of skin toxicity.

CD44v6 is also expressed in the stratum corneum at the surface of human skin. Consequently, the ADC attacked normal skin cells as well as tumor cells, exemplifying on-target, off-tumor toxicity.

This case highlighted a broader challenge: insufficient targeting specificity can make toxicity a major obstacle. Future approaches include selecting more specific CD44v isoforms, such as v9 or v5, or using bispecific ADCs to improve tumor selectivity.

When screening antibodies or designing ADCs, how can researchers ensure that a molecule recognizes CD44v9 on tumors without binding to CD44v6 in normal tissues? Addressing this question requires cell-based tools that can distinguish between isoforms.

Ⅳ. Reqbio hCD44v6/CHO and hCD44v9/CHO Cell Models

Reqbio has developed two CD44v overexpression cell models:

Both are stable overexpression cell lines displaying human CD44v6 and human CD44v9, respectively, on the cell surface.

Applications of These Cell Models

First, screening for antibody binding specificity. This is the most direct application. To determine whether a candidate antibody binds CD44v6 or CD44v9, researchers can evaluate the two cell models in parallel by flow cytometry. In view of the skin toxicity encountered during CD44v6 ADC development, this is an important step. Establishing early that an antibody does not bind v6 reduces risk in subsequent development.

Second, providing target cells for ADC internalization and cytotoxicity assays. Overexpression cells can serve as ADC target cells to evaluate specific binding, internalization, and subsequent killing of CD44v9-expressing cells. These two cell models provide a clean background for comparing the cytotoxic activity of different ADC candidates against v9-positive cells.

Third, supporting bispecific ADC development. Bispecific ADCs represent an important approach to improving safety. One arm may target CD44v9 while the other targets another tumor-associated antigen. These two cell models can be used to evaluate the binding activity of the CD44v9-targeting arm, while the CHO-K1 background does not express other interfering antigens, enabling cleaner data.

Fourth, serving as control cells. Wild-type CHO-K1 cells can be used as a negative control alongside the overexpression cells to rule out nonspecific binding. Such controls are important for both data publication and regulatory submissions.

One of the most compelling features of CD44v is its dual role as a cancer stem cell marker and a participant in drug resistance mechanisms. CD44v-targeted ADCs may therefore act against both the cells that sustain tumor growth and the mechanisms that protect them.

However, the diversity of CD44v isoforms and their expression in normal tissues make specificity critical to successful development. The failure of the CD44v6 ADC reflects insufficient precision in targeting rather than an inherently unsuitable target. Future progress will depend on molecules that can distinguish finely between isoforms, supported by tools capable of resolving those differences.

These two cell models were developed to support that objective. They cannot directly establish whether a molecule will become a successful drug, but they can help identify and eliminate insufficiently specific candidates early, potentially avoiding months of unproductive work later in development.

If you are working on a CD44v-related ADC program involving v6, v9, or another isoform, we welcome the opportunity to discuss your project, including the specifications of these two cell models and guidance on their use.