Antibody-drug conjugates or ADCs, which link an antibody to a potent, small-molecule, cytotoxic, cell-killing, chemotherapeutic agent, use the target-specificity of monoclonal antibodies or antibody fragments, to adhere exclusively to specific membrane receptors that are characteristic of tumor cells. Following internalization, the potent, anti-cancer drugs, are releases and kill the malignant cell.

This approach creates an excellent control mechanism of drug activation, resulting in an increase of the therapeutic window and, thereby, increasing the use attached drug. However, the therapeutic window of antibody-drug conjugates is still quite narrow, making research in developing a more safe and efficacious ADC technology with a wider therapeutic window a viable field of study.

Current technology
Antibody-drug Conjugates are currently used for the treatment of lymphoma and metastatic breast cancer. Today, four Antibody-drug Conjugates have been approved by the U.S. Food and Drug Administration (FDA). These agents include brentuximab vedotin (Adcetris®; Seattle Genetics) for Hodgkin and anaplastic large cell lymphoma, ado-trastuzumab emtansine (Kadcyla®; Genentech/Roche) for HER2-positive metastatic breast cancer, gemtuzumab ozogamicin (Mylotarg®; Pfizer) for acute myeloid leukemia and inotuzumab ozogamicin (Besponsa®; Pfizer) for the treatment of acute lymphoblastic leukemia.

Unique properties
The unique properties of an antibody-drug conjugate are determined by the balance of its components. One key part is the cytotoxic payload. The efficacy of of the payload are determined by the drug-to-antibody ratio (DAR) – the number of attached drug molecules to the antibody.

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Homogeneous DAR = 8 antibody-drug conjugate can be easily prepared by conjugation to the four accessible antibody hinge cystines. And antibody-drug conjugates with higher drug-to-antibody ratios have greater in vitro potency than the current clinically approved ADCs with a DAR of 2 – 4. Furthermore,  the higher DAR antibody-drug conjugates are especially effective against cells with low copy numbers of the target antigen. [1]

However, many clinical programs with higher a DAR have suffered from unwanted toxicity and insufficient efficacy against tumors. In turn, the use of hydrophobic payloads has effectively permitted only DAR = 2–4. This is, in part, due to poor pharmacokinetics and aggregation and systemic exposure. For example, DAR = 8 monomethyl auristatin E (MMAE) antibody-drug conjugates have been shown to be inferior to both ADCs with a drug loading of DAR of 2 and DAR of 4 in vivo. [1][2]

Researchers have shown that by reducing hydrophobicity of homogeneous antibody-drug conjugates it is possible to improve the pharmacokinetics and therapeutic index. These researchers demonstrated that by masking the hydrophobic payloads by hydrophilic linker moieties,antibody-drug conjugates with a drug loading of DAR = 8 and improved in vivo biodistribution and efficacy can be achieved [3].

A novel payload
Tero Satomaa and colleagues at  Glykos Finland, in Helsinki, Finland and OcellO, in Leiden, The Netherlands, generated a novel homogeneous antibody-drug conjugate (MC-Val-Cit-PABC-MMAU) with a DAR = 8.  Their antibody-drug conjugate includes a novel monomethyl auristatin β-D-glucuronide (MMAU).[4]

Their glycoside payload contributed to overall hydrophilicity of the antibody-drug conjugates, reducing aggregation. Furthermore, compared to standard drug loading of DAR 2 and 4, the cytotoxicity of the homogeneous ADC with a DAR of 8 was improved to low-picomolar IC50 values against cancer cells in vitro.

Although unconjugated MMAU was relatively non-toxic to cells, the bystander efficacy was restored after internalization and subsequent cleavage of the glycoside. The researchers concluded that their novel monomethyl auristatin β-D-glucuronide (MMAU) antibody-drug conjugate with a DAR of 8 was effective against target antigen-expressing xenograft tumors. Furthermore, studied in 3D in vitro patient-derived xenograft (PDX) assays, these antibody-drug conjugates outperformed clinically used ADC.

Overall, the researchers found that MMAU is a promising novel payload and can overcome many challenges of ADC technology. They demonstrated that the increased hydrophilicity of the payload contributed to the hydrophilicity and stability of antibody-drug conjugates as well as the safety to non-target cells, while significantly improving cytotoxicity to malignant cells and enabling bystander efficacy.

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