Targeting Human Epidermal Growth Factor Receptors with Drug Conjugates Based on Affibody Molecules

Abstract: Cancer is a major public health challenge and the second leading cause of death in the world, with millions of new cases being diagnosed each year. Traditional cancer treatments such as surgery, radiation therapy, and chemotherapy are many times effective, but may also cause damage to healthy cells, leading to side effects. Targeted therapy is a more precise and focused approach to cancer treatment, where the aim is to target the cancer cells while leaving the normal cells unaffected. It is particularly effective in cancers where specific molecular targets are known, such as the subset of breast cancer patients with HER2 over-expression or in the subset of patients with pancreatic cancer with HER3 over-expression. Antibody-drug conjugates (ADCs) are an important addition to tumor-targeted therapy, with twelve drugs approved for clinical use by the FDA. They utilize the high specificity of monoclonal antibodies conjugated with highly cytotoxic small molecules to enhance the accumulation of the drugs in the tumor, for highly specific and efficient killing. However, traditional ADCs may not be the optimal delivery format for the directed delivery of cytotoxic drugs. They are limited by their relatively large molecular weights, resulting in relatively low penetration of solid tumors. Recently, a novel type of drug conjugates, affibody-drug conjugates, has been described. These combined an engineered scaffold affinity protein, an affibody molecule, with an albumin binding domain (ABD) for half-life extension, to which a cytotoxic payload has been conjugated. Previous studies show that these novel drug conjugates have a potent and tumor-cell-specific cytotoxic effect. In the future, they may therefore become complementary or alternatives to current targeted cancer therapies. This thesis focuses on the optimization of affibody-drug conjugates targeting HER2 and HER3, members of the human epidermal growth factor receptor family. The thesis presents in vitro and in vivo preclinical data, showing the potential for further clinical development. In paper I, we investigated the influence of the drug-to-affibody ratio (DAR) on the pharmacokinetic profile of affibody-drug conjugates targeting HER2. Increasing the drug load resulted in an elevated delivery of the DM1 drug to the tumors; however, it also led to increased uptake by the liver. Further optimization of the molecular design is necessary to enable highly efficient delivery to tumors while minimizing the uptake in normal organs and tissues. In paper II, the effect of the length and composition of the linker between the HER2 targeting affibody molecule and the ABD was investigated. The use of a 12 amino acids linker reduced hepatic uptake compared with the use of a 5 amino acids linker. This finding offers an important insight into the influence of the linker on the properties of the affibody drug conjugates. In paper III, we investigated the influence of different cytotoxic payloads, as part of an affibody-drug conjugate targeting HER2, on binding properties, cytotoxicity, biodistribution, and anti-tumor effect. The combination of a potent cytotoxic effect in vitro, and a high tumor uptake in vivo, resulted in a superior anti-tumor effect for ZHER2- ABD-mcMMAF at lower doses compared to the previously investigated ZHER2-ABD- mcDM1. Importantly, it maintained a favorable toxicity profile with lower liver uptake compared to ZHER2-ABD-mcDM1. The affibody-drug conjugate ZHER2-ABD- mcMMAF holds great promise as a valuable agent for HER2-targeted cancer therapy. In paper IV, we generated a series of HER2-targeted affibody-drug conjugates fused with different PAS or XTEN polypeptides. We evaluated the ability of the XTEN and PAS polypeptides to extend the plasma half-life, and their influence on tumor uptake, and tissue biodistribution. We compared our new constructs with the previously developed construct, ZHER2-ABD-mcDM1, where an albumin binding domain was used for half-life extension. It was found that the ABD-fused affibody-drug conjugate demonstrated superior tumor uptake and tumor-to-normal-organ ratios compared to the PASylated and XTENylated affibody-drug conjugates. It is possible that ABD is better also for other cancer-targeting strategies where a high tumor uptake while maintaining comparable accumulation in normal tissues is desired. In paper V, we compared the binding properties and cytotoxic potential of a monovalent and a bivalent HER3-targeting affibody-drug conjugate. The biodistribution and therapeutic potential of the bivalent drug construct were evaluated. We found that the bivalent ZHER3-ABD-ZHER3- mcDM1 is a highly potent drug conjugate with favorable biodistribution and anti-tumor efficacy. These results suggest that ZHER3-ABD-ZHER3-mcDM1 holds promise for future clinical development as a potential therapeutic option for patients with HER3 over-expressing cancer. In summary, the potential for modification and optimization through the design of diverse components within HER2 and HER3-targeting affibody-drug conjugates significantly enhances therapeutic effectiveness, thereby encouraging prospective advancements in the development of targeted drug conjugates.