What is an Affibody?
Affibody Molecules are small proteins that can be engineered to bind with high affinity to a target protein, e.g., HER2 or TNF-alpha. Affibody Molecules work much like antibodies, yet there are striking differences between the two. An Affibody Molecule has a molecular weight of 6 kDa as compared with an antibody, which weighs 150 kDa. Despite its small size, the binding site of an Affibody Molecule is equivalent in surface area to that of an antibody. Their small size, simple structure, specific target recognition, ease of production, and high stability give Affibody Molecules valuable advantages over antibodies.

Affibody Molecules are composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. The protein domain consists of 58 amino acids, 13 of which are randomized to generate Affibody libraries with a large number of ligand variants (e.g., 8 x 10¹⁶ Affibody members). All library members have an identical backbone but variable surface-binding properties.

Affibody Molecules for Molecular Imaging
For molecular imaging applications, small size is the key: It allows for rapid kinetics and high-contrast imaging within hours after injection. This is especially pertinent for cancer patients. An Affibody Molecule coupled with an imaging agent permits patients to be injected in the morning and have their tumor imaged that afternoon. Affibody Molecules with specificity and selectivity for tumor markers such as HER2 can be used for early detection of tumors. Later, they can monitor disease by detecting tumor progression or regression in response to therapy. The Affibody's small size also confers efficient access into solid tumors and a short half-life.

As described in a recent publication (Orlova et al. 2006), Affibody constructed a library for affinity maturation. One selected Affibody Molecule, Z_HER2:342, showed a marked increase in affinity (2,000-fold), after a single-library affinity maturation step. When radioiodinated, Z_HER2:342 allowed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity), with 9% of the injected dose per gram of tissue in the tumor.

Can an Affibody Molecule Serve as a Targeted Therapeutic?
Affibody Molecules are also a highly suitable carrier for directing radioisotopes and other toxins to tumor cells. Affibody Molecules can be produced as fusion proteins and site-specifically modified to conjugate with various toxic molecules. According to Nilsson, the production cost is significantly less than for a conjugated monoclonal antibody.

During his presentation, Nilsson also described an Affibody program targeting breast cancer. The company has already demonstrated the concept in animal models using an Affibody Molecule carrying a radioactive load to the tumor. The putative drug targets a well-defined patient population using a validated cancer marker, such as HER2. Z_HER2:342 and Herceptin (a HER2-targeted monoclonal antibody) have completely different modes of action and therefore do not compete with one another.

However, the company saw off-the-charts uptake of the conjugated Affibody Molecule by the kidneys—indeed, surpassing uptake by the tumor itself. As Nilsson noted, "Such uptake by the kidneys is acceptable for an imaging event, but not desirable for sustained therapeutic treatment."

What's the solution? Albumin binding can be used to tailor blood kinetics. Affibody coupled the beta-emitter ¹⁷⁷Lutetium to an Affibody construct consisting of two domains of Z_HER2:342 and one albumin-binding domain (ABD). ¹⁷⁷Lutetium yields a short range of radiation that can be detected with gamma camera. The ABD fusion with the Affibody prolonged the serum half-life from minutes to days and drastically reduced kidney uptake (while increasing dose to the tumor by 5-fold), thereby optimizing for ¹⁷⁷Lutetium therapy and simultaneously sparing the kidneys.

Nilsson concluded that the HER2-specific Affibody monomer has very favorable properties for imaging. And further, the HER2-specific Affibody-ABD fusion can be used for efficient radiotherapy with ¹⁷⁷Lutetium.

Reference: Orlova A, Magnusson M, Eriksson TLJ, et al. Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Research. 2006;66:4339–4348

© Copyright 2006, Cambridge Healthtech Institute. All Rights Reserved.