Alphabody

Alphabodies, also known as Cell-Penetrating Alphabodies or CPAB for short, are small 10 kDa

Development

Alphabodies are developed as scaffolds with a set of

The three α-helices (A, B, and C) were designed to remain stable even when some residues are modified. Residues in the groove between helices A and C can be modified to bind convex targets, while residues on the outside of helix C can be modified to bind concave protein targets. There are currently 3 libraries containing 1.0 to 1.7 × 10⁸ variations each that can be screened using phage display for target affinity.

Structure

Standard

The standard Alphabody scaffold contains three α-helices, composed of four heptad repeats (stretches of 7 residues) each, connected via glycine/serine-rich linkers. The standard heptad sequence is "IAAIQKQ". Alanines are associated with α-helix formation, while isoleucines are known to induce coiled-coil formation.^[5] Specific residues on the A and C helices can be modified to bind targets, but only variants that retain thermostability are used for further research.

Specifically, the reference scaffold structure is N–HRS1–L1–HRS2–L2–HRS3–C.

HRS = IEEIQKQIAAIQKQIAAIQKQIYRM; L = TGGSGGGSGGGSGGGSGMS

The linker length is long enough to allow helices to fold in parallel or anti-parallel conformations, but experiments suggest only anti-parallel folding occurs.^[1]

Truncated version

An Alphabody scaffold variant with shorter linkers can be produced without the loss of thermostability. However, decreasing the number of heptad repeats per α-helix reduces the thermostability of the Alphabody by around 40 °C.

Properties

Alphabodies have low molecular weight (~10 kDa) and very high thermostability (T_m = ~120 °C). Moreover, circular dichroism experiments suggest that Alphabodies can refold correctly after being denatured. These properties allow Alphabody-based drugs to be administered in ways other than injection.^[6] They also make the molecule stable enough to allow modification of residues on the scaffold itself – rather than only loop regions – increasing the possible variations and target selectivity.^[1]

Alphabodies' high binding affinity and ability to target both extracellular and intracellular proteins allows them to be used to reach difficult targets that cannot be treated by therapeutic antibodies or small molecule drugs.

Targets

Autoimmune disease

Alphabody CMPX-1023 has been successfully developed to target the p19 subunit of

Complix N.V. used phage display to create Alphabodies that could bind IL-23, and then employed several affinity maturation strategies to increase affinity to sub-nanomolar levels. They determined increased affinity resulted in increased functional inhibition of IL-23 and thus selected top 20 strongest binding Alphabodies as drug candidates. Mouse studies and X-ray crystallography studies on IL-23 in complex with the Alphabody confirmed specific binding to p19 only.

Cancer

Using a similar drug development strategy, Complix N.V. is developing Alphabodies capable of binding intracellular targets in cancer cells that can induce apoptosis. According to a 2012 article, Complix has had a degree of success in doing so:

"These results show that Alphabodies can be designed to efficiently enter human cells and bind to targets of interest, allowing them to modulate intracellular protein-to-protein interactions and induce apoptosis in cancer cells. Complix expects to report further break-through data from this important program over the course of 2012."^[9]

Funding

The research on IL-23-specific Alphabodies was supported by grants from IWT-O&O, Ghent University, and the Hercules foundation (Belgium).

Complix N.V. is funded by

• Affibody
• Affimer
• Affitin
• Anticalin
• DARPin
• Monobody

References