# Alphabody

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**Alphabodies**, also known as **Cell-Penetrating Alphabodies** or **CPAB** for short, are small 10 kDa [proteins](/source/Proteins) engineered to bind to a variety of [antigens](/source/Antigens). Despite their name, they are not structurally similar to [antibodies](/source/Antibodies), which makes them a type of [antibody mimetic](/source/Antibody_mimetic). Alphabodies are different from many other antibody mimetics in their ability to reach and bind to [intracellular](/source/Intracellular) protein targets.[1] Their single chain alpha-helical structure is designed by computer modelling, inspired by naturally existing [coiled-coil](/source/Coiled-coil) protein structures. Alphabodies are being developed by the [Belgian](/source/Belgium) [biotechnology](/source/Biotechnology) company Complix N.V. as potential new [pharmaceutical drugs](/source/Pharmaceutical_drug) against [cancer](/source/Cancer) and [autoimmune disease](/source/Autoimmune_disease).[2] In 2012, a [collaboration](/source/Collaboration) agreement was signed with [Monsanto](/source/Monsanto) to develop the technology for [agricultural](/source/Agriculture) applications as well.[3]

## Development

Alphabodies are developed as scaffolds with a set of [amino acid residues](/source/Amino_acid_residues) that can be modified to bind protein targets, while maintaining correct folding and [thermostability](/source/Thermostability).

The Alphabody scaffold is computationally designed based on coiled-coil structures, but it has no known counterpart in nature. Initially, the scaffold was made of three [peptides](/source/Peptide) that associated [non-covalently](/source/Non-covalent_interactions) to form a parallel coiled-coil [trimer](/source/Protein_trimer).[4] However, the scaffold was later redesigned as a single peptide chain containing three [α-helices](/source/Alpha-helix) connected by linker regions. The new structure allows for [concentration](/source/Concentration)-independent assembly and [cost-effective](/source/Cost-effectiveness_analysis) scaling in bacterial [expression systems](/source/Gene_expression#Expression_system).[1]

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](/source/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](/source/Glycine)/[serine](/source/Serine)-rich linkers. The standard heptad sequence is "IAAIQKQ". [Alanines](/source/Alanine) are associated with α-helix formation, while [isoleucines](/source/Isoleucine) 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](/source/Protein_structure#Tertiary_structure), 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 (Tm = ~120 °C). Moreover, [circular dichroism](/source/Circular_dichroism) experiments suggest that Alphabodies can refold correctly after being [denatured](/source/Denaturation_(biochemistry)). 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](/source/Binding_selectivity).[1]

Alphabodies' high [binding affinity](/source/Ligand_(biochemistry)#Receptor.2Fligand_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](/source/Small_molecule) drugs.

## Targets

### Autoimmune disease

Further information: [Interleukin 23](/source/Interleukin_23)

Alphabody *CMPX-1023* has been successfully developed to target the p19 subunit of [Interleukin 23](/source/Interleukin_23) (IL-23) and has entered preclinical trials as of October 2011.[6][7] In brief, IL-23 is a [pro-inflammatory](/source/Inflammation) [cytokine](/source/Cytokine) that has been implicated in autoimmune inflammatory diseases like [psoriasis](/source/Psoriasis), [rheumatoid arthritis](/source/Rheumatoid_arthritis) and [Crohn's disease](/source/Crohn's_disease).[8] There are anti-IL-23 drugs available, which work by targeting the [p40](/source/Interleukin_12_subunit_beta) subunit. However, the p40 subunit is also present in [Interleukin 12](/source/Interleukin_12) (IL-12) and causes serious [side-effects](/source/Side-effects) when [antagonized](/source/Receptor_antagonist), such as increased susceptibility to infection.

Complix N.V. used phage display to create Alphabodies that could bind IL-23, and then employed several [affinity maturation](/source/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](/source/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](/source/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](/source/Ghent_University), and the Hercules foundation (Belgium).

Complix N.V. is funded by [equity](/source/Private_equity) [shareholders](/source/Shareholders) Baekeland Fund, [Biotech Fund Flanders](/source/Biotech_Fund_Flanders), CRP-Santé, Edmond de Rothschild Investment Partners, Gemma Frisius Fund, [Gimv](/source/Gimv), LRM, OMNES Capital, TrustCapital, Vesalius Biocapital, and Vinnof.[10]

## See also

- [Affibody](/source/Affibody_molecule)
- [Affimer](/source/Affimer)
- [Affitin](/source/Affitin)
- [Anticalin](/source/Anticalin)
- [DARPin](/source/DARPin)
- [Monobody](/source/Monobody)

## References

1. Desmet, J.; Verstraete, K.; Bloch, Y.; Lorent, E.; Wen, Y.; Devreese, B.; Vandenbroucke, K.; Loverix, S.; Hettmann, T.; Deroo, S.; Somers, K.; Henderikx, P.; Lasters, I.; Savvides, S. N. (5 Feb 2014). "Structural basis of IL-23 antagonism by an Alphabody protein scaffold". *Nature Communications*. **5**: 5237. [doi:10.1038/ncomms6237](https://doi.org/10.1038/ncomms6237). [PMC 4220489](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4220489). [PMID 25354530](https://pubmed.ncbi.nlm.nih.gov/25354530)

1. ["Complix - About Complix"](https://web.archive.org/web/20150214192533/http://www.complix.com/about-complix/). *Complix*. Archived from [the original](http://www.complix.com/about-complix/) on 14 February 2015. Retrieved 9 March 2015.

1. Powers, K. ["Monsanto Company and Complix Nv Sign Collaboration to Bring New Technologies to Agriculture"](https://web.archive.org/web/20150402102601/http://news.monsanto.com/press-release/monsanto-company-and-complix-nv-sign-collaboration-bring-new-technologies-agriculture). *Monsanto*. Archived from [the original](http://news.monsanto.com/press-release/monsanto-company-and-complix-nv-sign-collaboration-bring-new-technologies-agriculture) on 2 April 2015. Retrieved 26 March 2015.

1. Desmet, J. & Lasters, I. (2010). ["Non-natural proteinaceous scaffold made of three non-covalently associated peptides"](https://www.google.com.ar/patents/US20100305304). *US Patents*. '***(US20100305304 A1)***

1. Suzuki, K.; Hiroaki, H.; Kohda, D.; Tanaka, T. (1998). "An isoleucine zipper peptide forms a native-like triple stranded coiled coil in solution.". *Protein Engineering*. **11** (11): 1051–1055. [doi:10.1093/protein/11.11.1051](https://doi.org/10.1093/protein/11.11.1051)

1. ["Complix selects first Alphabody™ development candidate"](http://flandersbio.be/news/complix-selects-first-alphabody-development-candidate/). FlandersBio. 25 October 2011. Retrieved 26 March 2015.

1. ["CMPX-1023"](https://www.biocentury.com/products/cmpx-1023). *BioCentury BCIQ*. Retrieved 26 March 2015.

1. Benson, J.M.; Sachs, C.; Treacy, G.; Zhou, H.; Pendley, C.E.; Brodmerkel, C.M.; Shankar, G.; Mascelli, M.A. (2011). "Therapeutic targeting of the IL-12/23 pathways: generation and characterization of ustekinumab". *Nat. Biotechnol.*. **29** (7): 615–624. [doi:10.1038/nbt.1903](https://doi.org/10.1038/nbt.1903). [PMID 21747388](https://pubmed.ncbi.nlm.nih.gov/21747388)

1. ["Complix' Alphabody™ Protein Therapeutics Demonstrate Activity against both Undruggable Extracellular and Intracellular Disease Targets"](https://www.prnewswire.com/news-releases/complix-alphabody-protein-therapeutics-demonstrate-activity-against-both-undruggable-extracellular-and-intracellular-disease-targets-149021165.html). PR Newswire. April 26, 2012. Retrieved 26 March 2015.

1. ["Complix NV Raises $15.5 Million in Series B Round"](http://www.clinicaspace.com/News/complix-nv-raises-15-5-million-in-series-b-round/301099). ClinicaSpace. 26 June 2013. Retrieved 26 March 2015.

## External links

- [Complix N.V.](http://www.complix.com/)

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Adapted from the Wikipedia article [Alphabody](https://en.wikipedia.org/wiki/Alphabody) by Wikipedia contributors ([contributor history](https://en.wikipedia.org/wiki/Alphabody?action=history)). Available under [Creative Commons Attribution-ShareAlike 4.0 International](https://creativecommons.org/licenses/by-sa/4.0/). Changes may have been made.