Monobody

Last updated
The tenth fibronectin type III domain (human, PDB: 1TTG ) PDB 1ttg EBI.jpg
The tenth fibronectin type III domain (human, PDB: 1TTG ​)
Variable domain of an antibody's lambda light chain (human, PDB: 2RHE ) PDB 2rhe EBI.jpg
Variable domain of an antibody's lambda light chain (human, PDB: 2RHE ​)

Monobodies are synthetic binding proteins constructed using a fibronectin type III domain (FN3) as a molecular scaffold. Specifically, this class of binding proteins are built upon a diversified library of the 10th FN3 domain of human fibronectin. Monobodies are a simple and robust alternative to antibodies for creating target-binding proteins. The hybrid term monobody was coined in 1998 by the Koide group who published the first paper demonstrating the monobody concept using the tenth FN3 domain of human fibronectin. [1]

Contents

Monobodies are generated from combinatorial libraries in which portions of the FN3 scaffold are diversified using molecular display and directed evolution technologies such as phage display, mRNA display and yeast surface display. [2] [3] A large number of monobodies that have high affinity and high specificity to their respective targets have been reported. [4] [5] [6] [7] [8]

Monobodies belong to the class of molecules collectively called antibody mimics (or antibody mimetics) and alternative scaffolds that aim to overcome shortcomings of natural antibody molecules. A major advantage of monobodies over conventional antibodies is that monobodies can readily be used as genetically encoded intracellular inhibitors, that is you can express a monobody inhibitor in a cell of choice by simply transfecting the cell with a monobody expression vector. [9] [10] This is because of the characteristics of the underlying FN3 scaffold: small (~90 residues), stable, easy to produce, and its lack of disulfide bonds that makes it possible to produce functional monobodies regardless of the redox potential of the cellular environment, including the reducing environment of the cytoplasm and nucleus. [11] In contrast, most antibodies and antibody fragments depend on disulfide bonds formation and they must be produced under an oxidizing environment.

The monobody technology has been adopted in the biotechnology industry, most notably by Adnexus, a biotechnology company which has been part of Bristol-Myers Squibb since 2007 under the name of Adnectins (originally as Trinectins by its predecessor, Phylos [12] ). An example is pegdinetanib (Angiocept), an antagonist of vascular endothelial growth factor receptor 2 (VEGFR-2), which has entered Phase II clinical trials investigating the treatment of glioblastoma in October 2007. [13] [14]

Structure

The native FN3 scaffold consists of 94 amino acids and has a molecular mass of about 10 kDa, fifteen times smaller than an IgG type antibody and comparable to the size of a single variable domain of an antibody. They are based on the structure of human fibronectin, more specifically on its tenth extracellular type III domain. This domain has a structure similar to antibody variable domains, with seven beta sheets forming a beta-sandwich and three exposed loops on each side corresponding to the three complementarity-determining regions. [15] [16] [17] Monobodies lack binding sites for metal ions and the central disulfide bond.

Monobody library designs

Monobodies with high affinity and specificity for different target molecules can be generated from combinatorial libraries in which portions of the FN3 scaffold are diversified. There are two distinct designs of monobody libraries that have been successful. The first type modifies some or all of the loops BC (between the second and third beta sheets), DE (between the fourth and fifth beta sheets) and FG (between the sixth and seventh sheets). [18] [19] This design creates diversified positions on a convex surface that is suitable for targeting concave surfaces such as enzyme active sites. The second type modifies positions in some or all of the C, D, F and G (or the 3rd, 4th, 6th and 7th) strands in addition to the CD and FG loops. [20] This design creates a flatter, slightly concave surface that is suitable for targeting surfaces typically involved in protein-protein interactions.

See also

Related Research Articles

<span class="mw-page-title-main">Philadelphia chromosome</span> Genetic abnormality in leukemia cancer cells

The Philadelphia chromosome or Philadelphia translocation (Ph) is a specific genetic abnormality in chromosome 22 of leukemia cancer cells. This chromosome is defective and unusually short because of reciprocal translocation, t(9;22)(q34;q11), of genetic material between chromosome 9 and chromosome 22, and contains a fusion gene called BCR-ABL1. This gene is the ABL1 gene of chromosome 9 juxtaposed onto the breakpoint cluster region BCR gene of chromosome 22, coding for a hybrid protein: a tyrosine kinase signaling protein that is "always on", causing the cell to divide uncontrollably by interrupting the stability of the genome and impairing various signaling pathways governing the cell cycle.

<span class="mw-page-title-main">ABL (gene)</span> Human protein-coding gene on chromosome 9

Tyrosine-protein kinase ABL1 also known as ABL1 is a protein that, in humans, is encoded by the ABL1 gene located on chromosome 9. c-Abl is sometimes used to refer to the version of the gene found within the mammalian genome, while v-Abl refers to the viral gene, which was initially isolated from the Abelson murine leukemia virus.

<span class="mw-page-title-main">B-cell receptor</span> Transmembrane protein on the surface of a B cell

The B-cell receptor (BCR) is a transmembrane protein on the surface of a B cell. A B-cell receptor is composed of a membrane-bound immunoglobulin molecule and a signal transduction moiety. The former forms a type 1 transmembrane receptor protein, and is typically located on the outer surface of these lymphocyte cells. Through biochemical signaling and by physically acquiring antigens from the immune synapses, the BCR controls the activation of the B cell. B cells are able to gather and grab antigens by engaging biochemical modules for receptor clustering, cell spreading, generation of pulling forces, and receptor transport, which eventually culminates in endocytosis and antigen presentation. B cells' mechanical activity adheres to a pattern of negative and positive feedbacks that regulate the quantity of removed antigen by manipulating the dynamic of BCR–antigen bonds directly. Particularly, grouping and spreading increase the relation of antigen with BCR, thereby proving sensitivity and amplification. On the other hand, pulling forces delinks the antigen from the BCR, thus testing the quality of antigen binding.

<span class="mw-page-title-main">BCR (gene)</span>

The breakpoint cluster region protein (BCR) also known as renal carcinoma antigen NY-REN-26 is a protein that in humans is encoded by the BCR gene. BCR is one of the two genes in the BCR-ABL fusion protein, which is associated with the Philadelphia chromosome. Two transcript variants encoding different isoforms have been found for this gene.

<span class="mw-page-title-main">Adapter molecule crk</span> Protein-coding gene in the species Homo sapiens

Adapter molecule crk also known as proto-oncogene c-Crk is a protein that in humans is encoded by the CRK gene.

The PHLPP isoforms are a pair of protein phosphatases, PHLPP1 and PHLPP2, that are important regulators of Akt serine-threonine kinases and conventional/novel protein kinase C (PKC) isoforms. PHLPP may act as a tumor suppressor in several types of cancer due to its ability to block growth factor-induced signaling in cancer cells.

<span class="mw-page-title-main">GRB10</span> Protein-coding gene in the species Homo sapiens

Growth factor receptor-bound protein 10 also known as insulin receptor-binding protein Grb-IR is a protein that in humans is encoded by the GRB10 gene.

<span class="mw-page-title-main">GAB2</span> Protein-coding gene in the species Homo sapiens

GRB2-associated-binding protein 2 also known as GAB2 is a protein that in humans is encoded by the GAB2 gene.

<span class="mw-page-title-main">PTPN6</span> Protein-coding gene in humans

Tyrosine-protein phosphatase non-receptor type 6, also known as Src homology region 2 domain-containing phosphatase-1 (SHP-1), is an enzyme that in humans is encoded by the PTPN6 gene.

<span class="mw-page-title-main">CBL (gene)</span> Mammalian gene

Cbl is a mammalian gene family. CBL gene, a part of the Cbl family, encodes the protein CBL which is an E3 ubiquitin-protein ligase involved in cell signalling and protein ubiquitination. Mutations to this gene have been implicated in a number of human cancers, particularly acute myeloid leukaemia.

<span class="mw-page-title-main">HNRPU</span> Protein-coding gene in the species Homo sapiens

Heterogeneous nuclear ribonucleoprotein U is a protein that in humans is encoded by the HNRNPU gene.

<span class="mw-page-title-main">VAV1</span> Human protein and coding gene

Proto-oncogene vav is a protein that in humans is encoded by the VAV1 gene.

<span class="mw-page-title-main">NCK1</span> Protein-coding gene in the species Homo sapiens

Cytoplasmic protein NCK1 is a protein that in humans is encoded by the NCK1 gene.

<span class="mw-page-title-main">BCAR1</span> Protein-coding gene in the species Homo sapiens

Breast cancer anti-estrogen resistance protein 1 is a protein that in humans is encoded by the BCAR1 gene.

<span class="mw-page-title-main">ABL2</span> Protein-coding gene in the species Homo sapiens

Tyrosine-protein kinase ABL2 also known as Abelson-related gene (Arg) is an enzyme that in humans is encoded by the ABL2 gene.

<span class="mw-page-title-main">SH2B1</span> Protein-coding gene in the species Homo sapiens

SH2B adapter protein 1 is a protein that in humans is encoded by the SH2B1 gene.

A non-receptor tyrosine kinase (nRTK) is a cytosolic enzyme that is responsible for catalysing the transfer of a phosphate group from a nucleoside triphosphate donor, such as ATP, to tyrosine residues in proteins. Non-receptor tyrosine kinases are a subgroup of protein family tyrosine kinases, enzymes that can transfer the phosphate group from ATP to a tyrosine residue of a protein (phosphorylation). These enzymes regulate many cellular functions by switching on or switching off other enzymes in a cell.

Antibody mimetics are organic compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.

Bcr-Abl tyrosine-kinase inhibitors (TKI) are the first-line therapy for most patients with chronic myelogenous leukemia (CML). More than 90% of CML cases are caused by a chromosomal abnormality that results in the formation of a so-called Philadelphia chromosome. This abnormality was discovered by Peter Nowell in 1960 and is a consequence of fusion between the Abelson (Abl) tyrosine kinase gene at chromosome 9 and the break point cluster (Bcr) gene at chromosome 22, resulting in a chimeric oncogene (Bcr-Abl) and a constitutively active Bcr-Abl tyrosine kinase that has been implicated in the pathogenesis of CML. Compounds have been developed to selectively inhibit the tyrosine kinase.

Synthetic antibodies are affinity reagents generated entirely in vitro, thus completely eliminating animals from the production process. Synthetic antibodies include recombinant antibodies, nucleic acid aptamers and non-immunoglobulin protein scaffolds. As a consequence of their in vitro manufacturing method the antigen recognition site of synthetic antibodies can be engineered to any desired target and may extend beyond the typical immune repertoire offered by natural antibodies. Synthetic antibodies are being developed for use in research, diagnostic and therapeutic applications. Synthetic antibodies can be used in all applications where traditional monoclonal or polyclonal antibodies are used and offer many inherent advantages over animal-derived antibodies, including comparatively low production costs, reagent reproducibility and increased affinity, specificity and stability across a range of experimental conditions.

References

  1. ↑ Koide A, Bailey CW, Huang X, Koide S (December 1998). "The fibronectin type III domain as a scaffold for novel binding proteins". J. Mol. Biol. 284 (4): 1141–51. doi: 10.1006/jmbi.1998.2238 . PMID   9837732.
  2. ↑ Koide S, Koide A, Lipovšek D (2012). "Target-Binding Proteins Based on the 10th Human Fibronectin Type III Domain (10Fn3)". Protein Engineering for Therapeutics, Part B. Methods in Enzymology. Vol. 503. pp. 135–56. doi:10.1016/B978-0-12-396962-0.00006-9. ISBN   9780123969620. PMID   22230568.
  3. ↑ Koide A, Wojcik J, Gilbreth RN, Hoey RJ, Koide S (2012). "Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold". J. Mol. Biol. 415 (2): 393–405. doi:10.1016/j.jmb.2011.12.019. PMC   3260337 . PMID   22198408.
  4. ↑ Wojcik J, Hantschel O, Grebien F, Kaupe I, Bennett KL, Barkinge J, Jones RB, Koide A, Superti-Furga G, Koide S (2010). "A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain". Nat. Struct. Mol. Biol. 17 (4): 519–27. doi:10.1038/nsmb.1793. PMC   2926940 . PMID   20357770.
  5. ↑ Gilbreth RN; Truong K; Madu I; et al. (May 2011). "Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design". Proc. Natl. Acad. Sci. U.S.A. 108 (19): 7751–6. Bibcode:2011PNAS..108.7751G. doi: 10.1073/pnas.1102294108 . PMC   3093456 . PMID   21518904.
  6. ↑ Grebien F, Hantschel O, Wojcik J, Kaupe I, Kovacic B, Wyrzucki AM, Gish GD, Cerny-Reiterer S, Koide A, Beug H, Pawson T, Valent P, Koide S, Superti-Furga G (2011). "Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis". Cell. 147 (2): 306–19. doi:10.1016/j.cell.2011.08.046. PMC   3202669 . PMID   22000011.
  7. ↑ Sha F, Gencer EB, Georgeon S, Koide A, Yasui N, Koide S, Hantschel O (2013). "Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains". Proc. Natl. Acad. Sci. U.S.A. 110 (37): 14924–9. Bibcode:2013PNAS..11014924S. doi: 10.1073/pnas.1303640110 . PMC   3773763 . PMID   23980151.
  8. ↑ Stockbridge RB, Koide A, Miller C, Koide S (2014). "Proof of dual-topology architecture of Fluc F- channels with monobody blockers". Nat Commun. 5: 5120. doi:10.1038/ncomms6120. PMC   4265568 . PMID   25290819.
  9. ↑ Grebien F, Hantschel O, Wojcik J, Kaupe I, Kovacic B, Wyrzucki AM, Gish GD, Cerny-Reiterer S, Koide A, Beug H, Pawson T, Valent P, Koide S, Superti-Furga G (2011). "Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis". Cell. 147 (2): 306–19. doi:10.1016/j.cell.2011.08.046. PMC   3202669 . PMID   22000011.
  10. ↑ Sha F, Gencer EB, Georgeon S, Koide A, Yasui N, Koide S, Hantschel O (2013). "Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains". Proc. Natl. Acad. Sci. U.S.A. 110 (37): 14924–9. Bibcode:2013PNAS..11014924S. doi: 10.1073/pnas.1303640110 . PMC   3773763 . PMID   23980151.
  11. ↑ Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J.; Zhang, Xiaoying (2016-03-03). "Antibody mimetics: promising complementary agents to animal-sourced antibodies". Critical Reviews in Biotechnology. 36 (2): 268–275. doi:10.3109/07388551.2014.958431. ISSN   0738-8551. PMID   25264572. S2CID   367423.
  12. ↑ Xu L, Aha P, Gu K, Kuimelis RG, Kurz M, Lam T, Lim AC, Liu H, Lohse PA, Sun L, Weng S, Wagner RW, Lipovsek D (2002). "Directed evolution of high-affinity antibody mimics using mRNA display". Chem. Biol. 9 (8): 933–42. doi: 10.1016/s1074-5521(02)00187-4 . PMID   12204693.
  13. ↑ Clinical trial number NCT00562419 for "CT-322 in Treating Patients With Recurrent Glioblastoma Multiforme and Combination Therapy With Irinotecan" at ClinicalTrials.gov
  14. ↑ Bloom L, Calabro V (July 2009). "FN3: a new protein scaffold reaches the clinic". Drug Discov. Today. 14 (19–20): 949–55. doi:10.1016/j.drudis.2009.06.007. PMID   19576999.
  15. ↑ Koide A, Koide S (2007). "Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain". Protein Engineering Protocols. Methods Mol. Biol. Vol. 352. pp. 95–109. doi:10.1385/1-59745-187-8:95. ISBN   978-1-59745-187-1. PMID   17041261.
  16. ↑ Wojcik J, Hantschel O, Grebien F, Kaupe I, Bennett KL, Barkinge J, Jones RB, Koide A, Superti-Furga G, Koide S (2010). "A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain". Nat. Struct. Mol. Biol. 17 (4): 519–27. doi:10.1038/nsmb.1793. PMC   2926940 . PMID   20357770.
  17. ↑ Gilbreth RN; Truong K; Madu I; et al. (May 2011). "Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design". Proc. Natl. Acad. Sci. U.S.A. 108 (19): 7751–6. Bibcode:2011PNAS..108.7751G. doi: 10.1073/pnas.1102294108 . PMC   3093456 . PMID   21518904.
  18. ↑ Koide A, Bailey CW, Huang X, Koide S (December 1998). "The fibronectin type III domain as a scaffold for novel binding proteins". J. Mol. Biol. 284 (4): 1141–51. doi: 10.1006/jmbi.1998.2238 . PMID   9837732.
  19. ↑ Wojcik J, Hantschel O, Grebien F, Kaupe I, Bennett KL, Barkinge J, Jones RB, Koide A, Superti-Furga G, Koide S (2010). "A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain". Nat. Struct. Mol. Biol. 17 (4): 519–27. doi:10.1038/nsmb.1793. PMC   2926940 . PMID   20357770.
  20. ↑ Koide A, Wojcik J, Gilbreth RN, Hoey RJ, Koide S (2012). "Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold". J. Mol. Biol. 415 (2): 393–405. doi:10.1016/j.jmb.2011.12.019. PMC   3260337 . PMID   22198408.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.