Single domain antibodies

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Structural model of a single domain antibody (ribbon model)

Single domain antibodies , also called nanobodies or nanoantibodies , are antibody fragments that are built up from a single, monomeric variable domain of an antibody . In the simplest case, they consist of the monomeric variable domains of heavy chain antibodies , which are produced by members of the camel family and cartilaginous fish , and are also known as V H H or V NAR fragments (Variable New Antigen Receptor). Alternatively, single-domain antibodies can also be obtained by monomerizing the dimeric variable domains of conventional mouse or human antibodies with the aid of genetic engineering methods. With a molecular mass of around 12–15  kDa , they are the smallest antibody fragments that are capable of recognizing antigens . Because of their low molecular weight and their special physicochemical properties, there is hope that single-domain antibodies can be used as novel drugs for the treatment of diseases such as acute coronary syndrome or rheumatoid arthritis .

properties

The variable domains of heavy chain antibodies, which consist of one amino acid chain, differ from those of conventional antibodies, which consist of two amino acid chains, in that they are less lipophilic , and therefore have good solubility in aqueous media and high heat resistance. In contrast to classic antibodies, which can be inactivated by heat, single domain antibodies retain their ability to bind antigens even after heat treatment at 90 ° C. Single-domain antibodies are more resistant to gastric acid and, thanks to a lower number of cleavage sites, more resistant to proteolytic enzymes than classic antibodies. This resistance can be increased by further optimizing the structure of the single domain antibodies so that they can also pass through the gastrointestinal tract and are suitable for local-oral application. However , a low absorption rate characteristic of peptide drugs limits possible systemic application. Thanks to their resistance in the presence of detergents , they are also suitable for use in shampoos . Associated with the small molecular size of single domain antibodies is an improved tissue permeability compared to conventional antibodies. Since their molecular mass is well below the kidney threshold , they have a very short plasma half-life and can be excreted via the kidneys and the urine . Likewise, due to the absence of the F C fragment, they do not show any cytotoxicity that can be attributed to activation of the complement system .

Single domain antibodies obtained from heavy chain antibodies from camels or cartilaginous fish have pronounced antigen-recognizing loop structures compared to the variable domains of classic antibodies. Thanks to this, single-domain antibodies are able to recognize hidden antigen structures that remain inaccessible for classical antibodies. These include, for example, the catalytic centers of enzymes and the ligand binding domains of G protein-coupled receptors .

Manufacturing

Single domain antibodies from heavy chain antibodies

Structural structure of a heavy chain antibody from shark (left, IgNAR) and camel (middle, hcIgG) compared to a conventional antibody (right, IgG).

The generation and selection of antigen- specific single domain antibodies derived from heavy chain antibodies is usually carried out by immunizing dromedaries, camels, llamas or alpacas and then isolating the mRNA coding for heavy chain antibodies . Even sharks have established themselves as a donor heavy chain antibodies and their encoding mRNA. With the help of molecular biological methods such as reverse transcription and polymerase chain reaction , an immune library of single domain antibodies containing several million clones is created from the entirety of the mRNA isolated in this way . With the help of a screening using so-called display techniques, such as phage display or ribosome display , the antigen-binding clones are identified.

Antigen-binding clones can also be selected from non-immune or naive libraries (which are created from the mRNA of animals that are not explicitly immunized). Since the affinity of the single domain antibodies obtained in this way for the antigen is usually very low, an additional affinity maturation step is carried out in vitro using random mutagenesis , especially when using naive libraries .

Once the most potent clones have been identified, their DNA sequence is optimized, for example to increase their enzymatic stability. Humanization, which is considered unproblematic due to the homology between V H H fragments of camels and V H fragments of humans, is also intended to reduce the likelihood of immune reactions against single domain antibodies when used in the human body. The individual domain antibodies optimized in this way are finally produced in E. coli , Saccharomyces cerevisiae or other suitable organisms.

Single domain antibodies from conventional antibodies

Alternatively, single domain antibodies can be derived from conventional mouse or human IgG antibodies consisting of four amino acid chains. Display techniques using immune or naive libraries can also be used to generate them. The generation of single domain antibodies from conventional antibodies is more complex than that from heavy chain antibodies, since the variable domains of conventional antibodies are present as dimers (V H -V L fragments). Since monomeric variable domains in mice or humans tend to dimerize or aggregate due to their lipophilicity, they are usually monomerized by exchanging lipophilic for hydrophilic amino acids. Frequently, however, a loss of affinity can also be observed after separation of the V H - from the V L - fragments. Once these difficulties have been overcome, single-domain antibodies can also be obtained from conventional antibodies using the usual production organisms such as E. coli or S. cerevisiae .

To distinguish it from such single domain antibodies are scFv fragments ( engl. Single-chain variable fragments ) which, while also consist of a single amino acid chain, but the two binding domains (V H and V L containing).

history

The history of the development of single domain antibodies goes back to 1989. At that time, biologists in the research group led by Raymond Hamers from the Vrije Universiteit Brussel examined the immune system of dromedaries . To their surprise, they identified not only classic antibodies consisting of two heavy and two light chains, but also more simply constructed antibodies consisting only of the heavy chains. This discovery was published in the 1993 journal Nature . In 1995, the discovery of antibodies, also consisting only of heavy chains, in cartilaginous fish. The single domain antibodies obtained from these heavy chain antibodies could be isolated with the aid of the polymerase chain reaction in the following years.

Individual evidence

  1. ↑ Ablynx - Nanobodies in drug development ( Memento from February 28, 2015 in the Internet Archive )
  2. ↑ van der Linden RH, Frenken LG, de Geus B, et al. : Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies . In: Biochim. Biophys. Acta . 1431, No. 1, April 1999, pp. 37-46. PMID 10209277 .
  3. ↑ a b Harmsen MM, van Solt CB, Hoogendoorn A, van Zijderveld FG, Niewold TA, van der Meulen J: Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhea . In: Vet. Microbiol. . 111, No. 1-2, November 2005, pp. 89-98. doi : 10.1016 / j.vetmic.2005.09.005 . PMID 16221532 .
  4. ↑ Harmsen MM, van Solt CB, van Zijderveld-van Bemmel AM, Niewold TA, van Zijderveld FG: Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy . In: Appl. Microbiol. Biotechnol. . 72, No. 3, September 2006, pp. 544-51. doi : 10.1007 / s00253-005-0300-7 . PMID 16450109 .
  5. ↑ Dolk E, van der Vaart M, Lutje Hulsik D, et al. : Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo . In: Appl. Environ. Microbiol. . 71, No. 1, January 2005, pp. 442-50. doi : 10.1128 / AEM.71.1.442-450.2005 . PMID 15640220 . PMC 544197 (free full text).
  6. ↑ a b Harmsen MM, De Haard HJ: Properties, production, and applications of camelid single-domain antibody fragments . In: Appl. Microbiol. Biotechnol. . 77, No. 1, November 2007, pp. 13-22. doi : 10.1007 / s00253-007-1142-2 . PMID 17704915 . PMC 2039825 (free full text).
  7. ↑ Jähnichen S, Blanchetot C, Maussang D, et al. : CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells . In: Proc. Natl. Acad. Sci. USA . 107, No. 47, November 2010, pp. 20565-70. doi : 10.1073 / pnas.1012865107 . PMID 21059953 . PMC 2996674 (free full text).
  8. ↑ a b Ghahroudi MA, Desmyter A, Wyns L, Hamers R, Muyldermans S: Selection and identification of single domain antibody fragments from camel heavy-chain antibodies . In: FEBS Lett . . 414, No. 3, September 1997, pp. 521-6. PMID 9323027 .
  9. ↑ a b Saerens D, Ghassabeh GH, Muyldermans S: Single-domain antibodies as building blocks for novel therapeutics . In: Curr Opin Pharmacol . 8, No. 5, October 2008, pp. 600-8. doi : 10.1016 / j.coph.2008.07.006 . PMID 18691671 .
  10. ↑ Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM: Domain antibodies: proteins for therapy . In: Trends Biotechnol . . 21, No. 11, November 2003, pp. 484-90. PMID 14573361 .
  11. ↑ Borrebaeck CA, Ohlin M: Antibody evolution beyond nature . In: Nat. Biotechnol. . 20, No. 12, December 2002, pp. 1189-90. doi : 10.1038 / nbt1202-1189 . PMID 12454662 .
  12. ↑ Hamers-Casterman C, Atarhouch T, Muyldermans S, et al. : Naturally occurring antibodies devoid of light chains . In: Nature . 363, No. 6428, June 1993, pp. 446-8. doi : 10.1038 / 363446a0 . PMID 8502296 .
  13. ↑ Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF: A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks . In: Nature . 374, No. 6518, March 1995, pp. 168-73. doi : 10.1038 / 374168a0 . PMID 7877689 .
  14. ↑ Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R: Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains . In: Protein Eng. . 7, No. 9, September 1994, pp. 1129-35. PMID 7831284 .