Difference between revisions of "Part:BBa K3410001"

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Nanobody scaffold suitable for grafting.

cAbBCII-10 is a nanobody, originally isolated as an antibody capturing and neutralizing enzymes of the β-lactamase B class [1].
As CDR acceptor (also called "scaffold") we used the nanobody cAbBCII-10 [1] (or also NbBcII10 [2] or 3DWT [3], from here on called "3DWT"). This Nanobody is also available as part of a composite part in the iGEM parts reg (BBa_K2082001). However, the nanobody is not available as basic part, so we also contributed in submitting the basic part of cAbBCII-10 (BBa_K3410001). We used the sequence deposited in the PDB by Vincke et al. [2, 3].
Originally, 3DWT was isolated as an antibody capturing and neutralizing enzymes of the β-lactamase B class [1].

Figure 1: Map of the used nanobody scaffold 3DWT. Marked in purple are the CDRs 1 to 3 and in grey the framework regions. This map was generated with SnapGene.

A single-domain antibody, also known as nanobody, is an antibody consisting of a single variable antibody domain. First described in 1993 [6], nanobodies are an important research field for multiple pharmaceutical applications today. Mainly, nanobodies are divided in two types of different structures. The most common nanobodies are the VHH fragments, which consist only of a variable domain of a heavy chain and are mostly found in Camelidae species [8]. Another type of nanobodies are the VNAR fragments based on the IgNAR (“Immunoglobin new antigen receptor”) from cartilaginous fish [9]. Having a molecular mass of ∼12 kDa, the vNAR domain is the smallest antibody-like antigen binding domain known so far in the animals [10]. That is why VNAR domains have only two complementarity determining regions CDR1 and CDR3, in contrast to variable mammalian domains [11].

Accordingly, nanobodies are the smallest known antigen-binding proteins with a length of 4 nm and a diameter of 2.5 nm [12]. However, like a whole antibody, they are still able to recognize and bind specifically their target molecules using the single variable domain. Their compact structure and light molecular weight of only 12-15 kDa (compared to conventional antibodies with 150-160 kDa) make nanobodies the smallest active antigen-binding fragments [13]. Due to their versatile binding properties, high stability, manipulable characteristics as well as improved tissue penetration ability the nanobodies are crucial in the field of biotechnology or medicine today [14], [15].

Figure 2: Schematic overview of a conventional Ig-antibody (left side). Enlarged overview over the characteristic features of a VHH nanobody on the right side.

Antibodies consist of two glycosylated heavy chains (H) with a molecular weight of 50 kDA each and two light chains of 25 kDA each, which are covalently linked via disulfide bridges. [16]. On the one hand, two N-terminal variable domains (V) of the heavy (VH) and light chain (VL) form the antigen-binding domain, also called paratrope. On the other hand, the C-terminal regions form the three constant domains CH1, CH2 and CH3. Compared to common antibodies, the nanobody family consist of conventional heterotetramic antibodies with low-affinity binders and as well as unique functional heavy(H)-chain antibodies (HCAbs) with high affinity binders [13]. The H chain of these homodimeric antibodies consist of one antigen-binding domain, the VHH, and two constant domains. The HCAbs fail to incorporate light(L)-chains owing to the deletion of the first constant domain and a reshaped surface of the VHH side, which normally associates with the L-chain in common antibodies [17]. This structural change leads to this enlargement allowing the nanobodies to bind their antigen with a high affinity Furthermore, the hydrophobic amino acids within the conserved region (frame region; FR), which ensure the interaction of VH and VL, are replaced by hydrophilic amino acids [12], [18].

The field of application of nanobodies is very diverse. Their use in diagnostic test systems and therapeutic options is increasing. In contrast to conventional antibodies, nanobodies merge as a new technical achievement due to their low molecular weight (half the size of single-chain variable elements and ten times lighter than conventional antibodies) and small size (2-3 nm) [17]. Moreover, a high expression rate of nanobodies was shown in E. coli with high functionality and stability even in absence of conserved disulphide bonds [19]. The loss of the L-chain leads to missing disulphide modifications in nanobodies, which makes them suitable for production in different bacteria and yeast [20], [21]. Additionally, their high tolerance against acid conditions and temperature compared to mouse monoclonal antibodies [8], high solubility due to a tetrahedron of highly conserved hydrophilic substitutions [22] and very few cleavage sites for enzymes [13] make the nanobodies very promising and an interesting research subject for new applications in the biotechnology. Furthermore, the recombinant nature and single domain of the nanobodies allows easy generation, production and molecular biological manipulation. These include sequential modifications, the generation of fusion proteins with other substances (e.g. pharmaceuticals), the transfer of antigen specificities as well as the transfer of affinity from one nanobody to another [23].

Due to their low mass, the nanobodies are able to easily cross the blood-brain barrier and make them able to quickly leave the human body's circulation via urine after resolving their function inside the cell. Furthermore, nanobodies show a higher availability to tissue penetration and due to their smaller size, they trigger less immunological reaction, which leads to better pharmacokinetics [24]. All stated points are an important property for therapeutic options (cancer therapies) and diagnostic processes (molecular imaging) [23]. Nanobody Grafting Typically, a camelid immune library is needed for the generation of nanobodies, besides ethical concerns, you would have to keep animals and take care of them. One can also obtain nanobodies from synthetic libraries, but this requires large libraries and sophisticated selection mechanisms. An alternative is the so-called grafting of nanobodies. In this method, the CDRs of any antibody are transferred to an existing nanobody to create a mixed nanobody [4]. However, not only the three CDR regions 1 to 3 are transferred, but also amino acids important for the structure and function are transferred from the donor antibody to the nanobody scaffold [25].

Figure 3: Schematic representation of the in silico grafting process. To start grafting, an acceptor and a suitable CDR donor must be selected. The CDRs 1 to 3 are transferred from the donor (any antibody) to the acceptor (a nanobody suitable for grafting). The resulting initial graft is usually a weak binder. The grafted sequence must therefore be subjected to a suitable affinity maturation method, for example a phage display. After affinity maturation a new synthetic nanobody with high affinity is obtained. Abbreviations: CDR, complementarity-determining region; VH, variable domain of the heavy chain; VHH, single variable domain on a heavy chain antibody; VL, variable domain of the light chain. Created with BioRender.

The grafting itself is carried out in silico, therefore the amino acid residues from the VH domain from the donor antibody and from the accepting nanobody have to be numbered according to a numbering scheme like Kabat or AHo [26] and CDR1 to 3 have to be defined accordingly [25]. The correct structure of the graft can be checked with the help of visualization programs such as ChimeraX [27]. Here you can see the results of our grafting. Error Prone PCR For means of affinity maturation we needed a library of our grafted sequence. To introduce the required diversity into the grafted sequence, we chose a random mutagenesis approach by polymerase chain reaction (PCR), first described by Cadwell and Joyce in 1992 [28]. This variant of the PCR-technique is termed error prone PCR (epPCR). In this technique, the natural error rate of the Taq polymerase is increased by unfavorable reaction conditions. This is accomplished by: 1) Increased concentration of Taq DNA polymerase; 2) extended polymerase extension time; 3) increased concentration of MgCl2 ions; 4) unbalanced rate of dNTPs; and 5) addition of MnCl2 ions [29].

References
[1] K. E. Conrath et al., “Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae,” Antimicrobial Agents and Chemotherapy, vol. 45, no. 10, pp. 2807–2812, 2001, doi: 10.1128/AAC.45.10.2807-2812.2001.
[2] C. Vincke, R. Loris, D. Saerens, S. Martinez-Rodriguez, S. Muyldermans, and K. Conrath, “General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold,” The Journal of biological chemistry, vol. 284, no. 5, pp. 3273–3284, 2009, doi: 10.1074/jbc.M806889200.
[3] C. e. a. Vincke, 3DWT: Structure of CabBCII-10 nanobody.
[4] S. W. Fanning and J. R. Horn, “An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop,” Protein science : a publication of the Protein Society, vol. 20, no. 7, pp. 1196–1207, 2011, doi: 10.1002/pro.648.
References [1] K. E. Conrath et al., “Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae,” Antimicrobial Agents and Chemotherapy, vol. 45, no. 10, pp. 2807–2812, 2001, doi: 10.1128/AAC.45.10.2807-2812.2001. [2] C. Vincke, R. Loris, D. Saerens, S. Martinez-Rodriguez, S. Muyldermans, and K. Conrath, “General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold,” The Journal of biological chemistry, vol. 284, no. 5, pp. 3273–3284, 2009, doi: 10.1074/jbc.M806889200. [3] C. e. a. Vincke, 3DWT: Structure of CabBCII-10 nanobody. [4] S. W. Fanning and J. R. Horn, “An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop,” Protein science : a publication of the Protein Society, vol. 20, no. 7, pp. 1196–1207, 2011, doi: 10.1002/pro.648. [5] D. Saerens et al., “Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies,” Journal of molecular biology, vol. 352, no. 3, pp. 597–607, 2005, doi: 10.1016/j.jmb.2005.07.038.
[6] C. Hamers-Casterman u. a., „Naturally occurring antibodies devoid of light chains“, Nature, Bd. 363, Nr. 6428, S. 446–448, Juni 1993, doi: 10.1038/363446a0 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[7] A. L. Nelson, „Antibody fragments“, mAbs, Bd. 2, Nr. 1, S. 77–83, 2010.

[8] R. H. van der Linden u. a., „Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies“, Biochim. Biophys. Acta, Bd. 1431, Nr. 1, S. 37–46, Apr. 1999, doi: 10.1016/s0167-4838(99)00030-8 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[9] R. L. Stanfield, H. Dooley, M. F. Flajnik, und I. A. Wilson, „Crystal structure of a shark single-domain antibody V region in complex with lysozyme“, Science, Bd. 305, Nr. 5691, S. 1770–1773, Sep. 2004, doi: 10.1126/science.1101148 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[10] C. Barelle, D. S. Gill, und K. Charlton, „Shark novel antigen receptors--the next generation of biologic therapeutics?“, Adv. Exp. Med. Biol., Bd. 655, S. 49–62, 2009, doi: 10.1007/978-1-4419-1132-2_6 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[11] S. Zielonka, M. Empting, J. Grzeschik, D. Könning, C. J. Barelle, und H. Kolmar, „Structural insights and biomedical potential of IgNAR scaffolds from sharks“, mAbs, Bd. 7, Nr. 1, S. 15–25, Dez. 2014, doi: 10.4161/19420862.2015.989032 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[12] W. Wang, J. Yuan, und C. Jiang, „Applications of nanobodies in plant science and biotechnology“, Plant Mol. Biol., Okt. 2020, doi: 10.1007/s11103-020-01082-z Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[13] M. M. Harmsen und H. J. De Haard, „Properties, production, and applications of camelid single-domain antibody fragments“, Appl. Microbiol. Biotechnol., Bd. 77, Nr. 1, S. 13–22, Nov. 2007, doi: 10.1007/s00253-007-1142-2 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[14] P. Chames und U. Rothbauer, „Special Issue: Nanobody“, Antibodies, Bd. 9, Nr. 1, Art. Nr. 1, März 2020, doi: 10.3390/antib9010006 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[15] X. Yu u. a., „Nanobodies derived from Camelids represent versatile biomolecules for biomedical applications“, Biomater. Sci., Bd. 8, Nr. 13, S. 3559–3573, 2020, doi: 10.1039/D0BM00574F Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[16] J. Charles A Janeway, P. Travers, M. Walport, und M. J. Shlomchik, „The structure of a typical antibody molecule“, Immunobiol. Immune Syst. Health Dis. 5th Ed., 2001, Zugegriffen: Okt. 27, 2020. [Online]. Verfügbar unter: https://www.ncbi.nlm.nih.gov/books/NBK27144/.

[17] S. Muyldermans, „Nanobodies: natural single-domain antibodies“, Annu. Rev. Biochem., Bd. 82, S. 775–797, 2013, doi: 10.1146/annurev-biochem-063011-092449 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[18] „Nanobodies: Chemical Functionalization Strategies and Intracellular Applications - Schumacher - 2018 - Angewandte Chemie International Edition - Wiley Online Library“. https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201708459 Titel anhand dieser DOI in Citavi-Projekt übernehmen (zugegriffen Okt. 27, 2020).

[19] D. Saerens, „Isolation and optimization of camelid single-domain antibodies: Dirk Saerens’ work on nanobodies“, World J. Biol. Chem., Bd. 1, Nr. 7, S. 235–238, Juli 2010, doi: 10.4331/wjbc.v1.i7.235 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[20] F. Rahbarizadeh, D. Ahmadvand, und Z. Sharifzadeh, „Nanobody; an Old Concept and New Vehicle for Immunotargeting“, Immunol. Invest., Bd. 40, Nr. 3, S. 299–338, Jan. 2011, doi: 10.3109/08820139.2010.542228 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[21] „Anwendungsgebiete für rekombinante Antikörper | SpringerLink“. https://link.springer.com/chapter/10.1007/978-3-662-50276-1_5 Titel anhand dieser DOI in Citavi-Projekt übernehmen (zugegriffen Okt. 27, 2020).

[22] M. Arbabi Ghahroudi, A. Desmyter, L. Wyns, R. Hamers, und S. Muyldermans, „Selection and identification of single domain antibody fragments from camel heavy-chain antibodies“, FEBS Lett., Bd. 414, Nr. 3, S. 521–526, Sep. 1997, doi: 10.1016/s0014-5793(97)01062-4 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[23] S. Schoonooghe u. a., „Novel applications of nanobodies for in vivo bio-imaging of inflamed tissues in inflammatory diseases and cancer“, Immunobiology, Bd. 217, Nr. 12, S. 1266–1272, Dez. 2012, doi: 10.1016/j.imbio.2012.07.009 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[24] „The Therapeutic Potential of Nanobodies | SpringerLink“. https://link.springer.com/article/10.1007/s40259-019-00392-z Titel anhand dieser DOI in Citavi-Projekt übernehmen (zugegriffen Okt. 27, 2020).

[25] S. Ewert, A. Honegger, und A. Plückthun, „Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering“, Methods San Diego Calif, Bd. 34, Nr. 2, S. 184–199, Okt. 2004, doi: 10.1016/j.ymeth.2004.04.007 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[26] M. Dondelinger u. a., „Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition“, Front. Immunol., Bd. 9, Okt. 2018, doi: 10.3389/fimmu.2018.02278 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[27] „UCSF ChimeraX Home Page“. https://www.cgl.ucsf.edu/chimerax/ (zugegriffen Okt. 27, 2020).

[28] R. C. Cadwell und G. F. Joyce, „Randomization of genes by PCR mutagenesis“, PCR Methods Appl., Bd. 2, Nr. 1, S. 28–33, Aug. 1992, doi: 10.1101/gr.2.1.28 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[29] E. O. McCullum, B. A. R. Williams, J. Zhang, und J. C. Chaput, „Random mutagenesis by error-prone PCR“, Methods Mol. Biol. Clifton NJ, Bd. 634, S. 103–109, 2010, doi: 10.1007/978-1-60761-652-8_7 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[30] G. P. Smith, „Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface“, Science, Bd. 228, Nr. 4705, S. 1315–1317, Juni 1985, doi: 10.1126/science.4001944 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[31] J. McCafferty, A. D. Griffiths, G. Winter, und D. J. Chiswell, „Phage antibodies: filamentous phage displaying antibody variable domains“, Nature, Bd. 348, Nr. 6301, Art. Nr. 6301, Dez. 1990, doi: 10.1038/348552a0 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[32] A. N. Zacher, C. A. Stock, J. W. Golden, und G. P. Smith, „A new filamentous phage cloning vector: fd-tet“, Gene, Bd. 9, Nr. 1, S. 127–140, Apr. 1980, doi: 10.1016/0378-1119(80)90171-7 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[33] M. Better, C. P. Chang, R. R. Robinson, und A. H. Horwitz, „Escherichia coli secretion of an active chimeric antibody fragment“, Science, Bd. 240, Nr. 4855, S. 1041–1043, Mai 1988, doi: 10.1126/science.3285471 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[34] A. Skerra und A. Plückthun, „Assembly of a functional immunoglobulin Fv fragment in Escherichia coli“, Science, Bd. 240, Nr. 4855, S. 1038–1041, Mai 1988, doi: 10.1126/science.3285470 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[35] S. Bass, R. Greene, und J. A. Wells, „Hormone phage: an enrichment method for variant proteins with altered binding properties“, Proteins, Bd. 8, Nr. 4, S. 309–314, 1990, doi: 10.1002/prot.340080405 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[36] L. J. Garrard, M. Yang, M. P. O’Connell, R. F. Kelley, und D. J. Henner, „F AB Assembly and Enrichment in a Monovalent Phage Display System“, Bio/Technology, Bd. 9, Nr. 12, Art. Nr. 12, Dez. 1991, doi: 10.1038/nbt1291-1373 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[37] H. R. Hoogenboom, A. D. Griffiths, K. S. Johnson, D. J. Chiswell, P. Hudson, und G. Winter, „Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains.“, Nucleic Acids Res., Bd. 19, Nr. 15, S. 4133–4137, Aug. 1991.

[38] L. Ledsgaard, M. Kilstrup, A. Karatt-Vellatt, J. McCafferty, und A. H. Laustsen, „Basics of Antibody Phage Display Technology“, Toxins, Bd. 10, Nr. 6, Juni 2018, doi: 10.3390/toxins10060236 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[39] „Repertoires of aggregation-resistant human antibody domains | Protein Engineering, Design and Selection | Oxford Academic“. https://academic.oup.com/peds/article/20/8/413/1435168 (zugegriffen Okt. 27, 2020).

[40] C. F. Barbas, A. S. Kang, R. A. Lerner, und S. J. Benkovic, „Assembly of combinatorial antibody libraries on phage surfaces: the gene III site.“, Proc. Natl. Acad. Sci. U. S. A., Bd. 88, Nr. 18, S. 7978–7982, Sep. 1991.

[41] H. B. Lowman, S. H. Bass, N. Simpson, und J. A. Wells, „Selecting high-affinity binding proteins by monovalent phage display“, Biochemistry, Bd. 30, Nr. 45, S. 10832–10838, Nov. 1991, doi: 10.1021/bi00109a004 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[42] D. O’Connell, B. Becerril, A. Roy-Burman, M. Daws, und J. D. Marks, „Phage versus Phagemid Libraries for Generation of Human Monoclonal Antibodies“, J. Mol. Biol., Bd. 321, Nr. 1, S. 49–56, Aug. 2002, doi: 10.1016/S0022-2836(02)00561-2 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[43] U. Brinkmann, P. S. Chowdhury, D. M. Roscoe, und I. Pastan, „Phage display of disulfide-stabilized Fv fragments“, J. Immunol. Methods, Bd. 182, Nr. 1, S. 41–50, Jan. 1995, doi: 10.1016/0022-1759(95)00016-4 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[44] J. Yan, G. Li, Y. Hu, W. Ou, und Y. Wan, „Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications“, J. Transl. Med., Bd. 12, Nr. 1, S. 343, Dez. 2014, doi: 10.1186/s12967-014-0343-6 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[45] A. D. Griffiths u. a., „Isolation of high affinity human antibodies directly from large synthetic repertoires.“, EMBO J., Bd. 13, Nr. 14, S. 3245–3260, Juli 1994.

[46] T. Clackson, H. R. Hoogenboom, A. D. Griffiths, und G. Winter, „Making antibody fragments using phage display libraries“, Nature, Bd. 352, Nr. 6336, S. 624–628, Aug. 1991, doi: 10.1038/352624a0 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[47] J. D. Marks, H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. Griffiths, und G. Winter, „By-passing immunization: Human antibodies from V-gene libraries displayed on phage“, J. Mol. Biol., Bd. 222, Nr. 3, S. 581–597, Dez. 1991, doi: 10.1016/0022-2836(91)90498-U Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[48] R. E. Hawkins, S. J. Russell, und G. Winter, „Selection of phage antibodies by binding affinity: Mimicking affinity maturation“, J. Mol. Biol., Bd. 226, Nr. 3, S. 889–896, Aug. 1992, doi: 10.1016/0022-2836(92)90639-2 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

[49] S. R. Whaley, D. S. English, E. L. Hu, P. F. Barbara, und A. M. Belcher, „Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly“, Nature, Bd. 405, Nr. 6787, S. 665–668, Juni 2000, doi: 10.1038/35015043 Titel anhand dieser DOI in Citavi-Projekt übernehmen.

Sequence and Features