Single-chain variable fragment

<h2 id="purification">Purification</h2>
Single-chain variable fragments lack the constant <a href="/facts/Fc_region/hC7E3AMe">Fc region</a> found in complete antibody molecules, and, thus, the common binding sites (e.g., <a href="/facts/Protein_G/BswHV67v">protein G</a>) cannot be used to purify antibodies. These fragments can often be purified or immobilized using <a href="/facts/Protein_L/b8KxXqU7">protein L</a>, since protein L interacts with the variable region of kappa light chains. More commonly, scientists incorporate a six histidine tag on the c-terminus of the scFv molecule and purify them using immobilized metal affinity chromatography (IMAC). Some scFv can also be captured by <a href="/facts/Protein_A/6oQVH9Mh">protein A</a> if they contain a human VH3 domain.<a class="footnote-ref" id="fnref:5" href="#fn:5">5</a><a class="footnote-ref" id="fnref:6" href="#fn:6">6</a><a class="footnote-ref" id="fnref:7" href="#fn:7">7</a>

<h2 id="bivalent-and-trivalent-scfvs">Bivalent and trivalent scFvs</h2>

Divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs.<a class="footnote-ref" id="fnref:8" href="#fn:8">8</a><a class="footnote-ref" id="fnref:9" href="#fn:9">9</a> Another possibility is the creation of scFvs with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies.<a class="footnote-ref" id="fnref:10" href="#fn:10">10</a> Diabodies have been shown to have <a href="/facts/Dissociation_constant/uvaKXf26">dissociation constants</a> up to 40-fold lower than corresponding scFvs, meaning that they have a much higher <a href="/facts/Affinity_(pharmacology)/44QLL4eU">affinity</a> to their target. Consequently, diabody drugs could be dosed much lower than other therapeutic antibodies and are capable of highly specific targeting of tumors in vivo.<a class="footnote-ref" id="fnref:11" href="#fn:11">11</a> Still shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.<a class="footnote-ref" id="fnref:12" href="#fn:12">12</a>
All of these formats can be composed from variable fragments with specificity for two different antigens, in which case they are types of <a href="/facts/Bispecific_antibodies/7xpSyYS1">bispecific antibodies</a>.<a class="footnote-ref" id="fnref:13" href="#fn:13">13</a><a class="footnote-ref" id="fnref:14" href="#fn:14">14</a> The furthest developed of these are bispecific tandem di-scFvs, known as <a href="/facts/Bi-specific_T-cell_engager/T49u1kIQ">bi-specific T-cell engagers</a> (BiTE antibody constructs).

<h2 id="examples">Examples</h2>
<ul><li><a href="/facts/Pexelizumab/jglRDsYq">Pexelizumab</a>, a scFv binding to <a href="/facts/Complement_component_5/el4KIXMC">component 5</a> of the <a href="/facts/Complement_system/vRSrQDUv">complement system</a> and designed to reduce side effects of <a href="/facts/Cardiac_surgery/t34fz1hc">cardiac surgery</a><a class="footnote-ref" id="fnref:15" href="#fn:15">15</a></li>
<li>C6.5, a diabody targeting <a href="/facts/HER2%2fneu/xue3SgcA">HER2/neu</a><a class="footnote-ref" id="fnref:16" href="#fn:16">16</a> found in some <a href="/facts/Breast_cancer/6hME580v">breast cancers</a></li>
<li><a href="/facts/Brolucizumab/cIMpINkz">Brolucizumab</a>, a scFV binding to VEGF-A and used to treat wet age-related macular degeneration</li>
<li>G6A, a scFV binding to human Hsp70.1 and can be used to target this protein as a potential marker in vast cancers.<a class="footnote-ref" id="fnref:17" href="#fn:17">17</a></li></ul>

<h2 id="references">References</h2>

<ol>
<li id="fn:1">Huston, J. S.; Levinson, D.; Mudgett-Hunter, M.; Tai, M. S.; Novotný, J.; Margolies, M. N.; Crea, R. (1988). "Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli". Proceedings of the National Academy of Sciences of the United States of America. 85 (16): 5879–5883. Bibcode:1988PNAS...85.5879H. doi:10.1073/pnas.85.16.5879. PMC 281868. PMID 3045807. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC281868" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC281868</a> <a href="#fnref:1" class="footnote-back-ref">↩</a></li>
<li id="fn:2">Schirrmann, Thomas (8 November 2004). "Tumorspezifisches Targeting der humanen Natürlichen Killerzellinie YT durch Gentransfer chimärer Immunglobulin-T-Zellrezeptoren" (in German). Berlin. doi:10.18452/15246. {{cite journal}}: Cite journal requires |journal= (help) <a href="http://edoc.hu-berlin.de/dissertationen/schirrmann-thomas-2005-03-18/HTML/" target="_blank">http://edoc.hu-berlin.de/dissertationen/schirrmann-thomas-2005-03-18/HTML/</a> <a href="#fnref:2" class="footnote-back-ref">↩</a></li>
<li id="fn:3">Peterson, Eric; Owens, SM; Henry, RL (2006). "Monoclonal Antibody Form and Function: Manufacturing the Right Antibodies for Treating Drug Abuse". AAPS Journal. 8 (2): E383 – E390. doi:10.1208/aapsj080243. PMC 3231570. PMID 16796389. <a href="http://www.aapsj.org/view.asp?art=aapsj080243" target="_blank">http://www.aapsj.org/view.asp?art=aapsj080243</a> <a href="#fnref:3" class="footnote-back-ref">↩</a></li>
<li id="fn:4">Peterson, Eric; Owens, SM; Henry, RL (2006). "Monoclonal Antibody Form and Function: Manufacturing the Right Antibodies for Treating Drug Abuse". AAPS Journal. 8 (2): E383 – E390. doi:10.1208/aapsj080243. PMC 3231570. PMID 16796389. <a href="http://www.aapsj.org/view.asp?art=aapsj080243" target="_blank">http://www.aapsj.org/view.asp?art=aapsj080243</a> <a href="#fnref:4" class="footnote-back-ref">↩</a></li>
<li id="fn:5">Vostakolaei, Mehdi Asghari; Molavi, Ommoleila; Hejazi, Mohammad Saeid; Kordi, Shirafkan; Rahmati, Saman; Barzegari, Abolfazl; Abdolalizadeh, Jalal (September 2019). "Isolation and characterization of a novel scFv antibody fragments specific for Hsp70 as a tumor biomarker". Journal of Cellular Biochemistry. 120 (9): 14711–14724. doi:10.1002/jcb.28732. ISSN 0730-2312. PMID 30998271. S2CID 121351794. <a href="https://onlinelibrary.wiley.com/doi/10.1002/jcb.28732" target="_blank">https://onlinelibrary.wiley.com/doi/10.1002/jcb.28732</a> <a href="#fnref:5" class="footnote-back-ref">↩</a></li>
<li id="fn:6">Potter, K. N.; Li, Y.; Pascual, V.; Capra, J. D. (1997). "Staphylococcal protein a binding to VH3 encoded immunoglobulins". International Reviews of Immunology. 14 (4): 291–308. doi:10.3109/08830189709116521. PMID 9186782. <a href="https://pubmed.ncbi.nlm.nih.gov/9186782-staphylococcal-protein-a-binding-to-vh3-encoded-immunoglobulins/" target="_blank">https://pubmed.ncbi.nlm.nih.gov/9186782-staphylococcal-protein-a-binding-to-vh3-encoded-immunoglobulins/</a> <a href="#fnref:6" class="footnote-back-ref">↩</a></li>
<li id="fn:7">Kordi, Shirafkan; Rahmati-Yamchi, Mohammad; Asghari Vostakolaei, Mehdi; Barzegari, Abolfazl; Abdolalizadeh, Jalal (2019-02-21). "Purification of a Novel Anti-VEGFR2 Single Chain Antibody Fragmentand Evaluation of Binding Affinity by Surface Plasmon Resonance". Advanced Pharmaceutical Bulletin. 9 (1): 64–69. doi:10.15171/apb.2019.008. ISSN 2228-5881. PMC 6468230. PMID 31011559. <a href="https://apb.tbzmed.ac.ir/Abstract/apb-22975" target="_blank">https://apb.tbzmed.ac.ir/Abstract/apb-22975</a> <a href="#fnref:7" class="footnote-back-ref">↩</a></li>
<li id="fn:8">Xiong, Cheng-Yi; Natarajan, A; Shi, XB; Denardo, GL; Denardo, SJ (2006). "Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding". Protein Engineering Design and Selection. 19 (8): 359–367. doi:10.1093/protein/gzl020. PMID 16760193. <a href="https://doi.org/10.1093%2Fprotein%2Fgzl020" target="_blank">https://doi.org/10.1093%2Fprotein%2Fgzl020</a> <a href="#fnref:8" class="footnote-back-ref">↩</a></li>
<li id="fn:9">Kufer, Peter; Lutterbüse, Ralf; Baeuerle, Patrick A. (2004). "A revival of bispecific antibodies" (PDF). Trends in Biotechnology. 22 (5): 238–244. doi:10.1016/j.tibtech.2004.03.006. PMID 15109810. <a href="http://www.micromet.de/fileadmin/template/main/pdf/publications_147881aaf61df52304237f0ee7f0cf2a.pdf" target="_blank">http://www.micromet.de/fileadmin/template/main/pdf/publications_147881aaf61df52304237f0ee7f0cf2a.pdf</a> <a href="#fnref:9" class="footnote-back-ref">↩</a></li>
<li id="fn:10">Hollinger, Philipp; Prospero, T; Winter, G (July 1993). ""Diabodies": small bivalent and bispecific antibody fragments". Proceedings of the National Academy of Sciences of the United States of America. 90 (14): 6444–8. Bibcode:1993PNAS...90.6444H. doi:10.1073/pnas.90.14.6444. PMC 46948. PMID 8341653. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC46948" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC46948</a> <a href="#fnref:10" class="footnote-back-ref">↩</a></li>
<li id="fn:11">Adams, GP; Schier, R; McCall, AM; Crawford, RS; Wolf, EJ; Weiner, LM; Marks, JD (1998). "Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu". British Journal of Cancer. 77 (9): 1405–12. doi:10.1038/bjc.1998.233. PMC 2150193. PMID 9652755. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2150193" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2150193</a> <a href="#fnref:11" class="footnote-back-ref">↩</a></li>
<li id="fn:12">Le Gall, F.; Kipriyanov, SM; Moldenhauer, G; Little, M (1999). "Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding". FEBS Letters. 453 (1): 164–168. doi:10.1016/S0014-5793(99)00713-9. PMID 10403395. S2CID 20213440. <a href="https://doi.org/10.1016%2FS0014-5793%2899%2900713-9" target="_blank">https://doi.org/10.1016%2FS0014-5793%2899%2900713-9</a> <a href="#fnref:12" class="footnote-back-ref">↩</a></li>
<li id="fn:13">Dincq, S; Bosman, F; Buyse, MA; Degrieck, R; Celis, L; De Boer, M; Van Doorsselaere, V; Sablon, E (2001). "Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway". Protein Expression and Purification. 22 (1): 11–24. doi:10.1006/prep.2001.1417. PMID 11388794. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:13" class="footnote-back-ref">↩</a></li>
<li id="fn:14">Kellner, C (2008). Entwicklung und Charakterisierung bispezifischer Antikörper-Derivate zur Immuntherapie CD19-positiver Leukämien und Lymphome [Development and characterisation of bispecific antibody derivatives for the immunotherapy of CD19-positive leukaemia and lymphoma] (Thesis) (in German and English). Erlangen-Nürnberg: Friedrich-Alexander-Universität. <a href="http://www.opus.ub.uni-erlangen.de/opus/volltexte/2009/1235/" target="_blank">http://www.opus.ub.uni-erlangen.de/opus/volltexte/2009/1235/</a> <a href="#fnref:14" class="footnote-back-ref">↩</a></li>
<li id="fn:15">Mathew, JP; Shernan, SK; White, WD; Fitch, JC; Chen, JC; Bell, L; Newman, MF (2004). "Preliminary report of the effects of complement suppression with pexelizumab on neurocognitive decline after coronary artery bypass graft surgery". Stroke: A Journal of Cerebral Circulation. 35 (10): 2335–9. doi:10.1161/01.STR.0000141938.00524.83. PMID 15331798. <a href="https://doi.org/10.1161%2F01.STR.0000141938.00524.83" target="_blank">https://doi.org/10.1161%2F01.STR.0000141938.00524.83</a> <a href="#fnref:15" class="footnote-back-ref">↩</a></li>
<li id="fn:16">Adams, GP; Schier, R; McCall, AM; Crawford, RS; Wolf, EJ; Weiner, LM; Marks, JD (1998). "Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu". British Journal of Cancer. 77 (9): 1405–12. doi:10.1038/bjc.1998.233. PMC 2150193. PMID 9652755. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2150193" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2150193</a> <a href="#fnref:16" class="footnote-back-ref">↩</a></li>
<li id="fn:17">Vostakolaei, Mehdi Asghari; Molavi, Ommoleila; Hejazi, Mohammad Saeid; Kordi, Shirafkan; Rahmati, Saman; Barzegari, Abolfazl; Abdolalizadeh, Jalal (September 2019). "Isolation and characterization of a novel scFv antibody fragments specific for Hsp70 as a tumor biomarker". Journal of Cellular Biochemistry. 120 (9): 14711–14724. doi:10.1002/jcb.28732. ISSN 0730-2312. PMID 30998271. S2CID 121351794. <a href="https://onlinelibrary.wiley.com/doi/10.1002/jcb.28732" target="_blank">https://onlinelibrary.wiley.com/doi/10.1002/jcb.28732</a> <a href="#fnref:17" class="footnote-back-ref">↩</a></li>
</ol>

Single-chain variable fragment open-in-new

Single-chain variable fragment