Part:BBa_K5036016
Nanobody2
Part Description
nanobodies are a unique type of antibody derived from camelids. These single- domain antibodies are significantly smaller and more stable, making them ideal for applications in diagnostics and therapeutics. Nanobodies can be used to develop highly sensitive and specific tests for a variety of diseases, including cancer and autoimmune disorders. Their compact size and stability also make them promising candidates for targeted drug delivery and other biomedical applications
Usage
we inserted nanobodies in NSP3A and MCP to bind specifically to MMP9 so that when MMP9 level increases, Nanobodies will bind to it and mediate circularization and initiate translation of YAP-1
This figure illustrates the structure of MMP9 Nanobody in our TID switch .
Dry lab Characterization
We Started by making a decision about which Nanobodies are candidates to be used in our project. We had to nominate 2 of 3 available nanobodies for the MMP-9 so we started by validating the nanobodies binding affinity to the MMP-9
MMP9-NB2 complex binding stability
This figure confirmed that The second nanobody-MMP9 interaction scored a binding stability (ΔG) of -10.1 kcal mol-1, which is considered an accepted binding stability. .
Then we compared the binding stability between different nanobodies to MMP9
This figure shows that NB1 and NB3 have higher affinity over the NB2 which make them candidates to be used in our switch .
We performed molecular dynamics for MMP9-NB2 to measure its stability in normal physiological function
Using amber notebook, the RMSD of MMP-9-NB2 under similar physiological conditions shows mild deviation that varies between 3 Å and 4.5 Å. On the other hand, there is significant fluctuation especially at 2 and 7 nanoseconds and between 300 to 400 residues. We can conclude that MMP-9-NB2 is stable .
Characterization by Mathematical Modeling
The model provides the interaction kinetics of MMP-9 to Nanobody-2 that can be connected to MCP or NSP3A to mediate our TID switch circulation. The result shows an increase in the binding complex upon MMP-9 interaction based on parametric values from literature.
Graph(1). Illustrates the relation between decreasing free MMP-9 (Blue line) upon their binding to nanobody-2 (orange line) that results forming a binding complex (Green line) .
Literature Characterization
A two-stage method was investigated for creating variable heavy-chain fragments (VHHs). This approach involved transferring complementarity-determining regions (CDRs) from non-camel antibodies to VHH frameworks, followed by affinity maturation using a synthetic phage library. botulinum neurotoxin A (BoNT/A) Was selected and fluorescein as model antigens. antibodies and antibody fragments Was generated against both targets and used the variable heavy-chain domains of scFvs that recognized the light chain of BoNT/A (BoNT/A-LC) or fluorescein as CDR donors. The cAbBCII-10 nanobody and the enhancer nanobody were used as acceptor frameworks for the BoNT/A-LC and fluorescein-specific CDRs, respectively
the figure compares the sequences of the constructed proteins. Complementarity-determining regions (CDRs) and framework regions were identified using the Kabat numbering system. In addition to the CDRs, amino acids in the upper core of the variable domains, which influence CDR conformation and orientation, were aligned with the donor sequences. The resulting framework changes are highlighted in this figure .
The engineered VHHs were expressed in E. coli, purified using affinity chromatography and evaluated for their ability to bind to their target antigens. As shown in Figure A, the grafted BoNT/A-LC VHH exhibited slightly improved binding to its antigen compared to the negative control (fluorescein-conjugated BSA).
The figure shows that The grafted fluorescein-specific VHH also demonstrated enhanced binding to fluorescein-conjugated BSA compared to the non-conjugated form .
Reference
Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Fry TJ, Orentas R, Sabatino M, Shah NN, Steinberg SM. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. The Lancet. 2015 Feb 7;385(9967):517-28.
Shin YJ, Park SK, Jung YJ, Kim YN, Kim KS, Park OK, Kwon SH, Jeon SH, Trinh le A, Fraser SE, Kee Y, Hwang BJ. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins. Sci Rep. 2015 Sep 16;5:14269. doi: 10.1038/srep14269. PMID: 26373678; PMCID: PMC4571616.
Wagner HJ, Wehrle S, Weiss E, Cavallari M, Weber W. A Two-Step Approach for the Design and Generation of Nanobodies. Int J Mol Sci. 2018 Nov 2;19(11):3444. doi: 10.3390/ijms19113444. PMID: 30400198; PMCID: PMC6274671.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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