Coding

Part:BBa_K4829007

Designed by: Aditya Kamath Ammembal   Group: iGEM23_IISc-Bengaluru   (2023-10-05)


Sequence coding for a dAb against IL8

This sequence codes for a dAb against IL8, and you may use the sequence appended to a 6X Histag/ FLAG tag to purify the same. Please note that we have not tested this amino acid sequence by producing it. We have ONLY done the modelling. All the details of the modelling may be found on our dry lab page.

Usage and Biology

This specific dAb is expected to be a neutralising one. We have not used it for neutralising purposes ourselves yet. However, the potential use of this dAb would be to block IL8 either in disease processes or possibly even in cell biology assays/related studies. We have not included his tag/FLAG tag in this sequence. However, it would be prudent to do so, for purification purposes. This part has been introduced as a part of our mRNA-based therapeutic platform we are introducing on the biobricks registry. The sequence has been optimised for production by human cells. We therefore also encourage any users of this part to make a composite along the lines of BBa_K4829003 to use this and characterise it further.

  • This part is intended to be used as an mRNA therapeutic for endometriosis (for further information, you may look at our wiki). This part is also intended for conditions like triple-negative breast cancer, ovarian cancer and colorectal cancer. (Check the references).

dAb's: A Biological Overview

  • Domain antibodies (dAbs) are unique human variable domains (either VH or VL) that have been modified to prevent them from pairing up while keeping their specific antigen-binding capability.
  • This modification often uses a process called "camelisation," where hydrophobic parts typically seen in the VH/VL interface are replaced with hydrophilic parts similar to those in camelid VHH, along with an extension in the CDRH3.
  • These molecules, similar to nanobodies in size and structure, have properties like high stability, solubility, and a brief half-life. They are also easily fused with other proteins and produced in large quantities using microbes.
  • While dAbs themselves have some therapeutic uses, they're mainly explored as fusion proteins combined with other entities, such as full antibodies for dual specificity, an Fc domain, or an anti-albumin dAb, as seen in GSK/Domantis' AlbudAb®s.

In the design section, we will elaborate on the process of camelisation.

dAb's: A structural overview

dAb's are generally engineered antibody fragments, approximately 120 amino acids long. They are essentially the variable regions of the heavy chain of a human antibody with some key amino acid changes elucidated in the design part. However, in terms of a general structure, they have the following features:

  • In each of the two variable domains of the scFv, there are three distinct regions known as complementary determining regions (CDRs) that are interconnected by framework regions (FRs).
  • The CDRs play a key role in binding to antigens, with their structure tailored to match the epitope. On the other hand, the FRs serve primarily as a support structure and show minimal variability compared to the CDRs. Notably, each CDR contributes differently to antigen binding.
  • For example, the heavy chain's CDR3 is especially vital, contributing to 29% of the binding specificity, whereas the CDR2L's contribution is a mere 4%.
  • dAb's, similar to VH, contain nine beta-strands that create a standard IgV fold. The absence of the VL in nanobodies leads to significant structural differences, particularly in the FR2 region and hypervariable loops. In a standard VH region, the FR2 has four conserved hydrophobic amino acids that help in VL joining. However, in dAb's, these hydrophobic residues are replaced with hydrophilic ones to prevent unwanted exposure to solvents. This change, coupled with the rotation of nearby residues and the protective folding of the CDR3 domain over this interface, enhances the solubility of dAb's compared to VH domains and scFvs.

The specific features of this dAb have been modelled by our dry lab team, and the details may be found on our dry lab page. A brief summary of the details is mentioned below.

The image shown here is of the model of the nanobody, predicted using Alphafold

il8nbdock.png Figure 1. IL8(red) binding to our dAb(green)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 46
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 141


Functional Parameters

The functional parameters we have been able to measure are the Gibbs free energy of binding and the Kd of the interaction with IL8. These details have been obtained using the GRAMM software. A full and detailed explanation of how this tool was used may be found on our dry lab page, and we encourage any users of this part to go through the extensive documentation there. We will post a few images of the binding parameters obtained below:

  • il8nbdata.jpg
 Figure 2. The binding parameters obtained from the GRAMM software

References

  • Yue Wang, Rui Cheng Xu, Xiao Lei Zhang, Xiu Long Niu, Ye Qu, Ling Zhi Li, Xiang Yan Meng,Interleukin-8 secretion by ovarian cancer cells increases anchorage-independent growth, proliferation, angiogenic potential, adhesion and invasion,Cytokine,Volume 59, Issue 1,2012,Pages 145-155,ISSN 1043-4666
  • Yin, J., Zeng, F., Wu, N. et al. Interleukin-8 promotes human ovarian cancer cell migration by epithelial–mesenchymal transition induction in vitro. Clin Transl Oncol 17, 365–370 (2015). https://doi.org/10.1007/s12094-014-1240-4
  • Suyun Huang; Jubilee B. Robinson; Ariel DeGuzman; Corazon D. Bucana; Isaiah J. Fidler. Blockade of Nuclear Factor-κB Signaling Inhibits Angiogenesis and Tumorigenicity of Human Ovarian Cancer Cells by Suppressing Expression of Vascular Endothelial Growth Factor and Interleukin 8. Cancer Res (2000) 60 (19): 5334–5339.
  • J. Fujimoto, I. Aoki, S. Khatun, H. Toyoki, T. Tamaya. Clinical implications of expression of interleukin-8 related to myometrial invasion with angiogenesis in uterine endometrial cancers. Gynecologic tumors. https://doi.org/10.1093/annonc/mdf078.
  • Malihe Azadehrah, Shohre Vosoogh, Mahboobeh Azadehrah,The roles and therapeutic applications of cytokines in endometrial cancer,Journal of Reproductive Immunology,Volume 152,2022,103652,ISSN 0165-0378,https://doi.org/10.1016/j.jri.2022.103652.
  • RAZVAN CIORTEA1, DAN MIHU1 and CARMEN MIHAELA MIHU2. Association Between Visceral Fat, IL-8 and Endometrial Cancer. ANTICANCER RESEARCH 34: 379-384 (2014).
  • Lauren Ewington, Alexandra Taylor, Ruethairat Sriraksa, Yoshiya Horimoto, Eric W.-F. Lam, Mona A. El-Bahrawy,The expression of interleukin-8 and interleukin-8 receptors in endometrial carcinoma,Cytokine,Volume 59, Issue 2,2012,Pages 417-422,ISSN 1043-4666,https://doi.org/10.1016/j.cyto.2012.04.036.
  • Fang Deng, Yaguang Weng, Xian Li, Teng Wang, Mengtian Fan, Qiong Shi,Overexpression of IL-8 promotes cell migration via PI3K-Akt signaling pathway and EMT in triple-negative breast cancer,Pathology - Research and Practice,Volume 216, Issue 4,2020,152902,ISSN 0344-0338,https://doi.org/10.1016/j.prp.2020.152902.
  • Kim, S., Lee, J., Jeon, M. et al. MEK-dependent IL-8 induction regulates the invasiveness of triple-negative breast cancer cells. Tumor Biol. 3
  • Alraouji, N. N. and Aboussekhra, A. (2020). Tocilizumab inhibits il‐8 and the proangiogenic potential of triple negative breast cancer cells. Molecular Carcinogenesis, 60(1), 51-59. https://doi.org/10.1002/mc.23270
[edit]
Categories
Parameters
None