Coding

Part:BBa_K4829009

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


Sequence coding for a dAb against IL6

This sequence codes for a nanobody against IL6, and you may use the sequence with 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 and analysed the binding kinetics via the GRAMM software. All the details of the modelling ay be found on our dry lab page.

Usage and Biology

IL-6 Overview:

  • Structure and Production: IL-6 is a four-helix bundle protein produced by various cell types, including T cells, B cells, macrophages, endothelial cells, and fibroblasts.
  • Receptor: IL-6 exerts its effects by binding to its receptor IL-6R. The complex of IL-6 and IL-6R associates with glycoprotein 130 (gp130), leading to the activation of intracellular signaling pathways.
  • Signalling: Two major pathways are involved in IL-6 signalling: the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway and the MAPK pathway.
  • Functions: IL-6 is involved in immune regulation, inflammation, hematopoiesis, and neural function. It can act in both pro-inflammatory and anti-inflammatory capacities.

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.

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, contains 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

  • il6nb.jpg
 Figure 1. The modelling of the dAb using alphafold2
  • il6nbdock.jpg 
 Figure 2. IL6(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 XhoI site found at 13
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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 image of the binding parameters obtained below:

  • il6nbdata.jpg
 Figure 3. The binding parameters obtained from the GRAMM software

References

  • Browning L, Patel MR, Horvath EB, Tawara K, Jorcyk CL. IL-6 and ovarian cancer: inflammatory cytokines in promotion of metastasis. Cancer Manag Res. 2018 Dec 5;10:6685-6693. doi: 10.2147/CMAR.S179189. PMID: 30584363; PMCID: PMC6287645.
  • Zou, M., Zhang, X. & Xu, C. IL6-induced metastasis modulators p-STAT3, MMP-2 and MMP-9 are targets of 3,3′-diindolylmethane in ovarian cancer cells. Cell Oncol. 39, 47–57 (2016). https://doi.org/10.1007/s13402-015-0251-7
  • Joanna M. Watson, John L. Sensintaffar, Jonathan S. Berek, Otoniel Martínez-Maza; Constitutive Production of Interleukin 6 by Ovarian Cancer Cell Lines and by Primary Ovarian Tumor Cultures1. Cancer Res 1 November 1990; 50 (21): 6959–6965.
  • Patricia E. Ellis, Gemma A. Barron, Giovanna Bermano,Adipocytokines and their relationship to endometrial cancer risk: A systematic review and meta-analysis,Gynecologic Oncology,Volume 158, Issue 2,2020,Pages 507-516,ISSN 0090-8258, https://doi.org/10.1016/j.ygyno.2020.05.033.
  • Christine M. Friedenreich, Annie R. Langley, Thomas P. Speidel, David C.W. Lau,Kerry S. Courneya, Ilona Csizmadi, Anthony M. Magliocco, Yutaka Yasui and Linda S. Cook. Case–control study of inflammatory markers and the risk of endometrial cancer. European Journal of Cancer Prevention , JULY 2013, Vol. 22, No. 4 (JULY 2013),pp. 374-379. https://www.jstor.org/stable/10.2307/48504258
  • Shuwei Liang, Zhuojia Chen, Guanmin Jiang, Yan Zhou, Qiao Liu, Qiao Su, Weidong Wei, Jun Du, Hongsheng Wang, Activation of GPER suppresses migration and angiogenesis of triple negative breast cancer via inhibition of NF-κB/IL-6 signals, Cancer Letters,Volume 386,2017,Pages 12-23,ISSN 0304-3835,https://doi.org/10.1016/j.canlet.2016.11.003.
  • Tian, J., Chen, X., Fu, S. et al. Bazedoxifene is a novel IL-6/GP130 inhibitor for treating triple-negative breast cancer. Breast Cancer Res Treat 175, 553–566 (2019). https://doi.org/10.1007/s10549-019-05183-2
  • Zachary C. Hartman, Graham M. Poage, Petra den Hollander, Anna Tsimelzon, Jamal Hill, Nattapon Panupinthu, Yun Zhang, Abhijit Mazumdar, Susan G. Hilsenbeck, Gordon B. Mills, Powel H. Brown; Growth of Triple-Negative Breast Cancer Cells Relies upon Coordinate Autocrine Expression of the Proinflammatory Cytokines IL-6 and IL-8. Cancer Res 1 June 2013; 73 (11): 3470–3480. https://doi.org/10.1158/0008-5472.CAN-12-4524-T
  • Jin, K., Pandey, N.B. & Popel, A.S. Simultaneous blockade of IL-6 and CCL5 signaling for synergistic inhibition of triple-negative breast cancer growth and metastasis. Breast Cancer Res 20, 54 (2018). https://doi.org/10.1186/s13058-018-0981-3
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