Coding

Part:BBa_K4829008

Designed by: SGC Karolinska   Group: iGEM23_IISc-Bengaluru   (2023-10-05)


Sequence coding for Q-IL6-9, an scFv against IL6

This part, Q-IL6-9 is an antibody in scFv format that binds to and blocks IL6. It was designed by SGC Karolinska, and not by us. We have uploaded here, the optimised DNA sequence (optimised for human expression). The sequence optimised for bacterial expression may be found at https://www.addgene.org/166552/


Usage and Biology

  • This scFv is intended for use against TNBC in our project. However, it could be used for functional assays also!

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.

IL-6 in Triple-Negative Breast Cancer (TNBC): Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) expression. TNBC is known for its aggressive nature, poorer prognosis, and limited treatment options compared to other breast cancer subtypes.

  • Tumour Microenvironment: IL-6 is often elevated in the tumour microenvironment of TNBC. The cytokine is produced both by the tumour cells themselves and by the surrounding stromal cells.
  • Promotion of Proliferation and Survival: IL-6 can promote the proliferation and survival of TNBC cells through the activation of STAT3, which leads to the transcription of genes involved in cell cycle progression and anti-apoptosis.
  • Stemness: IL-6 has been linked to the maintenance and enhancement of cancer stem cell-like properties in TNBC. These properties are associated with increased tumor-initiating potential, resistance to therapy, and metastasis.
  • Angiogenesis: IL-6 can promote angiogenesis in TNBC, further supporting tumour growth and dissemination.
  • Therapeutic Resistance: Elevated levels of IL-6 have been associated with resistance to chemotherapy in TNBC.
  • Immune Evasion: IL-6 can suppress anti-tumour immune responses by recruiting and activating myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the tumour microenvironment.
  • Potential Therapeutic Implications of blocking IL6:

Given the critical role of IL-6 in promoting TNBC progression, targeting the IL-6/IL-6R/gp130 signalling axis has emerged as a potential therapeutic strategy for TNBC. Various agents, including monoclonal antibodies against IL-6 or IL-6R and small molecule inhibitors targeting the downstream signalling pathways, are being investigated for their efficacy in TNBC.

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 595
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

Docking data

Q-IL6-9:   This antibody (designed by SGC Karolinska) is one in a very special format called ‘scFv’, short for short chain Fragment Variable. Single-chain variable fragment (scFv) antibodies are engineered antibody fragments that retain the antigen-binding capability of full-length antibodies but in a smaller, single-chain format. scFv antibodies are composed of the variable regions of an antibody’s heavy and light chains, connected by a flexible peptide linker. This design allows scFv antibodies to be produced in microbial systems like bacteria or yeast, enabling easier and more cost-effective production compared to traditional antibody formats. scFv antibodies have many applications, including diagnostics, therapeutics, and research tools. Their small size, high specificity, and modifiability make them valuable in targeting specific molecules and studying protein interactions in various biological systems.

  • il6ab.jpg
 Figure 1:scFv model generated by AlphaFold2 (ColabFold with templates from PDB70)
  • il6abdock.jpg
 Figure 2: scFv (red) docked to IL6 (green). Docking generated using Free Docking by GRAMM   

 

The docking data of Q-IL6-9 to IL6 is as follows:

  • il6abdata.jpg
 Figure 3: The binding parameters obtained from the GRAMM software

The SGC Karolinska also got a lot of data on the probe: (PUBLISHED ON https://www.thesgc.org/biological-probes/il-6)

References

  • Kang S, Tanaka T, Narazaki M, Kishimoto T. Targeting Interleukin-6 Signaling in Clinic. Immunity. 2019 Apr 16;50(4):1007-1023.
  • H. Persson, C. Preger, E. Marcon, J. Lengqvist, and S. Gräslund. Antibody Validation by Immunoprecipitation Followed by Mass Spectrometry Analysis. Synth. Antibodies Methods Protoc. Methods Mol. Biol. 2017; 1575, 175–187.

For other diseases

  • 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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