Part:BBa_K4829014

This sequence, on In-vitro transcription, produces mRNA coding for an scFv against IL6

This composite biobrick is one of multiple combinations possible using our modular mRNA-based protein expression systems. We have not produced this using IVT. However, considering our success with BBa_K4829003, we can confidently say that this will produce the required antibody. We would like to emphasise further, that though this composite bio brick has used the CD33 signal peptide, we urge the users of the antibody for this application(mRNA protein production) to try out various different signal peptides. On transfection, the mRNA formed from this should make the cell produce the antibody and secrete into the cell culture supernatant.

Usage and Biology

• Sequence coding for Q-IL6-9, an scFv against IL6
• This sequence HAS a 3 X FLAG tag and a 6 X Histag.
• This part is designed for In Vitro Translation/Transcription. If this DNA fragment in a backbone is transfected into a mammalian cell, it WILL NOT produce a protein.
• We recommend that anyone using this construct use modified nucleotides: N1-Methylpseudouridine, and cap the mRNA with CleanCapAG, to improve translational efficiency and reduce immune response to the mRNA.
• It has a T7 promoter (BBa K4829000), a 5'UTR(BBa K4829004), a coding sequence (which has a signal peptide(BBa K4829001) along with the scFv antibody(BBa_K4829008), a 3'UTR(BBa K4829005), and a polyA tail(BBa K4829006).
• On In Vitro Transcription, followed by purification, the mRNA produced can be used to transfect HeLa cells with Lipofectamine messengerMax. After 6 hours, the protein will be detected in the supernatant.
• This part is intended to be used as an mRNA therapeutic for ovarian cancer and TNBC(papers referenced).

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 740
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

Antibody sequence:

Q-IL6-9:  

This antibody 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.

 Figure 1: scFv model generated by AlphaFold2 (ColabFold with templates from PDB70)   

  

In fact, the small size of this antibody makes it optimal for us to use in mRNA-based therapeutics, as larger mRNA sequences are harder to encapsulate in an LNP.  

 Figure 2: scFv (red) docked to IL6 (green); Docking generated using Free Docking by GRAMM   

The docking data of anti-IL6 to IL6 is as follows:

 Figure 3: The binding parameters obtained from the GRAMM software

SGC Karolinska lab has published a compilation of extensive data on the probe: (PUBLISHED ON https://www.thesgc.org/biological-probes/il-6)

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 Figure 4: The structure of the RNA of the CDS produced on IVT of this sequence. 'Loss' function value (AUP) = 130.82000000000005 

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 Scale of the AUP

For more information on the AUP, please check out out pages of BBa_K4829019 onwards

References

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182554/
• https://journals.sagepub.com/doi/full/10.1177/0300891618784790
• https://aacrjournals.org/clincancerres/article/23/23/7375/133458/The-Immune-microenvironment-Confers
• https://aacrjournals.org/cancerimmunolres/article/7/12/1944/469540/IL6-Modulates-the-Immune-Status-of-the-Tumor
• 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. Bre