Composite

Part:BBa_K4829003

Designed by: Aditya Kamath Ammembal, Dr. Raghavan Varadarajan, Dr. Debajyoti Chakraborty   Group: iGEM23_IISc-Bengaluru   (2023-10-04)


This sequence, on In-vitro transcription, produces mRNA coding for an scFv against IL8

This composite bio brick, on In vitro transcription, produces an mRNA construct that, on transfection into a mammalian cell, produces the scFv antibody O-Il8-15. This antibody will be secreted outside the cell, due to the CD33 signal peptide at the beginning.

Usage and Biology

  • 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 suing 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 K4829002)), a 3'UTR(BBa K4829005), and a polyA tail(BBa K4829006).
  • On In Vitro Translation, 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 endometriosis (for further information, you may look at our wiki). This part is also intended for conditions like triple-negative breast cancer. (Check the references).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters

Expression in HeLa cells:

  • hela-blot.png
 Figure 1. Expression of the protein noted in the supernatant 6 hours post transfection ]]

The signal peptide has been shown to work! We used the constructs, with 2ug of mRNA mixed with 2ul of Lipofectamine MessengerMAX, in each well of a 12 well plate, each well having 0.5 million cells. The expression was noticed 6 hours post-transfection in the supernatant. The cell lysate was not loaded, and the control was clear media. The blot results were as follows:

The 2 lanes beside the bands are controls, the blot was done in duplicates. There was an unfortunate amount of background, yet the band at 31kDa is quite clear. Antibody sequence:

O-IL8-15:  

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.

  • il8ab.jpg
 Figure 2: scFv model generated by AlphaFold2 (ColabFold with templates from PDB70) 

  

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

  

  • il8abdock.jpg 
 figure3: scFv (red) docked to IL8 (green); Docking generated using Free Docking by GRAMM   

 

The docking data of O-IL8-15 to IL8 is as follows:

  • il8ab-data.jpg
 Figure 4. The binding parameters obtained from the GRAMM software


The SGC Karolinska also got the following data on the probe: (TEXT COPIED AS PUBLISHED ON https://www.thesgc.org/biological-probes/il-8 )

Neutrophil Migration Assay

Neutrophil migration was tested against 10ng/ml IL-8 (R&D Systems) or empty buffer (-).

il8abneutromig.jpg 

 Figure 5. O-IL8-15 scFv almost completely neutralizes IL-8 and blocks migration (yellow bar) as compared to another inactive scFv (L-IL8-7)

O-IL8-15 scFv has got an IC50 value of ~0.6 µg/ml

Methods 

Neutrophil Migration Assay:

“Neutrophil granulocytes were isolated from heparinised peripheral blood of healthy donors using dextran sedimentation (3% dextran, added in 1:1 ratio) followed by Ficoll gradient separation. Residual red blood cells were lysed in hypotonic (0.2%) NaCl solution, followed by two washes in ice-cold PBS. The cells were thereafter suspended in Hanks’ balanced salt solution and 5% fetal bovine serum.

Cell migration was tested using 10 ng/ml recombinant human IL-8 (R&D Systems) as chemoattractant or empty buffer as negative control in Corning HTS Transwell 96-well permeable support plates with 3-micrometer pore size.

The number of migrated cells was studied following 60 minutes of incubation at 37 °C with the help of flow cytometry. Cell numbers were calculated by using AccuCheck counting beads (Thermo Fisher Scientific).”

Toxicity

As an essential part of any therapeutic, the same MUST be tested for evidence of toxicity. We have tested the toxicity of this antibody as follows: We utilise our amino acid sequence to estimate the toxicity of our anti-IL8 protein:

MPLLLLLPLLWAGALAEVQLLESGGGLVQPGGSLRLSCAASGFTFSYYGM

GWVRQAPGKGLEWVSGISYSGSGTYYADSVKGRFTISRDNSKNTLYLQMN

SLRAEDTAVYYCARDYVGNLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDI

QMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAS

SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDTPSTFGQGTK

LEIKRTDYKDHDGDYKDHDIDYKDDDDKAAALPETGGHHHHHH

To check for toxicity, we utilize CSM-Toxin, a web server for predicting protein toxicity, developed by Biosig Lab. CSM-Toxin provides a comprehensive suite for rapid identification of toxin proteins. Having uploaded our AA sequence to the server, the following result was obtained: toxicity-datail8.png


A detailed CSV was also provided as output, along with the following graph:

  • toxicity-graph.jpg
 Figure 6. The toxicity parameters obtained from the CSMtoxin software

Where a higher value indicates more toxicity of a given attention head. Clearly, our protein had close to no toxicity.

RNA Structure

  • il8abold.jpg
 Figure 7: The structure of the RNA of the CDS produced on IVT of this sequence. 'Loss' function value (AUP) = 337.67200000000014
  • scaleaup.jpg
 Scale of the AUP

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

BLI

  • We performed Bio Layer Interferometry, and got the following data at 6.25nM of IL8
  • 6-25il8bli.jpg
 Figure 8: The graph of BLI at 6.25nM of IL8. Kd=0.673nM

Biolayer Interferometry (BLI) is a label-free technology for measuring biomolecular interactions. It is similar in some respects to Surface Plasmon Resonance (SPR), but instead of measuring changes in refractive index on a metal surface, BLI measures the interference pattern of white light reflected from a biosensor tip.

Here's how BLI works:

  • Biosensor Tip Immersion: A biosensor tip, which is a fiber optic probe with a biocompatible layer, is immersed in a solution containing one of the binding partners (e.g., a protein).
  • Binding to the Tip: This molecule binds to the biocompatible layer, changing the optical thickness of the biosensor.
  • Interference Pattern Measurement: White light is directed down the fiber optic probe, and it reflects off both the tip's internal interface and the biolayer surface. When these reflected light waves overlap, they create an interference pattern.
  • Change in Interference Pattern: When molecules bind to or dissociate from the biosensor tip, the thickness of the biosensor's biolayer changes. This change in thickness alters the interference pattern.
  • Data Analysis: By monitoring these interference pattern shifts over time, BLI can provide real-time kinetics, affinity, and concentration data.

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


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