Part:BBa_K4829016

IVT of this sequence produces mRNA coding for an scFv antibody blocking PD1

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 Albumin(HSA) signal peptide, we urge the users of the antibody for this application(mRNA protein production) to try out various different signal peptides

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 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_K4829010), 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 scFv antibody is meant to block PD1 and act as a checkpoint inhibitor.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on the surface of activated T cells, B cells, and other immune cells. It plays a critical role in regulating immune responses, particularly in the context of preventing autoimmunity and controlling immune reactions in peripheral tissues.

Role of PD-1:

Immune Regulation: PD-1 is a key negative regulator of T cell activation and function. Its main role is to prevent excessive immune responses and autoimmunity.
Interaction with Ligands: PD-1 has two main ligands, PD-L1 (programmed death-ligand 1) and PD-L2. These ligands are expressed on a variety of cells, including tumour cells, antigen-presenting cells, and some non-immune cells in various tissues.
Tumor Immune Evasion: Many tumours upregulate the expression of PD-L1, which binds to PD-1 on T cells, leading to the inhibition of T cell function. This mechanism allows tumours to evade immune surveillance.

Single-Chain Variable Fragment (scFv) Antibodies:

Definition: scFv antibodies are composed of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, linked together by a short peptide linker. They represent the smallest functional fragment of an antibody that retains the antigen-binding capacity.
Advantages: Due to their small size, scFv antibodies can penetrate tissues more effectively than full-sized antibodies. They can be engineered easily and produced in bacteria or yeast, making them cost-effective. Their small size also means they are cleared from the body more rapidly, which can be advantageous in reducing potential side effects.

Use of scFv Antibodies in Blocking PD-1:

Targeting PD-1: scFv antibodies can be engineered to specifically target PD-1, blocking its interaction with its ligands (PD-L1 and PD-L2). By doing so, they can reactivate T cells that have been inhibited by tumours, enhancing the anti-tumour immune response.
Incorporation into Cell Therapies: scFv antibodies against PD-1 can be used to construct chimeric antigen receptor (CAR) T cells. In this approach, T cells are engineered to express CARs that incorporate scFv fragments targeting tumour antigens, along with intracellular signalling domains. By including a PD-1 blocking scFv, these CAR T cells can be made resistant to the inhibitory effects of PD-L1 expressed by tumours.
Combinatorial Therapies: PD-1 blocking scFv antibodies can be combined with other therapies, including other immunotherapies, radiation, or chemotherapy, to enhance the overall anti-tumor response.

In conclusion, PD-1 is a crucial checkpoint in immune regulation, and its blockade has emerged as a promising strategy in cancer immunotherapy. The use of scFv antibodies provides a versatile and efficient means to target PD-1 and can be integrated into various therapeutic modalities to boost anti-tumour immunity.

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

This sequence codes for an scFv against PD1. This sequence was designed by us, yet we have not produced this sequence. This has been modelled thoroughly, and its binding kinetics have been analysed using the GRAMM software.

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

  Figure 2:scFv (red) docked to PD1 (green).Docking generated using Free Docking by GRAMM

The docking data of the scFv to PD1 is as follows:

  Figure 3:The binding parameters obtained from the GRAMM software

 Figure 4: The structure of the RNA of the CDS produced on IVT of this sequence. 'Loss' function value (AUP) = 279.5870000000001

 Scale of the AUP

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

References

Świderska, J.; Kozłowski, M.; Kwiatkowski, S.; Cymbaluk-Płoska, A. Immunotherapy of Ovarian Cancer with Particular Emphasis on the PD-1/PDL-1 as Target Points. Cancers 2021, 13, 6063. https://doi.org/10.3390/cancers13236063
Purushottam Lamichhane, Lavakumar Karyampudi, Barath Shreeder, James Krempski, Deborah Bahr, Joshua Daum, Kimberly R. Kalli, Ellen L. Goode, Matthew S. Block, Martin J. Cannon, Keith L. Knutson; IL10 Release upon PD-1 Blockade Sustains Immunosuppression in Ovarian Cancer. Cancer Res 1 December 2017; 77 (23): 6667–6678. https://doi.org/10.1158/0008-5472.CAN-17-0740
Elina Khatoon, Dey Parama, Aviral Kumar, Mohammed S. Alqahtani, Mohamed Abbas, Sosmitha Girisa, Gautam Sethi, Ajaikumar B. Kunnumakkara,Targeting PD-1/PD-L1 axis as new horizon for ovarian cancer therapy,Life Sciences,Volume 306,2022,120827,ISSN 0024-3205,https://doi.org/10.1016/j.lfs.2022.120827.
Cathalijne C.B. Post, Anneke M. Westermann, Tjalling Bosse, Carien L. Creutzberg, Judith R. Kroep,PARP and PD-1/PD-L1 checkpoint inhibition in recurrent or metastatic endometrial cancer,Critical Reviews in Oncology/Hematology,Volume 152,2020, 102973,ISSN 1040-8428,https://doi.org/10.1016/j.critrevonc.2020.102973.
Barroso-Sousa R, Ott PA. PD-1 inhibitors in endometrial cancer. Oncotarget. 2017 Nov 21;8(63):106169-106170. doi: 10.18632/oncotarget.22583. PMID: 29290936; PMCID: PMC5739721.
Di Tucci Chiara,Capone Carmela, Galati Giulia, Iacobelli Valentina, Schiavi Michele C,Di Donato Violante,Muzii, Ludovico Panici, Pierluigi Benedetti. Immunotherapy in endometrial cancer: new scenarios on the horizon. doi: 10.3802/jgo.2019.30.e46
Yang, T.; Li, W.; Huang, T.; Zhou, J. Immunotherapy Targeting PD-1/PD-L1 in Early-Stage Triple-Negative Breast Cancer. J. Pers. Med. 2023, 13, 526. https://doi.org/10.3390/jpm13030526
Wang C. A Meta-Analysis of Efficacy and Safety of PD-1/PD-L1 Inhibitors in Triple-Negative Breast Cancer. J Oncol. 2022 Jan 21;2022:2407211. doi: 10.1155/2022/2407211. PMID: 35096057; PMCID: PMC8799355.
Matteo Santoni, Emanuela Romagnoli, Tiziana Saladino, Laura Foghini, Stefania Guarino, Marco Capponi, Massimo Giannini, Paolo Decembrini Cognigni, Gerardo Ferrara, Nicola Battelli,Triple negative breast cancer: Key role of Tumor-Associated Macrophages in regulating the activity of anti-PD-1/PD-L1 agents,Biochimica et Biophysica Acta (BBA) - Reviews on Cancer,Volume 1869,Issue 1,2018,Pages 78-84,ISSN 0304-419X,https://doi.org/10.1016/j.bbcan.2017.10.007.