Coding

Part:BBa_K4829011

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


This codes for a dAb against PD1

This sequence codes for a nanobody against PD-1, 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 may be found on our dry lab page

Usage and Biology

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 PD1 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.

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.

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, contain 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(dAb) binding to PD1

  • pd1nbdock.jpg 
 Figure 1. PD1(red) binding to our dAb(green)

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

The functional parameters we have been able to measure are the Gibbs free energy of binding and the Kd of the interaction with PD1. 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 images of the binding parameters obtained below:

  • pd1nb-data.jpg
 Figure 2. The binding parameters obtained from the GRAMM software
  • pd1nb.jpg
 Figure 3. The modelling of the dAb using alphafold2

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.
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