Difference between revisions of "Part:BBa K4175000"

Latest revision as of 16:51, 8 October 2022

anti-IL-6 scFv

Sequence and Features

Biology

Anti-IL-6 single chain variable fragment (scFv) can bind to interleukin 6 (IL-6). It comprises of a variable heavy chain domain and a variable light chain domain, with a GSG linker joining the two domains. The variable heavy and light chain domain are derived from the human anti-IL-6 monoclonal antibody AME-19a (Tan et al., 2020).

Usage

Interleukin-6 (IL-6) is a pro-inflammatory cytokine that is pivotal to immune response, hematopoiesis, and acute-phase reactions. However, it is also a culprit for chronic inflammation and auto-immune diseases (Kaur et al., 2020). In the context of chimeric antigen receptor-T (CAR-T) therapy, IL-6 is found to mediate cytokine release syndrome (CRS), a concerning side effect of CAR-T therapy which could lead to multi-organ failure in the worst case (Leclercq et al., 2022).

To ameliorate the symptoms of CRS (e.g., fever, hypotension, respiratory deficiency), Tan et al. designed a membrane-bound form of IL-6 receptor (mbaIL6). They joined anti-IL-6 scFv shown herein with the CD8α hinge and transmembrane domain. mbaIL6 was cloned into pMSCV-IRES-GFP plasmid together with anti–CD19-41BB-CD3ζ construct, and the plasmid was expressed in T cells (Tan et al., 2020) (Fig 1).

Figure 1. (Tan et al., 2020) The construction of mbaIL6-expressing cells.

They found that when mbaIL6 and anti-CD19 CAR were simultaneously expressed (dual-expressed) in T cells, IL-6 derived from monocyte cell lines (THP-1) was effectively neutralized. This is likely to be due to the capture of IL-6 by mbaIL6. Meanwhile, the cytotoxic effect of CAR-T cells was not affected (Fig 2).

Figure 2. (Tan et al., 2020) (A) The measure of cytotoxicity of CAR-T cells against OP-1 mCherry cells. mbaIL6/CAR dual-expressing T cells showed comparative killing capacity as CAR-T cells. (B) IL-6 concentration in the culture supernatant after 48h co-incubation.

As in immunodeficiency mice injected with leukemic cells (Nalm-6 cells), mbaIL6-expressing CAR-T cells also showed an efficient IL-6 neutralizing effect and a nearly uncompromised anti-leukemia capacity (Tan et al., 2020) (Fig 3).

Figure 3. (Tan et al., 2020) (upper) 1*10^6 Nalm/6-luciferase cells were injected into NOD/scid-IL2RGnull mice. These mice were then treated with either CAR-T or mbaIL6/CAR dual T cells. The remaining level of Nalm/6 cells were evaluated through ventral image after D-luciferin injection. (lower) Either CAR-T cells or mbaIL6/CAR dual T cells were injected into NOD/scid-IL2RGnull mice. 50 ng of human IL-6 was injected three days later. Two hours later, the mice were sacrificed and the serum IL-6 level was measured.

For our usage, we intend to take advantage of anti-IL-6 scFv to design a negative feedback loop for CAR-T cell. We joined anti-IL-6 scFv with Notch core domain (BBa_K4175001) and ZF_GAl4_KRAB (BBa_K2446037) and expressed this device in CAR-T cells (IL-6_scfv-Notch-Gal4KRAB). We hoped that this device would inhibit the expression of CAR when IL-6 concentration reaches a deteriorating high level during CRS. For more detailed information, please see BBa_K4175008.

Figure 4. The schematic of IL-6_scfv-Notch-Gal4KRAB

References

Kaur, S., Bansal, Y., Kumar, R., Bansal, G., 2020. A panoramic review of IL-6: Structure, pathophysiological roles and inhibitors. Bioorg. Med. Chem. 28, 115327. https://doi.org/10.1016/j.bmc.2020.115327

Leclercq, G., Steinhoff, N., Haegel, H., De Marco, D., Bacac, M., Klein, C., 2022. Novel strategies for the mitigation of cytokine release syndrome induced by T cell engaging therapies with a focus on the use of kinase inhibitors. Oncoimmunology 11, 2083479. https://doi.org/10.1080/2162402X.2022.2083479

Tan, A.H.J., Vinanica, N., Campana, D., 2020. Chimeric antigen receptor–T cells with cytokine neutralizing capacity. Blood Adv. 4, 1419–1431. https://doi.org/10.1182/bloodadvances.2019001287