Part:BBa_K4696505
(M1 CAR-M driver v1) Hu9f2-CAR-P2A-INF-gamma
The whole CAR construct starts with the single-chain variable fragment (scFv) [in green], this part would be protruding on the extracellular side and guide the CAR-M towards our target. The CD8 hinge [in blue] is the part that connects scFv with the intracellular component, in which CD28 [in purple]. As for the intracellular part, i.e. part of CD28 and CD3 [in orange], they mainly serves as the costimulatory domain which kickstarts downstream signaling that is responsible for CAR-M’s functionality. We designed two version of CAR construct, i.e. CAR per se and CAR – P2A – IFN-γ. The yellow part shown in the above diagram belongs to the latter design, where the translation will generate IFNγ, which was separated from the CAR structure by P2A, a self-cleaving protein. The IFN-γ would subsequently enhance the CAR-M’s function. Only the pivotal elements for the CAR to function are listed above, other elements on the plasmid we designed are omitted for conciseness.
Figure 1. The DNA sequence of hu9f2 scFv [3].
In the CAR-MA, 2 scfv are used, and both of the scfv we used are humanized. The first one is hu9F2, which was shown above in the plasmid. It was derived from a CAR-T structure patented by the Shanghai Cancer Institute in the European Patent Office (EP3789486A1) [3]. Another one is hYP7, which was derived from a high-affinity mouse anti-GPC3 antibody, humanized through grafting dual CDRs onto its most similar human germline sequence, reducing immunogenic reaction [2]. First, CD8 is separated into 2 parts of CD8α and CD8β. CD8α is a flexible CD8 hinge that provides a spatial separation between the scfv and intracellular domain, facilitating optimal antigen recognition and therefore enhancing the specificity and affinity towards the target antigen. The CD8β serves as a transmembrane domain connecting the extra- and intracellular sections of CAR[4]. Second, CD28 here is a co-stimulatory section. CD28 is mature in stimulating multiple cellular activation signaling pathways. he molecular mechanism lies within the presence of tyrosine and proline-based motifs on the intracellular domain of CD28, which can bind to src homology 2 and 3 domains [5]. Thrid, CD3 is selected partly for our design, CD3ζ within the CD3ζ with the Immunoreceptor Tyrosine-based Activation Motif (ITAM). The CD3ζ will be phosphorylated once the scfv is engaged to the GPC3 antigen, which lead to downstream signaling molecules release. Overall, we created a simple but efficient CAR construct that activates the macrophage target GP3C-rich cancer cells,
The problem of Macrophage polarization state
However, the CAR construct alone wouldn't contribute a mature immunotherapy. Despite macrophages being one of the richest immuno-species in the tumor microenvironment(TME), they are mainly M2-polarized tumor-associated macrophages(TAMs) [6]. In fact, in most solid tumors, TAMs can constitute up to 80% volume [1]. Therefore, TAMs are the key to creating a successful CAR-M.
IFN-γ, a cytokine that originated from helper T-cells or natural killer cells, is the key to macrophage M1 activation [7].To enhance the function of CAR-Ma, we designed a system that expresses IFN-γ along with CAR molecules that ensure the engineered CAR-Ma will maintain the M1 polarization state once it infiltrates into the pro-M2 TME.
Reference
[1] Lundstrom K. Self-Replicating RNA Viruses for RNA Therapeutics. Molecules. 2018;23(12):3310. Published 2018 Dec 13. doi:10.3390/molecules23123310
[2] Zhang Y F , Ho M .Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma[J].Scientific Reports, 2016, 6:33878.DOI:10.1038/srep33878.
[3] Hua J H, Li W Z. Immune effector cell and use thereof. Shanghai Cancer Institute in the European Patent Office (EP3789486A1), 2019, https://patents.google.com/patent/EP3789486A1/en#citedBy
[4] Classon, B. J., Brown, M. H., Garnett, D., Somoza, C., Barclay, A. N., Willis, A. C., & Williams, A. F. (1992). The hinge region of the CD8α chain: Structure, antigenicity, and utility in expression of immunoglobulin superfamily domains. International Immunology, 4(2), 215–225. https://doi.org/10.1093/intimm/4.2.215
[5] Prasad, K. V., Cai, Y. C., Raab, M., Duckworth, B., Cantley, L., Shoelson, S. E., & Rudd, C. E. (1994). T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif. Proceedings of the National Academy of Sciences of the United States of America, 91(7), 2834–2838. https://doi.org/10.1073/pnas.91.7.2834
[6] Sloas, C., Gill, S., & Klichinsky, M. (2021). Engineered car-macrophages as adoptive immunotherapies for solid tumors. Frontiers in Immunology, 12. https://doi.org/10.3389/fimmu.2021.783305
[7] Allavena, P., & Mantovani, A. (2012). Immunology in the clinic review series; focus on cancer: Tumour-associated macrophages: undisputed stars of the inflammatory tumour microenvironment. Clinical and Experimental Immunology, 167(2), 195–205. https://doi.org/10.1111/j.1365-2249.2011.04515.x
[8] Martinez, F. O., & Gordon, S. (2014). The M1 and M2 paradigm of macrophage activation: Time for reassessment. F1000Prime Reports, 6, 13. https://doi.org/10.12703/P6-13
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