Part:BBa_K4696507
(CAR-MA version 1) TMP controlled, srRNA-based CAR-M cell promoting system
Overview
CAR-Ma is a composite of parts BBa_K4696504andBBa_K4696505, which contribute to efficient and strong long-term expression of the Chimeric Antigen Receptor (CAR) and transfected macrophage M1 polarization in a scalable and controlled manner.
The final design for Phase I CAR-Ma evolved from a viral vector-based in vitro system. The Phase I design combines our 3rd generation CAR complex encoded by our advanced srRNA vector, with our Cas9-mediated SIRPa knockout. This combines the best of both worlds, allowing for non-integrative, long-term, and modulated expression into a system that would be applicable both in vitro and eventually in vivo, to provide maximal efficiency and efficacy.
NOTICE: Characterization of the part is in progress as of the Wiki Freeze 2023. Results will be expounded upon during the Presentation Video and the Giant Jamboree
The Scalable Controlled Amplifying Replicon System (SCARS) BBa_K4696504
Figure 1. The original design of Control over Protein Translation from a TMP-Responsive Replicon mRNA [8]. (1) nsP1-4 are translated as a complete replicon. (2) The replicon recognise the 2 Subgenomic Promoter(SGP), (3)amplify the expression of DD-L7Ae and Leu/mEPO protein. (4) the DD-L7Ae is degraded rapidly due to the destabilization domain(DD). (5) The presence of TMP stabilizes the L7Ae-DD from being degraded so it binds on the 2x k-turn upstream of the luc/mEPO ori, therefore, Leu/mEPO translation is suspended.
The Scalable Controlled Amplifying Replicon System (SCARS) involves two major components: DD-L7Ae protein complexes and 2x k-Turn motifs. The L7Ae protein, derived from an archaic bacteria, is a ribosomal binding protein that selectively binds to the 2x K-Turn sequence, inducing a strong conformational change within the KTurn. When the KTurn is positioned directly upstream of an ORF (Open Reading Frame), the conformational change triggered by L7Ae binding prevents any downstream translation.
By combining L7Ae with DDd, a degradation domain evolved from an enzyme found in E. coli, the L7Ae-DDd complexes are specifically designed to degrade rapidly when left untreated. However, the addition of TMP (Trimethoprim) treatment stabilizes DDd and prevents its degradation, as well as the degradation of any other protein attached to it. In our system, the DD-L7Ae control section is constantly expressed, but it quickly degrades due to the degradation capability of DDd, rendering it inert. Under TMP treatment, DD-L7Ae is effectively stabilized. This allows DD-L7Ae to bind to the 2x K-Turn site and shut down all downstream expression [9].
NOTICE: The DD-L7Ae complex can be placed in three places depending on the needs of the system designed. They can be placed before the replication complex upstream of nsP1, within the replication complex between nsP3 and nsP4 and finally alongside the gene of interest which is the CAR construct in our case.
M1 CAR-M driver BBa_K4696505
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 4. The DNA sequence of hYP7 scFv [2].
We linked the previously mentioned hu9F2 and hYP7 scFvs separately to other components of the CAR construct, resulting in two distinct anti-GPC3 CARs. The CAR construct components are arranged in a cluster of differentiation (CD) format. Each CAR consists of a scFv-CD8-CD28-CD3 configuration.
First, the CD8 component is divided into two parts: CD8α and CD8β. CD8α serves as a flexible hinge, providing spatial separation between the scFv and the intracellular domain. This spatial separation optimizes antigen recognition, enhancing specificity and affinity towards the target antigen. CD8β, on the other hand, functions as a transmembrane domain, connecting the extracellular and intracellular sections of the CAR [4].
Second, CD28 is included as a co-stimulatory domain. CD28 plays a crucial role in stimulating multiple cellular activation signaling pathways. This stimulatory effect is attributed to tyrosine and proline-based motifs present in the intracellular domain of CD28, which can bind to src homology 2 and 3 domains [5].
Third, CD3 is incorporated into our design, specifically CD3ζ with the Immunoreceptor Tyrosine-based Activation Motif (ITAM). Upon engagement of the scFv with the GPC3 antigen, CD3ζ is phosphorylated, leading to the release of downstream signaling molecules.
In summary, we have developed a simple yet efficient CAR construct that activates macrophages to target GP3C-rich cancer cells.
The problem of Macrophage polarization state
Figure 5. The M1 macrophage polarization pathway. Activation by PMA and M1 polarization by IFN-γ
However, the CAR construct alone would not be sufficient for a fully mature immunotherapy approach. Despite macrophages being abundant in the tumor microenvironment (TME), they are predominantly M2-polarized tumor-associated macrophages (TAMs) [6]. In fact, TAMs can constitute up to 80% of the tumor volume in most solid tumors [7]. Therefore, the key to achieving a successful CAR-Ma lies in reprogramming TAMs to adopt an M1-polarized state.
Interferon-gamma (IFN-γ), a cytokine produced by helper T-cells or natural killer cells, plays a crucial role in macrophage M1 activation [8]. To enhance the functionality of CAR-Ma, we have designed a system that incorporates the expression of IFN-γ alongside the CAR molecules. This ensures that the engineered CAR-Ma cells maintain an M1 polarization state upon infiltration 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
[8] Mc Cafferty S, De Temmerman J, Kitada T, et al. In Vivo Validation of a Reversible Small Molecule-Based Switch for Synthetic Self-Amplifying mRNA Regulation. Mol Ther. 2021;29(3):1164-1173. doi:10.1016/j.ymthe.2020.11.010
[9] Yoshioka, N., Gros, E., Li, H.-R., Kumar, S., Deacon, D. C., Maron, C., Muotri, A. R., Chi, N. C., Fu, X.-D., Yu, B. D., & Dowdy, S. F. (2013). Efficient generation of human ipscs by a synthetic self-replicative RNA. Cell Stem Cell, 13(2), 246–254. https://doi.org/10.1016/j.stem.2013.06.001
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2171
Illegal EcoRI site found at 10689
Illegal SpeI site found at 2119
Illegal PstI site found at 4500
Illegal PstI site found at 8861 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2171
Illegal EcoRI site found at 10689
Illegal NheI site found at 5371
Illegal NheI site found at 6559
Illegal NheI site found at 7376
Illegal NheI site found at 9604
Illegal SpeI site found at 2119
Illegal PstI site found at 4500
Illegal PstI site found at 8861 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2171
Illegal EcoRI site found at 10689
Illegal BglII site found at 2293
Illegal BamHI site found at 54
Illegal BamHI site found at 2155
Illegal BamHI site found at 2518
Illegal BamHI site found at 5807
Illegal BamHI site found at 7529
Illegal XhoI site found at 5535
Illegal XhoI site found at 5596
Illegal XhoI site found at 5637
Illegal XhoI site found at 8323 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2171
Illegal EcoRI site found at 10689
Illegal SpeI site found at 2119
Illegal PstI site found at 4500
Illegal PstI site found at 8861 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2171
Illegal EcoRI site found at 10689
Illegal SpeI site found at 2119
Illegal PstI site found at 4500
Illegal PstI site found at 8861
Illegal NgoMIV site found at 2960
Illegal AgeI site found at 60
Illegal AgeI site found at 5076
Illegal AgeI site found at 5445
Illegal AgeI site found at 5813
Illegal AgeI site found at 7535 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1046
Illegal BsaI site found at 5634
Illegal BsaI site found at 5652
Illegal BsaI site found at 8951
Illegal BsaI.rc site found at 2423
Illegal BsaI.rc site found at 3070
Illegal BsaI.rc site found at 5161
Illegal BsaI.rc site found at 9363
Illegal SapI.rc site found at 9568
| None |