Targeted Cancer Immunotherapy Using CAR-T Cells and LNP-Based Methods

Chimeric antigen receptors (CARs) are recombinant receptors that are designed as having extracellular part obtained from a monoclonal antibody’s single-chain variable fragment, which allows binding of the specific antigen (1, 2). CARs that consist tumor- specific antibody are expressed in activated T cells (CAR-T cells) ex vivo by using viral vectors, so that T cells are directly targeted to tumor cells by synthesizing specific CAR on their surfaces (3). In the human immune system, antigens are presented by specific HLA molecules for T cell activation (2). However, because CAR-T cells have antigen-specific antibodies on their surfaces, they do not use HLA for interacting with antigens (2, 3). They are not HLA-restricted, so they are successful in severe cancers that reduce HLA expression (2).

The CAR-T cells’ construction is based on the modification of the patient’s T cells to express CAR (4). Artificially activated T cells are isolated from the blood of a patient, and antigen-specific CAR construction is inserted into T cells ex vivo (1, 4). This insertion can be done by viral transduction of transposase systems (1). CAR-T cells are proliferated in flasks or plates until an adequate number of CAR-T cells are obtained (1, 4). Then the purified CAR- T cells are injected back into the patient (4). B- cell acute lymphoblastic leukemia (B-ALL) is the most common type of childhood cancer, characterized by the presence of transformed precursor B cells that have genetic abnormalities like chromosomal translocation or point mutation (5, 6). If CAR-T cells are designed to target the CD-19 antigen, which is present on the malignant B cells, they will deal with B-ALL (5, 6).

CAR-T manufactorization. Source: Faeq, M., H., et al. (2023). Medical Oncology.

On-target off-tumor response is one of the main safety concerns of using CARs (3). It arises when the targeted antigen of CAR-T cells is expressed on both healthy and targeted tissues (4). In this case, CAR-T eliminates both healthy and severe targets and can be lethal if these healthy tissues are the heart or lungs (4). The other disadvantage of CAR-T cells is cytokine release syndrome (CRS) (2, 1). It happens when a vast amount of cytokines is produced shortly after the infusion of CAR-T cells because of the large number of activated tumor-related T cells after meeting with the antigen (1, 2, 4). It causes hypotension, fever, and eventually organ failure (3).

Preparation of CAR-T cells ex vivo takes up to three weeks, and because it needs patient-specific arrangement, mass production is unacceptable (7). In addition, as mentioned before, because of not only a high ratio of suffering from CRS, adverse inflammation, neurotoxicity, and off- target effects in patients but also its dependence on viral vectors, manufacturing time length, and biosafety concerns, scientists are motivated to find alternative pathways instead of viral transduction and ex vivo T cell engineering (7, 8). The delivery of CAR-encoding messenger RNA (mRNA) to T cells came as a solution to evade these severe effects. mRNA does not integrate into the genome, which allows only transient CAR expression to prevent long-term effects of the CAR-T cell activity, like differentiation of T- cells (8, 9, 10).

In mRNA CAR-T therapy, T cells from the patient are taken and become competent by electroporation for delivering negatively charged CAR mRNA (9). However, electric pulses that are used in the electroporation method cause cell death and are conducted ex vivo, which makes this delivery system inefficient. The other more efficient way of delivery is encapsulating CAR mRNA in cationic nanoparticles like lipid nanoparticles (LNP) and polymer nanoparticles, which can reduce cytotoxicity and stabilize mRNA cargo (8, 9). Because polymer nanoparticles have a higher molecular weight than LNP, they can damage the tissues and cause off-target effects (7). On the other hand, LNP is composed high rate of phospholipids, so it cannot accumulate in the body (7). Lastly, using LNPs in a drug delivery system is widely studied, as used in the COVID-19 BioNTech vaccines (7, 8, 9).

LNP structure. Source: Bio-Techne (2024). Lipid Nanoparticles for mRNA Delivery.

LNPs are composed of four main parts. One of them is ionizable lipids, which are positively charged at low pH allows LNPs to escape from endosomes and promote cellular uptake (7, 8); whereas neutral at neutral pH allows them to be stable in the blood (7). Lipid- anchored polyethylene glycol (PEG) reduces aggregation (10). While cholesterol enables stability of LNP and membrane fusion, phospholipid ensures the LNP structure (8, 10). LNP encapsulates mRNA by its amorphous lipid matrix characteristics (10). Considering all these surface features of LNPs, changing their content ratio and length allows us to arrange where the LNP resides (10, 11). Also, LNP structure prevents mRNA from RNases and mechanical stress (10) while enabling the in vivo production of CAR-T cells by carrying mRNA to T cells (11).

Traditional method. Source: Metzloff, A., E., et al. (2024), Advanced Materials.

New method. Source: Metzloff, A., E., et al. (2024), Advanced Materials.

Legends

To conduct successful delivery of mRNA to T cells, T cells must be activated (9). In other words, firstly, major Histocompatibility Complex (MHC) proteins on the antigen-presenting cells (APC) must engage with CD3/T cell receptor (TCR) on the T-cell surface (9, 10). Secondly, CD80 and CD86 proteins on dendritic cells (DC), which are a type of APC, interact with CD28 proteins on the T-cells (8, 10). For this purpose, antibodies against CD3 and CD28 are linked to beads and used to target T cells, which are then removed (8). After activation of T cells, mRNA LNPs are added (8). The only difference is that while LNP carries CAR mRNA in this technique, viruses are carriers of CAR DNA in the traditional (previous) technique, and both of them conduct ex vivo. According to the new nanotechnology method, LNPs are chemically conjugated with CD3 and CD28 antibodies and create an active LNP (aLNP) form similar to APC (8,10). This method eliminates the need to activate the bead and directly activates T-cells using LNP (8, 10). aLNP encapsulates CAR mRNA, and aLNP binds T cells through its CD3 and CD28 antibodies. This binding triggers endocytosis, and aLNP is integrated into a T cell in an endosome (8, 9, 10). As the pH of the endosome decreases, ionizable lipids are charged positively (7, 8). Due to the negative charge of the endosome, the LNP is disintegrated and releases the mRNA (9, 10).

Interactions for activation of T cells. Created by Toni Kosanovic

To sum up, since ex vivo T-cell engineering is expensive and time-consuming, and dependency on viral vectors raises concerns about toxicity, inflammation, and off-target effects, LNP-based mRNA delivery in vivo has been developed. In vivo CAR-T engineering also has some drawbacks. An unpredictable off-target side effect and inadequate mRNA expression arise if the system is not optimized carefully (10, 11). To increase the specificity of the target, targeting strategies could be refined (12). Also, CAR-T cells should be designed to reach an adequate threshold quantity (10). In addition, mRNA insertion is not guaranteed due to pH-dependent release of mRNA (11). Because of that, new release strategies should be found in the future.

References:

1. Faeq, M. H., Al-Haideri, M., Mohammad, T. a. M., Gharebakhshi, F., Marofi, F., Tahmasebi, S., & Modaresahmadi, S. (2023). CAR-modified immune cells as a rapidly evolving approach in the context of cancer immunotherapies. Medical Oncology, 40(5), 155. https://doi.org/10.1007/s12032-023-02019-4

2. Davila, M. L., & Brentjens, R. J. (2016, October 1). CD19-Targeted CAR T cells as novel cancer immunotherapy for relapsed or refractory B-Cell acute lymphoblastic leukemia. https://pmc.ncbi.nlm.nih.gov/articles/PMC5536094/

3. Sadelain, M., Brentjens, R., & Rivière, I. (2013). The basic principles of chimeric antigen receptor design. Cancer Discovery, 3(4), 388–398. https://doi.org/10.1158/2159-8290.cd-12-0548

4. Kandra, P., Nandigama, R., Eul, B., Huber, M., Kobold, S., Seeger, W., Grimminger, F., & Savai, R. (2022). Utility and drawbacks of chimeric antigen receptor T cell (CAR-T) therapy in lung cancer. Frontiers in Immunology, 13, 903562. https://doi.org/10.3389/fimmu.2022.903562

5. Garcia, C., Miller-Awe, M. D., & Witkowski, M. T. (2024). Concepts in B-cell acute lymphoblastic leukemia pathogenesis. Journal of leukocyte biology, 116(1), 18–32. https://doi.org/10.1093/jleuko/qiae015

6. DeKoter, R. P., & Gibson, P. J. (2016). B-cell leukemia and lymphoma. In M. J. H. Ratcliffe (Ed.), Encyclopedia of immunobiology (pp. 279–286). Academic Press. https://doi.org/10.1016/B978-0-12-374279-7.09019-6

7. Zhou, J. E., Sun, L., Jia, Y., Wang, Z., Luo, T., Tan, J., Fang, X., Zhu, H., Wang, J., Yu, L., Yan, Z., & Yan, Z. (2022). Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo. Journal of Controlled Release, 350, 298–307. https://doi.org/10.1016/j.jconrel.2022.08.033

8. Billingsley, M. M., Hamilton, A. G., Mai, D., Patel, S. K., Swingle, K. L., Sheppard, N. C., June, C. H., & Mitchell, M. J. (2021). Orthogonal design of experiments for optimization of lipid nanoparticles for mRNA engineering of CAR T cells. Nano Letters, 22(1), 533–542. https://doi.org/10.1021/acs.nanolett.1c02503

9. Metzloff, A. E., Padilla, M. S., Gong, N., Billingsley, M. M., Han, X., Merolle, M., Mai, D., Figueroa‐Espada, C. G., Thatte, A. S., Haley, R. M., Mukalel, A. J., Hamilton, A. G., Alameh, M., Weissman, D., Sheppard, N. C., June, C. H., & Mitchell, M. J. (2024). Antigen presenting cell mimetic lipid nanoparticles for rapid mRNA CAR T cell cancer immunotherapy. Advanced Materials, 36(26), e2313226. https://doi.org/10.1002/adma.202313226

10. Ang, M. J. Y., Metzloff, A. E., Thatte, A. S., & Mitchell, M. J. (2025). Lipid nanoparticles for engineering next generation CAR T cell immunotherapy. Nanoscale Horizons, 11(1), 22–36. https://doi.org/10.1039/d5nh00432b

11. Khawar, M. B., Afzal, A., Si, Y., & Sun, H. (2024). Steering the course of CAR T cell therapy with lipid nanoparticles. Journal of Nanobiotechnology, 22(1), 380. https://doi.org/10.1186/s12951-024-02630-1

12. Tombácz, I., Laczkó, D., Shahnawaz, H., Muramatsu, H., Natesan, A., Yadegari, A., Papp, T. E., Alameh, M., Shuvaev, V., Mui, B. L., Tam, Y. K., Muzykantov, V., Pardi, N., Weissman, D., & Parhiz, H. (2021). Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs. Molecular Therapy, 29(11), 3293–3304. https://doi.org/10.1016/j.ymthe.2021.06.004