By Jana Hennelová
Chimeric antigen receptor (CAR) T-cell therapy is a type of immunotherapy which has gained a lot of attention in recent years. This therapy relies on a chimeric T-cell receptor first introduced in 1987 (Kuwana et al., 1987). This discovery led to a development of the first-generation CARs by an Israeli immunologist Zelig Eshhar in 1993 (Eshhar et al., 1993). Since then, genetically engineered T-cells possessing this chimeric receptor have been used to develop an effective, targeted treatment for various types of cancer. This extensive research resulted in the first FDA-approved CAR T-cell therapy for B-cell lymphomas in 2017 (Braendstrup, Levine, Ruella, 2020). However, this treatment is mostly effective for hematological cancers while solid tumours are particularly difficult to treat (Marofi et al., 2021). Nevertheless, in recent years there have been many advantages in this field that aimed to modify this therapy for solid tumour treatment. Such methods involve either the combination of CAR T-cell therapy with other cancer immunotherapies, or the use of other types of immune cells, such as natural killer (NK) cells and macrophages. This review article first outlines the principle of CAR T-cell therapy and then discusses new developments in this field, with a particular focus on solid tumours.
1. Introduction: CAR T-cell therapy overview and side effects
CAR T-cells are genetically engineered T-cells that have an ability to recognise a specific protein (June & Sadelain, 2018). The process of producing such cells, as illustrated in Figure 1, relies on the genetic modification of T-cells to express CARs on their surface. The extracellular domain of these receptors is derived from antibodies, which are normally produced by B-cells, and can therefore bind to a specific antigen. Meanwhile, the intracellular T-cell activating domain of CARs triggers T-cells’ effector functions. This type of CARs, consisting only of these two domains, have been termed first generation CARs. Multiple studies have shown that these types of receptors are not sufficient to fully activate a corresponding T-cell (Brocker & Karjalainen, 1995). This is caused by a stepwise activation process of naïve T-cells described in Figure 2. To enhance the activation of CAR T-cells, co-stimulatory domains, such as CD28, have been implemented into CARs. These domains not only enhance the signalling capacity of T-cells, but also drive their expansion and prolong their lifespan in the bloodstream. This type of CARs are therefore referred to as second-generation CARs which are preferred in current forms of such therapy (June & Sadelain, 2018).
Figure 1. Principle of CAR T-cell therapy in cancer treatment.
T-cells are isolated from either a patient (autologous) or a donor (allogeneic) (1) and are genetically modified to express CARs on their surface. This modification can be done by various means, such as by using retroviral vectors or gene editing technologies like CRISPR-Cas9 (2). These cells are then cloned (3) and reinjected into the patient to target a specific type of cancer (4). Adapted from: (Albinger et al., 2021)
Figure 2. Naïve CD4 T-cell activation requires three signals.
The first step in CD4 T-cell activation is the recognition of a particular peptide displayed by class II major histocompatibility complex (MHC-II) on an antigen-presenting cell (APC) by a T-cell receptor (TCR). This interaction is stabilized by a co-receptor CD4 expressed on a T-cell (1). The second step is the interaction of a costimulatory molecule B7 on an APC and a co-receptor CD28 on a T-cell. B7 is upregulated only if the peptide displayed on MHC-II is a non-self antigen. If the peptide is a self antigen, the second signal is absent (i.e. B7 on APC is not upregulated), which leads to T-cell death or anergy (2). The final activation and T-cell differentiation into a particular subtype is regulated by cytokines (3). Adapted from: (O’Donnell & McSorley, 2014)
There are various antigens that engineered CAR T-cells could target. The first CAR T-cell therapy used CAR T-cells specific for B-lymphocyte antigen CD19. This molecule is exclusively present on all B-cells, therefore CD19 is an excellent target for immunotherapies against several types of lymphomas and leukaemias (Davila & Brentjens, 2016). Clinical studies that tested the efficiency of this therapy for B-cell acute lymphoblastic leukaemia (B-ALL) treatment showed that almost 90% of patients treated with anti-CD19 CAR T-cells went into complete remission (Davila et al., 2014). Despite such promising results, there have been various side effects associated with this type of therapy, which are described in Figure 3. The main issue observed in the aforementioned study was B-cell aplasia (i.e. the complete absence of B-cells in patients’ bloodstream) which lasted more than one year in some cases (Davila et al., 2014). B-cell aplasia occurs because CD19 is expressed on malignant and healthy B-cells, both of which are equally eliminated by anti-CD19 CAR T-cells. This condition, called on-target off-tumour toxicity, leaves patients vulnerable to various infections as there are no antibody-producing cells in the body. As a consequence, such individuals need to receive immunoglobulin infusions during this period (Davila & Brentjens, 2016).
Figure 3. Side effects associated with CAR T-cell therapy.
Cytokine release syndrome (CRS) is a consequence of a cytokine overproduction upon CAR T-cell administration and might lead to various serious pathological changes which are potentially lethal. Insertional oncogenesis might result from genetic modification of T-cells which could potentially cause their malignant transformation. The reason for neurological toxicity is not clear but could be associated with elevated cytokine levels. On-target off-tumour toxicity is a consequence of the presence of the target receptor on non-pathological tissues. The severity of this condition ranges from low and therefore manageable (e.g. B-cell aplasia) to lethal. Anaphylaxis develops when antigen recognition domains on CAR T-cells have been derived from murine antibodies and are therefore recognised as non-self by the immune system. This issue could be solved by using a humanised version of this domain. Adapted from: (Bonifant et al., 2016)
On-target off-tumour toxicity is one of the main reasons for which solid cancers are relatively difficult to treat by CAR T-cell therapy compared to haematological cancers. Solid tumours have a high level of heterogeneity, meaning that there is a large collection of surface molecules that varies between different tumour types and among patients (Sun & Yu, 2015). Unlike B-cells expressing CD19, for other cells in organs such as breasts, lung, and liver, there is no unique molecule expressed on their surface that CAR T-cells could target. This inability to target a specific structure on solid tumour cells usually results in a significant off-tumour toxicity which can lead to severe side effects, including death.
2. Combination of cancer immunotherapies for the treatment of solid tumours
A combination of two cancer immunotherapies, CAR T-cell therapy and oncolytic viruses, has a potential to reduce the side effects associated with on-target off-tumour toxicity of CAR T-cell therapy alone. The idea behind this is to specifically inject a bioengineered virus into tumour cells. The virus carries a gene for a modified, truncated version of CD19 (CD19t), inducing expression of this construct in solid tumour cells upon their infection. Following that, anti-CD19t CAR T-cells can be used to target only the tumour cells without damaging healthy tissues.
Park and colleagues (2020) tested the efficacy of this approach in vitro on human cancer cells, and in vivo on mouse tumour models. They engineered an oncolytic vaccinia virus to induce an expression of CD19t in tumour cells. The viral cell infection proved to be straightforward, resulting in almost 100% of tumour cells being CD19t-positive. The subsequent administration of anti-CD19t CAR T-cells showed a high immunological response against tumour cells, which resulted in up to 60% tumour regression. In comparison, oncolytic virus administration alone resulted in only 22% regression of tumours in mouse models. This could be explained not only by CAR T-cell cytolytic activity against tumour cells, but also by the enhanced propagation of the virus into tumour cells following the CAR T-cell mediated lysis of the nearby cells. Moreover, a long-term protection from cancer cell proliferation and a reduced chance of tumour relapse were observed. This could be a consequence of naïve T-cell activation by anti-CD19t CAR T-cells (Park et al., 2020).
Researchers further evaluated the differences in delivery systems of these therapies into patients. The most widely used protocol for the administration of oncolytic viruses is to directly inject a tumour with a virus. While this approach might be useful for ensuring that high viral concentration reaches the tumour, this approach is not very effective against metastatic tumours. Instead, they could benefit from a systemic administration of the therapies into patients. The current combination approach of immunotherapies developed by Park and colleagues (2020) is currently entering phase I clinical trials which will bring further information about the efficacy and safety of this approach for clinical settings.
3. Other immune cells in CAR therapies
3.1 NK cells and CAR NK therapy
Despite the promising results from the aforementioned study, there are some drawbacks with using T-cells in CAR therapies. One of them is graft-vs-host disease (GvHD), which is a transplant-related complication. It is caused by the incompatibility of human leukocyte antigens (HLAs) expressed on host and donor T-cells. Although the evidence about the role of NK cells in GvHD is contradictory (Simonetta, Alvarez, Segrin, 2017), it has been demonstrated that NK cells might be involved in suppressing GvHD and not contributing to GvHD themselves. Olson and colleagues (2010) showed that this is achieved by NK cell-mediated inhibition of activated T-cells. Therefore, the use of NK cells in CAR therapies might not only have fewer side effects, but also be faster and less costly, as NK cells from one donor could be administered into many patients without developing GvHD.
There are additional advantages in the use of NK cells in cancer immunotherapies. NK cells, unlike T-cells, are a part of the innate immune system. Therefore, they do not require activation by a specific antigen-presenting cell to destroy cancer cells (Abel, 2018). Instead, they become activated in response to atypical protein composition on a cell surface, releasing cytolytic proteins, perforin and granzyme, to destroy these cells. This means that NK cells, unlike T-cells, do not have to be modified to specifically respond to cancer cells. This offers a possibility to use NK cells to combat cancer in a CAR-independent manner, e.g. by increasing NK cell numbers in cancer patients without modifying them.
CAR-independent response of NK cells was studied by a biotechnological company Glycostem on a small number of patients with acute myeloid leukaemia. Those patients were administered with a single dose of NK cells derived from the umbilical cord blood. The results from phase I clinical trials were promising, showing improved survival rates in these patients (Dolstra et al., 2015).
However, there are body’s natural mechanisms that control the level of activation of unmodified NK cells to avoid immunopathology driven by their overactivation. Therefore, CAR-dependent NK cell therapies might increase the effectiveness of this type of cells in cancer immunotherapies. A study led by Rezvani and colleagues showed promising results when cancer patients were treated with CAR NK cells engineered to target native CD19 on malignant B-cells. 7 out of 11 patients went into a complete remission after the treatment, with no side effects observed after CAR T-cell therapy (Liu et al., 2020).
CAR-dependent and independent anti-tumour properties of NK cells make them great candidates not only for the treatment of hematological cancers, but also for heterogeneous solid tumours that cannot be specifically targeted and eliminated by T-cells.. Several pre-clinical and clinical trials have been conducted to test this approach. It has been shown that heterogeneous tumours that escaped the recognition and elimination by CAR T-cells due to impaired antigen presentation could be eliminated by CAR NK cells. The latter were engineered to recognise a programmed death ligand 1 (PD-L1) on tumour cells that were resistant to T-cell dependent killing and proved to be effective in eliminating these tumour cells (Lee et al., 2021). This promising study led to further research of the effectiveness of CAR NK cells against various solid cancer types, such as glioblastoma, breast, ovarian, and pancreatic cancer (Wrona, Borowiec, Potemski, 2021).
3.2 Macrophages and CAR MAC therapy
Macrophages is another type of innate immune cells that have been used in CAR therapies instead of T-cells, and have shown to be particularly effective against solid tumours. Macrophages (MAC) can recognise various non-self proteins on cancer cell surface, but unlike NK cells, they act as antigen presenting cells (APCs). This means that they present molecules to T-cells and prime them to attack a specific cell. One of the main advantages in using macrophages is that they are specifically attracted by tumour cells and therefore could be effective in their elimination. However, once macrophages are present in the tumour microenvironment (TME), they are reprogrammed by the tumour cells to facilitate their growth and migration, rather than recognition and elimination (DeNardo & Ruffell, 2019).
To overcome this issue, macrophages have to be genetically modified to eliminate tumours. The first attempt to do this was conducted by researchers at a biotechnological company Carisma Therapeutics at the University of Pennsylvania who genetically engineered the first CAR macrophages (Klichinsky et al., 2020). These cells targeted a human epidermal growth factor receptor 2 (HER2) present on various types of solid cancers. The main issue with engineering such macrophages was to develop a suitable vector to deliver the CAR receptor into the cells, as the most common vectors used in CAR T-cell therapies did not work. A co-founder of Carisma, Michael Klichinsky, identified an adenovirus that was not only suitable for this delivery, but also stimulated macrophages to stay in a pro-inflammatory state. This proved to be beneficial in overcoming the tumours’ abilities to suppress macrophage activation by the release of immunosuppressive factors (Klichinsky et al., 2020).
Klichinsky and colleagues (2020) further tested CAR MAC therapy on mouse tumour models and the results of their study were very promising. CAR macrophages injected into mice stayed in the pro-inflammatory state and showed a significant reduction of tumours. Moreover, when these engineered macrophages came into contact with other macrophages inside the tumour, they reprogrammed them from anti-inflammatory to pro-inflammatory state. By doing so, the TME became less immunosuppressive and more accessible to other immune cells, such as T-cells, that could further facilitate tumour elimination. Their data from mouse models also suggests that CAR MAC could induce “therapeutic vaccination” inside a host organism, meaning that they can retain a long-lasting memory against the tumour. This was concluded based on the observation that macrophages used to eliminate HER2-positive tumours were also active against HER2-negative versions of the same type of tumour that appeared later on (Klichinsky et al., 2020). Based on these promising preclinical data, CAR macrophages have been approved to be tested on a small number of human subjects in phase I clinical trials which are currently ongoing (Charisma Therapeutic, 2021).
CAR T-cell therapy has revolutionised the immunotherapeutic approach to treat cancer. It relies on the combination of biotechnological tools to modify T-cells and the intrinsic ability of patients’ immune system to destroy malignant cells. While clinical trials for certain types of cancer using T-cells have shown promising results, there is emerging evidence of severe side effects associated with CAR T-cell administration. Moreover, certain properties of T-cells make it difficult to eliminate solid tumours. Therefore, the current research focus is on combining other types of immunotherapies with CAR therapy to increase the specificity of T-cells or the use of other immune cells. Both NK cells and macrophages have not only proved to be effective in eliminating malignant cancer cells, including solid tumours, but also had fewer side effects compared to T-cells. Therefore, these new types of therapies have a great potential to be widely used in cancer treatment in the future.
Abel, A.M., Yang, C., Thakar, M.S. & Malarkannan, S. (2018). Natural killer cells: development, maturation, and clinical utilization. Frontiers in immunology, 9, p.1869. Available at: https://internal-journal.frontiersin.org/articles/10.3389/fimmu.2018.01869/full
Albinger, N., Hartmann, J., & Ullrich, E. (2021). Current status and perspective of CAR-T and CAR-NK cell therapy trials in Germany. Gene Therapy. Available at: https://doi.org/10.1038/s41434-021-00246-w
Bonifant, C. L., Jackson, H. J., Brentjens, R. J., & Curran, K. J. (2016). Toxicity and management in CAR T-cell therapy. Molecular Therapy – Oncolytics, 3. Available at: https://doi.org/10.1038/mto.2016.11
Brocker, T., & Karjalainen, K. (1995). Signals through T cell receptor-ζ chain alone are insufficient to prime resting T lymphocytes. Journal of Experimental Medicine, 181(5), p.1653–1659. Available at: https://doi.org/10.1084/jem.181.5.1653
Carisma Therapeutics (2021) CARISMA Therapeutics Announces First Patient Dosed in Landmark Clinical Study Evaluating Engineered Macrophages in Humans. Available at: https://www.prnewswire.com/news-releases/carisma-therapeutics-announces-first-patient-dosed-in-landmark-clinical-study-evaluating-engineered-macrophages-in-humans-301249652.html
Davila, M.L. and Brentjens, R.J., 2016. CD19-Targeted CAR T cells as novel cancer immunotherapy for relapsed or refractory B-cell acute lymphoblastic leukemia. Clinical advances in hematology & oncology: H&O, 14(10), p.802. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536094/
Davila, M. L., Riviere, I., Wang, X., Bartido, S., Park, J., Chung, S. S., Stefanski, J., Borquez-ojeda, O., Qu, J., Wasielewska, T., He, Q., Fink, M., Shinglot, H., Youssif, M., Satter, M., Wang, Y., Hosey, J., Quintanilla, H., Halton, E., Giralt, S. (2014). Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy. Sci Transl Med, 6(224), p.224–225. Available at: https://doi.org/10.1126/scitranslmed.3008226.Efficacy
DeNardo, D. G., & Ruffell, B. (2019). Macrophages as regulators of tumour immunity and immunotherapy. Nature Reviews Immunology, 19(6), p.369–382. Available at: https://doi.org/10.1038/s41577-019-0127-6
Dolstra, H., Roeven, M. W. H., Spanholtz, J., Hangalapura, B., Tordoir, M., Maas, F., Leenders, M., Bohme, F., Kok, N., Trilsbeek, C., Paardekooper, J., Van der Waart, A. B., Westerweel, P., Snijders, T. J. F., Bos, G. M. J., Cornelissen, J. J., Pruijt, H., Huls, G., de Graaf, A., Schaap, N. P. M. (2015). A Phase I Study of Allogeneic Natural Killer Cell Therapy Generated from Cord Blood Hematopoietic Stem and Progenitor Cells in Elderly Acute Myeloid Leukemia Patients. Blood, 126(23), p.1357–1357. Available at: https://doi.org/10.1182/blood.v126.23.1357.1357
Eshhar, Z., Waks, T., Gross, G., & Schindler, D. G. (1993). Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the γ or ζ subunits of the immunoglobulin and T-cell receptors. Proceedings of the National Academy of Sciences of the United States of America, 90(2), 720–724. Available at: https://doi.org/10.1073/pnas.90.2.720
June, C.H. and Sadelain, M., 2018. Chimeric antigen receptor therapy. New England Journal of Medicine, 379(1), p.64-73. Available at: https://www.nejm.org/doi/full/10.1056/NEJMra1706169?casa_token=r4-9GEwMvWEAAAAA:YswVQvBcK6G1DHx6msGQYTw8JybbnKXdNQyQLRrieNN0RxU-9f8MyJ9pf2W2SQfpB8u8rl3B_7jFYC0
Kerbauy, L., Overman, B., Thall, P., Kaplan, M. and Nandivada, V. (2020). Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. New England Journal of Medicine, 382(6), p.545-553. Available at: https://www.nejm.org/doi/full/10.1056/NEJMoa1910607
Klichinsky, M., Ruella, M., Shestova, O., Lu, X. M., Best, A., Zeeman, M., Schmierer, M., Gabrusiewicz, K., Anderson, N. R., Petty, N. E., Cummins, K. D., Shen, F., Shan, X., Veliz, K., Blouch, K., Yashiro-Ohtani, Y., Kenderian, S. S., Kim, M. Y., O’Connor, R. S., Gill, S. (2020). Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology, 38(8), p.947–953. Available at: https://doi.org/10.1038/s41587-020-0462-y
Kuwana, Y., Asakura, Y., Utsunomiya, N., Nakanishi, M., Arata, Y., Itoh, S., Nagase, F. and Kurosawa, Y., 1987. Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions. Biochemical and biophysical research communications, 149(3), p.960-968. Available at: https://www.sciencedirect.com/science/article/abs/pii/0006291X8790502X
Lee, M.Y., Robbins, Y., Sievers, C., Friedman, J., Sater, H.A., Clavijo, P.E., Judd, N., Tsong, E., Silvin, C., Soon-Shiong, P. and Padget, M.R., (2021). Chimeric antigen receptor engineered NK cellular immunotherapy overcomes the selection of T-cell escape variant cancer cells. Journal for immunotherapy of cancer, 9(3). Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986659/
Liu, E., Marin, D., Banerjee, P., Macapinlac, H.A., Thompson, P., Basar, R., Nassif Kerbauy, L., Overman, B., Thall, P., Kaplan, M. and Nandivada, V. (2020). Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. New England Journal of Medicine, 382(6), p.545-553. Available at: https://www.nejm.org/doi/full/10.1056/NEJMoa1910607
Marofi, F., Motavalli, R., Safonov, V. A., Thangavelu, L., Yumashev, A. V., Alexander, M., Shomali, N., Chartrand, M. S., Pathak, Y., Jarahian, M., Izadi, S., Hassanzadeh, A., Shirafkan, N., Tahmasebi, S., & Khiavi, F. M. (2021). CAR T cells in solid tumors: challenges and opportunities. Stem Cell Research and Therapy, 12(1), p.1–16. Available at: https://doi.org/10.1186/s13287-020-02128-1
O’Donnell, H., & McSorley, S. J. (2014). Salmonella as a model for non-cognate Th1 cell stimulation. Frontiers in Immunology, 5, p.1–13. Available at: https://doi.org/10.3389/fimmu.2014.00621
Olson, J.A., Leveson-Gower, D.B., Gill, S., Baker, J., Beilhack, A. and Negrin, R.S. (2010). NK cells mediate reduction of GVHD by inhibiting activated, alloreactive T cells while retaining GVT effects. Blood, The Journal of the American Society of Hematology, 115(21), p.4293-4301. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879101/
Park, A. K., Fong, Y., Kim, S. I., Yang, J., Murad, J. P., Lu, J., Jeang, B., Chang, W. C., Chen, N. G., Thomas, S. H., Forman, S. J., & Priceman, S. J. (2020). Effective combination immunotherapy using oncolytic viruses to deliver CAR targets to solid tumors. Science Translational Medicine, 12(559). Available at: https://doi.org/10.1126/SCITRANSLMED.AAZ1863
Simonetta, F., Alvarez, M. and Negrin, R.S. (2017). Natural killer cells in graft-versus-host-disease after allogeneic hematopoietic cell transplantation. Frontiers in immunology, 8, p.465. Available at: https://www.frontiersin.org/articles/10.3389/fimmu.2017.00465/full
Sun, X.X. and Yu, Q., 2015. Intra-tumor heterogeneity of cancer cells and its implications for cancer treatment. Acta Pharmacologica Sinica, 36(10), p.1219-1227. Available at: https://www.nature.com/articles/aps201592
Wrona, E., Borowiec, M., & Potemski, P. (2021). Car-nk cells in the treatment of solid tumors. International Journal of Molecular Sciences, 22(11), p.1–19. Available at: https://doi.org/10.3390/ijms22115899