CAR T therapy from haematological malignancies to aging‐related diseases: An ever‐expanding universe

Short but impactful, the two‐decade story of gene editing allowed a significant breakthrough in the treatment of haematological malignancies. However, despite different generations of chimeric antigen receptor T (CAR T), such a successful therapy has not yet been replicated in solid tumours and non‐oncological diseases.


| STATE-OF-ART IN CHIMERIC ANTIGEN RECEPTOR-ENGINEERED T (CAR T) CELL THERAPY AND TECHNOLOGY
Since the 90s, advances in gene editing techniques and a deeper understanding of adaptive cell therapy have fueled the development of CAR T cell therapy.CAR T therapy then have relatively short but impactful story, marking a significant breakthrough in the treatment of haematological malignancies. 1Originally conceived for redirecting T lymphocytes against human immunodeficiency virus, 2 CAR T is a technology that progressed to its fifth generation of engineered cells.They are designed to recognize and bind to two different antigens on the surface of cancer cells in addition to activate TCR signalling and a better control over cytokine release. 3Over the last two decades, CAR T cell engineering then move from a single CD3 signalling domain, to incorporating additional co-stimulatory signalling domains in the second to fourth generations.These advancements also include the ability to secrete therapeutic molecules, such as cytokines or antibodies. 4,5Currently CAR T cell are capable of triggering an immune cell response targeting specific antigen, thereby boosting proliferative capacity, enhancing central memory differentiation, intensifying antitumour activity, and prevent an excessive cytokine release. 6AR T cells have represented a cutting-edge discovery in the treatment of hematologic malignancies since 2017 when the Food and Drug Administration (FDA) approved the Tisagenlecleucel.In its first application, targeting CD19 in acute lymphoblastic B-cell leukaemia refractory to second (or more)-line treatments with Tisagenlecleucel boasted an approximately 70% complete remission rate, despite being limited by a similar incidence sever adverse events, mostly related to a cytokine release syndrome (CRS).Overtime, technological improvement led to a gradual enhancement in response rates, coupled with a reduction in adverse events.8][9] While two additional therapies targeting B-cell maturation antigen (BCMA) have been approved for the treatment of refractory multiple myeloma, 10,11 solid tumours non-oncological diseases still remain substantially uncovered by any CAR T cell treatment, although encouraging preclinical findings.
Despite such a promising potential of CAR T therapy, this broader application outside malignancies still faces several issues.This short narrative review aims at critically summarizing the potentially application of CAR T therapy in chronic, aging-related diseases without hiding current limitation and future challenges.

| CHALLENGES IN CAR T CELL APPLICATION OUTSIDE HAEMATOLOGICAL MALIGNANCIES: FROM SOLID TUMOURS TO NON-ONCOLOGICAL DISEASES
The success of CAR T therapy as rescue therapy has already posed it as a fifth pillar for cancer treatment complementing surgery, chemo-, radio-, and immunotherapy.While CAR T therapy is currently indicated from the third line of therapy onwards, earlier use is expected in a foreseeable future once efficacy/safety data become available and costs are lowered.Furthermore, CAR T cells are often referred to as 'living drugs' capable to expand from a single cell and potential to persistence over a decade. 12This effect could be advantageous for preventing cancer recurrence, but it raises concerns about potential unknown and unpredictable consequences in non-oncological diseases.Even in the context of non-haematological malignancies, CAR T therapy faces challenges in overcoming the complexity of the tumour microenvironment (TME), which limits an effective cell penetration and persistence alongside with the lack of ideal target antigens and the risk of off-target/adverse effects (Figure 1).
Being all approved drugs selectively designed against B-cell markers, there is not specificity for solid tumours yet (Table 1).To address the lack of tumour-specific antigen engineering, alternative strategies have been devised.These include approaches, such as dividing the primary and co-stimulatory domains into separate CAR T constructs, as exemplified by the synthetic Notch approach 13 or the SUPRA CAR system. 14However, the utilization of CAR T therapy carries the inherited potential to worsen clinical conditions through on-target off-tumour toxicity 15 and CRS. 16any target antigens within solid tumours are indeed widely expressed in healthy viable cells, making them equally CAR T target and potentially precipitating severe, and potentially lethal, side effects. 17Furthermore, the expansion of a CAR T population required a lymphodepleting chemotherapy prior infusion.While this procedure sounds reasonable for rescue therapy of oncological diseases, it poses genotoxic risks that may not be justified for non-malignant diseases. 18 decade of immunotherapy has taught us a lot about the critical role of TME and its potent immunosuppressant role.Soluble factors such as TGF-beta, IL-4, and IL-10 promote the infiltration of immunosuppressant cells type like tumour-associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs).Their cytokine behaviour turns off T cell activation, while specific tumour-associated fibroblasts (TAFs) generate a dense extracellular matrix that physically prevents T cell infiltration into the tumour site.Thus, TME represents a real physical barrier around solid tumours. 19o overcome those obstacles, CAR T cells have been engineered to facilitate their trafficking into tumour milieu by enhancing the expression of CCR2 20 or improving cytokine secretion and on-site persistence and proliferation by deleting TGF-beta. 21Further alternative approaches under exploration includes the direct injection of CAR T cells into the tumour site, 22 the use of cell-targeted mRNA lipid nanoparticles for preventing their depletion 23,24 and the immunomodulation of TME with oncolytic viruses. 25his technical advance also has a transient nature that holds massive potential to streamline ex vivo production processes and implementing clinical application.A transient nature of CAR T cell also occurs without leucodepletion and is then privileged differently between oncological and nononcological diseases.It is preferable in non-oncological diseases-where physiological wound repair processes may be required-as it address ethical concerns related to a hardly legitimate chemotherapy and risk of CRS and on-target offdisease toxicity. 26This advancement may offer advantage in non-oncological disease as it would address ethical concerns.The expansion and persistence of CAR T cells are instead critical for achieving a long-term remission in oncological diseases.While leukodepletion effectively reduced host's immune response against the transgene product, 27 longterm persistence of CAR T cells may be insufficient, due to factors, such as the term recovery in host immune system response, the lack of target antigen to maintain CAR T cell population after remission, and downregulation/escape of CD19 the prevalent target antigen is approved therapies.
In this context, potential applications of CAR T cell therapies for non-oncological diseases have increasingly gained interest in recent years, especially for autoimmune and cardiovascular disease, as well as chronic infections (Table 2 and Figure 1).In this fields, efficacy/ safety requirements are fairly different than in oncological The scenario for future application of chimeric antigen receptor cell therapy in non-haematological diseases.Despite the success in haematological malignancies, future application of chimeric antigen receptor (CAR) T cells in solid tumours and non-oncological diseases still has to face many challenges.These are related to the different behaviour of aged cells, being both solid tumours and chronic disease more frequent in old individuals.Furthermore, both solid tumours and chronic diseases (cardiovascular and autoimmune) are characterized by a specific milieu (e.g.tumour microenvironment), where CAR cell penetration/interaction are different and somehow unpredictable (created with BioRe nder.com).

T A B L E 1
Ongoing phase 1 studies summarizing the application of CAR therapies in solid tumours.diseases: the expected effects are less strict and a disease control would be reasonably satisfactory, but a high safety profile is mandatory.Disease burden in malignancies is instead notably higher and necessitates a larger number of CAR T cells, which justifies a high risk of toxicity.In addition, chronic diseases are characterized by a lower antigenic heterogeneity with none or limited.Even disease sites are readily accessible to CAR T cells without physical of immunomodulating barriers or antigen escape mechanisms.In line, a growing number of clinical trials-even though limited by limited sample size and high costs-supports the effectiveness of CAR T cell therapy in immunological disease, mainly related to B-cells dysfunction.Recently published, anti-CD19 CAR T cells were proven to be effective in inducing a long-term resolution of symptoms and improvement of organ damage in five patients with refractory systemic lupus erythematosus. 28,29isease control persisted even after the swift regeneration of native B-cell after a few months, which is an intriguing observation opposite to the prolonged depletion observed in haematological malignancies.This finding opens up to potential differences in CAR T cell kinetic upon different disease clusters and/or burdens. 30Precision targeting of autoantigen-specific B cell by chimeric autoantibody receptors is the next frontier for applying CAR T cell to nononcological disease by reducing the risk of B cell aplasia. 31till few but interesting studies have focused on heart diseases, primarily targeting cardiac fibrosis, which is a significant concern in acute injury or aging, with currently limited treatment options.Once identified the fibroblast activation protein as a target, CAR T cells have been proved efficient in resolving cardiac fibrosis and subsequently improving cardiac function without relevant toxicity in mice. 24,32Being fibrosis a standardized process shared by different pathological conditions across different organs, these preliminary results call for future broader applications, such as in liver and lungs.

CONTEXT OF AGING
While advancements in standard care have contributed to increased life expectancy, the aging population is growing, burdening healthcare systems, particularly those with universal coverage.Senescence, characterized by irreversible proliferative arrest in cells, leads to the secretion of pro-inflammatory and proteolytic molecules, culminating in the manifestation of cellular phenotypes, notably senescence-associated secretory phenotypes (SASP). 33he decline of the immune system plays a pivotal role in the senescence process, with T cells exhibiting downregulation of CD27/CD28 costimulatory receptors and chemokine receptors like CCR7. 34Furthermore, senescent T cells, integral to SASP, can release cytokines and immunomodulatory chemokines, akin to those implicated in CRS. 35While all CAR T therapies approved for use thus far are primarily targeted towards paediatric or young adult populations, their effectiveness appears to diminish in elderly patients, although age alone may not be the sole determinant. 36As the average age of diagnosis for haematological diseases increases, the success rate of CAR T therapy declines.The diminishing physiological resilience of elderly patients, coupled with the biological deterioration of T-cell function, appears to be the primary contributing factor to CAR T therapy failure. 37Numerous studies indicate a strong correlation between impaired efficacy of CAR T cells in the elderly and the senescence of host T cells.CAR T cells exhibiting a lower response rate are prone to exhaustion and characterized by a more differentiated surface marker expression.Thus, the impaired response rate is not solely influenced by the selection of low-functioning T cells for CAR T production via apheresis in elderly patients, but CAR T cells may also become dysfunctional post-infusion due to immunosenescence and inflammaging.The use of heterologous transplants, employing young T-cell pool donors to produce CAR T cells for treating diseases prevalent in the elderly, warrants careful consideration.While offering a promising approach, it poses significant risks related to allogenicity and the development graft versus host disease.With caution, it still deserved to be further explored.Efforts are underway to address these limitations through interventions aimed at enhancing the fitness and rejuvenation of T cells.In addition to established interventions such as calorie restriction 38 and physical activity, various approaches have been explored in the literature.Metformin, a widely studied drug due to its diverse therapeutic properties and its ability to activate the AMPK pathway, has shown promise in enhancing T cell fitness.It exhibits anti-apoptotic effects, improves the memory of CD8 T cells, and inhibits SASP in macrophages 39 and fibroblasts, 40 although its efficacy appears to be dose-dependent. 41Another promising intriguing metabolite is adenosine, released during inflammation and hypoxia, which downregulates CD28 expression, induces apoptosis, and accelerates replicative senescence by reducing telomerase activity.In line, the lack of adenosine deaminase (ADA) has been proven to lead immunosuppressive effects and alterations in telomerase activity and CD28 expression, thus contributing to cell senescence process. 42The use of rapamycin, an mTOR pathway inhibitor, has been also investigated, demonstrating reduction of SASP and increased longevity in animal models. 43Finally, gene editing approaches to reprogram T cell senescence are under investigation with CMYC (OSKM) showing a reduced expression of senescence markers and prolonged lifespan. 44Building upon these strategies to enhance the fitness of senescent T cells, attempts have been made to apply them to improve the functions of CAR T cells.Pretreating these cells with rapamycin has been shown to upregulate CXCR4 expression, thereby enhancing the ability of CAR T cells to migrate within the bone marrow, a costeffective and straightforward approach. 45Additionally, modifying CAR T cells by either removing adenosine receptors 46 or overexpressing ADA 47 has demonstrated to improve anti-cancer efficacy.Further incorporating specific costimulatory signals into CAR T cells is another promising strategy, which recall past advance across CAR T generations.In senescent T cells costimulatory receptors are typically downregulated.As sustained expression of CD28 may slow the process of replicative senescence, 48 its engineered up-regulation in CAR T cell was proven effective in enhancing their tumour-killing properties by fostering activation, and differentiation into memory effector cells.This approach seems highly promising for addressing challenges in managing CAR T therapy in elderly, but alternative costimulatory molecules has been also proposed. 49Further progresses are expected by OSKM factor engineering in the production process of CAR T cells.
Recently, a direct senolytic potential for CAR T cell therapy have been demonstrated in two different preclinical models.Both the natural killer group 2 member D ligands (NKG2DLs) and senescence-associated protein urokinase plasminogen activator receptor (uPAR) have substantial potential to safely clear senescent cells in aged mice.Even more exciting, the senolytic activity of CAR T cell associated with an improvement of exercise capacity metabolic dysfunction in physiological aging. 50,51

OUTLOOK
Besides addressing current challenges in CAR T therapy, additional efforts are being directed towards engineering other cell populations, such as natural killer (NK) cells and macrophages.NK cells retain potent cytotoxic capabilities, while macrophages critically orchestrate inflammation processes with a leading role in tumour-and other-microenvironments. NK cells offer the advantage of being able to recognize target cells independently of major histocompatibility complex (MHC), thus reducing the risk of alloreactivity.CAR-NK production does not require autologous NK cells but NK92 cell lines, derived from umbilical cord blood, are currently utilized in clinical trials.This approach consistently reduces the risk of GVHD and make them readily available for use. 52Furthermore, CAR-NK seems to be burdened by a lower risk of CRS and neurotoxicity compared to CAR T, likely due to a limited cytokine storm upon activation.They harness both CARdependent and intrinsic cytotoxic properties, 53 while stimulation with IL-15 may further enhanced their long-term persistence and anti-tumour activity. 54The limited half-life of NK cell, approximately 12 months, also suggest a preferential use in non-malignant diseases, whereas frequent re-infusions are conceivable for a potential use in cancer therapy.However, it theoretically allows for a prompt resolution of adverse effects.Despite a growing number of ongoing clinical trials and a supposed advantage on CAR T, CAR-NK have not yet received institutional approval.Furthermore, CAR-NK share similar with CAR T therapy in dealing with selecting target antigens, antigen heterogeneity, production and storage issues, limited migration within the tumour site, and the immunosuppressive nature of the TME.Similarly, macrophages are under the lens for their unique ability to readily access the tumour site, overcoming the functional/physical barrier of TME that poses challenges for CAR T and CAR-NK cells.Moreover, macrophages exhibit high plasticity, allowing them to polarize into different phenotypes, including TAMs.Despite belonging to the anti-inflammatory M2 phenotype, TAMs contribute to the development of the immunosuppressive TME. 55ecruitment and infiltration of macrophages into the TME are mediated by cytokines secreted by tumour cells, particularly CCL2, CXCL12 and VEGF. 56Unlike T cells, which may infiltrate the tumour site but often face exhaustion due to the immunosuppressive TME, macrophages are less susceptible to these effects, as they are integral components of TME formation. 55Macrophage CARs are structurally similar to CAR T cells but engineered for signalling domains enhancing phagocytosis like phosphoinositide 3-kinase. 57owever, macrophage CAR face with similar limitations in selecting specific antigens, addressing antigen escape, and managing toxicity.An adenoviral vector has been recently used for engineering macrophage CAR targeting HER2expressing cells with high efficacy and reproducibility.A shift towards macrophage polarization from the predominant M2 phenotype to a pro-inflammatory M1 phenotype has been observed within TME and associated with increased survival in mouse studies. 58The hypothesis that engineered macrophage CARs have the potential to modulate the immunosuppressive milieu of the tumour microenvironment (TME), serve as antigen-presenting cells, effectively activate cytotoxic T cell responses, and ultimately overcome resistance to treatment, such as immunotherapy and other CAR cell therapies, is intriguing and awaits further investigation.With this rationale a phase 1 clinical trial is currently ongoing with a term for enrollment set for the end of 2024.

| CONCLUSION
While the use of CAR T therapy has achieved significant validation in haematological malignancies, its potential in solid tumours and non-oncological disease is upfront and no therapies are yet approved.Relevant limitations indeed persist, including challenges in selecting target antigens, which are often heterogeneous in diseases unrelated to B-cell lineages, and difficulties in accessing the disease site within hostile and immunosuppressive microenvironments.Moreover, unlike haematological diseases where the average age of patient candidate to CAR T cell therapy is relatively low, different class of disease primarily affect the elderly.This poses additional challenges related to the impaired fitness and senescence of aged T cells.Aware of this, the most recent clinical trials are focusing on enhancing the specificity of CAR-cell actions, including the use of alternative cell populations such as NK cells and macrophages to address these limitations.A comprehensive assessment of the patient's immunological state can aid in tailoring the production of CAR cells to be increasingly personalized.By integrating knowledge of cellular aging and employing methods to mitigate senescence, there is potential to improve treatment success rates in non-haematological diseases while reducing side effects.This approach holds promise for advancing the efficacy of CAR-cell therapy across diverse disease contexts.