Rational Alternatives to Fludarabine and Cyclophosphamide-Based Pre-CAR Lymphodepleting Regimens in the Pediatric and Young Adult B-ALL Setting.

PURPOSE OF REVIEW: Lymphodepleting chemotherapy (LD) has emerged as a key determinant of chimeric antigen receptor T cell (CAR) efficacy across pediatric/adult B cell malignancies. Clinical trials demonstrate the superiority of fludarabine/cyclophosphamide (Flu/Cy) regimens, resulting in the adoption of Flu/Cy as the pre-CAR LD standard. In the context of a global fludarabine shortage, consideration of alternative regimens is timely, yet limited clinical data exists, specifically in the pediatric B-ALL CAR setting. RECENT FINDINGS: Bendamustine has been used as an effective LD prior to CD19-CAR in adult lymphoma. Although use in the pediatric CAR setting is limited, tolerability has been established in pediatric Hodgkin's lymphoma. Clofarabine is a purine nucleoside analog with mechanistic overlap with fludarabine; however, toxicity is high in the upfront leukemia setting, and thus use as an LD pre-CAR should be pursued with caution. We review the experience using bendamustine and clofarabine to serve as a resource when considering LD regimens as an alternative to fludarabine for pediatric B-ALL.

Influence of Adipose Tissue Distribution, Sarcopenia, and Nutritional Status on Clinical Outcomes After CD19 CAR T-cell Therapy.

Although CD19-directed chimeric antigen receptor T-cell therapy (CD19.CAR-T) has proven clinical efficacy for multiple refractory B-cell malignancies, over 50% of patients ultimately relapse. Recent evidence has underlined the critical role of the host in determining treatment responses. In this retrospective observational study of 106 patients with relapsed/refractory large B-cell lymphoma receiving standard-of-care CD19.CAR-T, we analyzed the impact of immunometabolic host features and detailed body composition measurements on post-CAR T clinical outcomes. We extracted muscle and adipose tissue distributions from prelymphodepletion CT images and assessed laboratory-based immuno-nutritional scores. Early responders displayed increased total abdominal adipose tissue deposits (TAT: 336 mm3 vs. 266 mm3, P = 0.008) and favorable immuno-nutritional scores compared to nonresponding patients. On univariate Cox regression analysis, visceral fat distribution, sarcopenia, and nutritional indices significantly impacted both progression-free (PFS) and overall survival (OS). Patients with a low skeletal muscle index (SMI; e.g.<34.5), a sarcopenia indicator, exhibited poor clinical outcomes (mOS 3.0 months vs. 17.6 months, log-rank P = 0.0026). Prognostically adverse immuno-nutritional scores were linked to inferior survival [low PNI: HROS, 6.31; 95% confidence interval (CI), 3.35-11.90; P < 0.001]. In a multivariable analysis adjusting for baseline Eastern Cooperative Oncology Group performance status, C-reactive protein, and lactate dehydrogenase, increased TAT was independently associated with improved clinical outcomes (adjusted HROS, 0.27; 95% CI, 0.08-0.90; P = 0.03). We noted particularly favorable treatment outcomes in patients with both increased abdominal fat and muscle mass (TAThigh/SMIhigh: 1-year PFS 50%, 1-year OS 83%). These real-world data provide evidence for a role of body composition and immuno-nutritional status in the context of CD19.CAR-T and suggest that the obesity paradox may extend to modern T cell-based immunotherapies. See related Spotlight by Nawas and Scordo, p. 704.

A drug screening to identify novel combinatorial strategies for boosting cancer immunotherapy efficacy.

BACKGROUND: Chimeric antigen receptor (CAR) T cells and immune checkpoint blockades (ICBs) have made remarkable breakthroughs in cancer treatment, but the efficacy is still limited for solid tumors due to tumor antigen heterogeneity and the tumor immune microenvironment. The restrained treatment efficacy prompted us to seek new potential therapeutic methods. METHODS: In this study, we conducted a small molecule compound library screen in a human BC cell line to identify whether certain drugs contribute to CAR T cell killing. Signaling pathways of tumor cells and T cells affected by the screened drugs were predicted via RNA sequencing. Among them, the antitumor activities of JK184 in combination with CAR T cells or ICBs were evaluated in vitro and in vivo. RESULTS: We selected three small molecule drugs from a compound library, among which JK184 directly induces tumor cell apoptosis by inhibiting the Hedgehog signaling pathway, modulates B7-H3 CAR T cells to an effector memory phenotype, and promotes B7-H3 CAR T cells cytokine secretion in vitro. In addition, our data suggested that JK184 exerts antitumor activities and strongly synergizes with B7-H3 CAR T cells or ICBs in vivo. Mechanistically, JK184 enhances B7-H3 CAR T cells infiltrating in xenograft mouse models. Moreover, JK184 combined with ICB markedly reshaped the tumor immune microenvironment by increasing effector T cells infiltration and inflammation cytokine secretion, inhibiting the recruitment of MDSCs and the transition of M2-type macrophages in an immunocompetent mouse model. CONCLUSION: These data show that JK184 may be a potential adjutant in combination with CAR T cells or ICB therapy.

Hypophosphatemia Due to Increased Effector Cell Metabolic Activity Is Associated with Neurotoxicity Symptoms in CD19-Targeted CAR T-cell Therapy.

A major complication of chimeric antigen receptor (CAR) T-cell therapy is immune effector cell-associated neurotoxicity syndrome (ICANS), which presents as aphasia, confusion, weakness, somnolence, seizures, and coma. This is similar to the neurologic manifestations of hypophosphatemia, which can result from sudden increases in metabolic demand for phosphorylated intermediates (e.g., refeeding syndrome and sepsis). Given these similarities, we investigated whether CAR T-cell effector metabolic activity is associated with increased extracellular phosphate consumption and a possible association between hypophosphatemia and ICANS. In vitro 4-1BB and CD28 CD19-targeted CAR T-cell effector activity was found to be associated with increased consumption of media phosphorus, which was temporally associated with increased single-cell effector secretomic activity and increased phosphorus-dependent metabolic demand of the CAR T cells. A clinical cohort of 77 patients treated with CD19-targeted CAR T-cell therapy demonstrated a significant anticorrelation between serum phosphorus and ICANS incidence and severity, with earlier onset of hypophosphatemia after CAR T-cell infusion more likely to result in neurotoxicity. These results imply phosphorous level monitoring could alert to the development of ICANS in clinical scenarios. See related Spotlight by Tobin et al., p. 1422.

Mucosal-associated invariant T (MAIT) cells, a new source of universal immune cells for chimeric antigen receptor (CAR)-cell therapy.

Treatment of hematological malignancies by autologous T cells expressing a chimeric antigen receptor (CAR) is a breakthrough in the field of cancer immunotherapy. As CAR-T cells are entering advanced phases of clinical development, there is a need to develop universal, ready-to-use products using immune cells from healthy donors, to reduce time to treatment, improve response rate and finally reduce the cost of production. Mucosal-associated invariant T cells (MAIT) are unconventional T cells which recognize microbial-derived riboflavin derivatives presented by the conserved MR1 molecule and are endowed with potent effector functions. Because they are not selected by classical MHC/peptide complexes and express a semi-invariant T cell receptor, MAIT cells do not mediate alloreactivity, prompting their use as a new source of universal effector cells for allogeneic CAR-T cell therapy without the need to inactivate their endogenous TCR. We produced CD19-CAR MAIT cells as proof-of-concept allowing subsequent head-to-head comparison with currently used CD19-CAR T cells. We demonstrated their anti-tumor efficacy in vitro and their capacity to engraft without mediating GVHD in preclinical immunodeficient mouse models. Universal, off-the-shelf CAR-MAIT cells could provide a suitable alternative to current autologous CAR-T cells to treat patients regardless of HLA disparity, without production delay, enabling a cost-effective manufacturing model for large-scale clinical application.

Chimeric Antigen Receptor T-Cell Therapy in Lung Cancer: Potential and Challenges.

Chimeric antigen receptor T (CAR-T) cell therapy has exhibited a substantial clinical response in hematological malignancies, including B-cell leukemia, lymphoma, and multiple myeloma. Therefore, the feasibility of using CAR-T cells to treat solid tumors is actively evaluated. Currently, multiple basic research projects and clinical trials are being conducted to treat lung cancer with CAR-T cell therapy. Although numerous advances in CAR-T cell therapy have been made in hematological tumors, the technology still entails considerable challenges in treating lung cancer, such as on-target, of-tumor toxicity, paucity of tumor-specific antigen targets, T cell exhaustion in the tumor microenvironment, and low infiltration level of immune cells into solid tumor niches, which are even more complicated than their application in hematological tumors. Thus, progress in the scientific understanding of tumor immunology and improvements in the manufacture of cell products are advancing the clinical translation of these important cellular immunotherapies. This review focused on the latest research progress of CAR-T cell therapy in lung cancer treatment and for the first time, demonstrated the underlying challenges and future engineering strategies for the clinical application of CAR-T cell therapy against lung cancer.

Gene-Edited Interleukin CAR-T Cells Therapy in the Treatment of Malignancies: Present and Future.

In recent years, chimeric antigen receptor T cells (CAR-T cells) have been faced with the problems of weak proliferation and poor persistence in the treatment of some malignancies. Researchers have been trying to perfect the function of CAR-T by genetically modifying its structure. In addition to the participation of T cell receptor (TCR) and costimulatory signals, immune cytokines also exert a decisive role in the activation and proliferation of T cells. Therefore, genetic engineering strategies were used to generate cytokines to enhance tumor killing function of CAR-T cells. When CAR-T cells are in contact with target tumor tissue, the proliferation ability and persistence of T cells can be improved by structurally or inductively releasing immunoregulatory molecules to the tumor region. There are a large number of CAR-T cells studies on gene-edited cytokines, and the most common cytokines involved are interleukins (IL-7, IL-12, IL-15, IL-18, IL-21, IL-23). Methods for the construction of gene-edited interleukin CAR-T cells include co-expression of single interleukin, two interleukin, interleukin combined with other cytokines, interleukin receptors, interleukin subunits, and fusion inverted cytokine receptors (ICR). Preclinical and clinical trials have yielded positive results, and many more are under way. By reading a large number of literatures, we summarized the functional characteristics of some members of the interleukin family related to tumor immunotherapy, and described the research status of gene-edited interleukin CAR-T cells in the treatment of malignant tumors. The objective is to explore the optimized strategy of gene edited interleukin-CAR-T cell function.

Antigen-Specific Regulatory T Cell Therapy in Autoimmune Diseases and Transplantation.

Regulatory T (Treg) cells are a heterogenous population of immunosuppressive T cells whose therapeutic potential for the treatment of autoimmune diseases and graft rejection is currently being explored. While clinical trial results thus far support the safety and efficacy of adoptive therapies using polyclonal Treg cells, some studies suggest that antigen-specific Treg cells are more potent in regulating and improving immune tolerance in a disease-specific manner. Hence, several approaches to generate and/or expand antigen-specific Treg cells in vitro or in vivo are currently under investigation. However, antigen-specific Treg cell therapies face additional challenges that require further consideration, including the identification of disease-relevant antigens as well as the in vivo stability and migratory behavior of Treg cells following transfer. In this review, we discuss these approaches and the potential limitations and describe prospective strategies to enhance the efficacy of antigen-specific Treg cell treatments in autoimmunity and transplantation.

Switchable CAR-T Cells Outperformed Traditional Antibody-Redirected Therapeutics Targeting Breast Cancers.

Various antibody-redirected immunotherapeutic approaches, including antibody-drug conjugates (ADCs), bispecific antibodies (bsAbs), and chimeric antigen receptor-T (CAR-T) cells, have been devised to produce specific activity against various cancer types. Using genetically encoded unnatural amino acids, we generated a homogeneous Her2-targeted ADC, a T cell-redirected bsAb, and a FITC-modified antibody capable of redirecting anti-FITC CAR-T (switchable CAR-T; sCAR-T) cells to target different Her2-expressing breast cancers. sCAR-T cells showed activity against Her2-expressing tumor cells comparable to that of conventional anti-Her2 CAR-T cells and superior to that of ADC- and bsAb-based approaches. To prevent antigen escape, we designed bispecific sCAR-T cells targeting both the Her2 receptor and IGF1R, which showed an overall improved activity against cancer cells with low Her2 expression. This study increases our understanding of various explored cancer therapeutics and underscores the efficient application of sCAR-T cells as a promising therapeutic option for breast cancer patients with low or heterogeneous antigen expression.

Applications of CRISPR Genome Editing to Advance the Next Generation of Adoptive Cell Therapies for Cancer.

Adoptive cell therapy (ACT) for cancer shows tremendous potential; however, several challenges preclude its widespread use. These include poor T-cell function in hostile tumor microenvironments, a lack of tumor-specific target antigens, and the high cost and poor scalability of cell therapy manufacturing. Creative genome-editing strategies are beginning to emerge to address each of these limitations, which has initiated the next generation of cell therapy products now entering clinical trials. CRISPR is at the forefront of this revolution, offering a simple and versatile platform for genetic engineering. This review provides a comprehensive overview of CRISPR applications that have advanced ACT. SIGNIFICANCE: The clinical impact of ACT for cancer can be expanded by implementing specific genetic modifications that enhance the potency, safety, and scalability of cellular products. Here we provide a detailed description of such genetic modifications, highlighting avenues to enhance the therapeutic efficacy and accessibility of ACT for cancer. Furthermore, we review high-throughput CRISPR genetic screens that have unveiled novel targets for cell therapy enhancement.

Comparative Effectiveness Research for CAR-T Therapies in Multiple Myeloma: Appropriate Comparisons Require Careful Considerations of Data Sources and Patient Populations.

BACKGROUND AND OBJECTIVE: Registrational trials for ciltacabtagene autoleucel [cilta-cel]) and idecabtagene vicleucel [ide-cel] chimeric antigen receptor T-cell (CAR-T) therapies were single-arm studies conducted with relapse refractory multiple myeloma (MM) patients who were triple-class-exposed (TCE) or triple-class-refractory (TCR). It is critical for researchers conducting comparative effectiveness research (CER) to carefully consider the most appropriate data sources and comparable patient populations. The aim of this study was to identify potential data sources and populations for comparing to single-arm CAR-T trials CARTITUDE-1 (cilta-cel) and KarMMa (ide-cel). METHODS: A 2-part global systematic literature search produced a review of (1) clinical trials of National Comprehensive Cancer Network (NCCN) guideline preferred regimens in previously treated MM, and (2) real-world data cohorts of TCE or TCR populations, published between 1/1/2015 and 12/10/2020, with sample sizes of > 50 patients and reporting survival-related outcomes. Implications on CER and accepted best practices are discussed. RESULTS: Nine clinical trials of NCCN preferred regimens were identified along with five real-world data-based publications. No clinical trials evaluated patients with TCE or TCR MM. Among the real-world data-based publications, two evaluated patients exclusively with TCR MM, two analyzed a mixed population of patients with TCE or TCR MM, and one publication assessed patients exclusively with TCE MM. Real-world data treatment patterns were heterogeneous. CONCLUSION: Current NCCN preferred regimens were not specifically studied in TCE or TCR MM patients, although some studies do include a proportion of these types of patients. Therefore, appropriate matching of populations using either real-world data or patient level clinical trial data is critical to putting trials of novel CAR-Ts (i.e., CARTITUDE-1 or KarMMa) into appropriate comparative context.

Induced pluripotent stem cell-derived natural killer cells gene-modified to express chimeric antigen receptor-targeting solid tumors.

The use of allogeneic, pluripotent stem cell-derived immune cells for cancer immunotherapy has been the subject of recent research, including clinical trials. The use of pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, regarding efficacy, safety, and producibility. In this review, we have described the characteristics of natural killer (NK) cells from multiple cell sources, including pluripotent stem cells, the chimeric antigen receptor (CAR)-modification method and strategy for these NK cells, and the current and planned clinical trials of CAR-modified induced pluripotent stem cell-derived NK cells.

Revving the CAR - Combination strategies to enhance CAR T cell effectiveness.

Chimeric antigen receptor (CAR) T cell therapy is currently approved for treatment of refractory B-cell malignancies. Response rates in these diseases are impressive by historical standards, but most patients do not have a durable response and there remains room for improvement. To date, CAR T cell activity has been even more limited in solid malignancies. These limitations are thought to be due to several pathways of resistance to CAR T cells, including cell-intrinsic mechanisms and the immunosuppressive tumor microenvironment. In this review, we discuss current experimental strategies that combine small molecules and monoclonal antibodies with CAR T cells to overcome these resistance mechanisms. We describe the biological rationale, pre-clinical data and clinical trials in progress that test the efficacy and safety of these combinations.

Preclinical evaluation of CD8+ anti-BCMA mRNA CAR T cells for treatment of multiple myeloma.

Chimeric antigen receptor (CAR) T-cell therapy remains limited to select centers that can carefully monitor adverse events. To broaden use of CAR T cells in community clinics and in a frontline setting, we developed a novel CD8+ CAR T-cell product, Descartes-08, with predictable pharmacokinetics for treatment of multiple myeloma. Descartes-08 is engineered by mRNA transfection to express anti-BCMA CAR for a defined length of time. Descartes-08 expresses anti-BCMA CAR for 1 week, limiting risk of uncontrolled proliferation; produce inflammatory cytokines in response to myeloma target cells; and are highly cytolytic against myeloma cells regardless of the presence of myeloma-protecting bone marrow stromal cells, exogenous a proliferation-inducing ligand, or drug resistance including IMiDs. The magnitude of cytolysis correlates with anti-BCMA CAR expression duration, indicating a temporal limit in activity. In the mouse model of aggressive disseminated human myeloma, Descartes-08 induces BCMA CAR-specific myeloma growth inhibition and significantly prolongs host survival (p < 0.0001). These preclinical data, coupled with an ongoing clinical trial of Descartes-08 in relapsed/refractory myeloma (NCT03448978) showing preliminary durable responses and a favorable therapeutic index, have provided the framework for a recently initiated trial of an optimized/humanized version of Descartes-08 (i.e., Descartes-11) in newly diagnosed myeloma patients with residual disease after induction therapy.

Donor-derived CD19 CAR-T cell therapy of relapse of CD19-positive B-ALL post allotransplant.

Safety and efficacy of allogeneic anti-CD19 chimeric antigen receptor T cells (CAR-T cells) in persons with CD19-positive B-cell acute lymphoblastic leukemia (B-ALL) relapsing after an allotransplant remain unclear. Forty-three subjects with B-ALL relapsing post allotransplant received CAR-T cells were analyzed. 34 (79%; 95% confidence interval [CI]: 66, 92%) achieved complete histological remission (CR). Cytokine release syndrome (CRS) occurred in 38 (88%; 78, 98%) and was ≥grade-3 in 7. Two subjects died from multiorgan failure and CRS. Nine subjects (21%; 8, 34%) developed ≤grade-2 immune effector cell-associated neurotoxicity syndrome (ICANS). Two subjects developed ≤grade-2 acute graft-versus-host disease (GvHD). 1-year event-free survival (EFS) and survival was 43% (25, 62%). In 32 subjects with a complete histological remission without a second transplant, 1-year cumulative incidence of relapse was 41% (25, 62%) and 1-year EFS and survival, 59% (37, 81%). Therapy of B-ALL subjects relapsing post transplant with donor-derived CAR-T cells is safe and effective but associated with a high rate of CRS. Outcomes seem comparable to those achieved with alternative therapies but data from a randomized trial are lacking.

Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

CAR T cells: Living HIV drugs.

Human immunodeficiency virus type 1 (HIV-1), the virus that causes AIDS (acquired immunodeficiency syndrome), is a major global public health issue. Although the advent of combined antiretroviral therapy (ART) has made significant progress in inhibiting HIV replication in patients, HIV-infected cells remain the principal cellular reservoir of HIV, this allows HIV to rebound immediately upon stopping ART, which is considered the major obstacle to curing HIV infection. Chimeric antigen receptor (CAR) cell therapy has provided new opportunities for HIV treatment. Engineering T cells or hematopoietic stem cells (HSCs) to generate CAR T cells is a rapidly growing approach to develop an efficient immune cell to fight HIV. Herein, we review preclinical and clinical data available for the development of CAR T cells. Further, the advantages and disadvantages of clinical application of anti-HIV CAR T cells will be discussed.

Evaluation of targetable biomarkers for chimeric antigen receptor T-cell (CAR-T) in the treatment of pancreatic cancer: a systematic review and meta-analysis of preclinical studies.

One of the cutting edge techniques for treating cancer is the use of the patient's immune system to prevail cancerous disease. The versatility of the chimeric antigen receptor (CAR) T-cell approach in conjugation with promising treatments in haematological cancer has led to countless cases of research literature for the treatment of solid cancer. A systematic search of online databases as well as gray literature and reference lists of retrieved studies were carried out up to March 2019 to identify experimental animal studies that investigated the antigens targeted by CAR T-cell for pancreatic cancer treatment. Studies were evaluated for methodological quality using the SYstematic Review Center for Laboratory Animal Experimentation bias risk tool (SYRCLE's ROB tool). Pooled cytotoxicity ratio/percentage and 95% confidence intervals were calculated using the inverse-variance method while random-effects meta-analysis was used, taking into account conceptual heterogeneity. Heterogeneity was assessed with the Cochran Q statistic and quantified with the I2 statistic using Stata 13.0. Of the 485 identified studies, 56 were reviewed in-depth with 16 preclinical animal studies eligible for inclusion in the systematic review and 11 studies included in our meta-analysis. CAR immunotherapy significantly increased the cytotoxicity assay (percentage: 65%; 95% CI: 46%, 82%). There were no evidence for significant heterogeneity across studies [P = 0.38 (Q statistics), I2 = 7.14%] and for publication bias. The quality assessment of included studies revealed that the evidence was moderate to low quality and none of studies was judged as having a low risk of bias across all domains. CAR T-cell therapy is effective for pancreatic cancer treatment in preclinical animal studies. Further high-quality studies are needed to confirm our finding and a standard approach of this type of studies is necessary according to our assessment.

SJI 2020 special issue: A catalogue of Ovarian Cancer targets for CAR therapy.

Ovarian Cancer (OC) is currently difficult to cure, mainly due to its late detection and the advanced state of the disease at the time of diagnosis. Therefore, conventional treatments such as debulking surgery and combination chemotherapy are rarely able to control progression of the tumour, and relapses are frequent. Alternative therapies are currently being evaluated, including immunotherapy and advanced T cell-based therapy. In the present review, we will focus on a description of those Chimeric Antigen Receptors (CARs) that have been validated in the laboratory or are being tested in the clinic. Numerous target antigens have been defined due to the identification of OC biomarkers, and many are being used as CAR targets. We provide an exhaustive list of these constructs and their current status. Despite being innovative and efficient, the OC-specific CARs face a barrier to their clinical efficacy: the tumour microenvironment (TME). Indeed, effector cells expressing CARs have been shown to be severely inhibited, rendering the CAR T cells useless once at the tumour site. Herein, we give a thorough description of the highly immunosuppressive OC TME and present recent studies and innovations that have enabled CAR T cells to counteract this negative environment and to destroy tumours.

New insights into the pharmacological, immunological, and CAR-T-cell approaches in the treatment of hepatocellular carcinoma.

The tyrosine kinase inhibitor (TKI) sorafenib continues to be the anchor drug in the treatment of advanced stage hepatocellular carcinoma (HCC). Other TKIs as well as immune checkpoint inhibitors (ICIs) have also been approved, however the response rates remain poor and heterogeneous among HCC patients, largely due to antitumor drug resistance. Studies aimed at identifying novel biomarkers and developing new strategies to improve the response to current treatment and to overcome drug resistance, are urgently needed. Germline or somatic mutations, neoantigens, and an immunotolerogenic state against constant inflammatory stimuli in the liver, are crucial for the anti-tumor response. A pharmacogenetic approach has been attempted considering germline polymorphisms in genes encoding for proteins involved in drug-targeted pathways. Single gene and comprehensive multi-gene somatic profiling approaches have been adopted in HCC to identify tumor sensitivity scores and immunogenic profiles that can be exploited for new biomarkers and innovative therapeutic approaches. However, the high genomic heterogeneity of tumors and lack of molecularly targeted agents, hamper the discovery of specific molecular markers of resistance to therapy. Adoptive cell therapy with chimeric antigen receptor redirected T (CAR-T) cells targeting specific tumor-associated antigens (TAAs) was proposed recently. The specificity of the chosen TAA, an efficient homing of CAR-T cells to the tumor site, and the ability of CAR-T cells to survive in the tumor microenvironment are central factors in the success of CAR-T therapy. The current review describes the principal systemic treatments for HCC and the molecular evidence regarding potential predictive host and somatic genetic markers, as well as the emerging strategy of liquid biopsy for disease monitoring. Novel immunotherapeutic approaches for HCC treatment, including the use of ICIs and CAR-T, as well as strategies to overcome drug resistance, are discussed.