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We have undertaken this review to explore the various developments and insights of CAR-T cell therapy during 1989-2023 and its advantages in the treatment of cancer and immune modulation. It is a chimeric antigen receptor T-cell therapy, which is an innovative form of immunotherapy that harnesses the power of the immune system to fight cancer. At first, T cells are extracted from the patient's blood through a process called leukapheresis. Then the modification has been done in T cells by genetically engineered to express chimeric antigen receptors (CARs) on their surface. These receptors are designed to recognize specific proteins, or antigens, that are found on the surface of cancer cells. Many conventional therapies available in the market for the treatment of cancer and Immuno modulation but most of them having Adverse Drug Reaction (ADR). But CAR-T cells possess upper hand on these conventional Formulations. Once a sufficient number of CAR-T cells have been produced, they are infused back into the patient's bloodstream. Once reach inside the body, the CAR-T cells recognize and bind to the cancer cells that express the specific antigen targeted by the CAR. This triggers the destruction of the cancer cells by the immune system. CAR-T cell therapy has shown remarkable success in treating certain types of blood cancers, such as acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL), and certain types of lymphoma. The content of this review will pave the way to work on CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy has made impressive advances in improving the outcome of patients affected by malignant diseases. Nonetheless, some limitations still need to be tackled to more efficiently and safely treat patients, in particular for those affected by solid tumors. One of the limitations is related to the immunosuppressive tumor microenvironment (TME), which impairs antitumor immunity. Efforts to identify targets able to turn the TME into a milieu more auspicious to current immuno-oncotherapy is a real challenge due to the high redundancy of the mechanisms involved. However, the insulin-like growth factor 1 receptor (IGF1R), an attractive drug target for cancer therapy, is emerging as an important immunomodulator and regulator of key immune cell functions. Here, after briefly summarizing the IGF1R signaling pathway in cancer, we review its role in regulating immune cells function and activity, and discuss IGF1R as a promising target to improve anti-cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Ovarian cancer is a devastating disease due to the late diagnosis of advanced stage disease and high recurrence rates. Thus, new long-lasting, efficient drugs are needed. Since chimeric antigen receptor (CAR)-expressing T cell treatments have led to efficient and persisting anti-tumor responses, our study aimed to design and characterize CAR T cells targeting ovarian cancer. We confirmed folate receptor 1 (FOLR1) as a promising tumor-associated antigen in ovarian cancer patient samples. After designing a library of FOLR1-directed CAR T cells, their functionality and specificity were assessed in vitro against cell lines as well as in vivo in a mouse model of ovarian cancer. Finally, we selected a lead candidate for further characterization. The anti-FOLR1 CAR was successfully tested, including assays, reflecting the solid tumor microenvironment. These characteristics will support the clinical translation of our new FOLR1-specific CAR T cell asset. Treatment options for ovarian cancer patients are limited, and a high unmet clinical need remains for targeted and long-lasting, efficient drugs. Genetically modified T cells expressing chimeric antigen receptors (CAR), are promising new drugs that can be directed towards a defined target and have shown efficient, as well as persisting, anti-tumor responses in many patients. We sought to develop novel CAR T cells targeting ovarian cancer and to assess these candidates preclinically. First, we identified potential CAR targets on ovarian cancer samples. We confirmed high and consistent expressions of the tumor-associated antigen FOLR1 on primary ovarian cancer samples. Subsequently, we designed a series of CAR T cell candidates against the identified target and demonstrated their functionality against ovarian cancer cell lines in vitro and in an in vivo xenograft model. Finally, we performed additional in vitro assays recapitulating immune suppressive mechanisms present in solid tumors and developed a process for the automated manufacturing of our CAR T cell candidate. These findings demonstrate the feasibility of anti-FOLR1 CAR T cells for ovarian cancer and potentially other FOLR1-expressing tumors. [ABSTRACT FROM AUTHOR]

Natural killer cell‐derived extracellular vesicles (NK‐EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune‐modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical‐grade NK‐EVs for pre‐clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed‐loop hollow‐fibre bioreactor to continuously produce NK‐EVs from the clinically relevant NK92‐MI cell line under serum‐free, Xeno‐free and feeder‐free conditions following GMP‐compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK‐EV biotherapeutics at the end of each production lot (over 109 viable cells and 1013 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro‐inflammatory cytokine (interferon‐gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off‐target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell‐based and NK‐EV‐based immunotherapies for cancer with GMP considerations. [ABSTRACT FROM AUTHOR]

Simple Summary: The canonical methods of TCR gene delivery in pre-clinical and clinical applications are based on viral transduction of full-coding sequences, including α- and β-chains recognizing tumor-specific antigens and tumor-associated antigens. As the transduced α- and β-chains may mispair with the endogenous α- and β-chains, the resultant new antigen specificities may cause auto-reactivity, potentially leading to graft-versus-host disease. The mispaired TCRs may also lose their function. We assessed the feasibility of endogenous TCR orthotopic replacement with a TCR containing a CHM1 targeting sequence via CRISPR/Cas9, evaluated tumor recognition and cytotoxicity function of the CRISPR/Cas9-engineered T cells; compared the prevention of endogenous TCR expression in CRISPR/Cas9 vs. retrovirally engineered T cells. We show that both engineered T cell products specifically recognize tumor cells and elicit cytotoxicity in vitro, with CRISPR/Cas9 engineered T cells providing a more prolonged cytotoxic activity. Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer. [ABSTRACT FROM AUTHOR]

T cell immunotherapy remains an attractive approach for cancer immunotherapy. T cell immunotherapy mainly employs chimeric antigen receptor (CAR)- and T cell receptor (TCR)-engineered T cells. CAR-T cell therapy has been an essential breakthrough in treating hematological malignancies. TCR-T cells can recognize antigens expressed both on cell surfaces and in intracellular compartments. Although TCR-T cells have not been approved for clinical application, a number of clinical trials have been performed, particularly for solid tumors. In this article, we summarized current TCR-T cell advances and their potential advantages for solid tumor immunotherapy. [ABSTRACT FROM AUTHOR]

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Hematopoietic stem cell transplantation is a potent form of immunotherapy, potentially life-saving for many malignant hematologic diseases. However, donor lymphocytes infused with the graft while exerting a graft versus malignancy effect can also cause potentially fatal graft versus host disease (GVHD). Our group has previously validated the inducible caspase-9 suicide gene in the haploidentical stem cell transplant setting, which proved successful in reversing signs and symptoms of GVHD within hours, using a non-therapeutic dimerizing agent. Cellular death pathways such as apoptosis and necroptosis are important processes in maintaining healthy cellular homeostasis within the human body. Here, we review two of the most widely investigated cell death pathways active in T-cells (apoptosis and necroptosis), as well as the emerging strategies that can be exploited for the safety of T-cell therapies. Furthermore, such strategies could be exploited for the safety of other cellular therapeutics as well. [ABSTRACT FROM AUTHOR]

The field of adoptive cell transfer includes chimeric antigen receptor (CAR) engineered T cells, constructs that emerged from basic research into principles of immunology and have transformed into clinically effective therapies for haematological malignancies. T cells engineered to express these artificial receptors hold great promise, but also carry significant risk. While permanent genetic modification of mature T cells appears safe, modulating their in vivo function is difficult, partly because the robust response can trigger other arms of the immune system. Suicide systems and toxicity management with cytokine blockade or signal transduction modulators have emerged as a new frontier in this field, a far cry from early problems getting CAR T cells to work at all. Currently, clinical trials in patients with relapsed or refractory B cell malignancies treated with CD19-specific CAR T cells have induced durable remissions in adults and children. Results from these trials indicate that more work needs to be done to understand biomarkers of efficacy, the role of T cell persistence and how to integrate this care into standard practice. Cell therapy will not be a 'one size fits all' class of medicine, and here we will discuss the development of this therapy and important questions for its future. [ABSTRACT FROM AUTHOR]

Tumors originate from a number of genetic events that deregulate homeostatic mechanisms controlling normal cell behavior. The immune system, devoted to patrol the organism against pathogenic events, can identify transformed cells, and in several cases cause their elimination. It is however clear that several mechanisms encompassing both central and peripheral tolerance limit antitumor immunity, often resulting into progressive diseases. Adoptive T-cell therapy with either allogeneic or autologous T cells can transfer therapeutic immunity. To date, genetic engineering of T cells appears to be a powerful tool for shaping tumor immunity. In this review, we discuss the most recent achievements in the areas of suicide gene therapy, and TCR-modified T cells and chimeric antigen receptor gene-modified T cells. We provide an overview of current strategies aimed at improving the safety and efficacy of these approaches, with an outlook on prospective developments. [ABSTRACT FROM AUTHOR]

While opening new frontiers for the cure of malignant and non-malignant diseases, the increasing use of cell therapy poses also several new challenges related to the safety of a living drug. The most effective and consolidated cell therapy approach is allogeneic hematopoietic stem cell transplantation (HSCT), the only cure for several patients with high-risk hematological malignancies. The potential of allogeneic HSCT is strictly dependent on the donor immune system, particularly on alloreactive T lymphocytes, that promote the beneficial graft-versus-tumor effect (GvT), but may also trigger the detrimental graft-versus-host-disease (GvHD). Gene transfer technologies allow to manipulate donor T-cells to enforce GvT and foster immune reconstitution, while avoiding or controlling GvHD. The suicide gene approach is based on the transfer of a suicide gene into donor lymphocytes, for a safe infusion of a wide T-cell repertoire, that might be selectively controlled in vivo in case of GvHD. The herpes simplex virus thymidine kinase (HSV-TK) is the suicide gene most extensively tested in humans. Expression of HSV-TK in donor lymphocytes confers lethal sensitivity to the anti-herpes drug, ganciclovir. Progressive improvements in suicide genes, vector technology and transduction protocols have allowed to overcome the toxicity of GvHD while preserving the antitumor efficacy of allogeneic HSCT. Several phase I-II clinical trials in the last 20 years document the safety and the efficacy of HSV-TK approach, able to maintain its clear value over the last decades, in the rapidly progressing horizon of cancer cellular therapy. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptors (CARs) are recombinant receptors that combine the specificity of an antigen-specific antibody with the T-cell's activating functions. Initial clinical trials of genetically engineered CAR T cells have significantly raised the profile of T cell therapy, and great efforts have been made to improve this approach. In this review, we provide a structural overview of the development of CAR technology and highlight areas that require further refinement. We also discuss critical issues related to CAR therapy, including the optimization of CAR T cells, the route of administration, CAR toxicity and the blocking of inhibitory molecules. [ABSTRACT FROM AUTHOR]

The adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a relatively new but promising approach in the field of cancer immunotherapy. This therapeutic strategy is based on the genetic reprogramming of T cells with an artificial immune receptor that redirects them against targets on malignant cells and enables their destruction by exerting T cell effector functions. There has been an explosion of interest in the use of CAR T cells as an immunotherapy for cancer. In the pre-clinical setting, there has been a considerable focus upon optimizing the structural and signaling potency of the CAR while advances in bio-processing technology now mean that the clinical testing of these gene-modified T cells has become a reality. This review will summarize the concept of CAR-based immunotherapy and recent clinical trial activity and will further discuss some of the likely future challenges facing CAR-modified T cell therapies. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) is a stem cell disease in which BCR/ABL plays an important role as an oncoproteins and a molecular and immunogenic target. Despite the success of targeted therapy using tyrosine kinase inhibitors (TKIs), CML remains largely incurable, most likely due to the treatment resistance of leukemic stem cells. Several immunotherapies have been developed for CML in different stages and relapse after allogeneic stem cell transplantation. In the this review, several specific immunotherapeutic approaches for CML, including vaccination and adoptive cellular immunotherapy, are discussed along with results from clinical trials, and the value of such immunotherapies in the era of imatinib and leukemia-associated antigens (LAAs), which are capable of inducing specific T cell responses and are appropriate target structures for the immunological targeting of CML cells, are also summarized. [ABSTRACT FROM AUTHOR]

Background: Recent preclinical adoptive immunotherapy studies in murine models prompt to employ "proper" rather than "as many as possible" antigen-specific T cells to gain better therapeutic results. Ideally, "proper" T cells are poorly differentiated in vitro, but retain the capacity to fully differentiate into effector cells in vivo, where they can undergo long-term survival and strong proliferation. Such requirements can be achieved by modifying culture conditions, namely using less "differentiating" cytokines than IL-2. Methods: To evaluate this issue in human T cell cultures, we exploited a well characterized and clinical-grade protocol finalized at generating EBV-specific CTL for adoptive immunotherapy. In particular, we studied the impact of IL-7, IL-15 and IL-21 compared to IL-2 on different aspects of T cell functionality, namely growth kinetics, differentiation/activation marker expression, cytokine production, and short-term and long-term cytotoxicity. Results: Results disclosed that the culture modifications we introduced in the standard protocol did not improve activity nor induce substantial changes in differentiation marker expression of EBV-specific CTL. Conclusions: Our data indicated that the addition of γ-chain cytokines other than IL-2 for the generation of EBV-specific T cell cultures did not produce the improvements expected on the basis of recent published literature. This fact was likely due to the intrinsic differences between murine and human models and highlights the need to design ad hoc protocols rather than simply modify the cytokines added in culture. [ABSTRACT FROM AUTHOR]

Adoptive immunotherapy with in vitro expanded antigen-specific cytotoxic T lymphocytes (CTLs) may be an effective approach to prevent, or even treat, Aspergillus (Asp) infections. Such lines can be generated using monocyte-derived dendritic cells (DC) as antigen-presenting cells (APC) but requires a relatively high volume of starting blood. Here we describe a method that generates Asp-specific CTL responses more efficiently using a protocol of antigen presented on DC followed by Epstein–Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) as APC. Peripheral blood mononuclear cells were stimulated weekly (2–5×) with a complete pool of pentadecapeptides (PPC) spanning the coding region of Asp f16 pulsed onto autologous mature DC. Cultures were split and stimulated subsequently with either PPC-DC or autologous PPC-pulsed BLCL (PPC-BLCL). Lines from the DC/BLCL arm demonstrated Asp f16-specific cytotoxicity earlier and to a higher degree than lines generated with PPC-DC alone. The DC/BLCL-primed lines showed a higher frequency of Asp f16-specific interferon (IFN)-γ producing cells but an identical effector cell phenotype and peptide specificity compared to PPC-DC-only-primed lines. Tumour necrosis factor (TNF)-α, but not IL-10, appeared to play a role in the effectiveness of BLCL as APC. These results demonstrate that BLCL serve as highly effective APC for the stimulation of Asp f16-specific T cell responses and that a culture approach using initial priming with PPC-DC followed by PPC-BLCL may be a more effective method to generate Asp f16-specific T cell lines and requires less starting blood than priming with PPC-DC alone. [ABSTRACT FROM AUTHOR]

A variety of malignancies express Fas ligand (FasL), which can induce apoptosis in effector lymphocytes and may limit the success of cellular immunotherapy. Our laboratory has been investigating a population of ex vivo activated T cells, termed cytokine-induced killer (CIK) cells. These cells share functional and phenotypic properties with natural killer cells and a subset of cytolytic cells have the phenotype CD3+CD56+. CIK cells expand in culture, have significant antitumor activity and are presently being tested in phase I/II clinical trials. In this study, we investigated the sensitivity of CIK cells to Fas-mediated apoptosis. Fas engagement leads to apoptosis in small numbers of CIK cells and does not significantly influence antitumor cytotoxicity. CIK cells will undergo apoptosis following Fas engagement when protein synthesis is inhibited, suggesting the expression of antiapoptotic genes. Evaluation of antiapoptotic gene transcripts shows an up-regulation in the expression of cFLIP, Bcl-2, Bcl-xL, DAD1 and survivin. Resistance to Fas-mediated apoptosis may come about through an in vitro selection for Fas resistance, since CIK cells synthesize FasL and supernatant from CIK cultures contains biologically active soluble FasL, which can be inhibited with Fas:Fc. These results indicate that CIK cells are a suitable form of immunotherapy against FasL-positive tumors. [ABSTRACT FROM AUTHOR]

Renal cell carcinomas belong to the small group of tumors that are able to induce antitumor responses. Here we describe two general types of cytotoxic effector lymphocytes that can eliminate autologous tumor cells and discuss the role that major histocompatibility complex encoded molecules play in governing their specificities. Improved understanding of the cellular and molecular basis of renal cell carcinoma recognition opens new avenues of research with the potential to develop better immunotherapies for patients with metastatic disease. [ABSTRACT FROM AUTHOR]

Interleukin-2 (IL-2) is a cytokine produced by activated T cells. Its stimulatory activity allows T cells, B cells and natural killer cells to proliferate and to release cytokines and antibodies which protect the host against invading organisms. IL-2 plays a critical role in the prevention of apoptosis of HIV-infected cells, and the addition of IL-2 to a culture medium will increase the survival of T cells and will upregulate IL-2 receptor function. Clinical studies of the administration of exogenous IL-2 to HIV-infected patients have demonstrated that it can be given in well tolerated doses and that it can increase and sustain the number of CD4+ cells while only transiently affecting viral proliferation, especially when given to patients with CD4+ counts >200 cells/mm. Further investigations are required to determine the optimal use of exogenous IL-2 in HIV-infected patients. There may also be an important role for IL-2 as an adjunct to gene therapy and preventive vaccines against HIV infection. [ABSTRACT FROM AUTHOR]

Freshly isolated tumor-infiltrating lymphocytes (TIL) from stage IV melanoma patients were cultured for 2 weeks with low doses of interleukin-2 (IL-2; 120 IU/ml), to select potentially for tumor-specific lymphocytes present in the neoplastic lesion, followed by high doses (6000 IU/ml) to achieve lymphocyte expansion. TIL were serially analyzed for their expansion, phenotype and cytotoxic activity against autologous and allogeneic tumor cells. A preferential lysis of autologous melanoma cells was obtained in long-term cultures of 7/13 cases (54%), while the remaining ones showed a major-histocompatibility-complex-unrestricted, lymphokine-activated-killer(LAK)-like activity at the time of in vivo injection. Sixteen patients with metastatic melanoma were infused with TIL (mean number: 6.8×10, range: 0.35 × 10−20 × 10) and IL-2 (mean dose: 130 × 10 IU, range: 28.8 × 10−231 × 10 IU); 1 complete and 3 partial responses were observed in 12 evaluable patients (response rate 33%). In all responding patients, injected TIL showed an in vitro preferential lysis of autologous tumor cells, while in no cases were TIL with LAK-like activity associated with a clinical response. The mean autologous tumor cytotoxic activity of TIL at the time of in vivo injection was significantly higher in responding patients in comparison to nonresponding ones, suggesting that a marked and preferential cytolysis of autologous tumor cells is associated with the therapeutic efficacy of TIL. [ABSTRACT FROM AUTHOR]

T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci. [ABSTRACT FROM AUTHOR]