"Off-The-Shelf" allogeneic chimeric antigen receptor T-cell therapy for B-cell malignancies: current clinical evidence and challenges.

Chimeric antigen receptor T-cell therapy (CAR T) has revolutionized the treatment landscape for hematologic malignancies, notably B-cell non-Hodgkin lymphoma (B-NHL) and B-cell acute lymphoblastic leukemia (B-ALL). While autologous CAR T products have shown remarkable efficacy, their complex logistics, lengthy manufacturing process, and high costs impede widespread accessibility and pose therapeutic challenge especially for patients in rapid need for therapy. "Off-the-shelf" allogeneic CAR T-cell therapy (alloCAR T) has emerged as a promising alternative therapy, albeit experimental to date. AlloCARTs are derived from healthy donors, manufactured by batches and stored, making them available off-the-shelf which lowers financial burden. Various gene editing techniques have been employed to mitigate graft-versus-host disease (GVHD) and host-versus-graft (HvG) to enhance alloCAR T persistence. In this review, we summarize available manufacturing techniques, current evidence, and discuss challenges faced with the use of alloCAR Ts.

Chimeric antigen receptor T cells targeting the GM3(Neu5Gc) ganglioside.

Chimeric antigen receptor (CAR) T cell technology has ushered in a new era of immunotherapy, enabling the targeting of a broad range of surface antigens, surpassing the limitations of traditional T cell epitopes. Despite the wide range of non-protein tumor-associated antigens, the advancement in crafting CAR T cells for these targets has been limited. Owing to an evolutionary defect in the CMP-Neu5Ac hydroxylase (CMAH) that abolishes the synthesis of CMP-Neu5Gc from CMP-Neu5Ac, Neu5Gc is generally absent in human tissues. Despite this, Neu5Gc-containing antigens, including the ganglioside GM3(Neu5Gc) have consistently been observed on tumor cells across a variety of human malignancies. This restricted expression makes GM3(Neu5Gc) an appealing and highly specific target for immunotherapy. In this study, we designed and evaluated 14F7-28z CAR T cells, with a targeting unit derived from the GM3(Neu5Gc)-specific murine antibody 14F7. These cells exhibited exceptional specificity, proficiently targeting GM3(Neu5Gc)-expressing murine tumor cells in syngeneic mouse models, ranging from B cell malignancies to epithelial tumors, without compromising safety. Notably, human tumor cells enhanced with murine Cmah were effectively targeted and eliminated by the 14F7 CAR T cells. Nonetheless, despite the detectable presence of GM3(Neu5Gc) in unmodified human tumor xenografts, the levels were insufficient to trigger a tumoricidal T-cell response with the current CAR T cell configuration. Overall, our findings highlight the potential of targeting the GM3(Neu5Gc) ganglioside using CAR T cells across a variety of cancers and set the stage for the optimization of 14F7-based therapies for future human clinical application.

Has the shortage of fludarabine altered the current paradigm of lymphodepletion in favor of bendamustine?

The most common lymphodepletion regimen used prior to infusion of chimeric antigen receptor-T cells (CAR-T) is cyclophosphamide (CY) in combination with fludarabine (Flu) (CY-FLU). While cyclophosphamide (CY) possesses lymphotoxic effects, it concurrently preserves regulatory T cell activity, potentially affecting the efficacy of CAR-T cells. Moreover, the use of fludarabine (FLU) has been linked to neurotoxicity, which could complicate the early detection of immune effector cell-associated neurotoxicity syndrome (ICANS) observed in CAR-T cell therapy. Given the ongoing shortage of FLU, alternative lymphodepleting agents have become necessary. To date, only a limited number of studies have directly compared different lymphodepleting regimens, and most of these comparisons have been retrospective in nature. Herein, we review the current literature on lymphodepletion preceding CAR-T cell therapies for lymphoid hematologic malignancies, with a specific focus on the use of bendamustine (BEN). Recent evidence suggests that administering BEN before CAR-T cell infusion yields comparable efficacy, possibly with a more favorable toxicity profile when compared to CY-FLU. This warrants further investigation through randomized prospective studies.

How can Cytokine-induced killer cells overcome CAR-T cell limits.

The successful treatment of patients affected by B-cell malignancies with Chimeric Antigen Receptor (CAR)-T cells represented a breakthrough in the field of adoptive cell therapy (ACT). However, CAR-T therapy is not an option for every patient, and several needs remain unmet. In particular, the production of CAR-T cells is expensive, labor-intensive and logistically challenging; additionally, the toxicities deriving from CAR-T cells infusion, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), have been documented extensively. Alternative cellular therapy products such as Cytokine-induced killer (CIK) cells have the potential to overcome some of these obstacles. CIK cells are a heterogeneous population of polyclonal CD3+CD56+ T cells with phenotypic and functional properties of NK cells. CIK cell cytotoxicity is exerted in a major histocompatibility complex (MHC)-unrestricted manner through the engagement of natural killer group 2 member D (NKG2D) molecules, against a wide range of hematological and solid tumors without the need for prior antigen exposure or priming. The foremost potential of CIK cells lies in the very limited ability to induce graft-versus-host disease (GvHD) reactions in the allogeneic setting. CIK cells are produced with a simple and extremely efficient expansion protocol, which leads to a massive expansion of effector cells and requires a lower financial commitment compared to CAR-T cells. Indeed, CAR-T manufacturing involves the engineering with expensive GMP-grade viral vectors in centralized manufacturing facilities, whereas CIK cell production is successfully performed in local academic GMP facilities, and CIK cell treatment is now licensed in many countries. Moreover, the toxicities observed for CAR-T cells are not present in CIK cell-treated patients, thus further reducing the costs associated with hospitalization and post-infusion monitoring of patients, and ultimately encouraging the delivery of cell therapies in the outpatient setting. This review aims to give an overview of the limitations of CAR-T cell therapy and outline how the use of CIK cells could overcome such drawbacks thanks to their unique features. We highlight the undeniable advantages of using CIK cells as a therapeutic product, underlying the opportunity for further research on the topic.

Cell metabolism-based optimization strategy of CAR-T cell function in cancer therapy.

Adoptive cell therapy (ACT) using chimeric antigen receptor (CAR)-modified T cells has revolutionized the field of immune-oncology, showing remarkable efficacy against hematological malignancies. However, its success in solid tumors is limited by factors such as easy recurrence and poor efficacy. The effector function and persistence of CAR-T cells are critical to the success of therapy and are modulated by metabolic and nutrient-sensing mechanisms. Moreover, the immunosuppressive tumor microenvironment (TME), characterized by acidity, hypoxia, nutrient depletion, and metabolite accumulation caused by the high metabolic demands of tumor cells, can lead to T cell "exhaustion" and compromise the efficacy of CAR-T cells. In this review, we outline the metabolic characteristics of T cells at different stages of differentiation and summarize how these metabolic programs may be disrupted in the TME. We also discuss potential metabolic approaches to improve the efficacy and persistence of CAR-T cells, providing a new strategy for the clinical application of CAR-T cell therapy.

Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors.

Background: T cells that are genetically modified with chimeric antigen receptor (CAR) hold promise for immunotherapy of cancer. Currently, there are intense efforts to improve the safety and efficacy of CAR T cell therapies against liquid and solid tumors. Earlier we designed a novel CAR backbone (FiCAR) where the spacer is derived from immunoglobulin (Ig) -like domains of the signal-regulatory protein alpha (SIRPα). However, the analysis of novel CAR using primary T cells is slow and laborious. Methods: To explore the versatility of the CAR backbone, we designed a set of variant FiCARs with different spacer lengths and targeting antigens. To expedite the analysis of the novel CARs, we transduced the FiCAR genes using lentiviruses into Jurkat reporter T cells carrying fluorescent reporter genes. The expression of fluorescent markers in response to FiCAR engagement with targets was analyzed by flow cytometry, and cytotoxicity was evaluated using killing assays. Furthermore, the killing mechanisms that are employed by FiCAR-equipped Jurkat T cells were investigated by flow cytometry, and the intracellular pathways involved in signaling by FiCAR were analyzed by phosphoproteomic analysis using mass spectrometry. Results: Seven different CARs were designed and transduced into Jurkat reporter cells. We show that the SIRPα derived FiCARs can be detected by flow cytometry using the SE12B6A4 antibody recognizing SIRPα. Furthermore, FiCAR engagement leads to robust activation of NFκß and NFAT signaling, as demonstrated by the expression of the fluorescent reporter genes. Interestingly, the Jurkat reporter system also revealed tonic signaling by a HER-2 targeting FiCAR. FiCAR-equipped Jurkat T cells were cytotoxic in cocultures with target cells and target cell engagement lead to an upregulation of CD107a on the Jurkat reporter T cell surface. Phosphoproteomic analyses confirmed signal transduction via the intracellular CD28/CD3ζ sequences upon the interaction of the FiCAR1 with its antigen. In addition, downstream signaling of CD3ζ/ZAP70- SLP-76-PLCγ, PI3K-AKT-NFκB pathways and activation of NFAT and AP-1 were observed. Conclusion: We conclude that the FiCAR backbone can be shortened and lengthened at will by engineering it with one to three SIRPα derived Ig-like domains, and the FiCARs are functional when equipped with different single chain variable fragment target binding domains. The Jurkat reporter system expedites the analysis of novel CARs as to their expression, signaling function, evaluation of tonic signaling issues and cytotoxic activity.

Multiple CAR-T cell therapy for acute B-cell lymphoblastic leukemia after hematopoietic stem cell transplantation: A case report.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood malignancy. The cure rate has reached 90% after conventional chemotherapy and hematopoietic stem cell transplantation (HSCT), but the prognosis of patients with relapsed and refractory (R/R) leukemia is still poor after conventional treatment. Since FDA approved CD19 CAR-T cell (Kymriah) for the treatment of R/R B-ALL, increasing studies have been conducted on CAR-T cells for R/R ALL. Herein, we report the treatment of a patient with ALL who relapsed after allogeneic HSCT, had a complete remission (CR) to murine scFv CD19 CAR-T but relapsed 15 months later. Partial response was achieved after humanized CD19 CAR-T treatment, and the patient finally achieved disease-free survival after sequential CD22 CAR-T treatment. By comparing the treatment results of different CAR-T cells in the same patient, this case suggests that multiple CAR-T therapies are effective and safe in intramedullary and extramedullary recurrence in the same patient, and the expansion of CAR-T cells and the release of inflammatory cytokines are positively correlated with their efficacy. However, further clinical studies with large sample sizes are still needed for further clarification.

Targeting TGF-ß signaling in the multiple myeloma microenvironment: Steering CARs and T cells in the right direction.

Multiple myeloma (MM) remains a lethal hematologic cancer characterized by the expansion of transformed plasma cells within the permissive bone marrow (BM) milieu. The emergence of relapsed and/or refractory MM (RRMM) is provoked through clonal evolution of malignant plasma cells that harbor genomic, metabolic and proteomic perturbations. For most patients, relapsed disease remains a major cause of overall mortality. Transforming growth factors (TGFs) have pleiotropic effects that regulate myelomagenesis as well as the emergence of drug resistance. Moreover, TGF-ß modulates numerous cell types present with the tumor microenvironment, including many immune cell types. While numerous agents have been FDA-approved over the past 2 decades and significantly expanded the treatment options available for MM patients, the molecular mechanisms responsible for drug resistance remain elusive. Multiple myeloma is uniformly preceded by a premalignant state, monoclonal gammopathy of unknown significance, and both conditions are associated with progressive deregulation in host immunity characterized by reduced T cell, natural killer (NK) cell and antigen-presenting dendritic cell (DC) activity. TGF-ß promotes myelomagenesis as well as intrinsic drug resistance by repressing anti-myeloma immunity to promote tolerance, drug resistance and disease progression. Hence, repression of TGF-ß signaling is a prerequisite to enhance the efficacy of current and future immunotherapeutics. Novel strategies that incorporate T cells that have been modified to express chimeric antigen receptor (CARs), T cell receptors (TCRs) and bispecific T cell engagers (BiTEs) offer promise to block TGF-ß signaling, overcome chemoresistance and enhance anti-myeloma immunity. Here, we describe the effects of TGF-ß signaling on immune cell effectors in the bone marrow and emerging strategies to overcome TGF-ß-mediated myeloma growth, drug resistance and survival.

New insights into CAR T cell-mediated killing of tumor cells.

Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CAR) has demonstrated striking efficacy for the treatment of several hematological malignancies, including B-cell lymphoma, leukemia, and multiple myeloma. However, CAR T-cell efficacy has been very limited in most solid tumors. In this context, it is of paramount importance to understand the determinants that condition CAR T-cell success versus failure. To control tumor growth, CAR T cells need to form conjugates with their targets via the assembly of an immunological synapse. Here, we review recent advances showing that the adhesion between CAR T cells and cancer cells from solid tumors strengthens over time in an IFNγ- and ICAM-1-dependent manner, resulting in CAR T cell-mediated killing. We discuss how these findings can be exploited to increase the efficacy of the CAR T-cell strategy against solid tumors.

Case Report: Subtotal Lymphoid and Total Marrow Irradiation as Bridge Therapy to CD19-Directed CAR T Cells in a Chemorefractory DLBCL With Leukemic Involvement.

CAR T cell therapy has transformed the salvage approach for relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). Maintaining disease control before CAR T cell infusion during product manufacturing (so-called bridging therapy) is an important step to optimizing outcome. Among possible bridging therapies, radiation therapy (RT) represents a valuable option, particularly when the disease is limited. Here, we report for the first time on a patient with chemorefractory-transformed DLBCL showing nodal, extranodal, and massive bone marrow (BM) lymphoma infiltration associated with leukemic involvement, a successful bridge therapy to CD19-directed CAR T cell therapy by subtotal lymphoid/total marrow irradiation plus thiothepa followed by reinfusion of CD34+ autologous hematopoietic stem cells. Such a novel bridging regimen allowed a significant reduction of nodal and BM tumor volume while improving blood cell count before CAR T cell infusion. The PET-CT scan and BM evaluation performed at 1, 3, and 6 months after treatment showed complete remission of the disease. A relapse occurred at almost 1 year in lymph nodes because of CD19 antigen escape while the BM remained free of disease. This extended radiotherapy approach may be an effective bridging therapy for chemorefractory DLBCL patients eligible for CAR T cells who present with a high tumor burden, including massive BM involvement associated with leukemic involvement. This preliminary evidence is worth confirming in additional patients.

Recent Advances in IL-13Rα2-Directed Cancer Immunotherapy.

Interleukin-13 receptor subunit alpha-2 (IL-13Rα2, CD213A), a high-affinity membrane receptor of the anti-inflammatory Th2 cytokine IL-13, is overexpressed in a variety of solid tumors and is correlated with poor prognosis in glioblastoma, colorectal cancer, adrenocortical carcinoma, pancreatic cancer, and breast cancer. While initially hypothesized as a decoy receptor for IL-13-mediated signaling, recent evidence demonstrates IL-13 can signal through IL-13Rα2 in human cells. In addition, expression of IL-13Rα2 and IL-13Rα2-mediated signaling has been shown to promote tumor proliferation, cell survival, tumor progression, invasion, and metastasis. Given its differential expression in tumor versus normal tissue, IL-13Rα2 is an attractive immunotherapy target, as both a targetable receptor and an immunogenic antigen. Multiple promising strategies, including immunotoxins, cancer vaccines, and chimeric antigen receptor (CAR) T cells, have been developed to target IL-13Rα2. In this mini-review, we discuss recent developments surrounding IL-13Rα2-targeted therapies in pre-clinical and clinical study, including potential strategies to improve IL-13Rα2-directed cancer treatment efficacy.

Update for Advance CAR-T Therapy in Solid Tumors, Clinical Application in Peritoneal Carcinomatosis From Colorectal Cancer and Future Prospects.

Latest advances in the field of cancer immunotherapy have developed the (Chimeric Antigen Receptor) CAR-T cell therapy. This therapy was first used in hematological malignancies which obtained promising results; therefore, the use of CAR-T cells has become a popular approach for treating non-solid tumors. CAR-T cells consist of T-lymphocytes that are engineered to express an artificial receptor against any surface antigen of our choice giving us the capacity of offering precise and personalized treatment. This leaded to the development of CAR-T cells for treating solid tumors with the hope of obtaining the same result; however, their use in solid tumor and their efficacy have not achieved the expected results. The reason of these results is because solid tumors have some peculiarities that are not present in hematological malignancies. In this review we explain how CAR-T cells are made, their mechanism of action, adverse effect and how solid tumors can evade their action, and also we summarize their use in colorectal cancer and peritoneal carcinomatosis.

A Model Perspective Explanation of the Long-Term Sustainability of a Fully Human BCMA-Targeting CAR (CT103A) T-Cell Immunotherapy.

Different from canonical drugs, CAR T-cells are "living drugs", which derived from patient's own blood. Studies of the pharmacokinetics of CAR T-cells could improve our understanding of their efficacy, safety, optimal dosage, and other characterizes. We previously reported a phase I study of a novel fully human BCMA-targeting CAR (CT103A) in 18 patients with relapsed/refractory multiple myeloma. CT103A exhibited extraordinary persistence with low anti-drug antibody positivity. To figure out the pharmacokinetic characterizes and investigate the potential reason of CT103A's long-term persistence, we established a population pharmacokinetic (PopPK) model of CT103A based on 18 patients cohort by applying nonlinear mixed-effects modeling and analyzed the CAR T-cell clonal evolution. The results suggested that extramedullary spreading was found to impair Cmax and was therefore added as a covariate to the modified model. The model revealed tocilizumab and corticosteroids showed no impact on the CT103A expansion rate. No dominant clone existed in patients with persistently high peripheral CT103A by CAR integration sites analysis. Finally, patients with lower contraction rate constants and higher Cmax as well as memory CT103A fraction could achieve better clinical responses. Taken together, this study developed a PopPK model of a fully human anti-BCMA CAR T-cell therapy, and summarized its model characteristics. We suggested that the long-term persistence of CT103A was attributed to the memory CAR T-cell fraction but not the clonal evolution. This study will improve people's understanding of pharmacokinetics and PopPK of CAR T-cell immunotherapy.

Targets for chimeric antigen receptor T-cell therapy of acute myeloid leukemia.

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy associated with high mortality rates (less than 30% 5-year survival). Despite advances in our understanding of the molecular mechanisms underpinning leukemogenesis, standard-of-care therapeutic approaches have not changed over the last couple of decades. Chimeric Antigen Receptor (CAR) T-cell therapy targeting CD19 has shown remarkable clinical outcomes for patients with acute lymphoblastic leukemia (ALL) and is now an FDA-approved therapy. Targeting of myeloid malignancies that are CD19-negative with this promising technology remains challenging largely due to lack of alternate target antigens, complex clonal heterogeneity, and the increased recognition of an immunosuppressive bone marrow. We carefully reviewed a comprehensive list of AML targets currently being used in both proof-of-concept pre-clinical and experimental clinical settings. We analyzed the expression profile of these molecules in leukemic as well normal tissues using reliable protein databases and data reported in the literature and we provide an updated overview of the current clinical trials with CAR T-cells in AML. Our study represents a state-of-art review of the field and serves as a potential guide for selecting known AML-associated targets for adoptive cellular therapies.

Vitamin C, From Supplement to Treatment: A Re-Emerging Adjunct for Cancer Immunotherapy?

Vitamin C (VitC), in addition to its role as a general antioxidant, has long been considered to possess direct anti-cancer activity at high doses. VitC acts through oxidant and epigenetic mechanisms, which at high doses can exert direct killing of tumor cells in vitro and delay tumor growth in vivo. Recently, it has also been shown that pharmacologic-dose VitC can contribute to control of tumors by modulating the immune system, and studies have been done interrogating the role of physiologic-dose VitC on novel adoptive cellular therapies (ACTs). In this review, we discuss the effects of VitC on anti-tumor immune cells, as well as the mechanisms underlying those effects. We address important unanswered questions concerning both VitC and ACTs, and outline challenges and opportunities facing the use of VitC in the clinical setting as an adjunct to immune-based anti-cancer therapies.

Pushing Past the Blockade: Advancements in T Cell-Based Cancer Immunotherapies.

Successful cancer immunotherapies rely on a replete and functional immune compartment. Within the immune compartment, T cells are often the effector arm of immune-based strategies due to their potent cytotoxic capabilities. However, many tumors have evolved a variety of mechanisms to evade T cell-mediated killing. Thus, while many T cell-based immunotherapies, such as immune checkpoint inhibition (ICI) and chimeric antigen receptor (CAR) T cells, have achieved considerable success in some solid cancers and hematological malignancies, these therapies often fail in solid tumors due to tumor-imposed T cell dysfunctions. These dysfunctional mechanisms broadly include reduced T cell access into and identification of tumors, as well as an overall immunosuppressive tumor microenvironment that elicits T cell exhaustion. Therefore, novel, rational approaches are necessary to overcome the barriers to T cell function elicited by solid tumors. In this review, we will provide an overview of conventional immunotherapeutic strategies and the various barriers to T cell anti-tumor function encountered in solid tumors that lead to resistance. We will also explore a sampling of emerging strategies specifically aimed to bypass these tumor-imposed boundaries to T cell-based immunotherapies.