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Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells. When used in CAR-T cell processing, the plant alkaloid berbamine prolongs the survival of leukemic animals by boosting CAR-T cell viability, memory phenotype, and persistence by modulating cellular metabolism, including mitochondrial respiration. [ABSTRACT FROM AUTHOR]

CAR-T-cell therapy has shown promise in treating hematological malignancies but faces challenges in treating solid tumors due to impaired T-cell function in the tumor microenvironment. To provide optimal T-cell activation, we developed a B7 homolog 3 protein (B7H3)-targeting CAR construct consisting of three activation signals: CD3ζ (signal 1), 41BB (signal 2), and the interleukin 7 receptor alpha (IL7Rα) cytoplasmic domain (signal 3). We generated B7H3 CAR-T cells with different lengths of the IL7Rα cytoplasmic domain, including the full length (IL7R-L), intermediate length (IL7R-M), and short length (IL7R-S) domains, and evaluated their functionality in vitro and in vivo. All the B7H3-IL7Rα CAR-T cells exhibited a less differentiated phenotype and effectively eliminated B7H3-positive glioblastoma in vitro. Superiority was found in B7H3 CAR-T cells contained the short length of the IL7Rα cytoplasmic domain. Integration of the IL7R-S cytoplasmic domain maintained pSTAT5 activation and increased T-cell proliferation while reducing activation-induced cell death. Moreover, RNA-sequencing analysis of B7H3-IL7R-S CAR-T cells after coculture with a glioblastoma cell line revealed downregulation of proapoptotic genes and upregulation of genes associated with T-cell proliferation compared with those in 2nd generation B7H3 CAR-T cells. In animal models, compared with conventional CAR-T cells, B7H3-IL7R-S CAR-T cells suppressed tumor growth and prolonged overall survival. Our study demonstrated the therapeutic potential of IL7Rα-incorporating CAR-T cells for glioblastoma treatment, suggesting a promising strategy for augmenting the effectiveness of CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept. [ABSTRACT FROM AUTHOR]

Pancreatic cancer remains a challenging disease with limited treatment options, resulting in high mortality rates. The predominant approach to managing pancreatic cancer patients continues to be systemic cytotoxic chemotherapy. Despite substantial advancements in immunotherapy strategies for various cancers, their clinical utility in pancreatic cancer has proven less effective and durable. Whether administered as monotherapy, employing immune checkpoint inhibitors, tumor vaccines, chimeric antigen receptors T cells, or in combination with conventional chemoradiotherapy, the clinical outcomes remain underwhelming. Extensive preclinical experiments and clinical trials in the realm of pancreatic cancer have provided valuable insights into the complexities of immunotherapy. Chief among the hurdles are the immunosuppressive tumor microenvironment, limited immunogenicity, and the inherent heterogeneity of pancreatic cancer. In this comprehensive review, we provide an overview and critical analysis of current clinical immunotherapy strategies for pancreatic cancer, emphasizing their endeavors to overcome immunotherapy resistance. Particular focus is placed on strategies aimed at reshaping the immunosuppressive microenvironment and enhancing T cell-mediated tumor cell killing. Ultimately, through deeper elucidation of the underlying pathogenic mechanisms of pancreatic cancer and the refinement of therapeutic approaches, we anticipate breakthroughs that will pave the way for more effective treatments in this challenging disease. [ABSTRACT FROM AUTHOR]

Engineered T cells have emerged as highly effective treatments for hematological cancers. Hundreds of clinical programs are underway in efforts to expand the efficacy, safety, and applications of this immuno-therapeutic modality. A primary challenge in developing these "living drugs" is the complexity of their pharmacology, as the drug product proliferates, differentiates, traffics between tissues, and evolves through interactions with patient immune systems. Using publicly available clinical data from Chimeric Antigen Receptor (CAR) T cells, we demonstrate how mathematical models can be used to quantify the relationships between product characteristics, patient physiology, pharmacokinetics and clinical outcomes. As scientists work to develop next-generation cell therapy products, mathematical models will be integral for contextualizing data and facilitating the translation of product designs to clinical strategy. [ABSTRACT FROM AUTHOR]

Background: Aggressive B cell lymphoma with secondary central nervous system (CNS) involvement (SCNSL) carries a dismal prognosis. Chimeric antigen receptor (CAR) T cells (CAR-T) targeting CD19 have revolutionized the treatment for B cell lymphomas; however, only single cases with CNS manifestations successfully treated with CD19 CAR-T have been reported. Methods: We prospectively enrolled 4 patients with SCNSL into our study to assess clinical responses and monitor T cell immunity. Results: Two of four SNCSL patients responded to the CD19-targeted CAR-T. Only one patient showed a substantial expansion of peripheral (PB) CAR-T cells with an almost 100-fold increase within the first week after CAR-T. The same patient also showed marked neurotoxicity and progression of the SNCSL despite continuous surface expression of CD19 on the lymphoma cells and an accumulation of CD4+ central memory-type CAR-T cells in the CNS. Our studies indicate that the local production of chemokine IP-10, possibly through its receptor CXCR3 expressed on our patient's CAR-T, could potentially have mediated the local accumulation of functionally suboptimal anti-tumor T cells. Conclusions: Our results demonstrate expansion and homing of CAR-T cells into the CNS in SNCSL patients. Local production of chemokines such as IP-10 may support CNS infiltration by CAR-T cells but also carry the potential of amplifying local toxicity. Future studies investigating numbers, phenotype, and function of CAR-T in the different body compartments of SNSCL patients receiving CAR-T will help to improve local delivery of "fit" and highly tumor-reactive CAR-T with low off-target reactivity into the CNS. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is an autoimmune disorder that affects the neuromuscular junction, leading to muscle weakness and fatigue. MG is caused by antibodies against the acetylcholine receptor (AChR), the muscle-specific kinase (MuSK) or other AChR-related proteins that are expressed in the postsynaptic muscle membrane. The standard therapeutic approach for MG has relied on acetylcholinesterase inhibitors, corticosteroids and immunosuppressants, which have shown good efficacy in improving MG-related symptoms in most people with the disease; however, these therapies can carry a considerable burden of long-term adverse effects. Moreover, up to 15% of individuals with MG exhibit limited or no response to these standard therapies. The emergence of molecular therapies, including monoclonal antibodies, B cell-depleting agents and chimeric antigen receptor T cell-based therapies, has the potential to revolutionize the MG treatment landscape. This Review provides a comprehensive overview of the progress achieved in molecular therapies for MG associated with AChR antibodies and MuSK antibodies, elucidating both the challenges and the opportunities these therapies present to the field. The latest developments in MG treatment are described, exploring the potential for personalized medicine approaches. This Review provides a comprehensive summary of the growing number of molecular therapies for the treatment of myasthenia gravis, including monoclonal antibodies, B cell-depleting agents and chimeric antigen receptor T cell-based therapies. [ABSTRACT FROM AUTHOR]

Recently, a breakthrough immunotherapeutic strategy of chimeric antigen receptor (CAR) T-cells has been introduced to hematooncology. However, to apply this novel treatment in solid cancers, one must identify suitable molecular targets in the tumors of choice. CEACAM family proteins are involved in the progression of a range of malignancies, including pancreatic and breast cancers, and pose attractive targets for anticancer therapies. In this work, we used a new CEACAM-targeted 2A3 single-domain antibody-based chimeric antigen receptor T-cells to evaluate their antitumor properties in vitro and in animal models. Originally, 2A3 antibody was reported to target CEACAM6 molecule; however, our in vitro co-incubation experiments showed activation and high cytotoxicity of 2A3-CAR T-cells against CEACAM5 and/or CEACAM6 high human cell lines, suggesting cross-reactivity of this antibody. Moreover, 2A3-CAR T-cells tested in vivo in the BxPC-3 xenograft model demonstrated high efficacy against pancreatic cancer xenografts in both early and late intervention treatment regimens. Our results for the first time show an enhanced targeting toward CEACAM5 and CEACAM6 molecules by the new 2A3 sdAb-based CAR T-cells. The results strongly support the further development of 2A3-CAR T-cells as a potential treatment strategy against CEACAM5/6-overexpressing cancers. [ABSTRACT FROM AUTHOR]

Multiple Myeloma remains incurable, and there is a need for therapies with novel mechanisms of action. Recently, B cell maturation antigen targeted therapy has demonstrated deep and durable responses in a largely treated population. However, the relapse rate of myeloma patients after anti-BCMA treatment strategies is increasing worldwide, and one of the most challenging issues for them is to choose the best therapy sequencing. After anti-BCMA treatment, retreatment with anti-BCMA drugs remains an option, but new targets are emerging strongly. One of them is G protein-coupled receptor, class C group 5 member D (GPRC5D), that due to the very promising data from the use of chimeric antigen receptor T-cells (CAR-T) and bispecific antibodies (BsAb) seems to be the ideal candidate in the relay of myeloma treatment at relapse. In this literature review, we discuss data from treatment with the new drugs at relapse after anti-BCMA therapies, observing an undeniable benefit from the use of drugs directed against GPRC5D. [ABSTRACT FROM AUTHOR]

Process development for transferring lab-scale research workflows to automated manufacturing procedures is critical for chimeric antigen receptor (CAR)-T cell therapies. Therefore, the key factor for cell viability, expansion, modification, and functionality is the optimal combination of medium and T cell activator as well as their regulatory compliance for later manufacturing under Good Manufacturing Practice (GMP). In this study, we compared two protocols for CAR-mRNA-modified T cell generation using our current lab-scale process, analyzed all mentioned parameters, and evaluated the protocols' potential for upscaling and process development of mRNA-based CAR-T cell therapies. [ABSTRACT FROM AUTHOR]

To clarify the role of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) in the chimeric antigen receptor T-cell therapy era, we analyzed the clinical characteristics and outcomes of 52 patients treated with allo-HSCT with relapsed/refractory diffuse large B cell lymphoma. Most enrolled patients had previously undergone intensive treatments, the median number of chemotherapy lines was 4, and the median time from diagnosis to allo-HSCT was 27.1 months. Patients were divided into remission-achieved (n = 30) and active-disease (n = 22) groups before allo-HSCT. Over a median follow-up period of 38.3 months, overall survival (OS) and event-free survival (EFS) rates were 38.4% and 30.6%, respectively. The cumulative incidence of relapse (CIR) and the non-relapsed mortality (NRM) were 36.7% and 32.7%, respectively. OS, EFS, and graft-versus-host disease-free, relapse-free survival (GRFS) outcomes were significantly superior in the remission-achieved group with lower CIR. In a multivariate analysis, a shorter interval from diagnosis to allo-HSCT reflected relatively rapid disease progression and showed significantly poor OS and EFS with higher CIR. Patients with active disease had significantly lower EFS, GRFS, and higher CIR. Previous autologous stem-cell transplantation was associated with better GRFS. Allo-HSCT is an established modality with a prominent group of cured patients and still has a role in the CAR T-cell era, particularly given its acceptable clinical outcomes in young patients with chemo-susceptible disease. [ABSTRACT FROM AUTHOR]

Chimeric Antigen Receptor (CAR) T-cell therapy is a highly effective treatment for B-cell malignancies but limited in use due to clinically significant hyperinflammatory toxicities. Understanding the pathophysiologic mechanisms which mediate these toxicities can help identify novel management strategies. Here we report a novel in vitro model of the macrophage-endothelial interface to study the effects of CAR T-cell-induced cytokine storm. Using this model, we demonstrate that macrophage-mediated inflammation is regulated by endothelial cell activity. Furthermore, endothelial inflammation occurs independently of macrophages following exposure to CAR T-cell products and the induced endothelial inflammation potentiates macrophage-mediated inflammatory signaling, leading to a hyperinflammatory environment. While corticosteroids, the current gold standard of care, attenuate the resulting macrophage inflammatory signaling, the endothelial activity remains refractory to this treatment strategy. Utilizing a network model, coupled to in vitro secretion profiling, we identified STAT3 programming as critical in regulating this endothelial behavior. Lastly, we demonstrate how targeting STAT3 activity can abrogate endothelial inflammation and attenuate this otherwise hyperinflammatory environment. Our results demonstrate that endothelial cells play a central role in the pathophysiology of CAR T-cell toxicities and targeting the mechanisms driving the endothelial response can guide future clinical management. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer is the most lethal of gynecological cancers. The therapeutic efficacy of chimeric antigen receptor (CAR) T cell directed against single antigens is limited by the heterogeneous target antigen expression in epithelial ovarian tumors. To overcome this limitation, we describe an engineered cell with both dual targeting and orthogonal cytotoxic modalities directed against two tumor antigens that are highly expressed on ovarian cancer cells: cell surface Muc16 and intracellular WT1. Muc16-specific CAR T cells (4H11) were engineered to secrete a bispecific T cell engager (BiTE) constructed from a TCR mimic antibody (ESK1) reactive with the WT1-derived epitope RMFPNAPYL (RMF) presented by HLA-A2 molecules. The secreted ESK1 BiTE recruited and redirected other T cells to WT1 on the tumor cells. We show that ESK1 BiTE-secreting 4H11 CAR T cells exhibited enhanced anticancer activity against cancer cells with low Muc16 expression, compared to 4H11 CAR T cells alone, both in vitro and in mouse tumor models. Dual orthogonal cytotoxic modalities with different specificities targeting both surface and intracellular tumor-associated antigens present a promising strategy to overcome resistance to CAR T cell therapy in epithelial ovarian cancer and other cancers. [ABSTRACT FROM AUTHOR]

Process development for transferring lab-scale research workflows to automated manufacturing procedures is critical for chimeric antigen receptor (CAR)-T cell therapies. Therefore, the key factor for cell viability, expansion, modification, and functionality is the optimal combination of medium and T cell activator as well as their regulatory compliance for later manufacturing under Good Manufacturing Practice (GMP). In this study, we compared two protocols for CAR-mRNA-modified T cell generation using our current lab-scale process, analyzed all mentioned parameters, and evaluated the protocols' potential for upscaling and process development of mRNA-based CAR-T cell therapies. [ABSTRACT FROM AUTHOR]

To clarify the role of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) in the chimeric antigen receptor T-cell therapy era, we analyzed the clinical characteristics and outcomes of 52 patients treated with allo-HSCT with relapsed/refractory diffuse large B cell lymphoma. Most enrolled patients had previously undergone intensive treatments, the median number of chemotherapy lines was 4, and the median time from diagnosis to allo-HSCT was 27.1 months. Patients were divided into remission-achieved (n = 30) and active-disease (n = 22) groups before allo-HSCT. Over a median follow-up period of 38.3 months, overall survival (OS) and event-free survival (EFS) rates were 38.4% and 30.6%, respectively. The cumulative incidence of relapse (CIR) and the non-relapsed mortality (NRM) were 36.7% and 32.7%, respectively. OS, EFS, and graft-versus-host disease-free, relapse-free survival (GRFS) outcomes were significantly superior in the remission-achieved group with lower CIR. In a multivariate analysis, a shorter interval from diagnosis to allo-HSCT reflected relatively rapid disease progression and showed significantly poor OS and EFS with higher CIR. Patients with active disease had significantly lower EFS, GRFS, and higher CIR. Previous autologous stem-cell transplantation was associated with better GRFS. Allo-HSCT is an established modality with a prominent group of cured patients and still has a role in the CAR T-cell era, particularly given its acceptable clinical outcomes in young patients with chemo-susceptible disease. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer (TNBC) is a subtype of breast cancer with the highest degree of malignancy and the worst prognosis. The application of immunotherapy for TNBC is limited. This study was to verify the potential application of chimeric antigen receptor-T cells (CAR-T cells) targeting CD24 named as 24BBz in treatment of TNBC. 24BBz was constructed by lentivirus infection and then was co-culture with breast cancer cell lines to evaluate the activation, proliferation and cytotoxicity of engineered T cells. The anti-tumor activity of 24BBz was verified in the subcutaneous xenograft model of nude mice. We found that CD24 gene was significantly up-regulated in breast cancer (BRCA), especially in TNBC. 24BBz showed antigen-specific activation and dose-dependent cytotoxicity against CD24-positive BRCA tumor cells in vitro. Furthermore, 24BBz showed significant anti-tumor effect in CD24-positive TNBC xenografts and T cells infiltration in tumor tissues, while some T cells exhibited exhaustion. No pathological damage of major organs was found during the treatment. This study proved that CD24-specific CAR-T cells have potent anti-tumor activity and potential application value in treatment of TNBC. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor macrophage (CAR-M) is a promising immunotherapy strategy of anti-tumor due to its high infiltration, direct phagocytosis of tumor cells, immunomodulation of tumor microenvironment (TME) and linkage of innate and adaptive immunity. Here a series of novelly designed CAR-Ms by targeting vascular endothelial growth factor receptor-2 (VEGFR2), which highly expressed in tumor cells and TME, were evaluated. Their activation signals were transduced by Tlr4 or Ifn-γ receptors either alone or in combination, which were designed to mediate M1 polarization of macrophages as the downstream of lipopolysaccharide or Ifn-γ that had been widely reported. Our results showed that VEGFR2-targeting CAR-Ms could be activated under the stimulation of VEGFR2-expressing cells. They exhibited higher expression of CD86, MHCII and TNF-α in vitro and enhanced tumor suppressive abilities in vivo. Implantation of these CAR-Ms into 4T1 breast cancer-bearing mice could obviously inhibit the progression of tumor without significant toxic side effects, especially the group of mmC in which constructed with Tlr4 as the intracellular domain of CAR. In conclusion, this research provides a promising design of CAR that induce macrophages activation by Tlr4 and/or Ifn-γ receptors, and these CAR-Ms could effectively inhibit tumor growth through targeting VEGFR2. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)-engineered immune effector cells constitute a promising approach for adoptive cancer immunotherapy. Nevertheless, on-target/off-tumor toxicity and immune escape due to antigen loss represent considerable challenges. These may be overcome by adaptor CARs that are selectively triggered by bispecific molecules that crosslink the CAR with a tumor-associated surface antigen. Here, we generated NK cells carrying a first- or second-generation universal CAR (UniCAR) and redirected them to tumor cells with so-called target modules (TMs) which harbor an ErbB2 (HER2)-specific antibody domain for target cell binding and the E5B9 peptide recognized by the UniCAR. To investigate differential effects of the protein design on activity, we developed homodimeric TMs with one, two or three E5B9 peptides per monomer, and binding domains either directly linked or separated by an IgG4 Fc domain. The adaptor molecules were expressed as secreted proteins in Expi293F cells, purified from culture supernatants and their bispecific binding to UniCAR and ErbB2 was confirmed by flow cytometry. In cell killing experiments, all tested TMs redirected NK cell cytotoxicity selectively to ErbB2-positive tumor cells. Nevertheless, we found considerable differences in the extent of specific cell killing depending on TM design and CAR composition, with adaptor proteins carrying two or three E5B9 epitopes being more effective when combined with NK cells expressing the first-generation UniCAR, while the second-generation UniCAR was more active in the presence of TMs with one E5B9 sequence. These results may have important implications for the further development of optimized UniCAR and target module combinations for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Immunotherapy has revolutionized cancer therapy. Two recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or chimeric antigen receptor T (CAR-T) cells. Blinatumomab, an FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating effector-target cell contact and T-cell activation that results in effective elimination of target B cells. Although CD19 is expressed by essentially all B-cell malignancies at clinical presentation, relapses with loss or reduction in CD19 surface expression are increasingly recognized as a cause of treatment failure. Therefore, there is a clear need to develop therapeutics for alternate targets. We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments. Target binding of the anti-CD22 and anti-CD3 moieties was confirmed by flow cytometry. CD22-BiTE promoted in vitro cell-mediated cytotoxicity in a dose and effector: target (E:T)-dependent fashion. Additionally, in an established acute lymphoblastic leukemia (ALL) xenograft mouse model, CD22-BiTE demonstrated tumor growth inhibition, comparable to blinatumomab. Further, the combination of blinatumomab and CD22-BiTE yielded increased efficacy in vivo when compared to the single agents. In conclusion, we report here the development of a new BiTE with cytotoxic activity against CD22+ cells which could represent an alternate or complementary therapeutic option for B-cell malignancies. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is still an incurable disorder despite improved antibody and cellular therapies against different MM antigens. Single targeted antigens have so far been ineffective against MM with most patients relapsing after initial response. Hence, sequential immunotherapies directed at different targets are expected to perform better than monotherapy alone. Here, we optimized and established in preclinical studies the therapeutic rationale of using targeted alpha therapy (TAT) directed against CD38 antigen (225Ac-DOTA-daratumumab) with CAR T cell therapy directed at CS1 antigen in a systemic MM model. The sequential therapies compared CAR T therapy followed by TAT to TAT followed by CAR T therapy. CAR T cell monotherapy increased median survival from 49 days (d) in untreated controls to 71d with a modest improvement to 89d for 3.7 kBq of TAT given 14d later. When CAR T was followed by 7.4 kBq of TAT 29d later, sequential therapy increased median survival from 47d in untreated controls to 106d, compared to 68d for CAR T monotherapy. When CAR T therapy was followed by untargeted alpha immunotherapy using 7.4 kBq of 225Ac-DOTA-trastuzumab (anti-HER2) antibody 29d later, there was only a slight improvement in response over CAR T monotherapy demonstrating the role of tumor targeting. TAT (7.4 kBq) followed by CAR T therapy was also effective when CAR T therapy was delayed for 21d vs 14d or 28d post TAT, highlighting the importance of timing sequential therapies. Sequential targeted therapies using CS1 CAR T or 225Ac-DOTA-CD38 TAT in either order shows promise over monotherapies alone. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptors (CARs) have improved cancer immunotherapy in recent years. Immune cells, such as Natural killer cells (NK-cells) or T cells, are used as effector cells in CAR-therapy. NK92-cells, a cell line with known cytotoxic activity, are of particular interest in CAR-therapy since culturing conditions are simple and anti-tumor efficacy combined with a manageable safety profile was proven in clinical trials. The major pathways of immune effector cells, including NK92-cells, to mediate cytotoxicity, are the perforin/granzyme and the death-receptor pathway. Detailed knowledge of CAR-effector cells' cytotoxic mechanisms is essential to unravel resistance mechanisms, which potentially arise by resistance against apoptosis-inducing signaling. Since mutations in apoptosis pathways are frequent in lymphoma, the impact on CAR-mediated cytotoxicity is of clinical interest. In this study, knockout models of CD19-CAR-NK92 cells were designed, to investigate cytotoxic pathways in vitro. Knockout of perforin 1 (Prf1) and subsequent abrogation of the perforin/granzyme pathway dramatically reduced the cytotoxicity of CD19-CAR-NK92 cells. In contrast, knockout of FasL and inhibition of TRAIL (tumor necrosis factor-related apoptosis-inducing ligands) did not impair cytotoxicity in most conditions. In conclusion, these results indicate the perforin/granzyme pathway as the major pathway to mediate cytotoxicity in CD19-CAR-NK92 cells. [ABSTRACT FROM AUTHOR]

Immunotherapy with chimeric antigen receptor T (CAR T) cells has changed the treatment of hematological malignances, but they are still a challenge for solid tumors, including pediatric sarcomas. Here, we report a switchable CAR T cell strategy based on anti-FITC CAR T cells and a switch molecule conjugated with FITC for targeting osteosarcoma (OS) tumors. As a potential target, we analyzed the expression of B7-H3, an immune checkpoint inhibitor, in OS cell lines. In addition, we evaluate the capacity of an anti-B7-H3 monoclonal antibody conjugated with FITC (anti-B7-H3-FITC mAb) to control the antitumor activity of anti-FITC CAR T cells. The effector functions of anti-FITC CAR T cells against OS, measured in vitro by tumor cell killing activity and cytokine production, are dependent on the presence of the anti-B7-H3-FITC mAb switch. Moreover, OS cells stimulate anti-FITC CAR T cells migration. In vivo, anti-B7-H3 mAb penetrates in the tumor and binds 143B OS tumor cells. Furthermore, anti-FITC CAR T cells reach tumor region and exert antitumor effect in an OS NSG mouse model only in the presence of the switch molecule. We demonstrate that anti-B7-H3-FITC mAb redirects the cytotoxic activity of anti-FITC CAR T cells against OS tumors suggesting that switchable CAR T cell platforms might be a plausible strategy against OS. [ABSTRACT FROM AUTHOR]

The rapid expansion of cancer immunology and immunotherapy builds upon the success of early immune checkpoint inhibitors (ICI) and chimeric antigen receptor T cells for some cancer types. Many gaps still exist, however, in the scientific knowledge of immune dysfunction in the tumour microenvironment and predicting clinical immunotherapy response to allow more cancer patients to benefit from immunotherapy. The Cancer Immunotherapy Collection within Scientific Reports describes pioneering preclinical and clinical studies addressing these concepts, representing significant insights and breakthroughs in the field. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising modality for anti-cancer treatment. Its efficacy is quite remarkable in hematological tumors. Owing to their excellent clinical results, gene- modified cell therapies, including T cells, natural killer (NK) cells, and macrophages, are being actively studied in both academia and industry. However, the protocol to make CAR immune cells is too complicated, so it is still unclear how to efficiently produce the potent CAR immune cells. To manufacture effective CAR immune cells, we need to be aware of not only how to obtain highly infective viral particles, but also how to transduce CAR genes into immune cells. In this paper, we provide detailed information on spinoculation, which is one of the best known protocols to transduce genes into immune cells, in a methodological view. Our data indicate that gene transduction is significantly dependent on speed and duration of centrifugation, concentration and number of viral particles, the concentration of polybrene, and number of infected immune cells. In addition, we investigated on the optimal polyethylene glycol (PEG) solution to concentrate the viral supernatant and the optimized DNA ratios transfected into 293T cells to produce high titer of viral particles. This study provides useful information for practical production of the gene-modified immune cells using viral vectors. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)-modified T (CAR-T) cell therapy has been proven to be a powerful tool for the treatment of cancer, however, the limits are obvious, especially for solid tumors. Therefore, constantly optimizing the structure of CAR to improve its therapeutic effect is necessary. In this study, we generated three different third-generation CARs targeting IL13Rα2, with the same scFv, but different transmembrane domains (TMDs) from CD4, CD8 or CD28 (IL13-CD4TM-28.BB.ζ, IL13-CD8TM-28.BB.ζ and IL13-CD28TM-28.BB.ζ). CARs were transduced into primary T cells using retroviruses. The anti-GBM efficacy of CAR-T cells was monitored by flow cytometry and real-time cell analysis (RTCA) in vitro and examined in two xenograft mouse models. The differentially expressed genes related to different anti-GBM activity were screened by high throughput RNA sequencing. We observed that T cells transduced with these three CARs have similar anti-tumor activity when co-cultured with U373 cells which expressed higher IL13Rα2 but exhibited different anti-tumor activity when co-cultured with U251 cells that expressed lower IL13Rα2. All the three groups of CAR-T cells can be activated by U373 cells, but only IL13-CD28TM-28.BB.ζ CAR-T cells could be activated and expressed increased IFN-γ after co-culturing with U251 cells. IL13-CD28TM-28.BB.ζ CAR-T cells exhibited the best anti-tumor activity in xenograft mouse models which can infiltrate into the tumors. The superior anti-tumor efficacy of IL13-CD28TM-28.BB.ζ CAR-T cells was partially owing to differentially expressed extracellular assembly, extracellular matrix, cell migration and adhesion-related genes which contribute to the lower activation threshold, increased cell proliferation, and elevated migration capacity. [ABSTRACT FROM AUTHOR]

Purpose: Identifying the temporal pattern of recurrence and prognostic biomarkers would further help improve the efficacy of chimeric antigen receptor (CAR) -T therapy. Methods: We examined the prognoses of 119 patients after sequential infusion of anti-CD19 and anti-CD22, a cocktail of 2 single-target CAR (CAR19/22) T cells in an open-label, single-center clinical trial (ChiCTR-OPN-16008526). And we, from a 70-biomarker panel, identified candidate cytokines that might predict the treatment failure, including primary non-response (NR) and early relapse (ER). Results: In our study, 3 (11.5%) patients with B-cell acute lymphoblastic leukemia (B-ALL) and 9 (12.2%) cases of B-cell non-Hodgkin lymphoma (NHL) failed to respond to sequential CAR19/22 T-cell infusion (NR). A total of 11 (42.3%) B-ALL patients and 30 (52.7%) B-NHL patients had relapses during follow-up. Most recurrence events (67.5%) occurred within six months of sequential CAR T-cell infusion (ER). We found that macrophage inflammatory protein (MIP)-3α was a highly sensitive and specific prognostic predictor for patients with NR/ER and those attaining over-6-month remission. Patients who had higher MIP3α levels after sequential CAR19/22 T-cell infusion had significantly favorable progression-free survival (PFS) than their counterparts with relatively lower MIP3α expression. Our experiments demonstrated that MIP3α could enhance the therapeutic effect of CAR-T cells by promoting T-cell infiltration into and enriching memory-phenotype T cells in the tumor environment. Conclusion: This study showed that relapse occurred mainly within six months after sequential CAR19/22 T-cell infusion. Moreover, MIP3α could act as a valuable post-infusion biomarker for identifying patients with NR/ER. [ABSTRACT FROM AUTHOR]

The metabolic stress present in the tumor microenvironment of many cancers can attenuate T cell antitumor activity, which is intrinsically controlled by the mitochondrial plasticity, dynamics, metabolism, and biogenesis within these T cells. Previous studies have reported that the complement C1q binding protein (C1QBP), a mitochondrial protein, is responsible for maintenance of mitochondrial fitness in tumor cells; however, its role in T cell mitochondrial function, particularly in the context of an antitumor response, remains unclear. Here, we show that C1QBP is indispensable for T cell antitumor immunity by maintaining mitochondrial integrity and homeostasis. This effect holds even when only one allele of C1qbp is functional. Further analysis of C1QBP in the context of chimeric antigen receptor (CAR) T cell therapy against the murine B16 melanoma model confirmed the cell-intrinsic role of C1QBP in regulating the antitumor functions of CAR T cells. Mechanistically, we found that C1qbp knocking down impacted mitochondrial biogenesis via the AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor gamma coactivator 1-alpha signaling pathway, as well as mitochondrial morphology via the phosphorylation of mitochondrial dynamics protein dynamin-related protein 1. In summary, our study provides a novel mitochondrial target to potentiate the plasticity and metabolic fitness of mitochondria within T cells, thus improving the immunotherapeutic potential of these T cells against tumors. [ABSTRACT FROM AUTHOR]

CAR T cell therapy is a rapidly growing area of oncological treatments having a potential of becoming standard care for multiple indications. Coincidently, CRISPR/Cas gene-editing technology is entering next-generation CAR T cell product manufacturing with the promise of more precise and more controllable cell modification methodology. The intersection of these medical and molecular advancements creates an opportunity for completely new ways of designing engineered cells to help overcome current limitations of cell therapy. In this manuscript we present proof-of-concept data for an engineered feedback loop. We manufactured activation-inducible CAR T cells with the help of CRISPR-mediated targeted integration. This new type of engineered T cells expresses the CAR gene dependent on their activation status. This artifice opens new possibilities to regulate CAR T cell function both in vitro and in vivo. We believe that such a physiological control system can be a powerful addition to the currently available toolbox of next-generation CAR constructs. T cells are modified to express the CAR gene dependent on activation status under the control of an engineered feedback loop and are shown to function in vitro and in vivo to kill tumour cells. [ABSTRACT FROM AUTHOR]

CD19-specific chimeric antigen receptor T (CAR T) immunotherapy is used to treat B-cell malignancies. However, antigen-escape mediated relapse following CAR T therapy has emerged as a major concern. In some relapsed cases, especially KMT2A rearrangement-positive B-acute lymphoblastic leukemia (KMT2A-r B-ALL), most of the B-cell antigens are lost via lineage conversion to the myeloid phenotype, rendering multi-B-cell-antigen-targeted CAR T cell therapy ineffective. Fms-related tyrosine kinase-3 (FLT3) is highly expressed in KMT2A-r B-ALL; therefore, in this study, we aimed to evaluate the antitumor efficacy of CAR T cells targeting both CD19 and FLT3 in KMT2A-r B-ALL cells. We developed piggyBac transposon-mediated CAR T cells targeting CD19, FLT3, or both (dual) and generated CD19-negative KMT2A-r B-ALL models through CRISPR-induced CD19 gene-knockout (KO). FLT3 CAR T cells showed antitumor efficacy against CD19-KO KMT2A-r B-ALL cells both in vitro and in vivo; dual-targeted CAR T cells showed cytotoxicity against wild-type (WT) and CD19-KO KMT2A-r B-ALL cells, whereas CD19 CAR T cells demonstrated cytotoxicity only against WT KMT2A-r B-ALL cells in vitro. Therefore, targeting FLT3-specific CAR T cells would be a promising strategy for KMT2A-r B-ALL cells even with CD19-negative relapsed cases. [ABSTRACT FROM AUTHOR]

Ovarian cancer is a major cause of death among all gynaecological cancers. Although surgery, chemotherapy and targeted therapy have yielded successful outcomes, the 5-year survival rate remains < 30%. Adoptive immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy, has demonstrated improved survival in acute lymphoblastic leukaemia with manageable toxicity. We explored CAR T-cell therapy in a preclinical mouse model of ovarian cancer. Second-generation CAR T cells were developed targeting mesothelin (MSLN), which is abundantly expressed in ovarian cancer. Cytotoxicity experiments were performed to verify the lethality of CAR T cells on target cells via flow cytometry. The in vivo antitumour activity of MSLN CAR T cells was also verified using a patient-derived xenograft (PDX) mouse model with human tumour-derived cells. We also evaluated the potency of CAR T cells directed to MSLN following co-expression of a dominant-negative transforming growth factor-β receptor type II (dnTGFβRII). Our data demonstrate that anti-MSLN CAR T cells specifically eliminate MSLN-expressing target cells in an MSLN density-dependent manner. This preclinical research promises an effective treatment strategy to improve outcomes for ovarian cancer, with the potential for prolonging survival while minimizing risk of on-target off-tumour toxicity. [ABSTRACT FROM AUTHOR]

T cell exhaustion is a main obstacle against effective cancer immunotherapy. Exhausted T cells include a subpopulation that maintains proliferative capacity, referred to as precursor exhausted T cells (TPEX). While functionally distinct and important for antitumor immunity, TPEX possess some overlapping phenotypic features with the other T-cell subsets within the heterogeneous tumor-infiltrating T-lymphocytes (TIL). Here we explore surface marker profiles unique to TPEX using the tumor models treated by chimeric antigen receptor (CAR)-engineered T cells. We find that CD83 is predominantly expressed in the CCR7+PD1+ intratumoral CAR-T cells compared with the CCR7-PD1+ (terminally differentiated) and CAR-negative (bystander) T cells. The CD83+CCR7+ CAR-T cells exhibit superior antigen-induced proliferation and IL-2 production compared with the CD83- T cells. Moreover, we confirm selective expression of CD83 in the CCR7+PD1+ T-cell population in primary TIL samples. Our findings identify CD83 as a marker to discriminate TPEX from terminally exhausted and bystander TIL. CD83, a member of the immunoglobulin superfamily, is a novel marker to discriminate precursor exhausted T cells from terminally exhausted and bystander tumor-infiltrating T-lymphocytes. [ABSTRACT FROM AUTHOR]

Engineered immune cell therapy to treat malignancies refractory to conventional therapies is modernizing oncology. Although αβ T cells are time-tested chassis for CAR, potential graft versus host disease (GvHD) apart from cytokine toxicity and antigen escape pose limitations to this approach. αβ T cell malignancy challenges isolation and expansion of therapeutic T cells. Moreover, αβ T cells may pose toxicity risk to inflammation sensitive vital tissues bearing the tumor. The HLA independent, multivalent, versatile and systemic anti-tumor immunity increases the desirability of γδ T cells as an alternate chassis for CAR. Indeed, CD19 γδ CAR T cell therapy to treat advanced lymphoma reached a milestone with the fast track status by FDA. However, reduced tumor-toxicity, homing, in vivo persistence and heterogeneity limits the translation of this therapy. The field is gaining momentum in recent years with optimization of gene delivery approaches and mechanistic insights into co-signaling requirements in γδ T cells. There is a renewed interest in customizing design of CAR guided by the biology of the host immune cells. Progress has been made in the current good manufacturing practice compatible expansion and engineering protocols for the δ1 and δ2 T cells. γδ CAR T cells may find its niche in the clinical situations wherein conventional CAR therapy is less suitable due to propensity for cytokine toxicity or off-tumor effect. As the therapy is moving towards clinical trials, this review chronicles the hitherto progress in the therapeutic engineering of γδ T cells for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Recently, chimeric antigen receptor T cell (CAR-T) therapy has received increasing attention as an adoptive cellular immunotherapy that targets tumors. However, numerous challenges remain for the effective use of CAR-T to treat solid tumors, including ovarian cancer, which is an aggressive and metastatic cancer with a poor therapeutic response. We screened for an effective anti-MSLN single-chain Fv antibody with comparable binding activity and non-off-target properties using human phage display library. A second-generation of anti-MSLN CAR was designed and generated. We demonstrated the efficacy of our anti-MSLN CAR-T cells for ovarian cancer treatment in an in vitro experiment to kill ovarian tumor cell lines. The anti-MSLN CAR-T cells impeded MSLN-positive tumor growth concomitant with a significant increase in cytokine levels compared with the control. Then, we demonstrated the efficacy of anti-MSLN CAR-T cells in an in vivo experiment against ovarian cancer cell-derived xenografts. Furthermore, we herein report three cases with ovarian cancer who were treated with autologous anti-MSLN CAR-T cells and evaluate the safety and effectiveness of adoptive cell therapy. In this investigator-initiated clinical trials, no patients experienced cytokine release syndrome or neurological symptoms over 2 grads. Disease stabilized in two patients, with progression-free survival times of 5.8 and 4.6 months. Transient CAR expression was detected in patient blood after infusion each time. The tumor partially subsided, and the patient's condition was relieved. In conclusion, this work proves the efficacy of the anti-MSLN CAR-T treatment strategy in ovarian cancer and provides preliminary data for the development of further clinical trials. [ABSTRACT FROM AUTHOR]

Background: Chimeric antigen receptor (CAR) T cells are used to treat refractory and recurrent B-cell lymphoma. When administered intravenously, CAR T cells can be detected in cerebrospinal fluid, and thus represent a promising method for the treatment of central nervous system lymphoma (CNSL). This meta-analysis aimed to clarify the effectiveness and safety of CAR T-cell therapy in the treatment of CNSL. Methods: Studies involving patients with CNSL who received CAR T-cell therapy that reported overall response (OR), complete response (CR), and partial response (PR) were included. A random-effects or fixed-effects model with double arcsine transformation was used for the pooled analysis and 95% confidence intervals (CI) were determined for all outcomes. Results: Eight studies, comprising 63 patients, were identified and were included in the meta-analysis. The pooled OR and CR rates after treatment with CAR T cells were 69% (95% CI, 56–81%) and 51% (95% CI, 37–64%), respectively. The pooled rate of progressive disease after remission was 38% (95% CI, 21–55%). The pooled rate for neurotoxicity grade 3 or above was 12% (95% CI, 3–24%, I2 = 0.00%, p = 0.53). No treatment-related deaths were reported. Conclusions: CAR T-cell therapy is a promising option for the treatment of CNSL owing to a high short-term remission rate and controllable side effects. However, the high recurrence rate after remission must be addressed. Long-term follow-up data with large sample sizes are also needed to better assess the effectiveness and safety of CAR T-cell therapy. Registration: This meta-analysis was registered in the international prospective register of systematic reviews (PROSPERO) (CRD42022301332). [ABSTRACT FROM AUTHOR]

The ability to image adoptively transferred T cells in the body and to eliminate them to avoid toxicity will be vital for chimeric antigen receptor (CAR) T cell therapy, particularly against solid tumors with higher risk of off-tumor toxicity. Previously, we have demonstrated the utility of somatostatin receptor 2 (SSTR2) for CAR T cell imaging, illustrating the expansion and contraction of CAR T cells in tumor as well as off-tumor expansion. Using intercellular adhesion molecule 1 (ICAM-1)-specific CAR T cells that secrete interleukin (IL)-12 as a model, herein we examined the potential of SSTR2 as a safety switch when combined with the SSTR2-specific maytansine-octreotate conjugate PEN-221. Constitutive secretion of IL-12 led to continuous expansion of CAR T cells after rapid elimination of tumors, causing systemic toxicity in mice with intact MHC expression. Treatment with PEN-221 rapidly reduced the abundance of CAR T cells, decreasing the severity of xenogeneic graft-versus-host disease (GvHD), and prolonged survival. Our study supports the development of SSTR2 as a single genetic marker for CAR T cells that is readily applicable to humans both for anatomical detection of T cell distribution and an image-guided safety switch for rapid elimination of CAR T cells. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is considered as one of the most malignant conditions of the bone marrow. Over the past few decades, despite substantial progresses in the management of AML, relapse remission remains a major problem. Natural killer cells (NK cells) are known as a unique component of the innate immune system. Due to swift tumor detection, distinct cytotoxic action, and extensive immune interaction, NK cells have been used in various cancer settings for decades. It has been a growing knowledge of therapeutic magnitudes ranging from adoptive NK cell transfer to chimeric antigen receptor NK cells, aiming to achieve better therapeutic responses in patients with AML. In this article, the potentials of NK cells for treatment of AML are highlighted, and challenges for such therapeutic methods are discussed. In addition, the clinical application of NK cells, mainly in patients with AML, is pictured according to the existing evidence. [ABSTRACT FROM AUTHOR]

Prediction of responses to chimeric antigen receptor (CAR)-T cell therapies is essential to maximize their therapeutic efficacy for diffuse large B-cell lymphoma (DLBCL). While several tumor-intrinsic risk factors of resistance and/or early relapse have been identified, clinically useful markers that determine potential activity of CAR-T cells have not been fully investigated. T-cell property at the time of leukapheresis may serve as such a marker. Therefore, we evaluated the clinical impact of CD3+ cell count in peripheral blood at leukapheresis on clinical outcomes of CAR-T cell therapy. In total, 44 patients with relapsed or refractory (r/r) DLBCL who received tisagenlecleucel at Kyoto University Hospital were included. According to CD3+ cell counts, patients were categorized into CD3LOW and CD3HIGH groups with a threshold of 553/μL, based on receiver operating characteristic curve analysis. 1-year progression-free survival was significantly higher in the CD3HIGH group than the CD3LOW group (68.3% vs. 17.3%; adjusted hazard ratio [aHR], 0.37; p = 0.042). Overall survival was also superior in the CD3HIGH group (aHR, 0.24; p = 0.043). Moreover, higher CD3+ cell counts at leukapheresis were associated with significantly higher lymphocyte counts in peripheral blood at day 7 after CAR-T cell infusion (median 860 vs. 420/μL, P = 0.021), suggesting more extensive expansion of infused CAR-T cells in vivo. In conclusion, we demonstrated that the CD3+ cell count at leukapheresis predicts both expansion of CAR-T cells after infusion and outcomes of CAR-T cell therapy, and are useful for building comprehensive therapeutic strategies at the time of leukapheresis. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cells remain unsatisfactory in treating solid tumors. The frequency of tumor-infiltrating T cells is closely related to the good prognosis of patients. Augmenting T cell accumulation in the tumor microenvironment is essential for tumor clearance. To overcome insufficient immune cell infiltration, innovative CAR designs need to be developed immediately. CXCL9 plays a pivotal role in regulating T cell migration and inhibiting tumor angiogenesis. Therefore, we engineered CAR T cells expressing CXCL9 (CART-CXCL9). The addition of CXCL9 enhanced cytokine secretion and cytotoxicity of CAR T cells and endowed CAR T cells with the ability to recruit activated T cells and antiangiogenic effect. In tumor-bearing mice, CART-CXCL9 cells attracted more T cell trafficking to the tumor site and inhibited angiogenesis than conventional CAR T cells. Additionally, CART-CXCL9 cell therapy slowed tumor growth and prolonged mouse survival, displaying superior antitumor activity. Briefly, modifying CAR T cells to express CXCL9 could effectively improve CAR T cell efficacy against solid tumors. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T-cell therapy achieves great success for hematological malignancies. However, clinical trials have revealed some limitations in both improving the efficacy and reducing the relapse, which calls for innovative strategies to engineer more powerful CAR-T cells. Promoting the formation of CAR clusters provides an alternative approach and potentially improves current CAR T-cell therapy against cancers. Here, we generated CARCys-T cells using a 4-1BB-derived hinge region including 11 cysteines residues. The cysteines in the hinge were found to facilitate CARCys clustering upon antigen stimulation and promote the antitumor activity of CAR-T cells. Compared with most conventionally used CAR-T cells with CD8α-derived hinge (CARconv-T cells), CARCys-T cells exhibited larger diameter of CAR clusters and enhanced antigen-specific tumor lysis both in vitro and in vivo. In addition, the CARCys-mediated enhancement could be applied to HER2, CD19 as well as GPC3-targeted CAR-T cells. More importantly, CARCys-T cells showed potent antitumor efficacy in clinically relevant patient-derived primary tumor cells and organoids. Thus, the novel hinge containing 11 cysteines provides a promising strategy to facilitate CAR clustering and maximize anti-tumor activity of CAR-T cells, which emphasizes the importance of CAR clustering to improve CAR T-cell therapy in the clinic. [ABSTRACT FROM AUTHOR]

Non-keratinizing nasopharyngeal carcinoma (NPC) is a malignancy with a poor prognosis for relapsing patients and those with metastatic disease. Here, we identify a novel disease mechanism of NPC which may be its Achilles' heel that makes it susceptible to immunotherapy. CD137 is a potent costimulatory receptor on activated T cells, and CD137 agonists strongly enhance anti-tumor immune responses. A negative feedback mechanism prevents overstimulation by transferring CD137 from T cells to CD137 ligand (CD137L)-expressing antigen presenting cells (APC) during cognate interaction, upon which the CD137-CD137L complex is internalized and degraded. We found ectopic expression of CD137 on 42 of 122 (34.4%) NPC cases, and that CD137 is induced by the Epstein-Barr virus latent membrane protein (LMP) 1. CD137 expression enables NPC to hijack the inbuilt negative feedback mechanism to downregulate the costimulatory CD137L on APC, facilitating its escape from immune surveillance. Further, the ectopically expressed CD137 signals into NPC cells via the p38-MAPK pathway, and induces the expression of IL-6, IL-8 and Laminin γ2. As much as ectopic CD137 expression may support the growth and spread of NPC, it may be a target for its immunotherapeutic elimination. Natural killer cells that express a CD137-specific chimeric antigen receptor induce death in CD137+ NPC cells, in vitro, and in vivo in a murine xenograft model. These data identify a novel immune escape mechanism of NPC, and lay the foundation for an urgently needed immunotherapeutic approach for NPC. [ABSTRACT FROM AUTHOR]

Chimeric Antigen Receptor (CAR) T cell therapies have received increasing attention, showing promising results in the treatment of acute lymphoblastic leukaemia and aggressive B cell lymphoma. Unlike typical cancer treatments, autologous CAR T cell therapies are patient-specific; this makes them a unique therapeutic to manufacture and distribute. In this work, we focus on the development of a computer modelling tool to assist the design and assessment of supply chain structures that can reliably and cost-efficiently deliver autologous CAR T cell therapies. We focus on four demand scales (200, 500, 1000 and 2000 patients annually) and we assess the tool's capabilities with respect to the design of responsive supply chain candidate solutions while minimising cost. [ABSTRACT FROM AUTHOR]

As the main immune checkpoint, PD-L1-PD-1 interaction plays a critical role in the dysregulation of effector T cells, which contributes to the failure of Chimeric Antigen Receptor T-cell (CAR-T) and other immunotherapies. Presently, most research focuses on the extracellular function of PD-L1. Membrane PD-L1 can interact with its receptor PD-1 and decrease T cell-induced cancer immunity. However, the function of PD-L1 in cancer cells is still unclear. Recent studies have shown the separated clinical significance of PD-L1 expression in various cancer types, showing the complexity of PD-L1 in cancer cell regulation. As a novel regulatory pathway, the nuclear translocation of PD-L1 in cancer cells receives more attention. Results of these preclinical studies demonstrated that nuclear PD-L1 has an essential role in cancer development and other immune checkpoint molecules transcription. Herein, we summarized the mechanisms involved in PD-L1 nuclear transportation and identify the key regulatory factors in this process. Furthermore, we also summarize the function of nuclear PD-L1 in cancer immunity. These findings suggested the novel PD-L1 regulation in cancer development, which showed that nuclear PD-L1 is a potential therapeutic target in future cancer therapy. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) redirected T-cells has shown efficacy in the treatment of B-cell leukemia/lymphoma, however, high numbers of relapses occur due to loss of targeted antigen or intrinsic failure of the CAR T-cells. In this situation modifications of the basic strategy are envisaged to reduce the risk of relapse, some of them are in early clinical exploration. These include simultaneous targeting of multiple antigens or combination of CAR T-cell therapy with other treatment modalities such as checkpoint inhibitors. The review evaluates and discusses these modified advanced therapies and pre-clinical approaches with respect to their potential to control leukemia and lymphoma in the long-term. [ABSTRACT FROM AUTHOR]

Background: CAR-T cell (chimeric antigen receptor T) therapy has emerged as an effective treatment of refractory hematological malignancies. Intensive care management is intrinsic to CAR-T cell therapy. We aim to describe and to assess outcomes in critically ill CAR-T cell recipients. Study design and methods: Hospital-wide retrospective study. Consecutive CAR-T cell recipients requiring ICU admission from July 2017 and December 2020 were included. Results: 71 patients (median age 60 years [37–68]) were admitted to the ICU 6 days [4–7] after CAR-T cell infusion. Underlying malignancies included diffuse large B cell lymphoma (n = 53, 75%), acute lymphoblastic leukemia (17 patients, 24%) and multiple myeloma (n = 1, 1.45%). Performance status (PS) was 1 [1–2]. Shock was the main reason for ICU admission (n = 40, 48%). Isolated cytokine release syndrome (CRS) was the most common complication (n = 33, 46%), while 21 patients (30%) had microbiologically documented bacterial infection (chiefly catheter-related infection). Immune effector cell-associated neurotoxicity syndrome was reported in 26 (37%) patients. At ICU admission, vasopressors were required in 18 patients (25%) and invasive mechanical ventilation in two. Overall, 49 (69%) and 40 patients (56%) received tocilizumab or steroids, respectively. Determinant of mortality were the reason for ICU admission (disease progression vs. sepsis or CRS (HR 4.02 [95%CI 1.10–14.65]), Performance status (HR 1.97/point [95%CI 1.14–3.41]) and SOFA score (HR 1.16/point [95%CI 1.01–1.33]). Conclusions: Meaningful survival could be achieved in up to half the CAR-T cell recipients. The severity of organ dysfunction is a major determinant of death, especially in patients with altered performance status or disease progression. [ABSTRACT FROM AUTHOR]

Novel CAR T cells delivered directly to the CNS could have therapeutic effects in recurrent glioblastoma, according to two early-stage trials. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising novel therapeutic approach. However, primary and secondary resistance to CAR-T cell therapy is commonly encountered in various clinical trials. Despite the comprehensive studies to elucidate the mechanisms of resistance, effective resolution in clinical practice is still elusive. Inadequate persistence and subsequent loss of infused CAR-T cells are proposed major resistance mechanism associated with CAR-T cell treatment failure. Thus, we generated CAR-T cells armored with IL-7 to prolong the persistence of infused T-cells, particularly CD4 + T cells, and enhanced anti-tumor response. IL-7 increased CAR-T-cell persistence in vivo and contributed to the distinct T-cell cytotoxicity profile. Using mass cytometry (CyTOF), we further assessed the phenotypic and metabolic profiles of IL-7-secreting CAR-T cells, along with conventional CAR-T cells at the single-cell level. With in-depth analysis, we found that IL-7 maintained CAR-T cells in a less differentiated T-cell state, regulated distinct metabolic activity, and prevented CAR-T-cell exhaustion, which could be essential for CAR-T cells to maintain their metabolic fitness and anti-tumor response. Our findings thus provided clinical rationale to exploit IL-7 signaling for modulation and metabolic reprogramming of T-cell function to enhance CAR-T cell persistence and induce durable remission upon CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs. [ABSTRACT FROM AUTHOR]

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine. [ABSTRACT FROM AUTHOR]

Anti-tumor therapies that seek to exploit and redirect the cytotoxic killing and effector potential of autologous or syngeneic T cells have shown extraordinary promise and efficacy in certain clinical settings. Such cells, when engineered to express synthetic chimeric antigen receptors (CARs) acquire novel targeting and activation properties which are governed and orchestrated by, typically, antibody fragments specific for a tumor antigen of interest. However, it is becoming increasingly apparent that not all antibodies are equal in this regard, with a growing appreciation that 'optimal' CAR performance requires a consideration of multiple structural and contextual parameters. Thus, antibodies raised by classical approaches and intended for other applications often perform poorly or not at all when repurposed as CARs. With this in mind, we have explored the potential of an in vitro phenotypic CAR library discovery approach that tightly associates antibody-driven bridging of tumor and effector T cells with an informative and functionally relevant CAR activation reporter signal. Critically, we demonstrate the utility of this enrichment methodology for 'real world' de novo discovery by isolating several novel anti-mesothelin CAR-active scFv candidates. [ABSTRACT FROM AUTHOR]

B7H6, a stress-induced ligand which binds to the NK cell receptor NKp30, has recently emerged as a promising candidate for immunotherapy due to its tumor-specific expression on a broad array of human tumors. NKp30 can function as a chimeric antigen receptor (CAR) extracellular domain but exhibits weak binding with a fast on and off rate to B7H6 compared to the TZ47 anti-B7H6 single-chain variable fragment (scFv). Here, directed evolution using yeast display was employed to isolate novel NKp30 variants that bind to B7H6 with higher affinity compared to the native receptor but retain its fast association and dissociation profile. Two variants, CC3 and CC5, were selected for further characterization and were expressed as soluble Fc-fusion proteins and CARs containing CD28 and CD3ς intracellular domains. We observed that Fc-fusion protein forms of NKp30 and its variants were better able to bind tumor cells expressing low levels of B7H6 than TZ47, and that the novel variants generally exhibited improved in vitro tumor cell killing relative to NKp30. Interestingly, CAR T cells expressing the engineered variants produced unique cytokine signatures in response to multiple tumor types expressing B7H6 compared to both NKp30 and TZ47. These findings suggest that natural CAR receptors can be fine-tuned to produce more desirable signaling outputs while maintaining evolutionary advantages in ligand recognition relative to scFvs. [ABSTRACT FROM AUTHOR]