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Post-transplant or hematological cancer patients have a higher risk of mortality after infection with ancestral and early variants of severe acute respiratory syndrome (SARS)-CoV-2. Adoptive cell therapy (ACT) with virus-specific T cells (VSTs) could augment endogenous T cell immunity to avoid disease deterioration before viral clearance. We established a third-party SARS-CoV-2-specific T cell (COVID-T) bank in 2020 (NCT04351659) using convalescent and/or vaccinated donors. In a phase I/II study (NCT04457726), 13 adult and pediatric patients, acutely positive for SARS-CoV-2 and predicted to have a high chance of mortality, were recruited from September 2021 to February 2022. Twelve patients received a single dose of COVID-T cells, matched on at least 1 HLA. A dose of either 75,000 or 150,000 IFN-γ+CD3+ cells/m2 SARS-COV-2-specific T cells did not cause cytokine release syndrome, acute respiratory distress syndrome, or graft-versus-host disease. In the 8 patients who had detectable donor SARS-COV-2-specific T cells after ACT, none progressed to severe disease or died with COVID-19. In contrast, among the other four patients without evidence of donor micro-chimerism, two died of COVID-19. Long-acting third-party VSTs from convalescent or vaccinated donors could be expediently produced and might be clinically useful in future pandemics, particularly before global vaccination is implemented. [ABSTRACT FROM AUTHOR]

Vγ9Vδ2 T cells are under investigation as alternative effector cells for adoptive cell therapy (ACT) in cancer. Despite promising in vitro results, anti-tumor efficacies in early clinical studies have been lower than expected, which could be ascribed to the complex interplay of tumor and immune cell metabolism competing for the same nutrients in the tumor microenvironment. To contribute to the scarce knowledge regarding gamma delta T-cell metabolism, we investigated the metabolic phenotype of 25-day-expanded Vγ9Vδ2 T cells and how it is intertwined with functionality. We found that Vγ9Vδ2 T cells displayed a quiescent metabolism, utilizing both glycolysis and oxidative phosphorylation (OXPHOS) for energy production, as measured in Seahorse assays. Upon T-cell receptor activation, both pathways were upregulated, and inhibition with metabolic inhibitors showed that Vγ9Vδ2 T cells were dependent on glycolysis and the pentose phosphate pathway for proliferation. The dependency on glucose for proliferation was confirmed in glucose-free conditions. Cytotoxicity against malignant melanoma was reduced by glycolysis inhibition but not OXPHOS inhibition. These findings lay the groundwork for further studies on manipulation of Vγ9Vδ2 T-cell metabolism for improved ACT outcome. [Display omitted] [ABSTRACT FROM AUTHOR]

Somatic cell therapy based on the infusion of donor-derived cytotoxic T lymphocytes (CTL) able to recognize patients' leukemia blasts (LB) is a promising approach to control leukemia relapse after allogeneic HSCT. The success of this approach strongly depends on the ex vivo generation of high-quality donor-derived anti-leukemia CTL in compliance with Good Manufacturing Practices (GMP). We previously described a procedure for generating large numbers of donor-derived anti-leukemia CTL through stimulation of CD8-enriched lymphocytes with dendritic cells (DCs) pulsed with apoptotic LB in the presence of interleukin (IL)-12, IL-7 and IL-15. Here we report that the use of IFN-DC and the addition of IFNα2b during the priming phase significantly improve the generation of an efficient anti-leukemia T cells response in vitro. Using this approach, 20 high-risk pediatric patients given haploidentical HSCT for high-risk acute leukemia were enrolled and 51 batches of advanced therapy medical products (ATMP), anti-leukemia CTL, were produced. Quality controls demonstrated that all batches were sterile, free of mycoplasma and conformed to acceptable endotoxin levels. Genotype analysis confirmed the molecular identity of the ATMP based on the starting biological material used for their production. The majority of ATMP were CD3+/CD8+ cells, with a memory/terminal activated phenotype, including T-central memory populations. ATMP were viable after thawing, and most ATMP batches displayed efficient capacity to lyse patients' LB and to secrete interferon-γ and tumor necrosis factor-α. These results demonstrated that our protocol is highly reproducible and allows the generation of large numbers of immunologically safe and functional anti-leukemia CTL with a high level of standardization. [ABSTRACT FROM AUTHOR]

With the objective of improving the ex vivo production of therapeutic chimeric antigen receptor (CAR) T cells, we explored the addition of three-dimensional (3D) polystyrene scaffolds to standard suspension cell cultures. We aimed to mimic the structural support given by the lymph nodes during in vivo lymphocyte expansion. We observed an increase in cell proliferation compared with standard suspension systems as well as an enhanced cytotoxicity toward cancer cells. Moreover, we directly obtained the CAR T cells from peripheral blood mononuclear cells, thus minimizing the ex vivo manipulation of the therapeutic cells and opening the way to synergies among different cell populations. We propose the use of commercially available 3D polystyrene systems to improve the current immune cell cultures and resulting cell products for emerging cellular (immuno)therapies. [Display omitted] [ABSTRACT FROM AUTHOR]

Adoptive cell therapy (ACT) using specific immune cells and stem cells has emerged as a promising treatment option that could complement traditional cancer therapies in the future. In particular, tumor-infiltrating lymphocytes (TILs) have been shown to be effective against solid tumors in various clinical trials. Despite the enormous disease burden and large number of premature deaths caused by colorectal cancer (CRC), studies on TILs isolated from tumor tissue of patients with CRC are still rare. To date, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations. We describe a procedure for generating patient-specific TILs, which are prerequisites for clinical trials of ACT in CRC. The manufacturing and characteristics of these TILs differ in important modalities from TILs commonly used for this therapeutic approach. Tumor tissue samples were obtained from 12 patients undergoing surgery for primary CRC, predominantly with low microsatellite instability (pMMR-MSI-L). Tumors in the resected specimens were examined pathologically, and an approved volume of tumor tissue was transferred to a disposable perfusion bioreactor. Tissue samples were subjected to an automatically controlled and highly reproducible cultivation process in a GMP-conform, closed perfusion bioreactor system using starting medium containing interleukin-2 and interleukin-12. Outgrowth of TIL from tissue samples was initiated by short-term supplementation with a specific activation cocktail. During subsequent expansion, TILs were grown in interleukin-2–enriched medium. Expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor under hyperoxic conditions resulted in a number of approximately 2 × 109 cells. The expanded TILs consisted mainly (73%) of the ACT-relevant CD3+/CD8+ effector memory phenotype (CD45RO+/CCR7−). TILs harvested under these conditions exhibited high functional potential, which was confirmed upon nonspecific stimulation (interferon-γ, tumor necrosis factor-α cytokine assay) [ABSTRACT FROM AUTHOR]

CD4+CD25+CD127lo regulatory T cells (Tregs) are responsible for maintaining immune homeostasis. Tregs can be rendered defective and deficient as a result of the immune imbalance seen in lung injury, and such dysfunction can play a major role in continued tissue inflammation. The authors hypothesized that adoptive therapy with healthy allogeneic umbilical cord blood (UCB)-derived Tregs may be able to resolve inflammation. Ex vivo -expanded UCB Tregs exhibited a unique phenotype with co-expression of CD45RA+CD45RO+ >80% and lung homing markers, including CD49d. UCB Tregs did not turn pathogenic when exposed to IL-6. Co-culture with increasing doses of dexamethasone led to a synergistic increase in UCB Treg-induced apoptosis of conventional T cells (Tcons), which translated into significantly higher suppression of proliferating Tcons, especially at a lower Treg:Tcon ratio. Multiple injections of UCB Tregs led to their preferential accumulation in lung tissue in an immune injury xenogenic model. A significant decrease in lung resident cytotoxic CD8+ T cells (P = 0.0218) correlated with a sustained decrease in their systemic distribution compared with controls (P < 0.0001) (n = 7 per arm) as well as a decrease in circulating human soluble CD40 ligand level (P = 0.031). Tissue architecture was preserved in the treatment arm, and a significant decrease in CD3+ and CD8+ burden was evident in immunohistochemistry analysis. UCB Treg adoptive therapy is a promising therapeutic strategy for treatment of lung injury. [ABSTRACT FROM AUTHOR]

Despite huge potentials of NK cells in adoptive cell therapy (ACT), formidable physical barriers of the tumor tissue and deficiency of recognizing signals on tumor cells severely prevent NK cell infiltrating, activating and killing performances. Herein, a nano-immunomodulator AuNSP@ αCD16 (CD16 antibody encoding plasmid) is explored to remodel the tumor microenvironment (TME) for improving the antitumor effects of adoptive NK cells. The as-prepared AuNSP, with a seaurchin-like gold core and a cationic polymer shell, exhibited a high gene transfection efficiency and a stable NIR-II photothermal capacity. The AuNSP could trigger mild photothermal intervention to partly destroy tumors and collapse the dense physical barriers, making a permeable TME for NK cell infiltration. What's more, the AuNSP could achieve αCD16 gene transfection to modify tumor surface with CD16 antibody, marking a unique structure on tumor cells for NK cell recognition and then lead to strong NK cell activation by CD16-mediated antibody-dependent cellular cytotoxicity (ADCC). As expected, the designed AuNSP@ αCD16 induced an immune-favorable TME for NK cell performing killing functions against solid tumors, increasing the release of cytolytic granules and proinflammatory cytokines, which ultimately achieved a robustly boosted NK cell-based immunotherapy. Hence, the AuNSP@ αCD16 -mediated TME reconstituting strategy provides a substantial perspective for NK-based ACT on solid tumors. In adoptive cell therapy (ACT), natural killer (NK) cells exhibit greater off-the-shelf utility and improved safety comparing with T cells, but the efficacy of NK cell therapy is severely compromised by formidable physical barriers of the tumor tissue and deficiency of NK cell recognizing signals on tumor cells. Herein, a nano-immunomodulator AuNSP@ αCD16 , with the abilities of inducing mild photothermal intervention and modifying the tumor cell surface with αCD16, is explored to reconstruct an infiltration-favorable and activation-facilitating tumor microenvironment for NK cells to perform killing functions. Such a simple and safe strategy is believed as a very promising candidate for future NK-based ACT. [Display omitted] [ABSTRACT FROM AUTHOR]

The success of chimeric antigen receptor (CAR) T cell therapy in solid tumors, unlike in hematologic malignancies, is limited by inadequate tumor infiltration and T cell dysfunction and exhaustion. Regional delivery of CAR T cells in patients with solid tumors is safe and feasible; promotes infiltration, proliferation, and trafficking; and ignites functionally persisting systemic immunity. [ABSTRACT FROM AUTHOR]

• High number of stable, pure and functional Tregs upon GMP-compliant cell culture • Tregs can be engineered by TCR-encoding mRNA electroporation • mRNA-engineered Tregs remain functional and phenotypically and epigenetically stable Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs. Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4+CD127−CD25hi Tregs and CD4+CD127−CD25+CD45RA+ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated. In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs. [ABSTRACT FROM AUTHOR]

• Persistent B19 infection can lead to aplastic crisis and graft rejection. • TCRαβ-depleted HSCT can treat secondary graft rejection. • The third-party CD45RA+ depleted adoptive cell therapy is an effective treatment. This is a case report of a 6-year-old girl with relapsed B cell acute lymphoblastic leukemia in which adoptive cell therapy was applied successfully to treat refractory human parvovirus (HPV) B19 infection. Allogenic chimeric antigen receptor (CAR) T-cell therapy (bispecific CD19/CD22) was bridged to hematopoietic stem cell transplantation (HSCT) using a haploidentical paternal donor. However, HPV B19 DNAemia progressed and transfusion-related graft versus host disease occurred. After finding a third-party related donor with a better HLA match, haploidentical HPV B19-seropositive CD45RA+ depleted cells (16.5 × 106/kg) were administered and paternal TCRαβ+ depleted stem cell were retransplanted. The HPV B19 DNAemia became negative within 1 week and the reticulocyte, neutrophil, hemoglobin, and platelet counts gradually normalized. The patient remained stable during the 1-year outpatient follow-up period. Thus, our case report highlights that persistent B19 infection can lead to pancytopenia, aplastic crisis, and graft rejection and TCRαβ+ depleted haplo-HSCT is an effective means of hematopoiesis recovery. CD45RO memory T-cell therapy is the key to treating and preventing the development of refractory severe HPV B19 infection. [ABSTRACT FROM AUTHOR]

• Bladder cancer recurrence after TUR remains a challenge, necessitating innovative approaches like Nectin-4 CAR-T cells. • Combining intravesical BCG or ICIs with CAR-T therapy may enhance efficacy against bladder cancer's immune suppression. • Nectin-4 CAR-T cells hold promise in revolutionizing bladder cancer treatment by preventing recurrence. Bladder cancer poses a significant global health challenge, particularly with its prevalence in males and the increasing incidence rates in Europe. While non-muscle invasive bladder cancer can be managed with localized treatments, its recurrence remains a concern. Intravesical Bacillus Calmette-Guérin therapy triggers an immune response against cancer cells, yet recurrences post- transurethral resection of bladder tumor persist. Thus, innovative strategies are needed. We propose the use of Nectin-4 targeted chimeric antigen receptor T cell therapy to prevent non-muscle invasive bladder cancer recurrence and progression. Nectin-4 is expressed in non-muscle invasive bladder cancer, making it an ideal target. CAR-T cells engineered with Nectin-4 receptors can specifically eliminate cancer cells. By directly applying these cells intravesically, the limitations of CAR-T cell access to solid tumors are circumvented. However, challenges persist, such as the immunosuppressive microenvironment of bladder cancer. This can be addressed by combining intravesical Bacillus Calmette-Guérin with CAR-T therapy or utilizing immune checkpoint inhibitors. CAR-T therapy-related toxicities, like cytokine release syndrome, can be managed with IL-6 and IL-1 receptor inhibitors. Our hypothesis suggests that Nectin-4 targeted CAR-T cells have the potential to revolutionize bladder cancer treatment. This therapy could not only eliminate cancer cells but also prevent recurrence and progression by inducing immune memory. Although further research is needed, this innovative approach shows promise in the treatment of non-muscle invasive bladder cancer. [ABSTRACT FROM AUTHOR]

Adoptive cell transfer therapy using CD8+ T lymphocytes showed promising results eradicating metastatic malignancies. However, several regulatory mechanisms limit its efficacy. We studied the role of the expression of the transcription factor FOXP3 on CD8+ T cell function and anti-tumor immunity. Here we show that suboptimal T cell receptor stimulation of CD8+ T cells upregulates FOXP3 in vitro. Similarly, CD8 T cells transferred into tumor-bearing mice upregulate FOXP3 in vivo. Cell-intrinsic loss of FOXP3 by CD8+ T cells resulted in improved functionality after TCR stimulation and better antitumor responses in vivo. Inhibition of the FOXP3/NFAT interaction likewise improved CD8+ T cell functionality. Transcriptomic analysis of cells after TCR stimulation revealed an enrichment of genes implicated in the response to IFN-γ, IFN-α, inflammatory response, IL-6/JAK/STAT, G2M checkpoint and IL-2/STAT signaling in FOXP3-deficient CD8+ T cells with respect to FOXP3-wt CD8+ T cells. Our results suggest that transient expression of FOXP3 by CD8+ T cells in the tumor microenvironment restrains their anti-tumor activity, with clear implications for improving T cell responses during immunotherapy. [ABSTRACT FROM AUTHOR]

• CAR T cells administered intrapleurally in 31 participants with pleural cancers. • Intrapleural delivery of CAR T cells using intracavitary/intratumoral routes is safe. • Repeated administration of therapeutic agents to the pleural cavity is feasible. We describe techniques and results of image-guided delivery of mesothelin-targeted chimeric antigen receptor (CAR) T cells in patients with pleural malignancies in a phase I/II trial (ClinicalTrials.gov: NCT02414269). Patients without a pleural catheter or who lack effusion for insertion of a catheter (31 of 41) were administered intrapleural CAR T cells by interventional radiologists under image guidance by computed tomography or ultrasound. CAR T cells were administered through a needle in an accessible pleural loculation (intracavitary) or following an induced loculated artificial pneumothorax. In patients where intracavitary infusion was not feasible, CAR T cells were injected via percutaneous approach either surrounding and/or in the pleural nodule/thickening (intratumoral). Pre- and post-procedural clinical, laboratory, and imaging findings were assessed. CAR T cells were administered intrapleurally in 31 patients (33 procedures, 2 patients were administered a second dose) with successful delivery of planned dose (10–186 mL); 14/33 (42%) intracavitary and 19/33 (58%) intratumoral. All procedures were completed within 2 h of T-cell thawing. There were no procedure-related adverse events greater than grade 1 (1 in 3 patients had prior ipsilateral pleural fusion procedures). The most common imaging finding was ground glass opacities with interlobular septal thickening and/or consolidation, observed in 12/33 (36%) procedures. There was no difference in the incidence of fever, CRP, IL-6, and peak vector copy number in the peripheral blood between infusion methods. Image-guided intrapleural delivery of CAR T cells using intracavitary or intratumoral routes is feasible, repeatable and safe across anatomically variable pleural cancers. [ABSTRACT FROM AUTHOR]

• SARS-CoV-2 subtly disrupts the equations of immune responses in COVID-19 patients. • The virus activates infected macrophages and CD4+ T cells to induce cytokine storm. • Cytokine storm and direct interaction of viruses inhibit NK cell cytolytic effect. • Exhausted NK cell fails to lyse infected cells to avoid their unleashed cytokine release. • Adoptive transfer of cytolytic NK cell may reduce cytokine storm while decreasing viral load. The global outbreak of coronavirus-2019 (COVID-19) still claims more lives daily around the world due to the lack of a definitive treatment and the rapid tendency of virus to mutate, which even jeopardizes vaccination efficacy. At the forefront battle against SARS-CoV-2, an effective innate response to the infection has a pivotal role in the initial control and treatment of disease. However, SARS-CoV-2 subtly interrupts the equations of immune responses, disrupting the cytolytic antiviral effects of NK cells, while seriously activating infected macrophages and other immune cells to induce an unleashed "cytokine storm", a dangerous and uncontrollable inflammatory response causing life-threatening symptoms in patients. Notably, the NK cell exhaustion with ineffective cytolytic function against the sources of exaggerated cytokine release, acts as an Achilles' heel which exacerbates the severity of COVID-19. Given this, approaches that improve NK cell cytotoxicity may benefit treatment protocols. As a suggestion, adoptive transfer of NK or CAR-NK cells with proper cytotolytic potentials and the lowest capacity of cytokine-release (for example CD56dim NK cells brightly express activating receptors), to severe COVID-19 patients may provide an effective cure especially in cases suffering from cytokine storms. More intriguingly, the ongoing evidence for persistent clonal expansion of NK memory cells characterized by an activating phenotype in response to viral infections, can benefit the future studies on vaccine development and adoptive NK cell therapy in COVID-19. Whether vaccinated volunteers or recovered patients can also be considered as suitable candidates for cell donation could be the subject of future research. [ABSTRACT FROM AUTHOR]

T cell based immunotherapies can be applicable to acute myeloid leukemia (AML). Therefore, the selection of optimal T cells, cell manufacturing, and therapeutic T cell engineering are essential for the development of effective adoptive T cell therapies for AML. Autologous tumor-infiltrating lymphocytes (TILs) have been in clinical trials to treat solid malignancies. Herein, we assessed whether TILs can be isolated from the bone marrow (BM) of AML patients, expanded ex vivo and utilized as a novel therapeutic strategy for AML. To this end, firstly we analyzed the immunophenotypes of a series of primary BM samples from AML patients (N = 10) by flow cytometry. We observed a variable amount of CD3+ TILs (range ∼2.3–∼32.6% of mononuclear cells) among BM samples. We then developed a novel protocol that produced a three-log ex vivo expansion of TILs isolated from AML patient BM (N = 10) and peripheral blood (PB) (N = 10), including from patients with a low number of CD3+ T cells, within 3, 4 weeks. Further, we identified previously described naïve T cells (CCR7+CD95-/or CD62L+CD45RA+) in AML BM and PB samples, which seemed to be required for a successful TILs ex vivo expansion. Finally, we showed that the expanded TILs could: (1) cause cytotoxicity to autologous AML blasts ex vivo (90.6% in control without T cell treatment vs. 1.89% in experimental groups with PB derived T cells and 1.77% in experimental groups with BM derived TILs, p < 0.01), (2) be genetically engineered to express CYP27B1 gene, and (3) infiltrate the BM and reside in close proximity to pre-injected autologous AML blasts of engrafted immunodeficiency mice. Altogether, these results provide a rationale for further studies of the therapeutic use of TILs in AML. [ABSTRACT FROM AUTHOR]

Immunotherapeutic approaches using γδ T cells have emerged as the function of γδ T cells in tumor surveillance and clearance has been discovered. In vitro expansion methods of γ9δ2 T cells have been based on phosphoantigens and cytokines, but expansion methods using feeder cells to generate larger numbers of γδ T cells have also been studied recently. However, there are no studies that directly compare γδ T cells cultured with phosphoantigens with those cultured with feeder cells. Therefore, this study aimed to compare the expansion, characteristics and effector functions of γδ T cells stimulated with K562-based artificial antigen-presenting cells (aAPCs) (aAPC-γδ T cells) and γδ T cells stimulated with only zoledronic acid (ZA) (ZA-γδ T cells). Peripheral blood mononuclear cells were stimulated with ZA for 7 days, and aAPC-γδ T cells were stimulated weekly with K562-based aAPCs expressing CD32, CD80, CD83, 4-1BBL, CD40L and CD70, whereas ZA-γδ T cells were stimulated with only IL-2. Cultured γδ T cells were analyzed by flow cytometry for the expression of co-stimulatory molecules, activating receptors and checkpoint inhibitors. Differentially expressed gene (DEG) analysis was also performed to determine the difference in gene expression between aAPC-γδ T cells and ZA-γδ T cells. In vitro cytotoxicity assay was performed with calcein AM release assay, and in vivo anti-tumor effect was compared using a U937 xenograft model. Fold expansion on day 21 was 690.7 ± 413.1 for ZA-γδ T cells and 1415.2 ± 1016.8 for aAPC- γδ T cells. Moreover, aAPC-γδ T cells showed continuous growth, whereas ZA-γδ T cells showed a decline in growth after day 21. The T-cell receptor Vγ9+δ2+ percentages (mean ± standard deviation) on day 21 were 90.0 ± 2.7% and 87.0 ± 4.5% for ZA-γδ T cells and aAPC-γδ T cells, respectively. CD25 and CD86 expression was significantly higher in aAPC-γδ T cells. In DEG analysis, aAPC-γδ T cells and ZA-γδ T cells formed distinct clusters, and aAPC-γδ T cells showed upregulation of genes associated with metabolism and cytokine pathways. In vitro cytotoxicity revealed superior anti-tumor effects of aAPC-γδ T cells compared with ZA-γδ T cells on Daudi, Raji and U937 cell lines. In addition, in the U937 xenograft model, aAPC-γδ T-cell treatment increased survival, and a higher frequency of aAPC-γδ T cells was shown in bone marrow compared with ZA-γδ T cells. Overall, this study demonstrates that aAPC-γδ T cells show long-term proliferation, enhanced activation and anti-tumor effects compared with ZA-γδ T cells and provides a basis for using aAPC-γδ T cells in further studies, including clinical applications and genetic engineering of γδ T cells. [ABSTRACT FROM AUTHOR]

Lymphodepletion with non-myeloablative (NMA) chemotherapy is currently a prerequisite for adoptive cell therapy (ACT). ACT based on tumor-infiltrating lymphocytes has long been used in malignant melanoma (MM), but with the advance of ACT into new cancer diagnoses, the patient predisposition will change. The authors here evaluate the bone marrow (BM) toxicity of NMA in combination with checkpoint inhibition and a priori risk factors in a wide range of cancer diagnoses. Thirty-one non-MM and MM patients were included from two different clinical trials with ACT. The treatment history was extracted from the medical records, together with the hematology data. Immune monitoring with flow cytometry was performed before and at several time points after therapy. NMA induced reversible myelosuppression in all patients. No significant differences in BM toxicity between MM and non-MM patients were found. The overall hematology counts were reconstituted within 3–6 months but with great individual heterogeneity, including eight patients who developed a second phase of neutropenia after hospital discharge. A performance status >0 was found, and shorter overall survival and sex were statistically associated with longer duration of anemia. By contrast, high expression of co-stimulatory markers CD28+ and CD27+ on T cells at baseline was significantly correlated with shorter duration of neutropenia (P = 0.010 and P = 0.009, respectively), anemia (P = 0.001 and P = 0.001, respectively) and thrombocytopenia (P = 0.017 and P = 0.030, respectively). In addition, following NMA, the authors saw a significant differentiation of T-cell phenotype associated with old age. ACT with NMA and checkpoint inhibition is tolerable in patients with multiple cancer diagnoses and therapy backgrounds but comes with substantial transient BM toxicity that is comparable in both non-MM and MM patients. Baseline T-cell CD28/CD27 expression level is predictive of duration of BM toxicity. Furthermore, NMA conditioning induces changes in the immune system that may affect a patient's immunocompetence for many months following therapy. [ABSTRACT FROM AUTHOR]

• Tumor antigen-targeted CAR T-cell therapy is effective for leukemia and lymphoma. • CAR T-cell therapy is in phase I/II clinical trials for pleural mesothelioma. • Regional delivery of CAR T cells achieve systemic immunity. • Combination of CAR T cells and anti-PD1 strategies is under investigation. The aim of adoptive T-cell therapy is to promote tumor-infiltrating immune cells following the transfer of either tumor-harvested or genetically engineered T lymphocytes. A new chapter in adoptive T-cell therapy began with the success of chimeric antigen receptor (CAR) T-cell therapy. T cells harvested from peripheral blood are transduced with genetically engineered CARs that render the ability to recognize cancer cell-surface antigen and lyse cancer cells. The successes in CAR T-cell therapy for B-cell leukemia and lymphoma have led to efforts to expand this therapy to solid tumors. Herein, we discuss the rationale behind the preclinical development and clinical trials of T-cell therapies in patients with malignant pleural mesothelioma. Furthermore, we highlight the ongoing investigation of combination immunotherapy strategies to synergistically potentiate endogenous as well as adoptively transferred immunity. [ABSTRACT FROM AUTHOR]

Adoptive cell therapy (ACT) is an emerging powerful cancer immunotherapy, which includes a complex process of genetic modification, stimulation and expansion. During these in vitro or ex vivo manipulation, sensitive cells are inescapability subjected to harmful external stimuli. Although a variety of cytoprotection strategies have been developed, their application on ACT remains challenging. Herein, a DNA network is constructed on cell surface by rolling circle amplification (RCA), and T cell-targeted trivalent tetrahedral DNA nanostructure is used as a rigid scaffold to achieve high-efficient and selective coating for T cells. The cytoprotective DNA network on T-cell surface makes them aggregate over time to form cell clusters, which exhibit more resistance to external stimuli and enhanced activities in human peripheral blood mononuclear cells and liver cancer organoid killing model. Overall, this work provides a novel strategy for in vitro T cell-selective protection, which has a great potential for application in ACT. T cell-selective coating and protection strategy with a flexible DNA network was formed by rolling circle amplification, which promotes T-cell aggregation and activation for anti-tumor therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Immunocompetent Cancer-on-Chips (iCoCs) can replicate complex tumor microenvironments. • iCoCs provide powerful tools for immuno-oncology research and immunotherapy testing. • Various iCoCs have been applied for mechanistic research and efficacy assessment. • Flexibility in design and readouts paves the way for further applications of iCoCs. The advances in cancer immunotherapy come with several obstacles, limiting its widespread use and benefits so far only to a small subset of patients. One of the underlying challenges remains to be the lack of representative nonclinical models that translate to human immunity and are able to predict clinical efficacy and safety outcomes. In recent years, immunocompetent Cancer-on-Chip models emerge as an alternative human-based platform that enables the integration and manipulation of complex tumor microenvironment. In this review, we discuss novel opportunities offered by Cancer-on-Chip models to advance (mechanistic) immuno-oncology research, ranging from design flexibility to multimodal analysis approaches. We then exemplify their (potential) applications for the research and development of adoptive cell therapy, immune checkpoint therapy, cytokine therapy, oncolytic virus, and cancer vaccines. [ABSTRACT FROM AUTHOR]

• Low-dose radiation upregulates T cell chemokines and iRGD receptors. • Low-dose radiation improves infiltration and antitumor potency of transferred T cells. • Combining low-dose radiation with adoptive cell therapy is beneficial in solid tumors. Poor penetration of transferred T cells represents a critical factor impeding the development of adoptive cell therapy in solid tumors. We demonstrated that iRGD-antiCD3 modification promoted both T cell infiltration and activation in our previous work. Interest in low-dose radiotherapy has recently been renewed due to its immuno-stimulatory effects including T cell recruitment. This study aims to explore the synergistic effects between low-dose radiotherapy and iRGD-antiCD3-modified T cells. Flow cytometry was performed to assess the expression of iRGD receptors and chemokines. T cell infiltration was evaluated by immunohistofluorescence and in vivo real-time fluorescence imaging and antitumor effects were investigated by in vivo bioluminescence imaging in the gastric cancer peritoneal metastasis mouse model. We found that 2 Gy irradiation upregulated the expression of all three iRGD receptors and T-cell chemokines. The addition of 2 Gy low-dose irradiation boosted the accumulation and penetration of iRGD-antiCD3-modified T cells in peritoneal tumor nodules. Combining 2 Gy low-dose irradiation with iRGD-antiCD3-modified T cells significantly inhibited tumor growth and prolonged survival in the peritoneal metastasis mouse model with a favorable safety profile. Altogether, we demonstrated that low-dose radiotherapy could improve the antitumor potency of iRGD-antiCD3-modified T cells by promoting T cell infiltration, providing a rationale for exploring low-dose radiotherapy in combination of other adoptive T cell therapies in solid tumors. [ABSTRACT FROM AUTHOR]

Tumor-infiltrating lymphocytes (TILs) is a type of adoptive cell therapy that addresses the challenges associated with cancer treatment. In this approach, T cells from a patient's tumor are harvested and stimulated to activate and expand them. Once a significant number of these cells have been generated, and reintroduced into the patient's body, where they can effectively recognize and eliminate cancer cells. However, conducting research in this field whit various challenges, including limited access to necessary technological devices for TILs isolation and expansion. To address this issue, a search was conducted to identify technological devices that could be used in adoptive cell therapy, using patent exploration strategies. The search began by identifying the best-known patent databases platforms and selecting four to explore. Over 300,000 documents were initially found, but applying various filters to reduce the number of relevant documents to approximately less than 500. The information obtained was then cross-referenced and analyzed to identify the top some technologies with the highest potential for use in the isolation and expansion of TILs. Finally, the interfaces of the search platforms were compared to identify their differences. [Display omitted] [ABSTRACT FROM AUTHOR]

• Resistance to ICIs is mediated by tumor intrinsic and tumor extrinsic mechanisms. • Targeting co-inhibitory or co-stimulatory signals raises antitumour immune response. • Tumour microenvironment conditions the immune infiltrate composition and activity. • Defining an immune profile of tumour, TME and host may help in selecting I–O therapy. The adoption of Immune checkpoint inhibitors (ICIs) allowed the achievement of impressive long-term survival results in non-small cell lung cancer (NSCLC), but most patients develop resistance to ICI treatment over time. Resistance to ICIs is mediated by several complex mechanisms affecting, but not limited to, tumour cell-intrinsic alterations and the tumour microenvironment. The possibility of modulating the immune response by interfering with specific alternative immune receptors, pathways and mediators might provide additional strategies to delay or prevent the development of resistance. Therefore, a greater in-depth investigation and understanding of these mechanisms aims to identify novel classes of immune targets and subsequently to evaluate potential new strategies for overcoming resistance, which will be assessed in this review. [ABSTRACT FROM AUTHOR]

Tumours employ a variety of immune-evasion and suppression mechanisms to impair development of functional tumor-specific T cells and subvert T cell-mediated immunity in the tumour microenvironment. Adoptive T cell therapy (ACT) aims to overcome these barriers and overwhelm tumor defenses with a bolus of T cells that were selectively expanded ex vivo. Although this strategy has been effective in liquid tumors and melanomas, many tumors appear to be resistant to ACT. Several factors are thought to play into this resistance, including poor engraftment and persistence of transferred cells, tumour cell heterogeneity and antigen loss, poor immune cell recruitment and infiltration into the tumour, and susceptibility to local immunosuppression in the tumor microenvironment. Oncolytic viruses (OV) have been identified as powerful stimulators of the anti-tumour immune response. As such, OVs are inherently well-positioned to act in synergy with ACT to bolster the anti-tumour T cell response. Further, OV vaccines, wherein tumour-associated antigens are encoded into the viral backbone, have proven to be remarkable in boosting antigen-specific T cell response. Pre-clinical studies have revealed remarkable therapeutic outcomes when OV vaccines are paired with ACT. In this scenario, OV vaccines are thought to function in a "push and pull" manner, where push refers to expanding T cells in the periphery and pull refers to recruiting those cells into the tumour that has been rendered amenable to T cell attack by the actions of the OV. In this review, we discuss barriers that limit eradication of tumors by T cells, highlight attributes of OVs that break down these barriers and present strategies for rational combinations of ACT with OV vaccines. [ABSTRACT FROM AUTHOR]

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application. [ABSTRACT FROM AUTHOR]

Genetically engineered immune cells with chimeric antigen receptors (CAR) or modified T cell receptors (TCR) have demonstrated their potential as a potent class of new cancer therapeutic strategy. Despite the clinical success of autologous CD19 CAR T cells in hematological malignancies, allogeneic T cells exhibit many advantages over their autologous counterparts and have recently gathered widespread attention due to the emergence of multiplex genome editing techniques, particularly CRISPR/Cas systems. Furthermore, genetically engineered T cells face a host of major challenges in solid tumors that are not as significant for blood cancers such as T cell targeted delivery, target specificity, proliferation, persistence, and the immunosuppressive tumor microenvironment. We take this opportunity to analyze recent strategies to develop allogeneic T cells, specifically in consideration of CRISPR/Cas and its delivery systems for multiplex gene editing. Additionally, we discuss the current methods used to delivery CRISPR/Cas systems for immunotherapeutic applications, and the challenges to continued development of novel delivery systems. We also provide a comprehensive analysis of the major challenges that genetically engineered T cells face in solid tumors along with the most recent strategies to overcome these barriers, with an emphasis on CRISPR-based approaches. We illustrate the synergistic prospects for how the combination of synthetic biology and immune-oncology could pave the way for designing the next generation of precision cancer therapy. Unlabelled Image [ABSTRACT FROM AUTHOR]

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) has been tested in advanced melanoma patients at various centers. We conducted a systematic review and meta-analysis to assess its efficacy on previously treated advanced metastatic cutaneous melanoma. The PubMed electronic database was searched from inception to 17 December 2018 to identify studies administering TIL-ACT and recombinant interleukin-2 (IL-2) following non-myeloablative chemotherapy in previously treated metastatic melanoma patients. Objective response rate (ORR) was the primary end point. Secondary end points were complete response rate (CRR), overall survival (OS), duration of response (DOR) and toxicity. Pooled estimates were derived from fixed or random effect models, depending on the amount of heterogeneity detected. Analysis was carried out separately for high dose (HD) and low dose (LD) IL-2. Sensitivity analyses were carried out. Among 1211 records screened, 13 studies (published 1988 − 2016) were eligible for meta-analysis. Among 410 heavily pretreated patients (some with brain metastasis), 332 received HD-IL-2 and 78 LD-IL-2. The pooled overall ORR estimate was 41% [95% confidence interval (CI) 35% to 48%], and the overall CRR was 12% (95% CI 7% to 16%). For the HD-IL-2 group, the ORR was 43% (95% CI 36% to 50%), while for the LD-IL-2 it was 35% (95% CI 25% to 45%). Corresponding pooled estimates for CRR were 14% (95% CI 7% to 20%) and 7% (95% CI 1% to 12%). The majority of HD-IL-2 complete responders (27/28) remained in remission during the extent of follow-up after CR (median 40 months). Sensitivity analyses yielded similar results. Higher number of infused cells was associated with a favorable response. The ORR for HD-IL-2 compared favorably with the nivolumab/ipilimumab combination following anti-PD-1 failure. TIL-ACT therapy, especially when combined with HD-IL-2, achieves durable clinical benefit and warrants further investigation. We discuss the current position of TIL-ACT in the therapy of advanced melanoma, particularly in the era of immune checkpoint blockade therapy, and review future opportunities for improvement of this approach. [ABSTRACT FROM AUTHOR]

Cancer is one of the most devastating complications of kidney transplantation and constitutes one of the leading causes of morbidity and mortality among solid organ transplantation (SOT) recipients. Immunosuppression, although effective in preventing allograft rejection, inherently inhibits immune surveillance against oncogenic viral infections and malignancy. Adoptive cell therapy, particularly immune effector cell therapy, has long been a modality of interest in both cancer and transplantation, though has only recently stepped into the spotlight with the development of virus-specific T-cell therapy and chimeric antigen receptor T-cell therapy. Although these modalities are best described in hematopoietic cell transplantation and hematologic malignancies, their potential application in the SOT setting may hold tremendous promise for those with limited therapeutic options. In this review, we provide a brief overview of the development of adoptive cell therapies with a focus on virus-specific T-cell therapy and chimeric antigen receptor T-cell therapy. We also describe the current experience of these therapies in the SOT setting as well as the challenges in their application and future directions in their development. [ABSTRACT FROM AUTHOR]

• Recent advances in using the patient's immune system to combat cancer show promising results. • Immune checkpoint blockade approved for various tumors, marking significant progress in the last decade. • Adoptive cell therapy (ACT) uses modified T cells to target tumor cells, showing encouraging outcomes. • Ongoing worldwide clinical trials seek to enhance ACT's efficacy in treating different tumor types. Although the concept of using the patient's immune system to combat cancer has been around for a while, it is only in recent times that substantial progress has been achieved in this field. Over the last ten years, there has been a significant advancement in the treatment of cancer through immune checkpoint blockade. This treatment has been approved for multiple types of tumors. Another approach to modifying the immune system to detect tumor cells and fight them off is adaptive cell therapy (ACT). This therapy involves using T cells that have been modified with either T cell receptors (TCR) or chimeric antigen receptors (CAR) to target the tumor cells. ACT has demonstrated encouraging outcomes in different types of tumors, and clinical trials are currently underway worldwide to enhance this form of treatment. This review focuses on the advancements that have been made in ACT from preclinical to clinical settings till now. [ABSTRACT FROM AUTHOR]

• Improving T cell co-stimulation enhances persistence and functionality of T cells. • Immunosuppressive tumour microenvironment modulation overcomes T cell exhaustion. • Low T cell trafficking into the tumours can be manipulated by chemotaxis. Ovarian cancer is amongst the ten most common cancer types in women, and it is one of the leading causes of death. Despite the promising results of targeted therapies, including anti-angiogenic agents and poly (ADP-ribose) polymerase inhibitors (PARPi), the majority of patients will relapse and develop treatment resistance, implying that novel therapeutic strategies are required. Adoptive cell therapy (ACT) refers to the process by which autologous immune cells are used to eliminate cancer. Examples include tumour infiltrating lymphocytes (TILs), T cells genetically engineered with T cell receptors (TCR), or chimeric antigen receptor (CAR)-T cells. Recently, ACT has revealed promising results in the treatment of haematological malignancies, however, its application to solid tumours is still limited due to lack of functionality and persistence of T cells, prevalence of an exhausted phenotype and impaired trafficking towards the tumour microenvironment (TME). In this review we explore the potential of ACT for the treatment of ovarian cancer and strategies to overcome its principal limitations. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy is a new and promising option for cancer treatment. Unlike traditional chemo- and radiotherapy, immunotherapy actives host immune system to attack malignancies, and this potentially offers long-term protection from recurrence with less toxicity in comparison to conventional chemo- and radiation therapy. In adoptive CD8+ T cell therapy (ACT), large numbers of tumor-specific T cells are sourced from patients and expanded in vitro and infused back to patients. T cells can be expanded from naturally-induced tumor-specific CD8+ T cells isolated from tumor infiltrating lymphocytes (TIL) or genetically-modified autologous circulating CD8+ T cells. The engineered T cells expressed tumor-specific antigen receptors including chimeric antigen receptors (CARs) and T cell receptors (TCRs), prepared from cultured B and T cell clones, respectively. The most successful ACT, anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy directed against B cell lymphoma, is already approved for use based on evidence of efficacy. Efficacy of solid tumors is not yet forthcoming. This review summarizes current technology developments using ACT in clinical trials. In this review, differences between various ACT approaches are discussed. Furthermore, resistance factors in the tumor microenvironment are also considered, as are immune related adverse effects, critical clinic monitoring parameters and potential mitigation approaches. [ABSTRACT FROM AUTHOR]

• CARs transmit modified information/signals of choice across the plasma membrane. • Unlike wildtype receptors, CARs can even invert signals into the opposite. • Now, the hunt is on for consensus signaling domains ensuring longevity of CAR cells. • Universal, off-the-shelf and broadly applicable CAR cell types would be desirable. • Diseases with nonmalignant polyclonal cellular expansions are future CAR targets. Genetically engineered T cells expressing chimeric antigen receptors (CAR) present a new treatment option for patients with cancer. Recent clinical trials of B cell leukemia have demonstrated a response rate of up to 90%. However, CAR cell therapy is frequently accompanied by severe side effects such as cytokine release syndrome and the development of target cell resistance. Consequently, further optimization of CARs to obtain greater long-term efficacy and increased safety is urgently needed. Here we high-light the various efforts of adjusting the intracellular signaling domains of CARs to these major requirements to eventually obtain high-level target cell cytotoxicity paralleled by the establishment of longevity of the CAR expressing cell types to guarantee for extended tumor surveillance over prolonged periods of time. We are convinced that it will be crucial to identify the molecular pathways and signaling requirements utilized by such 'efficient CARs' in order to provide a rational basis for their further hypothesis-based improvement. Furthermore, we here discuss timely attempts of how to: i) control 'on-tumor off-target' effects; ii) introduce Signal 3 (cytokine responsiveness of CAR cells) as an important building-block into the CAR concept; iii) most efficiently eliminate CAR cells once full remission has been obtained. We also argue that universal systems for the variable and pharmacokinetically-controlled attachment of extracellular ligand recognition domains of choice along with the establishment of 'off-the-shelf' cell preparations with suitability for all patients in need of a highly-potent cellular therapy may become future mainstays of CAR cell therapy. Such therapies would have the attraction to work independent of the patients' histocompatibility make-up and the availability of functionally intact patient's cells. Finally, we summarize the evidence that CAR cells may obtain a prominent place in the treatment of non-malignant and auto-reactive T and B lymphocyte expansions in the near future, e.g. , for the alleviation of autoimmune diseases and allergies. After the introduction of red blood cell transfusions, which were made possible by the landmark discoveries of the ABO blood groups by Karl Landsteiner , and the establishment of bone marrow transplantation by E. Donnall Thomas to exchange the entire hematopoietic system of a patient suffering from leukemia, the introduction of patient-tailored cytotoxic cellular populations to eradicate malignant cell populations in vivo pioneered by Carl H. June , represents the third major and broadly applicable milestone in the development of human cellular therapies within the rapidly developing field of applied biomedical research of the last one hundred years. [ABSTRACT FROM AUTHOR]

Abstract Immunotherapy constitutes an exciting and rapidly evolving field, and the demonstration that genetically modified T-cell receptors (TCRs) can be used to produce T-lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy. Overall, TCR-modified T cells have the ability to target a wide variety of self and non–self targets through the normal biology of a T cell. Although major histocompatibility complex (MHC)–restricted and dependent on co-receptors, genetically engineered TCRs still present a number of characteristics that ensure they are an important alternative strategy to chimeric antigen receptors (CARs), and high-affinity TCRs can now be successfully engineered with the potential to enhance therapeutic efficacy while minimizing adverse events. This review will focus on the main characteristics of TCR gene-modified cells, their potential clinical application and promise to the field of adoptive cell transfer (ACT), basic manufacturing procedures and characterization protocols and overall challenges that need to be overcome so that redirection of TCR specificity may be successfully translated into clinical practice, beyond early-phase clinical trials. [ABSTRACT FROM AUTHOR]

Abstract Immunotherapy has revolutionized therapy in both solid and liquid malignancies. The ability to cure acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) with an allogeneic hematopoietic stem cell transplant (HSCT) is proof of concept for the application of immunotherapy in AML and MDS. However, outside of HSCT, only the anti-CD33 antibody drug conjugate gemtuzumab ozogamicin is currently approved as an antibody-targeted therapy for AML. Several avenues of immunotherapeutic drugs are currently in different stages of clinical development. Here, we review recent advances in antibody-based therapy, immune checkpoint inhibitors, vaccines and adoptive cell-based therapy for patients with AML and MDS. First, we discuss different antibody constructs. Immune checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein-1 (PD-1) and CD47 as well as peptide, dendritic cell and dendritic/AML cell-based vaccines are reviewed next. Lastly, adoptive cell-based therapy including chimeric antigen receptor (CAR)-T cell and NK cell therapy is discussed. [ABSTRACT FROM AUTHOR]

• Anti-PD-L1 × anti-CD3 bispecific T-cell engagers (BTEs) were produced from CHO cells. • The BTEs were used to arm T cells to generate BTE-armed T cells (BATs). • BATs allow blockage of PD-L1/PD-1 and redirect T cells to kill PD-L1 + cancer cells. • Cryopreserved BATs retained stability, supporting their use and transportation. T cell-based immunotherapy has transformed cancer treatment. Nonetheless, T cell antitumor activity can be inhibited by an immune checkpoint molecule expressed on cancer cells, program death ligand 1 (PD-L1), which interacts with the PD-1 on T cells. We generated αPD-L1 × αCD3 bispecific T-cell engager-armed T cells (BATs) to prevent PD-L1/PD-1 interaction and hence to redirect T cells to kill cancer cells. αPD-L1 × αCD3 bispecific T-cell engagers (BTEs) were produced from Chinese hamster ovary (CHO) cells to arm human primary T cells. Flow cytometry was used to investigate BTE binding to BATs. The cytotoxicity of BATs against PD-L1-expressing breast cancer (BC) cell lines was assessed in 2-dimensional (2D) and 3-dimensional (3D) culture models. The binding stability of BTE on BATs and their efficacy after cryopreservation were also examined. The CHO cell BTE expression yield was 3.34 mg/ml. The binding ability on T cells reached 91.02 ± 4.2 %. BATs specifically lysed PD-L1-expressing BC cells, with 56.4 ± 15.3 % HCC70 cells and 70.67 ± 15.6 % MDA-MB-231 cells lysed at a 10:1 effector-to-target ratio. BATs showed slight, nonsignificant lysis of PD-L1-negative BC cells, MCF-7, and T47D. Moreover, BATs significantly disrupted MDA-MB-231 3D spheroids expressing PD-L1 after 48 and 72 h of coculture. Cryopreserved BATs maintained BTE binding stability, cell viability, and anticancer activity, comparable to fresh BATs. αPD-L1 × αCD3 BATs induced the cytolysis of PD-L1-expressing BC cells in 2D and 3D coculture assays. BATs can be prepared and preserved, facilitating their use and transportation. This study demonstrates the potential of αPD-L1 × αCD3 BATs in treating cancers with positive PD-L1 expression. [ABSTRACT FROM AUTHOR]

Immunotherapy has ignited hope to cure paediatric solid tumours that resist traditional therapies. Among the most promising methods is adoptive cell therapy (ACT). Particularly, ACT using T cells equipped with chimeric antigen receptors (CARs) has moved into the spotlight in clinical studies. However, the efficacy of ACT is challenged by ACT-intrinsic factors, like lack of activation or T cell exhaustion, as well as immune evasion strategies of paediatric solid tumours, such as their highly immunosuppressive microenvironment. Novel strategies, including ACT using innate-like lymphocytes, innovative cell engineering techniques, and ACT combination therapies, are being developed and will be crucial to overcome these challenges. Here, we discuss the main classes of ACT for the treatment of paediatric extracranial solid tumours, reflect on the available preclinical and clinical evidence supporting promising strategies, and address the challenges that ACT is still facing. Ultimately, we highlight state-of-the-art developments and opportunities for new therapeutic options, which hold great potential for improving outcomes in this challenging patient population. • Different paediatric solid tumours share targetable common antigens. • Expanded and/or engineered αβ T, γδ T, NK, and NKT cells are explored ACT sources. • Pronounced immunosuppression hinders ACT efficacy. • New engineering techniques and combination therapies could bypass immunosuppression. • A disparity exists between target identification and their clinical implementation. [ABSTRACT FROM AUTHOR]

The last decade has witnessed great progress in the field of adoptive cell therapies, with the authorization of Kymriah (tisagenlecleucel) in 2017 by the Food and Drug Administration (FDA) as a crucial stepstone. Since then, five more CAR-T therapies have been approved for the treatment of hematological malignancies. While this is a great step forward to treating several types of blood cancers, CAR-T cell therapies are still associated with severe side-effects such as Graft- versus -Host Disease (GvHD), cytokine release syndrome (CRS) and neurotoxicity. Because of this, there has been continued interest in Natural Killer cells which avoid these side-effects while offering the possibility to generate allogeneic cell therapies. Similar to T-cells, NK cells can be genetically modified to improve their therapeutic efficacy in a variety of ways. In contrast to T cells, viral transduction of NK cells remains inefficient and induces cytotoxic effects. Viral vectors also require a lengthy and expensive product development process and are accompanied by certain risks such as insertional mutagenesis. Therefore, non-viral transfection technologies are avidly being developed aimed at addressing these shortcomings of viral vectors. In this review we will present an overview of the potential of NK cells in cancer immunotherapies and the non-viral transfection technologies that have been explored to engineer them. [ABSTRACT FROM AUTHOR]

• The tumor microenvironment could induce immunosuppression, rendering the tumor immune-evasive. • Immune checkpoint inhibitors release the brakes on the immune system to eliminate cancer cells. • Immune check inhibitors have demonstrated impressive success in treating some but not all cancers. • Immunotherapy for pMMR-MSI-L and MSS phenotypes of colorectal cancer is still inadequate. The tremendous success of immunotherapy in clinical trials has led to its establishment as a new pillar of cancer therapy. However, little clinical efficacy has been achieved in microsatellite stable colorectal cancer (MSS-CRC), which constitutes most CRC tumors. Here, we discuss the molecular and genetic heterogeneity of CRC. We review the immune escape mechanisms, and focus on the latest advances in immunotherapy as a treatment modality for CRC. By providing a better understanding of the tumor microenvironment (TME) and the molecular mechanisms underlying immunoevasion, this review offers an insight into developing therapeutic strategies that are effective for patients with various subsets of CRC. [ABSTRACT FROM AUTHOR]

Peyronie's disease is a fibro-inflammatory disease of penile connective tissue. Fibrotic plaque formed in Peyronie's disease can cause penile curvature disorders, anxiety disorders, and disruption of sexual life. Medical and surgical regimens are available to treat Peyronie's disease. However, current pharmacological treatment modalities are ineffective and may not prevent recurrences. Tissue losses and recurrences may occur in Peyronie's surgery performed for treatment. This situation creates the need for new-generation therapeutic agents. This article hypothesized how adoptive cell therapy with CAR T-cells, a popular and effective methodology, can be used to treat Peyronie's disease. Our hypothesis is centered around the development of a T cell equipped with a chimeric antigen receptor specifically designed to target FAP, which serves as an indicator of activated fibroblasts. We think it will be a promising therapy model for Peyronie's disease in the future because adoptive cell therapy can prevent recurrences because it takes place in the body for a long time and can reduce plaque before fibrotic tissue grows. [ABSTRACT FROM AUTHOR]

Highlights • The MHC molecule polymorphism limits ACT applicability using conventional T cells. • Targeting non-polymorphic CD1 molecules on tumor cells overcome the HLA restriction. • CD1-restricted T and iNKT cells can be used as donor-unrestricted universal effector cells in ACT. • CD1 humanized mouse models can help assessing CD1-restricted T and NKT cell potential in ACT. Abstract Adoptive cell therapy (ACT) using tumor-specific "conventional" MHC-restricted T cells obtained from tumor-infiltrating lymphocytes, or derived ex vivo by either antigen-specific expansion or genetic engineering of polyclonal T cell populations, shows great promise for cancer treatment. However, the wide applicability of this therapy finds limits in the high polymorphism of MHC molecules that restricts the use in the autologous context. CD1 antigen presenting molecules are nonpolymorphic and specialized for lipid antigen presentation to T cells. They are often expressed on malignant cells and, therefore, may represent an attractive target for ACT. We provide a brief overview of the CD1-resticted T cell response in tumor immunity and we discuss the pros and cons of ACT approaches based on unconventional CD1-restricted T cells. [ABSTRACT FROM AUTHOR]

Abstract Targeted adoptive immunotherapy with engineered T cells is a promising treatment for refractory hematologic malignancies. However, many patients achieving early complete remissions ultimately relapse. Immunosuppressive ligands are expressed on tumor and supportive cells in the tumor microenvironment (TME). When activated, T cells express associated "checkpoint" receptors. Binding of co-inhibitory ligands and receptors may directly contribute to T-cell functional exhaustion. It is not known whether all T cells engineered to express chimeric antigen receptors (CARs) are subject to checkpoint-mediated regulation. It is also unknown whether distinct CAR signaling moieties modulate T-cell responsiveness to these inhibitory pathways. We have, therefore, directly compared functional co-inhibition in engineered T cells identically targeted to the tumor-associated antigen CD123, but distinct in their mode of T-cell activation: via the endogenous T-cell receptor (ENG), or downstream of CD28 or 41BB-containing CARs. In all cases, we have observed antigen-independent T-cell activation associated with upregulation of the co-inhibitory receptors programmed cell death protein 1 (PD-1, CD279), Tim-3 and Lag-3. Notably, CD28.CAR T cells were uniquely susceptible to PD-1/PD-L1 mediated checkpoint inhibition. Together, our data indicate that PD-1/PD-L1 checkpoint blocking agents may be considered clinically when CD28.CAR T cells do not perform optimally in human trials. [ABSTRACT FROM AUTHOR]

Summary Immune recognition of tumor-expressed antigens by cytotoxic CD8 + T cells is the foundation of adoptive T cell therapy (ACT) and has been shown to elicit significant tumor regression. However, therapy-induced selective pressure can sculpt the antigenicity of tumors, resulting in outgrowth of variants that lose the target antigen. We demonstrate that tumor relapse from ACT and subsequent oncolytic viral vaccination can be prevented using class I HDACi, MS-275. Drug delivery subverted the phenotype of tumor-infiltrating CD11b + Ly6C hi Ly6G – myeloid cells, favoring NOS2/ROS secretion and pro-inflammatory genes characteristic of M1 polarization. Simultaneously, MS-275 abrogated the immunosuppressive function of tumor-infiltrating myeloid cells and reprogrammed them to eliminate antigen-negative tumor cells in a caspase-dependent manner. Elevated IFN-γ within the tumor microenvironment suggests that MS-275 modulates the local cytokine landscape to favor antitumor myeloid polarization through the IFN-γR/STAT1 signaling axis. Exploiting tumor-infiltrating myeloid cell plasticity thus complements T cell therapy in targeting tumor heterogeneity and immune escape. [ABSTRACT FROM AUTHOR]

Following the landmark approvals by the United States Food and Drug Administration, the adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells has now entered mainstream clinical practice for patients with chemotherapy-resistant or refractory B-cell malignancies. These approvals have followed on from a prolonged period of pre-clinical evaluation, informing the design of clinical trials that have demonstrated unprecedented efficacy in this difficult to treat patient population. However, the delivery of autologous CAR-engineered T-cell therapy is complex, costly and not without significant risk. Here we summarize the key themes of CAR T-cell preclinical development and highlight a number of innovative strategies designed to further address toxicity and improve efficacy. In concert with the emerging promise of precision genome editing, it is hoped these next generation products will increase the repertoire of clinical applications of CAR T-cell therapy in malignant and perhaps other disease settings. [ABSTRACT FROM AUTHOR]

Liver transplantation is accepted as the most reliable therapeutic option for patients with end-stage liver failure, but lifelong administration of immunosuppressive agents continues to be problematic due to various drug-induced morbidities and the risk of mortality. Complete cessation of immunosuppressive drugs while maintaining normal graft function and histology, called operational tolerance, has the potential to overcome these long-standing problems. Previously, we reported the results of a pilot study of anti- donor regulatory T cell therapy in 10 consecutive adult patients who underwent living donor liver transplantation (LDLT), of whom 7 patients successfully stopped immunosuppression for nearly 2 years. Described herein are the clinical follow-ups of these patients, a brief description of the protocol and its theoretical background, and a possible explanation for the immunological findings. [ABSTRACT FROM AUTHOR]

Immunological destruction of tumors is a multistep, coordinated process that can be modulated or targeted at several critical points to elicit tumor rejection. These steps in the cancer immunity cycle include: (i) generation of sufficient numbers of effector T cells with high avidity recognition of tumor antigens in vivo; (ii) trafficking and infiltration into the tumor; (iii) overcoming inhibitory networks in the tumor microenvironment; (iv) direct recognition of tumor antigens and generation of an effector anti-tumor response; and (v) persistence of the anti-tumor T cells. In an effort to understand whether the immune system plays a role in controlling ovarian cancer, our group and others demonstrated that the presence of tumor infiltrating lymphocytes (TILs) is associated with improved clinical outcome in ovarian cancer patients. Recently, we hypothesized that the quality of infiltrating T cells could also be a critical determinant of outcome in ovarian cancer patients. In the past decade, several immune-based interventions have gained regulatory approval in many solid tumors and hematologic malignancies. These interventions include immune checkpoint blockade, cancer vaccines, and adoptive cell therapy. There are currently no approved immune therapies for ovarian cancer. Immunotherapy in ovarian cancer will have to consider the immune suppressive networks within the ovarian tumor microenvironment; therefore, a major direction is to develop biomarkers that would predict responsiveness to different types of immunotherapies, and allow for treatment selection based on the results. Moreover, such biomarkers would allow rational combination of immunotherapies, while minimizing toxicities. In this review, the current understanding of the host immune response in ovarian cancer patients will be briefly reviewed, progress in immune therapies, and future directions for exploiting immune based strategies for long lasting durable cure. [ABSTRACT FROM AUTHOR]

Successes of immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T cell therapy in curing patients with otherwise lethal cancers have validated immunotherapy as a treatment for cancer and have inspired excitement for its broader potential. Most promising is the ability of each approach to eliminate bulky and advanced-stage cancers and to achieve durable cures. Despite this success, to date only a subset of cancer patients and a limited number of cancer types respond to these therapies. A major goal now is to expand the types of cancer and number of patients who can be successfully treated. To this end a multitude of immunotherapies are being tested clinically in new combinations, and many new immunomodulatory antibodies and CARs are in development. A third major immunotherapeutic approach with renewed interest is cancer vaccines. While over 20 years of therapeutic cancer vaccine trials have met with limited success, these studies have laid the groundwork for the use of therapeutic vaccines in combination with other immunotherapies or alone as prophylactic cancer vaccines. Prophylactic vaccines are now poised to revolutionize cancer prevention as they have done for the prevention of infectious diseases. In this review we examine three major cancer immunotherapy modalities: immunomodulatory antibodies, CAR T cell therapy and vaccines. For each we describe the current state of the art and outline major challenges and research directions forward. [ABSTRACT FROM AUTHOR]

Cytokine-Induced killer (CIK) cells are raising growing interest in cellular antitumor therapy, as they can be easily expanded with a straightforward and inexpensive protocol, and are safe requiring only GMP-grade cytokines to obtain very high amounts of cytotoxic cells. CIK cells do not need antigen-specific stimuli to be activated and proliferate, as they recognize and destroy tumor cells in an HLA-independent fashion through the engagement of NKG2D. In several preclinical studies and clinical trials, CIK cells showed a reduced alloreactivity compared to conventional T cells, even when challenged across HLA-barriers; only in a few patients, a mild GVHD occurred after treatment with allogeneic CIK cells. Additionally, their antitumor activity can be redirected and further improved with chimeric antigen receptors, clinical-grade monoclonal antibodies or immune checkpoint inhibitors. The evidence obtained from a growing body of literature support CIK cells as a very promising cell population for adoptive immunotherapy. In this review, all these aspects will be addressed with a particular emphasis on the role of the cytokines involved in CIK cell generation, expansion and functionalization. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy is now a powerful clinical reality, with a steady progression of new drug approvals and a massive pipeline of additional treatments in clinical and preclinical development. However, modulation of the immune system can be a double-edged sword: Drugs that activate immune effectors are prone to serious non-specific systemic inflammation and autoimmune side effects. Drug delivery technologies have an important role to play in harnessing the power of immune therapeutics while avoiding on-target/off-tumor toxicities. Here we review mechanisms of toxicity for clinically-relevant immunotherapeutics, and discuss approaches based in drug delivery technology to enhance the safety and potency of these treatments. These include strategies to merge drug delivery with adoptive cellular therapies, targeting immunotherapies to tumors or select immune cells, and localizing therapeutics intratumorally. Rational design employing lessons learned from the drug delivery and nanomedicine fields has the potential to facilitate immunotherapy reaching its full potential. [ABSTRACT FROM AUTHOR]

Therapeutic cells hold tremendous promise in treating currently incurable, chronic diseases since they perform multiple, integrated, complex functions in vivo compared to traditional small-molecule drugs or biologics. However, they also pose significant challenges as therapeutic products because (a) their complex mechanisms of actions are difficult to understand and (b) low-cost bioprocesses for large-scale, reproducible manufacturing of cells have yet to be developed. Immunotherapies using T cells and dendritic cells (DCs) have already shown great promise in treating several types of cancers, and human mesenchymal stromal cells (hMSCs) are now extensively being evaluated in clinical trials as immune-modulatory cells. Despite these exciting developments, the full potential of cell-based therapeutics cannot be realized unless new engineering technologies enable cost-effective, consistent manufacturing of high-quality therapeutic cells at large-scale. Here we review cell-based immunotherapy concepts focused on the state-of-the-art in manufacturing processes including cell sourcing, isolation, expansion, modification, quality control (QC), and culture media requirements. We also offer insights into how current technologies could be significantly improved and augmented by new technologies, and how disciplines must converge to meet the long-term needs for large-scale production of cell-based immunotherapies. [ABSTRACT FROM AUTHOR]

Background. Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the “semi-allogeneic” fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs. Methods. Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties. Results. HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4+HLA-Gpos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-Gpos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD). Discussion. We propose, in vitro generation of HLA-G–expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy. [ABSTRACT FROM AUTHOR]