Objective: Exploring the efficacy and safety of bridging blinatumomab (BiTE) in combination with chimeric antigen receptor T (CAR-T) cell therapy for the treatment of adult patients with acute B-cell lymphoblastic leukemia (B-ALL) . Methods: Clinical data from 36 adult B-ALL patients treated at the First Affiliated Hospital of Suzhou University from August 2018 to May 2023 were retrospectively analyzed. A total of 36 cases were included: 18 men and 18 women. The median age was 43.5 years (21-72 years). Moreover, 21 cases of Philadelphia chromosome-positive acute lymphoblastic leukemia were reported, and 16 of these cases were relapsed or refractory. Eighteen patients underwent blinatumomab bridging followed by CAR-T cell therapy, and 18 patients received CAR-T cell therapy. This study analyzed the efficacy and safety of treatment in two groups of patients. Results: In the BiTE bridge-to-CAR-T group, 16 patients achieved complete remission (CR) after BiTE immunotherapy, with a CR rate of 88.9%. One month after bridging CAR-T therapy, bone marrow examination showed a CR rate of 100.0%, and the minimal residual disease (MRD) negativity rate was higher than the nonbridging therapy group (94.4% vs. 61.1%, Fisher, P=0.041). The incidence of cytokine release syndrome and other adverse reactions in the BiTE bridge-to-CAR-T group was lower than that in the nonbridging therapy group (11.1% vs. 50.0%, Fisher, P=0.027). The follow-up reveals that 13 patients continued to maintain MRD negativity, and five patients experienced relapse 8.40 months (2.57-10.20 months) after treatment. Two of five patients with relapse achieved CR after receiving the second CAR-T cell therapy. In the nonbridging therapy group, 10 patients maintained continuous MRD negativity, 7 experienced relapse, and 6 died. The 1 year overall survival rate in the BiTE bridge-to-CAR-T group was higher than that in the nonbridging therapy group, with a statistically significant difference at the 0.1 level (88.9%±10.5% vs. 66.7%±10.9%, P=0.091) . Conclusion: BiTE bridging CAR-T cell therapy demonstrates excellent efficacy in adult B-ALL treatment, with a low recent recurrence rate and ongoing assessment of long-term efficacy during follow-up.
目的： 探讨贝林妥欧单抗桥接嵌合抗原受体T（CAR-T）细胞疗法治疗急性B淋巴细胞白血病（B-ALL）患者的疗效及安全性。 方法： 回顾性分析2018年8月至2023年5月在苏州大学附属第一医院住院治疗的36例成人B-ALL患者的临床资料。男18例，女18例，中位年龄为43.5（21~72）岁。其中费城染色体阳性急性淋巴细胞白血病（Ph(+)ALL）21例，复发/难治16例。18例患者接受贝林妥欧单抗桥接CAR-T细胞治疗，18例患者仅接受CAR-T细胞治疗，分析两组患者的疗效及安全性。 结果： 贝林妥欧单抗桥接CAR-T组中16例患者贝林妥欧单抗治疗后获得完全缓解（CR），CR率88.9%。桥接CAR-T治疗1个月后复查骨髓象，CR率为100.0%，微小残留病（MRD）阴性率高于未接受贝林妥欧单抗桥接治疗组（94.4%对61.1%，Fisher，P=0.041）。贝林妥欧单抗桥接CAR-T组患者细胞因子释放综合征（CRS）及其他不良反应发生率低于未接受贝林妥欧单抗桥接治疗组（11.1%对50.0%，Fisher，P=0.027）。截至随访终点，贝林妥欧单抗桥接CAR-T组中13例患者持续MRD阴性状态，5例患者（包含2例经贝林妥欧单抗治疗无效的病例）在治疗后2.57~10.20个月复发，2例复发患者后续接受第二次CAR-T细胞治疗后达CR。未接受贝林妥欧单抗桥接治疗组中10例患者持续MRD阴性状态，7例复发，6例死亡。贝林妥欧单抗桥接CAR-T组患者1年总生存率高于未接受桥接治疗组，差异在0.1水平有统计学意义[（88.9±10.5）%对（66.7±10.9）%，P=0.091]。 结论： 贝林妥欧单抗桥接CAR-T细胞治疗成人B-ALL疗效及安全性值得肯定，清除肿瘤残留效果好，近期疗效复发率低，不良反应少。.

Hotspot driver mutations presented by human leukocyte antigens might be recognized by anti-tumor T cells. Based on their advantages of tumor-specificity and immunogenicity, neoantigens derived from hotspot mutations, such as PIK3CAH1047L, may serve as emerging targets for cancer immunotherapies. NetMHCpan V4.1 was utilized for predicting neoepitopes of PIK3CA hotspot mutation. Using in vitro stimulation, antigen-specific T cells targeting the HLA-A*11:01-restricted PIK3CA mutation were isolated from healthy donor-derived peripheral blood mononuclear cells. T cell receptors (TCRs) were cloned using single-cell PCR and sequencing. Their functionality was assessed through T cell activation markers, cytokine production and cytotoxic response to cancer cell lines pulsed with peptides or transduced genes of mutant PIK3CA. Immunogenic mutant antigens from PIK3CA and their corresponding CD8+ T cells were identified. These PIK3CA mutation-specific CD8+ T cells were subsequently enriched, and their TCRs were isolated. The TCR clones exhibited mutation-specific and HLA-restricted reactivity, demonstrating varying degrees of functional avidity. Identified TCR genes were transferred into CD8+ Jurkat cells and primary T cells deficient of endogenous TCRs. TCR-expressing cells demonstrated specific recognition and reactivity against the PIK3CAH1047L peptide presented by HLA-A*11:01-expressing K562 cells. Furthermore, mutation-specific TCR-T cells demonstrated an elevation in cytokine production and profound cytotoxic effects against HLA-A*11:01+ malignant cell lines harboring PIK3CAH1047L. Our data demonstrate the immunogenicity of an HLA-A*11:01-restricted PIK3CA hotspot mutation and its targeting therapeutic potential, together with promising candidates of TCR-T cell therapy.

Induction of tumor cell senescence has become a promising strategy for anti-tumor immunotherapy, but fibrotic matrix severely blocks senescence inducers penetration and immune cells infiltration. Herein, we designed a cancer-associated fibroblasts (CAFs) triggered structure-transformable nano-assembly (HSD-P@V), which can directionally deliver valsartan (Val, CAFs regulator) and doxorubicin (DOX, senescence inducer) to the specific targets. In detail, DOX is conjugated with hyaluronic acid (HA) via diselenide bonds (Se-Se) to form HSD micelles, while CAFs-sensitive peptide is grafted onto the HSD to form a hydrophilic polymer, which is coated on Val nanocrystals (VNs) surface for improving the stability and achieving responsive release. Once arriving at tumor microenvironment and touching CAFs, HSD-P@V disintegrates into VNs and HSD micelles due to sensitive peptide detachment. VNs can degrade the extracellular matrix, leading to the enhanced penetration of HSD. HSD targets tumor cells, releases DOX to induce senescence, and recruits effector immune cells. Furthermore, senescent cells are cleared by the recruited immune cells to finish the integrated anti-tumor therapy. In vitro and in vivo results show that the nano-assembly remarkably inhibits tumor growth as well as lung metastasis, and extends tumor-bearing mice survival. This work provides a promising paradigm of programmed delivering multi-site nanomedicine for cancer immunotherapy.

Pyroptosis is an inflammatory form of programmed cell death (PCD) that is regulated by the Gasdermin protein family in response to various stimuli, playing a critical role in the development of tumor therapy strategies. However, cancers are generally known to escape from PCD via immunosuppressive pathways or other resistant mechanisms. In this study, an acid-responsive Fe/Mn bimetal-organic framework nanosystem carrying metal ions and immune adjuvant R848 (FeMn@R@H) was designed for combining pyroptosis and augmented immunotherapy. The FeMn@R@H would be triggered to disintegrate and release Fe3+ and Mn2+ ions in response to the acidic tumor microenvironment (TME), thereby initiating Fenton-like reactions for ROS-mediated pyroptosis. On the one hand, the pyroptosis-caused cell rupture would induce the release of proinflammatory cytokines and immunogenic constituents from tumor cells, further resulting in immunogenic cell death (ICD) to promote antitumor immune responses. On the other hand, the co-delivered R848 could reverse suppressive tumor immune microenvironment (TIME) and induce inflammatory responses by activating the TLR7/8 pathway. In conclusion, this tumor-specific therapy system can co-deliver metal ions and R848 to tumor tissues to perform pyroptosis-mediated PCD and augmented anti-tumor immunotherapy.

Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response.

The aim of this study was to evaluate the association between Ferredoxin 1 (FDX1) expression and the prognostic survival of tumor patients and predict the efficacy of immunotherapy response to antitumor drug sensitivity. FDX1 plays an oncogenic role in thirty-three types of tumors, based on TCGA and GEO databases, and further experimental validation in vitro was provided through multiple cell lines. FDX1 was expressed highly in multiple types of cancer and differently linked to the survival prognosis of tumorous patients. A high phosphorylation level was correlated with the FDX1 site of S177 in lung cancer. FDX1 exhibited a significant association with infiltrated cancer-associated fibroblasts and CD8+ T cells. Moreover, FDX1 demonstrated correlations with immune and molecular subtypes, as well as functional enrichments in GO/KEGG pathways. Additionally, FDX1 displayed relationships with the tumor mutational burden (TMB), microsatellite instability (MSI), DNA methylation, and RNA and DNA synthesis (RNAss/DNAss) within the tumor microenvironment. Notably, FDX1 exhibited a strong connection with immune checkpoint genes in the co-expression network. The validity of these findings was further confirmed through Western blotting, RT-qPCR, and flow cytometry experiments conducted on WM115 and A375 tumor cells. Elevated FDX1 expression has been linked to the enhanced effectiveness of PD-L1 blockade immunotherapy in melanoma, as observed in the GSE22155 and GSE172320 cohorts. Autodocking simulations have suggested that FDX1 may influence drug resistance by affecting the binding sites of antitumor drugs. Collectively, these findings propose that FDX1 could serve as a novel and valuable biomarker and represent an immunotherapeutic target for augmenting immune responses in various human cancers when used in combination with immune checkpoint inhibitors.

OBJECTIVE: To compare 3 commercial immunogenic cell death (ICD) inducers, namely Chlorin e6 (Ce6), Neutral Red (NR), and Rose Bengal Sodium salt (RB), for their photosensitive properties, efficacy for photodynamic therapy (PDT) and ICD induction efficiency in antitumor immunotherapy.
METHODS: Reactive oxygen species (ROS) probes were used to evaluate the photosensitivity of the 3 ICD inducers, and their capacity for inducing intracellular ROS production was evaluated using a DCFH-DA probe. The cytotoxicity and biocompatibility of the 3 photosensitizers were compared using a CCK-8 kit, and their ICD-inducing efficiency was assessed by detecting the levels of surface-exposed calreticulin (ecto-CRT), high mobility group protein 1 (HMGB1) and adenosine triphosphate (ATP). In the animal experiment, BALB/c mouse models bearing 4T1 cellderived subcutaneous tumor were given intratumoral injection of Ce6 or NR solution (30 μL, 5 mg/mL), followed 2 h later by white light irradiation for 10 min (400 mW/cm2). Body weight and tumor size changes of the mice were monitored, and the percentage of CD8+ T cells in the tumor and IFN-γ+ CD8+ T cells in the spleen were analyzed by flow cytometry 14 days after the treatment. HE and TUNEL staining was used to analyze tumor cell apoptosis in the mice.
RESULTS: Among the 3 photosensitizers, Ce6 exhibited the strongest ROS-inducing capability and killing effect on the tumor cells. The results of ectoCRT, HMGB1 and ATP level detection all demonstrated a stronger ICD induction ability of Ce6. In the tumor-bearing mice, the tumor growth in Ce6 and NR groups was significantly inhibited after the treatment. The percentages of CD8+ T cells and IFN-γ+ CD8+ T cells were 12.7% and 7.1% in Ce6 group, respectively, significantly higher than those in NR group (6.1% and 2.8%, respectively; P < 0.05). HE and TUNEL staining revealed obvious tumor cell apoptosis in the tumor tissues in both Ce6 and NR groups, but the therapeutic effect was more prominent in Ce6 group.
CONCLUSIONS: Among the 3 photosensitizers, Ce6 has the highest efficiency for inducing ROS production with the strongest PDT efficacy and ICD induction capability. Ce6 can also increase the number and function of CD8+ T cells in anti-tumor immunotherapy to initiate robust adaptive immune response.

Ferroptosis is a novel regulatory cell death, which is characterized by iron dependency and mainly caused by accumulation of intracellular lipid peroxides and reactive oxygen species. Ferroptosis plays an important role in the occurrence and development of a variety of malignant tumors, especially in anti-tumor treatment. As an emerging treatment method, the immunotherapy has been widely applied in the clinical practice, and the role of ferroptosis in tumor immunotherapy has been gradually explored. This study aims to illustrate the features of ferroptosis, and its role in anti-tumor immunotherapy and potential clinical application.

Immune checkpoint inhibitors (ICI) therapy is the main type of immunotherapy for cancer. Current clinical trials are focused on enhancing anti-tumor effects through combinations of multiple ICIs with agents that cause tumor cell death and release tumor antigens. In this study, weprepared nanobubbles (NBs) to load programmed death-ligand 1 (PD-L1) antibody andmiR-424gene to evaluate the combined anti-tumor activity of the targeted NBs.The miR-424 gene was chosen to be an anti-tumor gene, which can target PD-L1 and Bcl-2, through bioinformatics analysis and target gene verification. Then, PD-L1 Ab/miR-424-NBs were prepared by thin-film hydration. The optimal shape, size, and character of the NBs were determined by scanning electron microscopy and Zeta potential study. In addition, the antibody binding rate and gene loading of the targeted NBs were studied by agarose gel electrophoresis and flow cytometry, respectively. The synergistic immunotherapeutic effect of anti-PD-L1 antibody andmiR-424in vivo and their mechanism were evaluated using an H22 hepatoma transplanted tumor model in mice,whichproved that the targeted NBs mediated the PD-L1 antibody toblock the PD-1/PD-L1 signaling pathway and the transfected miR-424gene to downregulate the PD-L1 expression of tumor cells, both of which enhanced the antitumor immune effect mediated by T cells. It was also found that the targeted NBs activated T cells, which released a large number of cytokines, such as IFN-γ and IL-2, to recruit and activate macrophages and NK cells. It is suggested that ultrasound-mediated PD-L1 antibody NBs delivering miR-424 can inhibit the growth of subcutaneously transplanted hepatocellular carcinoma in terms of apoptosis and immunity. Therefore, ultrasound-mediated targeted NBs are a potential effective carrier for liver cancer, and PD-L1 antibody and miR-424 have a synergistic anti-tumor immunotherapy effect.

We used mouse CRC cell line (MC38) to establish a heterotopic mouse model, and applied [89Zr]-labeled PD-L1 antibody KN035 for PET imaging. Attenuated Salmonella typhimurium 3261 was used as an anti-tumor vaccine, and the combined anti-tumor immunotherapy with bivalent genetic vaccine and anti-PD1 antibody Nivolumab was conducted. MicroPET was performed to observe the changes of tumor tissues and expression of PD-L1. We found that the recombinant double-gene plasmids were stably expressed in COS7 cells. Study results showed the combined immunotherapy improved the effectiveness over genetic vaccine alone. This study supports that combination of genetic vaccines and anti-immunocheckpoint immunotherapy can inhibit MC38 tumor growth.