Significant Cytokine Release Research Articles

Reverse vaccinology approach for identification of epitopes from E1 protein as peptide vaccine against HCV: A proof of concept

The development of effective vaccines against Hepatitis C Virus (HCV) remains a global health priority and challenge. In this study, we employed an integrative approach combining computational epitope prediction with experimental validation to identify immunogenic peptides targeting the E1 glycoprotein of HCV. In the present report, computational data from various epitope prediction algorithms such as IEDB and SYFPEITHI, followed by molecular dynamics (MD) simulations and immuno-informatics analysis is presented. Through computational screening, we identified potential epitope candidates, with QVRNSSGLY (P3) and QLFTFSPRH (P7) emerging as promising candidates. MD simulations revealed stable interactions between these epitopes and MHC molecule, further validated by free energy estimations using MMPBSA method. Immuno-informatics analysis supported these findings, showing high binding potential and immunogenicity scores for the selected peptides. Subsequent synthesis and characterization of epitope peptides confirmed their structural integrity and purity required for conducting immune activation assays. Experimental immunological assays carried out in this study involved epitope peptide induced activation of CD8 + and CD4 + T cells from healthy human subjects and HCV- recovered patients. Data from experimental validation revealed significant cytokine release upon exposure to epitope peptides, particularly TNF-a, IL-6, and GM-CSF, indicative of robust immune responses. Notably, peptides P3 and P7 exhibited the most pronounced cytokine induction profiles, underscoring their potential as vaccine candidates. Further investigations addressing the mechanism of action of these epitope peptides under preclinical and clinical settings may help in developing effective vaccine against HCV.

Abstract 3192: A novel long acting pegylated T cell engager targeting CD3 and cd19 with effective tumor killing and no potential CRS risk in CD19+ hematologic malignancies

Abstract The development of bispecific T cell engagers represents a highly effective immunotherapy strategy for the treatment of hematologic malignancies. However, Blinatumomab, a bispecific T cell engager of CD3/CD19, has disadvantages in clinical setting due to the adverse pharmacokinetics (PK), which resulted in less than 2 hours of in vivo elimination half-life, neurotoxicity, and cytokine release syndrome. In this study, a bispecific antibody JY108 was developed to target CD19 and CD3e with much reduced affinities towards both targets (revealed by Biacore assay results). JY108 was prepared by a patented pegylation bispecific linker technology, which could address the limitations of current therapeutic approaches. In vitro cytotoxicity assay results demonstrated that the EC50s of the drug specific cytotoxicity of T cells mediated by JY108 are in the concentrations of ng/ml, while blinatumomab mediated cytotoxicity to the same CD19+ cells are in the concentrations of pg/ml. Despite the weaker in vitro cytotoxicity than Blinatumomab, JY108 presents comparable tumor inhibition effects with Blinatumomab in different in vivo studies, which include the PBMC reconstructed models with established MEC-1 or Farage tumors, the delayed drug administration on human PBMCs/tumor co-transplanted model of Raji. In addition, JY108 prolonged the survival of animals with Nalm6 brain metastasis more significantly than Blinatumomab at the same dosage. Furthermore, the PK study revealed that at the different dosages, the circulation T1/2s of JY108 in mice are above 20 hours, which is expected to result in more significant efficacious benefits in clinical setting. Meanwhile, the completed package of GLP-toxicity study did not observe any drug related tox to the model animals (including no cytokine release in vivo, no neurotoxicity, no respiratory toxicity, etc.), and JY108 did not induce significant cytokine release than the blank control to the whole blood or PBMCs from healthy donor in vitro. In the efficacious concentrations of exerting cytotoxicity to CD19+ tumor cells in the presence of PBMC, JY108 did not induce significant cytokine release than the blank control, while blinatumomab always induced significant cytokine release. We postulated that the lower CD3 affinity of JY108 most probably resulted in the decoupling of the signaling for activating the gene expression of cytokines from the secretion of the polarized lytic granules by T cells, which resulted in a much milder T cell activation by JY108. Currently, JY108 is a phase I trial candidate and awaits further evaluations. Citation Format: Shumin (Sam) Liu, Yu (Yvonne) Wen, Weidong Lyu, Shuqiang Yin, Yang Lei, Dechun Wu, Zibin Wu, Qiudong Zhuo. A novel long acting pegylated T cell engager targeting CD3 and cd19 with effective tumor killing and no potential CRS risk in CD19+ hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3192.

Secretome analysis and virulence assessment in Abiotrophia defectiva

Background Abiotrophia defectiva, although infrequently occurring, is a notable cause of culture-negative infective endocarditis with limited research on its virulence. Associated with oral infections such as dental caries, exploring its secretome may provide insights into virulence mechanisms. Our study aimed to analyze and characterize the secretome of A. defectiva strain CCUG 27639. Methods Secretome of A. defectiva was prepared from broth cultures and subjected to mass spectrometry and proteomics for protein identification. Inflammatory potential of the secretome was assessed by ELISA. Results Eighty-four proteins were identified, with diverse subcellular localizations predicted by PSORTb. Notably, 20 were cytoplasmic, 12 cytoplasmic membrane, 5 extracellular, and 9 cell wall-anchored proteins. Bioinformatics tools revealed 54 proteins secreted via the ‘Sec’ pathway and 8 via a non-classical pathway. Moonlighting functions were found in 23 proteins, with over 20 exhibiting potential virulence properties, including peroxiredoxin and oligopeptide ABC transporter substrate-binding protein. Gene Ontology and KEGG analyses categorized protein sequences in various pathways. STRING analysis revealed functional protein association networks. Cytokine profiling demonstrated significant proinflammatory cytokine release (IL-8, IL-1β, and CCL5) from human PBMCs. Conclusions Our study provides a comprehensive understanding of A. defectiva‘s secretome, laying the foundation for insights into its pathogenicity.

A Phase 1 Study of CD38-Bispecific Antibody (XmAb18968) for Patients with Relapsed/Refractory T-Cell Acute Lymphoblastic Leukemia

Background: Outcomes of adults with relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has remained poor. CD38 is a transmembrane glycoprotein that is highly expressed in T-ALL and is potentially targetable with CD38 blocking agents [Naik J et al. Haematologica 2019, Bride KL et al. Blood 2018, Tembhare et al. J Immunother Cancer 2020]. XmAb18968 is a novel CD38-CD3 bi-specific T-cell engager with Fc domain modified to minimize Fcγ receptor binding and non-selective T-cell activation resulting in reduced cytokine release without compromising target cell killing. We designed a phase I clinical trial to assess the safety and tolerability of XmAb18968 in patients with T-ALL. Methods: We conducted a multicenter, open label, phase I dose escalation study of XmAb18968 (NCT05038644). Patients aged ≥18 years with relapsed/refractory T-ALL or T-lymphoblastic lymphoma (T-LBL), ECOG PS 0-2 and adequate organ function were eligible. Major exclusion criteria are hematopoietic cell transplantation within 6 months of enrollment, prior therapy with daratumumab in the last 6 months and active acute graft-versus-host disease. Dose escalation was conducted using 3+3 design, and 4 separate dose levels were studied [0.8mg, 1mg, 1.3mg, 1.5mg]. XmAb18968 was administered intravenously on days 1, 8, 15, 22 on a 28-day cycle. During cycle 1 alone, day 1 dose was fractionated and administered over 2 days to minimize the risk of significant cytokine release syndrome (CRS). Treatment was continued until progression or unacceptable toxicity. The primary objective was to determine the safety and dose limiting toxicity (DLT) and secondary objective was to determine the preliminary efficacy. Correlative studies included pharmacokinetics, characterizing changes in serum cytokines and phenotypic expression of activated T-cells and leukemic cells, correlation of the phenotypic expression with changes in transcriptome at the single-cell level, proteomics evaluation of cytokine secretion at the single-cell level and correlation of response with N-glycan profiling, quantitative site-occupancy, and direct glycopeptide analysis. Results: Four patients with relapsed/refractory T-ALL have been enrolled so far at dose level 1 (0.8mg) with 3 of them being evaluable for response (one patient came off study before DLT period). Median age was 62 years (34-74), all response evaluable patients had early T-precursor ALL (ETP-ALL), and one patient (25%) was previously treated with allogeneic HCT, CD7 CAR-T therapy and daratumumab. Grade 3 or higher adverse events are summarized in Table 1. CRS grades 1 was noted in 25% of patients, no grade ≥3 CRS or neurotoxicity (any grade) was seen. Of the 3 evaluable total patients, 2 patients had achieved complete response (CR) after 1 cycle of therapy (one patient had prior allogeneic HCT, CD7 CAR-T therapy and daratumumab) (Table 2). The two responders remain on treatment. Conclusions: XmAb18968, a novel CD-38 bispecific antibody appears safe and is showing response at the starting dose level in T-ALL. The study is currently enrolling additional patients with T-ALL. Updated analysis with additional patients and details on correlative studies examining mechanisms of therapeutic efficacy will be reported at the main meeting.

A Phase 1 Study of CD38-Bispecific Antibody (XmAb18968) for Patients with CD38 Expressing Relapsed/Refractory Acute Myeloid Leukemia

Background: Outcomes of adults with relapsed/refractory acute myeloid leukemia (RR-AML) have remained poor. CD38 is a transmembrane glycoprotein that is highly expressed in AML and is potentially targetable with CD38 blocking agents [Naik J et al. Haematologica 2019, Tembhare et al. J Immunother Cancer 2020]. XmAb18968 is a novel CD38-CD3 bi-specific T-cell engager with Fc domain modified to minimize Fcγ receptor binding and non-selective T-cell activation resulting in reduced cytokine release without compromising target cell killing. We designed a phase I clinical trial to assess the safety and tolerability of XmAb18968 in patients with RR-AML. Methods: We conducted a multicenter, open label, phase I dose escalation study of XmAb18968 (NCT05038644). Patients aged ≥18 years with relapsed/refractory AML, ECOG PS 0-2 and adequate organ function were eligible. Major exclusion criteria are hematopoietic cell transplantation within 6 months of enrollment, active acute graft-versus-host disease, and acute promyelocytic leukemia. Dose escalation was conducted using 3+3 design and 4 separate dose levels were studied [0.8mg, 1mg, 1.3mg, 1.5mg]. XmAb18968 was administered intravenously on days 1, 8, 15, 22 on a 28-day cycle. During cycle 1 alone, day 1 dose was fractionated and administered over 2 days to minimize the risk of significant cytokine release syndrome (CRS). Treatment was continued until progression or unacceptable toxicity. The primary objective was to determine the safety and dose limiting toxicity (DLT) and secondary objective was to determine the preliminary efficacy. Measurable residual disease (MRD) status was assessed using multicolor flow cytometry with 0.01% sensitivity. Correlative studies included pharmacokinetics, characterizing changes in serum cytokines and phenotypic expression of activated T-cells and leukemic cells, correlation of the phenotypic expression with changes in transcriptome at the single-cell level, proteomics evaluation of cytokine secretion at the single-cell level and correlation of response with N-glycan profiling, quantitative site-occupancy, and direct glycopeptide analysis. Results: Thirteen patients with RR-AML were enrolled, 12 of whom were evaluable (1 patient came off study before DLT period). Median age was 63 years (45-77), 77% with adverse risk disease, 38% with secondary AML, and received median two (range 2-7) prior lines of therapy. Twelve of 13 patients (92%) were previously treated with venetoclax based regimen and 31% had prior allogeneic HCT. Adverse risk mutations included TP53 (31%), RUNX1 (23%), and ASXL1 (15%). Grade 3 or higher adverse events included anemia (8%), neutropenia (15%), and thrombocytopenia (23%) (Table 1). CRS grades 1-2 were noted in 62% of patients, no grade ≥3 CRS or neurotoxicity (any grade) was seen. There was no dose limiting toxicity (DLT). Of the 12 evaluable total patients, clinically meaningful response was noted in two patients who were MRD positive by flow cytometry prior to study entry and achieved MRD negative CR (Table 2). These two patients who achieved MRD negative CR subsequently proceeded to allogeneic HCT. Median time to response was 1 cycle. Median OS of the cohort was 6.2 months (1.8-NR). Conclusions: XmAb18968 is safe and well tolerated in patients with RR-AML. Dose escalation yielded MRD negative CR in RR-AML patients who were MRD positive at study entry. Details on correlative studies examining mechanisms of therapeutic efficacy and resistance will be reported in the main meeting.

Significant Cytokine Release Syndrome Risk Model with T-Cell Engaging Therapies

Background and Objectives: Cytokine release syndrome (CRS) is an adverse event associated with T-cell engaging (TCE) therapies and multi-specific antibody therapies, like BiTEs or DARTs. We developed a risk model to predict the pre-infusion risk of significant CRS for patients treated with TCE therapies. A supplementary exploratory analysis evaluated the role of tumor burden and the use of bridging chemotherapy prior to the first dose of TCE infusion on the rate of sCRS. Methods: The TCE dataset was sourced from the Medidata Enterprise Data Store, an anonymized repository of data from completed clinical trials. The outcome of interest was the first sCRS occurring within 10 days of TCE therapy. sCRS was defined as CRS grade ≥ 2 and non-sCRS was defined as CRS grade <2 using the CTCAE 4.0 scale. CTCAE 4.0 was used as these trials gathered data using this version of the scale. Important risk factors of sCRS were identified from the literature and preliminary data analysis. All the features were measured at the time of, or prior to, the first TCE treatment. Patients were included in the analysis dataset if they had a data element fill rate of >70% in the key features. Features were pruned by assessing the multicollinearity across features to increase the stability of the models. Tumor burden was explored as a potential feature, but was not selected for the final model, as the range of tumor burden in most of the patients selected was narrow (see below). In order to compare different TCE therapies, first treatment doses were normalized by dividing patients' first dose by the mean of the first dose of TCE administered in each study. Logistic regression and tree-based models were trained. Across 100 iterations with different train-test splits, the average Area Under the Receiver-Operator Characteristic Curve (AUROC) was calculated for each model-type. Results: A total of 715 patients were included in the analysis (115 sCRS; 600 non-sCRS); the majority of patients had acute lymphoblastic leukemia (81%) and 19% of patients had solid tumors or Non-Hodgkin's lymphoma. Patients who developed sCRS had a higher incidence of infections prior to first TCE infusion, at 38% vs. 28% (p = 0.03). They also received a higher dose of TCE therapy (p< 0.001). The features selected for the model are listed in table 1. The best model to predict sCRS had a mean AUROC of 0.69 (95% CI 0.66-0.72) on the test set. When patients were ranked based on their predicted probability of getting sCRS and divided into quartiles based on predicted sCRS risk (very low, low, high, very high), the very high-risk quartile developed sCRS at 4x the rate (38% (0.347-0.413)) compared to the lowest quartile (5.7% ( 0.05-0.07); sample average was 16% (0.153-0.167) (figure 1). Compared to patients with very low sCRS risk, patients with very high sCRS risk had mainly ALL as a disease type (99% vs. 67%, p < 0.001), received a higher TCE dose (1.00 vs. 0.61, p < 0.001) and had a higher rate of infections prior to TCE treatment (49% vs. 12%, p < 0.001). Given the lack of uniform presence of disease burden in the predictive model cohort, a separate cohort of 945 ALL patients was analyzed to evaluate the impact of tumor burden on sCRS. 98% of patients received pre-TCE bridging therapy, resulting in median decline in %blasts from a maximum of 87% (84% vs. 88% for sCRS v/s non-sCRS patients; p = 0.157) to 0% (1% vs. 0% for sCRS v/s non-sCRS patients; p = 0.005) prior to TCE infusion. In the setting of these small ranges in tumor burden, neither total burden or most recent value prior to dosing was found to be significantly correlated with sCRS. Conclusion: Using the sCRS risk model, it was possible to stratify patients by risk categories. This could allow better selection of patients to receive TCE therapy and tailored pre-treatment and monitoring of CRS, which could in turn allow better resource allocation. As tumor burden can be a critical determinant of sCRS risk in other clinical settings where CRS is a relevant risk, future research is needed to further evaluate the impact of tumor burden on sCRS across different indications.

Novel Bone Marrow/HSC-Targeted, Megakaryocyte-Derived Extracellular Vesicle Delivery Modality for In Vivo Gene Therapy

Gene therapy lacks tools to deliver in a safe and tissue-targeted manner. Limitations include toxicity, off-target effects due to targeting, and inability to repeat dose. We have developed a novel megakaryocyte-derived extracellular vesicle (MkEV)-based non-viral gene therapy delivery platform that preferentially targets bone marrow in vivo. Here we present work showing that STRM.BIO MkEVs: 1) pass through the liver and spleen to preferentially target bone marrow in mice and non-human primates (NHPs); 2) can selectively deliver pDNA cargo to long term HSCs to drive reporter protein expression exclusively in bone marrow following intravenous delivery in mice; and 3) present a platform to develop and deliver targeted gene therapies in vivo that are safe for repeat dosing. Our vision is to open the door to the future of medicine for patients living with rare diseases worldwide and bring gene therapy to life. Primary human CD34+ hematopoietic stem and progenitor cells (hHSPCs) were differentiated into megakaryocytes in vitro. MkEVs were harvested and loaded with pDNA via electroporation, followed by DNase treatment to remove free cargo. For in vivo biodistribution and cargo-mediated protein expression studies, STRM.BIO MkEVs were exogenously labeled with fluorescent dye (PKH26, Cell Tracker Deep Red, and/or DiD), loaded with cargo, and IV-injected into mouse tail veins (wild type or NSG) or NHPs (cynomolgus monkeys). Tissues were collected 16-48h after IV injection, with MkEV tissue biodistribution quantified by dye fluorescence (Mean Fluorescence Intensity (MFI), plate reader) normalized to tissue weight or genomic DNA input, and pDNA cargo quantified by qPCR. Additionally, quantification of MkEVs+ cells was performed by flow cytometry analysis of murine bone marrow derived sub-populations (lineage negative, c-Kit +, Sca-1 + (LSK); lineage positive (Lin +); and, long-term HSCs (LT-HSCs; Lin -/CD150 +/CD201 +)) cells. Protein expression from MkEV-loaded pDNA cargo was similarly determined by plate reader fluorescence data and confocal microscopy. In mouse biodistribution studies, DiD-labeled MkEVs were predominantly detected in bone marrow, followed by liver and spleen 16h-post tail vein injection (plate reader analyses: Bone Marrow - 1.1e 4 ± 0.2 SD; Liver - 9.6e 3 ± 1.0 SD; Spleen - 1.8e 4 ± 0.2 SD. MFI/g tissue; n=3mice/group). Flow cytometry analyses show MkEVs preferentially targeted the hematopoietic compartment (of the MkEV+ cells, 94 ± 1% CD45+ vs 6 ± 1% CD45-, respectively), specifically, the HSPCs (68%, LSK) and LT-HSCs (100%) 48h post intravenous delivery via tail vein injection. Pharmacokinetic studies show that while MkEVs can circulate through highly vascularized tissues like the liver and spleen, they accumulate in the bone marrow. Strikingly, miRFP encoded by the pDNA was exclusively detected in bone marrow (p<0.0001 vs. all other tissues; Fig. 1). In NHPs, dye-labeled MkEVSs were loaded with a MkEV-driven eGFP reporter plasmid. Studies showed preferential biodistribution to bone marrow, and preferential qPCR amplification of the pDNA cargo in bone marrow (both MFI/ ACTB and GFP/ ACTB were significantly higher vs. all other tissues: range p<0.05 through p<0.0001), Fig. 2. A tolerability and repeat dosing study was performed in NHPs. Monkeys were injected with 4 weekly doses of STRM.BIO MkEVs and serial bloodwork was obtained 6h post dose for cytokine evaluation and 5 days post dose for end organ damage assessment (n=4). The study showed no evidence of kidney, hematologic, or liver damage as evidenced by normal creatinine and electrolytes, no significant changes in complete blood counts, coagulation parameters, or liver function tests. There was no evidence of significant cytokine release or inflammatory changes on tissue histology. These data confirm that MkEVs are non-toxic and the platform is amenable to repeat dosing. Taken together, these data establish an advanced gene therapy platform that can target bone marrow specifically following in vivo administration in both mice and NHPs, potentially eliminating the need for current ex vivo approaches to treat rare blood disorders, and providing the critical ability to repeat dose as required in clinic. STRM.BIO is leveraging our platform to develop gene therapies for rare blood diseases in people.

Extracellular vesicles produced by avian pathogenic Escherichia coli (APEC) activate macrophage proinflammatory response and neutrophil extracellular trap (NET) formation through TLR4 signaling

BackgroundAvian pathogenic Escherichia coli (APEC) is the major pathogen causing important avian diseases in poultry. As an important subtype of extraintestinal pathogenic E. coli, APEC has zoonotic potential and is considered a foodborne pathogen. APEC extracellular vesicles (EVs) may play vital roles in the interaction of the pathogen with its host cells. However, the precise roles played by APEC EVs are still not completely clear, especially in immune cells.ResultsIn this study, we investigated the relationships between APEC EVs and immune cells. The production and characteristics of the EVs of APEC isolate CT265 were identified. Toll like receptor 4 (TLR4) triggered the cellular immune responses when it interacted with APEC EVs. APEC EVs induced a significant release of proinflammatory cytokines in THP-1 macrophages. APEC EVs induced the macrophage inflammatory response via the TLR4/MYD88/NF-κB signaling pathway, which participated in the activation of the APEC-EV-induced NLRP3 inflammasome. However, the loss of lipopolysaccharide (LPS) from APEC EVs reduced the activation of the NLRP3 inflammasome mediated by TLR4/MYD88/NF-κB signaling. Because APEC EVs activated the macrophage inflammatory response and cytokines release, we speculated that the interaction between APEC EVs and macrophages activated and promoted neutrophil migration during APEC extraintestinal infection. This study is the first to report that APEC EVs induce the formation of neutrophil extracellular traps (NETs) and chicken heterophil extracellular traps. Treatment with APEC EVs induced SAPK/JNK activation in neutrophils. The inhibition of TLR4 signaling suppressed APEC-EV-induced NET formation. However, although APEC EVs activated the immune response of macrophages and initiated NET formation, they also damaged macrophages, causing their apoptosis. The loss of LPS from APEC EVs did not prevent this process.ConclusionAPEC-derived EVs induced inflammatory responses in macrophages and NETs in neutrophils, and that TLR4 was involved in the APEC-EV-activated inflammatory response. These findings provided a basis for the further study of APEC pathogenesis.

Controlled Decompression Alleviates Motor Dysfunction by Regulating Microglial Polarization via the HIF-1α Signaling Pathway in Intracranial Hypertension.

Decompressive craniectomy (DC) is a major form of surgery that is used to reduce intracranial hypertension (IH), the most frequent cause of death and disability following severe traumatic brain injury (sTBI) and stroke. Our previous research showed that controlled decompression (CDC) was more effective than rapid decompression (RDC) with regard to reducing the incidence of complications and improving outcomes after sTBI; however, the specific mechanisms involved have yet to be elucidated. In the present study, we investigated the effects of CDC in regulating inflammation after IH and attempted to identify the mechanisms involved. Analysis showed that CDC was more effective than RDC in alleviating motor dysfunction and neuronal death in a rat model of traumatic intracranial hypertension (TIH) created by epidural balloon pressurization. Moreover, RDC induced M1 microglia polarization and the release of pro-inflammatory cytokines. However, CDC treatment resulted in microglia primarily polarizing into the M2 phenotype and induced the significant release of anti-inflammatory cytokines. Mechanistically, the establishment of the TIH model led to the increased expression of hypoxia-inducible factor-1α (HIF-1α); CDC ameliorated cerebral hypoxia and reduced the expression of HIF-1α. In addition, 2-methoxyestradiol (2-ME2), a specific inhibitor of HIF-1α, significantly attenuated RDC-induced inflammation and improved motor function by promoting M1 to M2 phenotype transformation in microglial and enhancing the release of anti-inflammatory cytokines. However, dimethyloxaloylglycine (DMOG), an agonist of HIF-1α, abrogated the protective effects of CDC treatment by suppressing M2 microglia polarization and the release of anti-inflammatory cytokines. Collectively, our results indicated that CDC effectively alleviated IH-induced inflammation, neuronal death, and motor dysfunction by regulating HIF-1α-mediated microglial phenotype polarization. Our findings provide a better understanding of the mechanisms that underlie the protective effects of CDC and promote clinical translational research for HIF-1α in IH.

IMMU-20. EVALUATION OF B7-H3 NANOBODY CAR-T CELLS IN PEDIATRIC GLIOBLASTOMA

Abstract Chimeric antigen receptor (CAR)-T cell therapies are an undoubted success in childhood acute lymphoblastic leukemia; however, this success has not translated clinically to any childhood solid tumor. B7-H3 has been found to be highly expressed in pediatric brain tumors and correlates with tumor progression and poor prognosis. We utilized a novel nanobody based CAR-T cell which can bind tumor antigens with high affinity. We hypothesize that anti-B7-H3 nanobody CAR-T cells mediate more potent antitumor effects both in vitro and in vivo compared to existing antibody-based B7-H3 CAR-T cells. High expression of B7-H3 was confirmed on the surface of two pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. B7-H3 nanobodies isolated from camel phage libraries were cloned into lentiviral CAR-T cell constructs. We manufactured CAR-T cells by lentiviral transduction of human T cells. Glioblastoma tumor cells were co-cultured with B7-H3 CAR-T cells vs. control T cells. We confirmed B7-H3 specific cytokine release by CAR-T cells via ELISA and effective tumor cell killing via two different cytotoxicity assays (impedance or luciferase based). Nanobody based B7-H3 CAR-T cells exhibited statistically significant higher cytokine release and cytotoxicity compared to antibody based B7-H3 CAR-T cells. In vivo studies are currently evaluating the efficacy of intracerebroventricular delivery of B7-H3 CAR-T cells against two orthotopic glioblastoma models utilizing multiple human donor T cells in immunocompromised mice. We are simultaneously developing a syngeneic orthotopic glioblastoma model to evaluate the nanobody based B7-H3 CAR-T cell cross-reactive in mice utilizing an immunocompetent model. Careful investigation of CAR-T cell therapy in appropriate immunocompetent in vivo models is needed to develop effective immunotherapy for clinical application.

Granulocyte transfusion during cord blood transplant for relapsed, refractory AML is associated with massive CD8+ T-cell expansion, significant cytokine release syndrome and induction of disease remission.

In high-risk myeloid malignancy, relapse is reduced using cord blood transplant (CBT) but remains the principal cause of treatment failure. We previously described T-cell expansion in CBT recipients receiving granulocyte transfusions. We now report the safety and tolerability of such transfusions, T-cell expansion data, immunophenotype, cytokine profiles and clinical response in children with post-transplant relapsed acute leukaemia who received T-replete, HLA-mismatched CBT and pooled granulocytes within a phase I/II trial (ClinicalTrials.Gov NCT05425043). All patients received the transfusion schedule without significant clinical toxicity. Nine of ten patients treated had detectable measurable residual disease (MRD) pre-transplant. Nine patients achieved haematological remission, and eight became MRD negative. There were five deaths: transplant complications (n = 2), disease (n = 3), including two late relapses. Five patients are alive and in remission with 12.7 months median follow up. Significant T-cell expansion occurred in nine patients with a greater median lymphocyte count than a historical cohort between days 7-13 (median 1.73 × 109 /L vs. 0.1 × 109 /L; p < 0.0001). Expanded T-cells were predominantly CD8+ and effector memory or TEMRA phenotype. They exhibited markers of activation and cytotoxicity with interferon-gamma production. All patients developed grade 1-3 cytokine release syndrome (CRS) with elevated serum IL-6 and interferon-gamma.

Major histocompatibility complex I-induced endoplasmic reticulum stress mediates the secretion of pro-inflammatory muscle-derived cytokines.

Major histocompatibility complex (MHC) I is an important component of intracellular antigen presentation. However, improper expression of MHC I upon the cell surface has been associated with several autoimmune diseases. Myositis is a rare acquired autoimmune disease which targets skeletal muscle, and MHC I overexpression on the surface of muscle fibres and immune cell infiltration are clinical hallmarks. MHC I overexpression may have an important pathogenic role, mediated by the activation of the endoplasmic reticulum (ER) stress response. Given the evidence that muscle is a diverse source of cytokines, we aimed to investigate whether MHC I overexpression can modify the profile of muscle-derived cytokines and what role the ER stress pathway may play. Using C2C12 myoblasts we overexpressed MHC I with a H-2kb vector in the presence or absence of salubrinal an ER stress pathway modifying compound. MHC I overexpression induced ER stress pathway activation and elevated cytokine gene expression. MHC I overexpression caused significant release of cytokines and chemokines, which was attenuated in the presence of salubrinal. Conditioned media from MHC I overexpressing cells induced in vitro T-cell chemotaxis, atrophy of healthy myotubes and modified mitochondrial function, features which were attenuated in the presence of salubrinal. Collectively, these data suggest that MHC I overexpression can induce pro-inflammatory cytokine/chemokine release from C2C12 myoblasts, a process which appears to be mediated in-part by the ER stress pathway.

Activation of Neutrophils by Mucin–Vaterite Microparticles

Nano- and microparticles enter the body through the respiratory airways and the digestive system, or form as biominerals in the gall bladder, salivary glands, urinary bladder, kidney, or diabetic pancreas. Calcium, magnesium, and phosphate ions can precipitate from biological fluids in the presence of mucin as hybrid nanoparticles. Calcium carbonate nanocrystallites also trap mucin and are assembled into hybrid microparticles. Both mucin and calcium carbonate polymorphs (calcite, aragonite, and vaterite) are known to be components of such biominerals as gallstones which provoke inflammatory reactions. Our study was aimed at evaluation of neutrophil activation by hybrid vaterite–mucin microparticles (CCM). Vaterite microparticles (CC) and CCM were prepared under standard conditions. The diameter of CC and CCM was 3.3 ± 0.8 µm and 5.8 ± 0.7 µm, with ƺ-potentials of −1 ± 1 mV and −7 ± 1 mV, respectively. CC microparticles injured less than 2% of erythrocytes in 2 h at 1.5 mg mL−1, and no hemolysis was detected with CCM; this let us exclude direct damage of cellular membranes by microparticles. Activation of neutrophils was analyzed by luminol- and lucigenin-dependent chemiluminescence (Lum-CL and Luc-CL), by cytokine gene expression (IL-6, IL-8, IL-10) and release (IL-1β, IL-6, IL-8, IL-10, TNF-α), and by light microscopy of stained smears. There was a 10-fold and higher increase in the amplitude of Lum-CL and Luc-CL after stimulation of neutrophils with CCM relative to CC. Adsorption of mucin onto prefabricated CC microparticles also contributed to activation of neutrophil CL, unlike mucin adsorption onto yeast cell walls (zymosan); adsorbed mucin partially suppressed zymosan-stimulated production of oxidants by neutrophils. Preliminary treatment of CCM with 0.1–10 mM NaOCl decreased subsequent activation of Lum-CL and Luc-CL of neutrophils depending on the used NaOCl concentration, presumably because of the surface mucin oxidation. Based on the results of ELISA, incubation of neutrophils with CCM downregulated IL-6 production but upregulated that of IL-8. IL-6 and IL-8 gene expression in neutrophils was not affected by CC or CCM according to RT2-PCR data, which means that post-translational regulation was involved. Light microscopy revealed adhesion of CC and CCM microparticles onto the neutrophils; CCM increased neutrophil aggregation with a tendency to form neutrophil extracellular traps (NETs). We came to the conclusion that the main features of neutrophil reaction to mucin–vaterite hybrid microparticles are increased oxidant production, cell aggregation, and NET-like structure formation, but without significant cytokine release (except for IL-8). This effect of mucin is not anion-specific since particles of powdered kidney stone (mainly calcium oxalate) in the present study or calcium phosphate nanowires in our previous report also activated Lum-CL and Luc-CL response of neutrophils after mucin sorption.

Targeted delivery of self-assembled nanocomplex between fusion peptides and siRNAs for breast cancer treatment

Breast cancer is one of the serious diseases and has the second-highest mortality in women worldwide. RNA interference has been developed as a promising way of specific cancer treatment by silencing oncogenes efficiently. However, small RNAs exhibit difficulties in specific cellular uptake and instability. Therefore, we developed novel self-assembled nanocomplexes between newly designed fusion peptides and small RNAs for an efficient, stable, targeted delivery to breast cancer cells. Designed R11-SP82 (RS) and R11-TT1 (RT) peptides could form self-assembled nanocomplexes via electrostatic attraction. As a result, RS nanocomplexes exhibited prolonged stability, enhanced cellular uptake, and target gene silencing by siRNAs to MDA-MB-231 breast cancer cells. Moreover, RS nanocomplexes successfully inhibited breast cancer cell growth via specific and efficient siRNA delivery. Furthermore, in vitro and in vivo safety tests showed negligible cytotoxicity and neither tissue damage nor significant inflammatory cytokine release. Therefore, the RS nanocomplex strategy could be expected to become a promising siRNA delivery platform for treating breast cancer or other cancers.

Validation and promise of a TCR mimic antibody for cancer immunotherapy of hepatocellular carcinoma

Monoclonal antibodies are at the vanguard of the most promising cancer treatments. Whereas traditional therapeutic antibodies have been limited to extracellular antigens, T cell receptor mimic (TCRm) antibodies can target intracellular antigens presented by cell surface major histocompatibility complex (MHC) proteins. TCRm antibodies can therefore target a repertoire of otherwise undruggable cancer antigens. However, the consequences of off-target peptide/MHC recognition with engineered T cell therapies are severe, and thus there are significant safety concerns with TCRm antibodies. Here we explored the specificity and safety profile of a new TCRm-based T cell therapy for hepatocellular carcinoma (HCC), a solid tumor for which no effective treatment exists. We targeted an alpha-fetoprotein peptide presented by HLA-A*02 with a highly specific TCRm, which crystallographic structural analysis showed binds directly over the HLA protein and interfaces with the full length of the peptide. We fused the TCRm to the γ and δ subunits of a TCR, producing a signaling AbTCR construct. This was combined with an scFv/CD28 co-stimulatory molecule targeting glypican-3 for increased efficacy towards tumor cells. This AbTCR + co-stimulatory T cell therapy showed potent activity against AFP-positive cancer cell lines in vitro and an in an in vivo model and undetectable activity against AFP-negative cells. In an in-human safety assessment, no significant adverse events or cytokine release syndrome were observed and evidence of efficacy was seen. Remarkably, one patient with metastatic HCC achieved a complete remission after nine months and ultimately qualified for a liver transplant.

Abstract 4240: In vivo assessment of AICD and cytokine release upon treatment of OKT3 and CD3 bispecific antibody in homozygous CD3EDG HuGEMM™

Abstract Introduction: CD3 bispecific antibodies have transformed the therapeutic landscape of immunotherapy for some hematological malignancies, however, the clinical therapeutic effects of CD3 bispecific therapies is still under investigation for solid tumors. Moreover, CD3 bispecific antibody-mediated tumor targeting as well as T cell engagement have resulted in cytokine release syndrome (CRS), increased on-target/off-tumor toxicities and poor T cell infiltration, which may affect safety and limit therapeutic efficacy. To better understand the relationship between T cell activation and systemic cytokine release in the context of CD3 bispecific antibody treatment, a homozygous human CD3EDG HuGEMM࣪ preclinical model was developed. Methods: The homozygous CD3EDG HuGEMM mice, which express human CD3ε/CD3δ/CD3γ on the surface of T cells, were subcutaneously inoculated with MC38 or MC38-hEpCAM HuCELL™ syngeneic tumors and treated with vehicle, anti-mPD-1 (RMP1-14), OKT3, and anti-hCD3×hEpCAM. For efficacy studies, mouse body weight and tumor volume were measured twice weekly and tumor growth inhibition (TGI%) was determined. To investigate OKT3 or CD3 bispecifics-induced cytokine release, mouse body weight was monitored daily and several inflammatory and proinflammatory cytokines in the serum were detected by MSD. Results: Firstly, anti-mPD-1 treatment (5 mg/kg and 15 mg/kg i.p.) of homozygous CD3EDG mice engrafted with MC38 syngeneic tumors led to 42% and 62% TGI, respectively. OKT3 treatment of MC38-hEpCAM bearing homozygous CD3EDG HuGEMM promoted tumor growth (TGI= -80%), likely due to activation-induced cell death (AICD) of T cells. In addition, CRS was induced within 24 hours of OKT3 dosing, with IL-2, IL-6, and TNF-α peaking 2 hr post the 1st dose, and IFN-γ, IL-10 and IL-5 peaking 6 hr post the 1st dose. In contrast, no significant cytokine release was observed upon anti-hCD3×hEpCAM treatment despite remarkable TGI, indicating that the delicate balance of in vivo T cell activation and T cell cytotoxicity is crucial for effective tumor killing by CD3 bispecific antibodies or engagers. Conclusions: Our CD3EDG HuGEMM and HuCELL models provide a predictive platform for in vivo efficacy and safety assessment of CD3 bispecific or trispecific antibodies. The uncoupling of systemic cytokine release and T cell cytotoxicity via CD3 bispecific or trispecific antibodies provides a biological rationale to clinically explore preventative treatment approaches to avoid or reduce CRS-related toxicity, as well as combination approaches to increase selectivity and efficacy. Citation Format: Daniel XF He. In vivo assessment of AICD and cytokine release upon treatment of OKT3 and CD3 bispecific antibody in homozygous CD3EDG HuGEMM™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4240.

Phase 1 dose escalation study of DSP107, a first-in-class CD47 and 4-1BB targeting multifunctional immune-recruitment protein, in patients with advanced solid tumors.

2647 Background: DSP107 is a bi-functional, trimeric, fusion protein composed of sequences from the extracellular domain of SIRPα and 4-1BBL. The SIRPα arm targets CD47 overexpressed on tumor cells, blocking the “don’t eat me” checkpoint and triggering tumor cell phagocytosis. The trimeric 4-1BBL arm, once cross-presented and immobilized by SIRPα binding to CD47, interacts with 4-1BB expressed on activated immune cells, mainly T- and NK cells in the tumor microenvironment, and stimulates their proliferation and activation. Thereby, DSP107 triggers both an innate and an adaptive immune response. Here we describe data from the completed DSP107 monotherapy dose escalation portion of study NCT04440735. Methods: Adult patients with advanced solid tumors were treated with weekly intravenous DSP107 infusions (0.01 - 10 mg/kg) during 3-week treatment cycles. An accelerated dose escalation in single patient cohorts (dose levels 1-3) was followed by a standard 3 + 3 design. The primary objective was to determine safety, tolerability and pharmacokinetic (PK) parameters. Paired biopsies were obtained from 8 patients (screening, after cycle 2). Restaging imaging was performed every two months and evaluated by RECIST criteria. Results: In 23 patients, DSP107 demonstrated no treatment related hematologic or hepatic adverse events (AEs) and no dose limiting toxicities. Grade 1-2 treatment related AEs were observed in 70% of patients (16/23). The most frequent AEs included infusion related reaction (IRR; 26%), diarrhea (17%), fatigue (17%), nausea (13%) and constipation (9%). IRRs were managed during subsequent infusions by reducing the infusion rate and administering IV fluids. No significant systemic cytokine release was measured at any dose level using a 10-plex, pro-inflammatory panel. PK analysis revealed 100% CD47 receptor engagement on peripheral T and NK cells at doses of 3 mg/kg and above. DSP107 did not bind CD47 on red blood cells at any dose. Preliminary histologic assessment of paired biopsies by an independent, blinded pathologist demonstrated increased tumor necrosis compared to screening in 3 out of 4 patients associated with immune cell infiltration. Immuno-profiling of tumor by immunofluorescence is on-going. Stable disease as best response was observed in 11/22 patients with treatment duration up to 26 weeks. Conclusions: DSP107 is a novel, CD47 and 4-1BB targeting fusion protein with a differentiated safety, binding and pharmacodynamic profile compared to other CD47 and 4-1BB targeting agents. Clinical trial information: NCT04440735.

Targeting CD38 in acute myeloid leukemia interferes with leukemia trafficking and induces phagocytosis

Targeting the interaction between leukemic cells and the microenvironment is an appealing approach to enhance the therapeutic efficacy in acute myeloid leukemia (AML). AML infiltration induces a significant release of inflammatory cytokines in the human bone marrow niche which accelerates leukemogenesis. As the transmembrane glycoprotein CD38 has been shown to regulate cytokine release, we assessed the anti-leukemic potential of CD38 inhibition in AML. CD38 expression in AML cells proved to depend on microenvironmental cues and could be significantly enforced through addition of tretinoin. In fact, the anti-CD38 antibody daratumumab showed significant cytostatic efficacy in a 3D in vitro triple-culture model of AML, but with modest cell-autonomous cytotoxic activity and independent of CD38 expression level. In line with a predominantly microenvironment-mediated activity of daratumumab in AML, CD38 inhibition significantly induced antibody-dependent phagocytosis and showed interference with AML cell trafficking in vivo in a xenograft transplantation model, but overall lacked robust anti-leukemic effects.

Highly sensitive in vitro cytokine release assay incorporating high-density preculture

Immunostimulatory effects of monoclonal antibodies (mAb) through binding to Fcγ receptors (FcγR) on immune cells are a likely cause of cytokine release syndrome. However, it is difficult to detect the potential risk of FcγR-dependent cytokine release associated with mAb in the current standard cytokine release assays (CRA), including the air-drying solid-phase method using human peripheral blood mononuclear cells (PBMC). To increase the sensitivity to detect FcγR-dependent cytokine release due to mAb, a high-density preculture (HDC) method was incorporated into the air-drying solid-phase CRA. Here, PBMC were exposed to panitumumab, trastuzumab, rituximab, or alemtuzumab at 0.1, 0.3, 1, and 3 μg/well for 24 or 48 hr under both non-HDC and HDC conditions. T-cell agonists (anti-CD3 mAb, anti-CD28 super-agonist [SA] mAb) were used as reference mAb. Panitumumab, trastuzumab, rituximab, or alemtuzumab induced cytokine release under both non-HDC and HDC conditions, and cytokine release caused by alemtuzumab was more pronounced under HDC conditions. To investigate FcγR involvement in cytokine release associated with panitumumab, trastuzumab, rituximab, and alemtuzumab, CRA of these four mAb were conducted with anti-FcγRI, -FcγRII, or -FcγRIII F(ab’)2 fragments. The results showed cytokine release caused by trastuzumab, rituximab, and alemtuzumab was significantly suppressed by anti-FcγRIII F(ab’)2 pretreatment, and slightly reduced by anti-FcγRI or anti-FcγRII pretreatment, indicating these mAb induced FcγR (especially FcγRIII)-dependent cytokine release from PBMC. Cytokine release caused by panitumumab was slightly suppressed by anti-FcγRIII F(ab’)2 pretreatment. Anti-CD3 mAb and anti-CD28 SA mAb also induced significant release of cytokines under HDC conditions compared with that under non-HDC conditions. In conclusion, CRA incorporating HDC into the air-drying solid-phase method using human PBMC could sensitively capture the FcγR-dependent cytokine release potential of mAb.

Synthesis of the pentasaccharide repeating unit from Ruminococcus gnavus and measurement of its inflammatory properties.

The roles played by the gut microbiome in human health are increasingly recognized, and the prevalence of specific microorganisms has been correlated with different diseases. For example, blooms of the Gram-positive bacterium Ruminococcus gnavus have been correlated with inflammatory bowel disease, and recently a polysaccharide produced by this organism was shown to stimulate release of inflammatory cytokines. This stimulation was proposed to signal through toll-like receptor 4 (TLR4). We have synthesized the pentasaccharide repeating unit of this polysaccharide and showed that it stimulates TNF-α and IL-6 release from bone marrow-derived dendritic cells (BMDCs) in a TLR4-dependent manner. A related glycan does not stimulate significant cytokine release, demonstrating TLR4 selectivity in glycan recognition.