CD40 Receptor Research Articles

Venous Thromboembolism (VTE) in Post-CAR-T Patients - a Meta-Analysis of Phase 2 & 3 Clinical Trials

Introduction: Chimeric Antigen Receptor T- cells (CAR-T) therapy is genetically engineered T cells that provide durable responses for patients with certain hematological malignancies. CAR-T therapy continues to expand as a treatment option for various malignant conditions. ICANS and CRS, a clinical syndrome of neurological symptoms, fever, hypotension, and hypoxia, are potentially serious complications of post-CAR-T. Both of these complications can, in turn, increase the risk of VTE. Only a few case reports and retrospective analyses have described the risk of VTE. The primary objective of this systematic review and meta-analysis is to review the data from phase 2 and 3 clinical trials to determine the incidence and risk of VTE post-CAR-T treatment. Material and methods: The National Institute of Health (NIH) library, Cochrane Library, and ClinicalTrials.gov database were scoured for all phase 2 and phase 3 clinical trials, completed by March 7, 2023, on CAR-T cell therapy. Two reviewers independently verified the results. Several data points were extracted. A biostatistician conducted the meta-analysis using the Meta package in the R version 4.1.2 (Balduzzi, Rücker, & Schwarzer, 2019; R Core Team, 2021). The endpoints of interest were the odds ratio and relative risk of developing VTE while in CRS using the Mantel-Haenszel method. Result: Nine phase 2 & 3 trials (Seven phase 2 and two phase 3 trials) were identified and were included in our study. The incidence of CRS, ICANS, and VTE was obtained from the toxicity list and supplemental data for analysis. Only KTE-X19 studying Brexucabtagene (Tecartus) for relapsed or refractory adult B-cell acute lymphoblastic leukemia and Relapsed or refractory Mantle-Cell Lymphoma mentioned the incidence of one and three cases of VTE, respectively, in patients getting CAR-T cell therapy. The total number of patients reviewed from these studies was 1784, of which 1017 received CAR-T cell therapy. The odds of VTE were 99.9995% lower than CRS, OR = 0.0005, 95% CI [0.0001, 0.0017] in patients receiving a CAR-T infusion, p < .0001. The Relative risk of VTE was 0.0050, 95% CI [0.0019, 0.0132]. The test of heterogeneity (p = .0430) suggests the presence of heterogeneous results. The heterogeneity statistic I2 is 49.86%, indicating moderate heterogeneity (percentage of variation across studies due to heterogeneity rather than chance). The odds of VTE were 99.9999% lower than no VTE, OR = 0.0001, 95% CI [0.0001, 0.0004], p <.0001 with a Relative risk of 0.0039, 95% CI [0.0015, 0.0105]. Discussion: CRS is a known risk factor for VTE. In patients with underlying malignancy undergoing CAR-T therapy, the risk can be much higher than those without malignancy who develop CRS for other reasons. In a retrospective single-center observational study, Hashmi et al. reported an 11% incidence of VTE in lymphoma patients treated with CAR-T directed against the CD-19 receptor. Our systematic review and metanalysis didn't find a statistically increased risk of VTE in the patients getting CAR-T cell therapy despite the risk of CRS/ ICANS and underlying malignancy. The findings suggest that the thrombotic risks are either low or were not reported in the interval specified in the studies. The need for thromboprophylaxis must be made on a case-by-case basis, factoring in other risk factors such as underlying disease, cytopenia, and other medical comorbidities. Conclusion: The Incidence of VTE is low following CAR-T therapy despite the risks, and the need for thromboprophylaxis should be based on individual patient characteristics. Patient selection, exclusion criteria, and short follow-up periods following phase 2 trials might impact our study results. In a long-term phase 3 trial, collecting data for VTE may be needed to appropriately stratify patients for their VTE risk so that an appropriate thromboprophylaxis can be recommended to prevent an additional serious complication post-CAR-T.

A Phase 1 Dose Escalation Trial of Third-Generation CD19-Directed CAR T-Cells Incorporating CD28 and Toll-like Receptor 2 (TLR2) Intracellular Domains for Relapsed or Refractory B-Cell Non-Hodgkin Lymphomas (ENABLE)

Introduction: Chimeric antigen receptor (CAR) T cell therapies directed against CD19 and incorporating either CD28 or 4-1BB intracellular co-stimulatory domains are a standard of care for relapsed or refractory (r/r) B-cell lymphomas. CD19-directed CAR T-cell products employing CD28 co-stimulation yield high response rates, but have been associated with higher rates of immune effector cell-associated neurotoxicity syndrome (ICANS) and severe cytokine release syndrome (CRS) than 4-1BB co-stimulated products. There is a need for CAR T-cell products that combine high efficacy with low toxicity. Toll-like receptor 2 (TLR2) is expressed by T-cells, and its engagement enhances T-cell expansion, modulates cytokine production, and promotes long-lived T-cell memory. In preclinical studies, interposition of an intracellular domain from TLR2 between CD28 and CD3ζ resulted in reduced CAR T-cell production of ICANS-associated cytokines GM-CSF and IFN-γ, while maintaining production of the homeostatic cytokine IL-7. We investigated the safety and efficacy of a novel third generation CD19-directed CAR T-cell product, which combines CD28 and TLR2 co-stimulatory domains ( Figure). Methods: We completed a first-in-human phase 1 dose escalation trial of WZTL-002, comprising autologous 1928T2z CAR T-cells (‘ENABLE’, NCT04049513) for patients with r/r B-cell non-Hodgkin lymphomas (B-NHL). A 3+3 dose escalation design was used, with doses from 5 × 10 4 to 1 × 10 6 viable CAR T-cells/kg body weight. Eligible participants had radiologically assessable disease, satisfactory organ function and no central nervous system (CNS) involvement by lymphoma. Bridging therapy was permitted after leukapheresis and pending CAR T-cell manufacture and release. WZTL-002 CAR T-cells were administered intravenously after 3 days of fludarabine (30mg/m 2/day) and cyclophosphamide (500mg/m 2/day) lymphodepletion. Adverse events (AEs) were graded by CTCAE 5.0 except CRS and ICANS (graded by American Society for Transplantation and Cellular Therapy criteria). Response assessment was by PET/CT at month 3, per Lugano 2014 criteria. Pharmacokinetic analyses were by droplet digital PCR for CAR transgenes in blood mononuclear cells. Results: Of 21 patients treated within the dose escalation trial, median age was 57 years (range 23 - 70); 10 (48%) were female; 4 (19%) Māori. Lymphomas were of large cell histology in 17 (81%, Table). Participants had received a median of 4 prior lines of therapy, including autograft in 11 (52%) and allograft in 1. Product phenotyping showed WZTL-002 CAR T-cells were median 49% CD4 +, 41% CD8 + and 34% CD62L +. As of June 14 2023, all patients had reached the primary follow-up timepoint (3 months after WZTL-002 administration). Grade ≥ 3 AEs occurring in ≥ 10% of recipients were: neutropenia (95%), lymphopenia (57%), hypogammaglobulinemia (57%), anaemia (43%), febrile neutropenia (43%), thrombocytopenia (24%) and tumor pain (19%). Grade 1 - 2 CRS occurred in 13 patients (62%); 6 received tocilizumab and 3 dexamethasone. No CRS of grade ≥ 3, and no ICANS of any grade, occurred. Two grade 4 cytopenia dose limiting toxicities occurred at day 21 (one each at 5 × 10 5 and 1 × 10 6 cells/kg), both resolved to grade 2 by day 90. Maximum tolerated dose was not reached. Responses were seen at all dose levels with complete metabolic response in 11 (52%) at month 3. WZTL-002 CAR T-cells expanded at all dose levels. Among recipients of 0.5 - 1.0 × 10 6 CAR T-cells/kg (n = 12), median peak CAR T-cell level (C max) was 93,950 transgene copies/μg genomic DNA (reached at median day 10) and CAR T-cells persisted in 9 of 11 assessed (82%) at day 90. A recommended phase 2 dose (RP2D) range of 0.5 - 1 × 10 6/kg was selected. Conclusions: In this dose escalation trial of a novel third-generation CD19-directed CAR T-cell product that combines CD28 and TLR2 co-stimulatory domains for r/r B-NHL, we observed no severe CRS, no ICANS of any grade and complete responses at all dose levels. At RP2D, in vivo CAR T-cell expansion was similar to or greater than other CD19-directed products. In conjunction with preclinical findings, this study suggests interposition of a TLR2 domain between CD28 and CD3ζ domains may reduce CAR T-cell-related ICANS risk while retaining efficacy. Enrolment to a dose expansion cohort has commenced and will assess outpatient management and the safety and efficacy of WZTL-002 CAR T-cells manufactured using a closed automated process.

Phase I Study of FT538 + Daratumumab for Treatment of r/r AML

Introduction: Relapsed and refractory (r/r) acute myeloid leukemia (AML) remains an unmet need with immune-therapy based treatments such as hematopoietic stem cell transplantation (HSCT) providing the longest relapse-free survival (RFS). Natural Killer (NK cells) have been shown to effect lysis of AML cells, both in vitro and in vivo across multiple platforms. In particular, “adaptive” NK cells induced in vivo in HSCT recipients after CMV reactivation have been shown to confer an RFS advantage. These cells are characterized phenotypically by the expression of NKG2C, CD57, KIR and downregulation of CD38. CD38 is an ectoenzyme broadly expressed on hematopoietic cells, including approximately 60% of AML. It catalyzes the conversion of nicotinamide-adenine dinucleotide (NAD+) and nicotinamide-adenine dinucleotide phosphate (NADP+) to cyclic adenosine diphosphate (cADP) ribosyl. CD38 knock-out NK cells have increased persistence by having enhanced metabolic fitness and resistance to oxidative stress via a reduction of reactive oxygen species. FT538 is an iPSC-derived NK cell product with a non-cleavable CD16 receptor to augment antibody-dependent cytotoxicity (ADCC), IL-15/IL-15R fusion to increase activation and persistence, and CD38 knockout to enhance its metabolic fitness and avoid fratricide when combined with CD38 monoclonal antibodies. Daratumumab (Dara) is a monoclonal antibody targeting CD38 on the surface of AML cells as well as activated lymphocytes. We hypothesized that the addition of Dara to fludarabine and cyclophosphamide would enhance lymphodepletion and augment ADCC for CD38+ AML leading to better response rates when combined with FT538. Methods: This (NCT04714372) is a phase I, dose-escalation study, utilizing FT538 in combination with fludarabine, cyclophosphamide, and Dara for the treatment of r/r AML. A study schema is presented in Figure 1A. Patients received 5 weekly doses of Dara starting day -12, 2 doses of fludarabine and cyclophosphamide at day -4, and 3 weekly doses of FT538. They were followed for DLT for 28 days after FT538 infusion. Patients with r/r AML after 2 or more lines of therapy and good organ function were enrolled. The primary objective was the safety of this combination, and the endpoints were the incidence of dose-limiting toxicities (DLTs) and incidence of adverse events. Secondary endpoint was the efficacy and predictors of efficacy. Patients evaluable for safety are those who received at least one dose of FT538. Patients evaluable for efficacy are those who completed all doses. Fast-track enrollment was utilized where one patient per dosing level (DL) was enrolled as long as no DLTs were seen with cohort expansion in the event of DLTs or related severe adverse events. Results: Six patients were enrolled on 4 DLs with increasing dose of FT538 from 1x10 8 - 1.5x10 9 cells. Median age was 70 years. Median number of prior lines is 3.5 (3-4). All patients were refractory to most recent therapy. One patient was enrolled on each DL1, DL2, and DL3. Three enrolled on DL4 with 1 completing therapy. Five patients are evaluable for safety and 4 for efficacy. There were no DLTs and we were able to enroll one patient per cohort. Eighty percent (4/5) experienced febrile neutropenia and infections. One patient with a prior biliary stent had choledocholithiasis requiring a complicated intervention. No prolonged cytopenias attributable to FT538 were seen. One patient experienced a G1 CRS lasting 1 day. There were no neurotoxicity events. One patient on DL2 achieved stable disease and one patient on DL4 achieved a CR for an ORR of 50% (2/4). Both went on to receive HSCT and continue to be alive and well without disease with a median follow up of 5 months. This is summarized in Figure 1.B. Macrochimerism (donor cells) was detected for up to 8 days post-infusion. Macrochimerism did not correlate with response but did show that Ki67 expression was present on donor cells suggesting proliferation. Conclusions: FT538 in combination with daratumumab has been tolerated in a highly pre-treated cohort of patients with expected toxicities and a signal of efficacy.

Generation and Screening of Various CD70 CAR NK Cells Identify the Most Effective Construct Against Hematologic Malignancies

The ability to re-direct the intrinsic power of the immune system against cancer has resulted in unprecedented outcomes in certain patients with otherwise incurable diseases. Natural killer (NK) cells, similar to T cells, are powerful immune effectors that possess intrinsic anti-tumor properties and are capable of generating a strong cytotoxic response upon engaging tumor cells. However, unlike T cells, NK cells do not identify specific antigens and do so in a way that is HLA-independent, posing minimal risk of GvHD. Thus, NK cells have are being explored as a possible allogeneic source for over-the-counter cellular immunotherapy. CD70, the ligand for the CD27 receptor, is an attractive “pan-cancer antigen”, since in addition to being expressed in hematologic malignancies such as acute myeloid leukemia (AML) and lymphoma, it is also expressed on many solid tumors, including bladder, lung, triple negative breast cancer, renal cell carcinoma, pancreatic cancer, and melanoma. Moreover, CD70 is only transiently found on activated T and B lymphocytes and on dendritic cells. To target CD70-expressing cancers, we designed and tested a number of different chimeric antigen receptors (CAR). Briefly, we used the natural interaction between CD70 and CD27 to make a CAR construct that incorporated the extracellular domain of human CD27, which is the natural receptor for CD70. We generated various second-generation CAR constructs that differed in their transmembrane domains (CD27 vs. CD28) and costimulatory domains. We included co-stimulatory domains that are NK-centric (DAP10 and DAP12), T-cell-centric (CD28), or bi-centric (4-1BB). The CD3ζ chain was used as a signaling endodomain, and all the CAR constructs also included IL-15 to enhance in vivo proliferation and persistence. Cord blood (CB)-derived NK cells were efficiently retrovirally transduced with the different CAR constructs and subjected to an in vitro functional screen against various cancer cells to evaluate the best constructs for in vivo validation (Figure 1). The top constructs from our in vitro screen were further evaluated in multiple in vivo models, including a Raji lymphoma model and a THP-1 AML model. The best CAR construct in terms of superior tumor control and prolongation of survival was selected for GMP manufacturing. Based on these preclinical findings, a Phase I/II clinical study is now being conducted at our center to assess the safety and effectiveness of CD70 CAR NK cells against CD70-expressing hematologic malignancies (NCT05092451). Figure 1: Flow chart showing the screening process to identify the most effective CAR construct.

Multiplex immunohistochemistry elucidates increased distance between cytotoxic T cells and plasma cells in relapsed myeloma, and identifies Lag-3 as the most common checkpoint receptor on cytotoxic T cells of myeloma patients.

A dysfunctional immune tumor microenvironment facilitates disease progression in multiple myeloma (MM). Using multiplex immunohistochemistry (mIHC), we describe the quantitative and qualitative changes in CD3+CD8+ cytotoxic T cells and assess their proximity to malignant plasma cells (PC) in patients with monoclonal gammopathy of undetermined significance (MGUS), and newly diagnosed (ND) and relapsed and/or refractory (RR) MM. Formalin-fixed, paraffin-embedded trephine sections from patients with MGUS (N=32), NDMM (N=65), and RRMM (N=59) were sequentially stained for CD138, CD3, CD8, and checkpoint receptors (CPR) Tim-3, Lag-3, and PD-1. The Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of cytotoxic T cells and analysis of proximity to PC. The percentage of CD8+ cytotoxic T cells in proximity to PC is greater in patients with NDMM than patients with RRMM (at 50 μm distance, 90.8% vs. 81.5%; P=0.038). There is a trend for more CD3+ T cells in MGUS (P=0.08) but no difference was observed in the prevalence of CD8+ cytotoxic T cells (P=0.48). Lag-3 is the most common CPR expressed on cytotoxic T cells in myeloma (P<0.0001), while PD-1 is the most common CPR on CD8- T cells of patients with MGUS and RRMM. Our study is the first to report on the spatial relationship between T cells and PC using mIHC on FFPE bone marrow trephine sections from patients with PC dyscrasia. The proximity of T cells to PC during early stages of MM, and overexpression of Lag-3, validate the move of immune therapeutic strategies, including T-cell engagers and checkpoint inhibitors, to upfront treatment or in early-line treatment of MM.

Teclistamab-cqyv in multiple myeloma.

Multiple myeloma (MM) is an incurable neoplasm characterized by significant morbidity and mortality. Despite advances in treatment, MM patients eventually experienced a relapse of the disease. Penta-drug refractory patients continue to be the hard core of relapsed/refractory (RR) settings. Teclistamab-cqyv is a humanized IgG4 antibody and a bispecific BCMA-director CD3 T-cell engager. It recruits endogenous T cells, by targeting CD3 receptors expressed on their surface, resulting in their activation against BCMA, an antigen expressed by plasma cells. US Food and Drug Administration (FDA) and European Medicines Agency (EMA) have approved Teclistamab-cqyv in monotherapy for the treatment of RRMM patients who have received at least three prior therapies, including immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), and anti-CD38 monoclonal antibodies (MoAbs) and have demonstrated disease progression during the last therapy. Its effectiveness was demonstrated in a pivotal clinical trial where the overall response rate (ORR) reached 60%. Other clinical studies are currently ongoing to investigate the association of the bispecific antibody with novel drugs with encouraging preliminary results, especially in the setting of heavily pretreated patients. In this review, the authors will provide a comprehensive overview of the drug, including its mechanism of action, major clinical trials, and future perspectives.

Emerging Immune Checkpoint Molecules on Cancer Cells: CD24 and CD200.

Cancer immunotherapy strategies are based on the utilization of immune checkpoint inhibitors to instigate an antitumor immune response. The efficacy of immune checkpoint blockade, directed at adaptive immune checkpoints, has been demonstrated in select cancer types. However, only a limited subset of patients has exhibited definitive outcomes characterized by a sustained response after discontinuation of therapy. Recent investigations have highlighted the significance of immune checkpoint molecules that are overexpressed in cancer cells and inhibit myeloid lineage immune cells within a tumor microenvironment. These checkpoints are identified as potential targets for anticancer immune responses. Notably, the immune checkpoint molecules CD24 and CD200 have garnered attention owing to their involvement in tumor immune evasion. CD24 and CD200 are overexpressed across diverse cancer types and serve as signaling checkpoints by engaging their respective receptors, Siglec-10 and CD200 receptor, which are expressed on tumor-associated myeloid cells. In this review, we summarized and discussed the latest advancements and insights into CD24 and CD200 as emergent immune checkpoint moieties, further delving into their therapeutic potentials for cancer treatment.

Computer screening of peptidomimetics and small-molecule ligands of B-cell membrane proteins for therapy of Burkitt lymphoma

The capabilities of molecular modelling and docking allow for the discovery of new potential drug agents to improve the treatment of diseases, which is a current concern. The objective of this study was to conduct in silico screening for antibody mimetics to B-cell membrane proteins for the treatment of Burkitt lymphoma through virtual screening. In this work, a standard protocol for structure-based virtual screening was employed, with the distinction that pharmacophores for screening were built not based on small-molecule ligands but on selected amino acid residues of antibodies. Based on literature data and the presence of a mechanism of direct cytotoxic action, as well as the availability of 3D structures of complexes, three monoclonal antibodies were selected: obinutuzumab, epratuzumab, and atezolizumab. The identification of biological targets was carried out by searching for 3D structures of selected complexes with target proteins in the Protein Data Bank. For virtual screening, the web service Pharmit was chosen. Using the Molecular Operating Environment program, pharmacophore models were constructed for three complexes: CD20 and obinutuzumab, CD22 and epratuzumab, and PD-L1 and atezolizumab. Docking with the CD20, CD22, and PD-L1 proteins was conducted at the binding sites recognised by the original antibody. Through in silico virtual screening using the Molecular Operating Environment software, a search for antibody mimetics to B-cell membrane proteins for Burkitt lymphoma treatment was conducted, resulting in the selection of 5 potential anti-lymphoma agents: CHEMBL505179 for the CD20 receptor, an antagonist of the melanocortin receptor for CD20 (PubChem-44406884), an inhibitor of blood clotting Factor Xa for CD22 (PubChem-136510605), and a blocker of epithelial Na+ channels for CD22 (PubChem-126761430), and an agonist of the melanocortin receptor for PD-L1 (PubChem-25078192). The obtained results can be applied in the pharmaceutical industry and oncological practice to enhance therapeutic outcomes in the treatment of patients with Burkitt lymphoma

Insect cell‐derived human CD200‐Fc increases zonula occludens‐1 tight junction protein in urothelial carcinoma cells

AbstractCD200 is a ligand that interacts with the CD200 receptor 1, with an anti‐inflammatory effect. CD200 inhibits the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathway, attenuating the inflammatory response and maintaining barrier function. In this study, human CD200 was fused with the fragment crystallizable (Fc) region of human immunoglobulin G (IgG) to generate the recombinant CD200‐Fc protein, produced in insect cells using the baculovirus expression vector system. The CD200‐Fc gene was cloned under the control of the polyhedrin promoter in the pFastBac‐1 vector of the baculovirus expression vector system. Immunoblot analysis confirmed the expression of CD200‐Fc in insect cells. The CD200‐Fc protein was successfully purified using protein A affinity chromatography. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay revealed that purified CD200‐Fc protein inhibited the proliferation of TCCSUP cells, a bladder epithelial cancer cell expressing CD200 receptor 1, at concentrations of 1, 10 and 100 ng/mL. Quantitative real‐time reverse transcription PCR (qRT‐PCR) and immunoblot analyses confirmed that the mRNA and protein levels of zonula occludens‐1, a tight junction protein for barrier protection in epithelial tissues, were increased in TCCSUP cells treated with insect cell‐derived CD200‐Fc. These results suggest that insect cell‐derived CD200‐Fc could alter zonula occludens‐1 expression in bladder epithelial cancer cells, enhancing the function of the cell barrier protein, which could inhibit cancer metastasis. Taken together, the baculovirus expression vector system can be applied to express antitumor therapeutic CD200‐Fc protein for the inhibition of bladder cancer.

Radiation Therapy Sensitizes Head-and-Neck Cancer Cells to Killing by Chimeric Antigen Receptor (CAR)-NK Cells Targeting CD70

CAR-T cell therapy is limited by toxicity, high cost, logistical manufacturing issues in the autologous setting and risk of GVHD in the allogeneic setting. Substitution of T cells with NK cells opens the possibility for an allogeneic off-the-shelf product with a better safety profile. However, the inadequate efficacy of CAR-NK cells against solid tumors can be extrapolated from experience with CAR-T cells. There is limited but promising preclinical evidence that radiation therapy (RT) enhances CAR-T cell tumoricidal activity against solid tumors. However, there is no data examining the potential synergy between RT and CAR-NK cell therapy. We engineered CAR-NK cells with CD27 receptor as extracellular domain to target its natural ligand CD70, which is overexpressed in head-and-neck cancers (HNSCC). CAR-NK cell killing was assessed real-time through xCELLigence cytotoxicity assays. CD70+ OQ01 human HNSCCs were used for most experiments. FaDu is a CD70- HNSCC (negative ctrl). UMRC3 is a CD70+ kidney cancer cell line (positive ctrl). CD70 expression pre- and post-RT was assessed by flow cytometry and Western blot. Ionizing RT was compared at 5 doses: 0, 1.75, 3.5, 7, and 14 Gy. A single dose of 3.5 Gy was used for most experiments. Post-radiation effects were generally assessed at 3 days or 9 days post-RT. Intracellular staining was used to assess NK cell expression of IFN-γ, CD107a, and TNF-α by flow cytometry. CD27/CD70 interaction blockade was through α-CD27 pre-treatment of CAR-NK cells. OQ01 HNSCCs heterogeneously express CD70 and are killed by CD70 CAR-NK cells in vitro. Pre-conditioning low-dose RT of 3.5 Gy applied to OQ01 HNSCCs 3 days prior to coculture with NK cells enhances CD70 CAR-NK cell killing, with ∼30% increased cytotoxicity against the tumor cells. Low-dose RT by itself did not induce acute cytolysis. As a possible mechanism for the increased sensitivity of irradiated OQ01 cells to CD70 CAR-NK cells, we found that RT enhances CD70 expression among HNSCCs in a dose-dependent manner. There was no increase in NK cell expression of IFN-γ, CD107a, and TNF-α with exposure to irradiated target cells. CD27/CD70 blockade does not solely abrogate RT-induced sensitization toward CAR-NK cell killing. Despite RT induction of transient increased expression of CD70, which normalizes by 9 days post-RT, there is persistent increase in RT-synergized target cell killing even at this later timepoint. Thus, altogether, RT sensitizes CD70-expressing HNSCC cells to CAR-NK cell killing in vitro. This work represents the first preclinical study to identify the synergy of RT and CAR-NK cell therapy in solid tumors and is the first demonstration of CAR-NK cell activity against human HNSCCs. We show significantly enhanced potency of CAR-NK cells against irradiated tumor cells in vitro. Collectively, this research will be vital to guide efforts expanding into other target antigens and tumor types.

P97/VCP Promotes the Recycling of Endocytic Cargo.

The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14, and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.

Positive effects of recombinant interferon α2b on the phenotype of CD16&lt;sup&gt;+&lt;/sup&gt;INFα/βR1&lt;sup&gt;-&lt;/sup&gt;CD119&lt;sup&gt;+&lt;/sup&gt;, CD16&lt;sup&gt;+&lt;/sup&gt;INFα/βR1&lt;sup&gt;+&lt;/sup&gt;CD119&lt;sup&gt;-&lt;/sup&gt; neutrophil granulocyte subset in patients with post-COVID syndrome and herpesvirus infections

Post-COVID syndrome (PCS) is a multisystem inflammatory condition with manifestations of chronic fatigue syndrome (CFS) and cognitive disorders (CD), along with reactivation of chronic herpesvirus infections (HVI). The PCS manifestations require studying the molecular mechanisms associated with the production of IFN and receptor functions of neutrophil granulocytes (NG), which is relevant and promotes the search for immunotherapeutic strategies in patients with PCS. Our objective was to study the in vitro effects of recombinant interferon α2b (recIFNα2b) on the phenotype of CD16+IFNα/βR1-CD119+, CD16+IFNα/ βR1+CD119+ subsets and functional activity of NG in patients with post-COVID syndrome and herpesvirus infections. Materials and methods: 45 patients (24-60 years old) with PCS and HVI (HSV 1, EBV, HHV6, CMV) comprised the study group 1 (SG1). A questionnaire was conducted to assess the severity of PCS symptoms using a point scale. We performed a study of the content and phenotype of NG subsets, i.e., the CD16+IFNα/βR1-CD119+, CD16+IFNα/βR1+CD119-, CD16+IFNα/βR1+CD119+ subpopulation, phagocytic and NADPH oxidase function of NG before and after in vitro incubation with recIFNα2b (50 IU/ µL, for 60 min, at 37 °C) in the study group 1a (SG1a). The comparison group (CG) of 30 volunteers examined during the pre-COVID period. Results: We revealed more pronounced clinical manifestations of CFS and CD in SG1 patients with mixed HVI, than in mono-HVI cases. Increased expression density of all receptors was registered on CD16+IFNα/βR1+CD119-NG and CD16+IFNα/βR1-CD119+ NG, thus suggesting the NG activation with initiation of cytotoxicity or NETosis, a decrease in phagocytic function and intensity of NADPH oxidase activity with depletion of NG reserve capacity in SG1. We have obtained some data on the positive effect of recIFNα2b in vitro (SG1a), e.g., decreased CD16 expression density and enhancement of IFNα/βR1 receptor expression in the CD16+IFNα/βR1+CD119- subset. In the CD16+IFNα/βR1-CD119+ subset, we have found persistence of increased MFI CD16 and MFI CD119 receptors, restoration of defective NG phagocytic function and reduced excessive activity of NADPH oxidases. Conclusion: The positive effects of the recIFNα2b influence on deficient function of NG in PCS patients suggest an oppoptunity of using immunotherapy with a recIFNα2b-based drug, combined with highly active antioxidants for treatment of various PCS manifestations including CFS, CD, HVI, thus, probably, ensuring adequate functioning of antiviral and regulatory mechanisms of the immune system.

Cell-surface photochemistry mediated calcium overload for synergistic tumor therapy

Calcium (Ca2+) is essential for mitochondrial homeostasis and function coordination, particularly in cancer cells that metabolize frequently to sustain their growth. Photochemistry mediated calcium overload has attracted lots of attention as an effective way to achieve tumor suppression. Herein, we developed a photonanomedicine to synergistically induce calcium overload via cell-surface photochemistry and thus tumor suppression. Specifically, the photosensitizer, protoporphyrin IX (PpIX) was loaded onto upconversion nanoparticles (UCNP), which was subsequently modified by a polymer bearing photo-crosslinking cinnamate (CA) groups. The resulting nanoparticle was further functionalized by anti-CD20 aptamers (Apt), to give photonanomedicine. The interaction between CD20 receptors and anti-CD20 aptamers allowed photonanomedicine to accurately attach onto the Raji cell surface after an intravenous injection. Following the local application of a 980 nm NIR laser, the photonanomedicine was able to capture the NIR light and convert it into ultraviolet (UV) light. On one hand, the converted UV light led the crosslinking of cinnamate groups in photonanomedicine, further stimulating the clustering of CD20 receptors and causing Ca2+ influx. On the other hand, the UV light could simultaneously excited PpIX to generate reactive oxygen species (ROS) in situ to break down the integrity of cell membrane and lead to an influx of Ca2+. The synergistic Ca2+ overload mediated by photonanomedicine exhibited an enhanced and superior anti-tumor efficacy. We believe this photonanomedicine expands the toolbox to manipulate intracellular Ca2+ concentration and holds a great potential as an anti-tumor therapy.

Different mechanisms of CD200-CD200R induce diverse outcomes in cancer treatment

The CD200 is a cell membrane protein expressed by tumor cells, and its receptor CD200 receptor (CD200R) is expressed by immune cells including macrophages and dendritic cells. The formation of CD200-CD200R inhibits the cellular functions of the targeted immune cells, so CD200 is one type of the immune checkpoint and blockade CD200-CD200R formation is a potential cancer treatment. However, the CD200 blockade has opposite treatment outcomes in different types of cancers. For instance, the CD200R deficient mice have a higher tumor load than the wild type (WT) mice in melanoma suggesting that CD200-CD200R inhibits melanoma. On the other hand, the antibody anti-CD200 treatment in pancreatic ductal adenocarcinoma (PDAC) and head and neck squamous cell carcinoma (HNSCC) significantly reduces the tumor load indicating that CD200-CD200R promotes PDAC and HNSCC. In this work, we hypothesize that different mechanisms of CD200-CD200R in tumor microenvironment could be one of the reasons for the diverse treatment outcomes of CD200 blockade in different types of cancers. We create one Ordinary Differential Equations (ODEs) model for melanoma including the inhibition of CCL8 and regulatory T cells and the switching from M2 to M1 macrophages by CD200-CD200R to capture the tumor inhibition by CD200-CD200R. We also create another ODEs model for PDAC and HNSCC including the promotion of the polarization and suppressive activities of M2 macrophages by CD200-CD200R to generate the tumor promotion by CD200-CD200R. Furthermore, we use these two models to investigate the treatment efficacy of the combination treatment between the CD200-CD200R blockade and the other immune checkpoint inhibitor, anti-PD-1. Our result shows that different mechanisms of CD200-CD200R can induce different treatment outcomes in combination treatments, namely, only the CD200-CD200R blockade reduces tumor load in melanoma and only the anti-PD-1 and CD200 knockout decrease tumor load in PDAC and HNSCC. Moreover, in melanoma, the CD200-CD200R mainly utilizes the inhibitions on M1 macrophages and dendritic cells to inhibit tumor growth, instead of M2 macrophages.

Nutrition impact on ILC3 maintenance and function centers on a cell-intrinsic CD71-iron axis.

Iron metabolism is pivotal for cell fitness in the mammalian host; however, its role in group 3 innate lymphoid cells (ILC3s) is unknown. Here we show that transferrin receptor CD71 (encoded by Tfrc)-mediated iron metabolism cell-intrinsically controls ILC3 proliferation and host protection against Citrobacter rodentium infection and metabolically affects mitochondrial respiration by switching of oxidative phosphorylation toward glycolysis. Iron deprivation or Tfrc ablation in ILC3s reduces the expression and/or activity of the aryl hydrocarbon receptor (Ahr), a key ILC3 regulator. Genetic ablation or activation of Ahr in ILC3s leads to CD71 upregulation or downregulation, respectively, suggesting Ahr-mediated suppression of CD71. Mechanistically, Ahr directly binds to the Tfrc promoter to inhibit transcription. Iron overload partially restores the defective ILC3 compartment in the small intestine of Ahr-deficient mice, consistent with the compensatory upregulation of CD71. These data collectively demonstrate an under-appreciated role of the Ahr-CD71-iron axis in the regulation of ILC3 maintenance and function.

Exploiting the CD200-CD200R immune checkpoint axis in multiple myeloma to enhance CAR T-cell therapy

AbstractPatients with multiple myeloma (MM) treated with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells usually relapse with BCMA+ disease, indicative of CAR T-cell suppression. CD200 is an immune checkpoint that is overexpressed on aberrant plasma cells (aPCs) in MM and is an independent negative prognostic factor for survival. However, CD200 is not present on MM cell lines, a potential limitation of current preclinical models. We engineered MM cell lines to express CD200 at levels equivalent to those found on aPCs in MM and show that these are sufficient to suppress clinical-stage CAR T-cells targeting BCMA or the Tn glycoform of mucin 1 (TnMUC1), costimulated by 4-1BB and CD2, respectively. To prevent CD200-mediated suppression of CAR T cells, we compared CRISPR-Cas9–mediated knockout of the CD200 receptor (CD200RKO), to coexpression of versions of the CD200 receptor that were nonsignaling, that is, dominant negative (CD200RDN), or that leveraged the CD200 signal to provide CD28 costimulation (CD200R-CD28 switch). We found that the CD200R-CD28 switch potently enhanced the polyfunctionality of CAR T cells, and improved cytotoxicity, proliferative capacity, CAR T-cell metabolism, and performance in a chronic antigen exposure assay. CD200RDN provided modest benefits, but surprisingly, the CD200RKO was detrimental to CAR T-cell activity, adversely affecting CAR T-cell metabolism. These patterns held up in murine xenograft models of plasmacytoma, and disseminated bone marrow predominant disease. Our findings underscore the importance of CD200-mediated immune suppression in CAR T-cell therapy of MM, and highlight a promising approach to enhance such therapies by leveraging CD200 expression on aPCs to provide costimulation via a CD200R-CD28 switch.

TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

ABSTRACT A preliminary study investigating immunotherapy strategies for aggressive triple negative breast cancer (TNBC) revealed an overexpression of genes involved in the release of extracellular vesicles (EVs). Proteins expressed by EVs play a role in reprogramming the tumor microenvironment and impeding effective responses to immunotherapy. Galectin 3 (Gal3), found in the extracellular space of breast cancer cells, downregulates T-cell receptor expression. Gal3 binds to several receptors, including CD45, which is required for T-cell receptor activation. Previously, we reported a novel tumor escape mechanism, whereby TNBC cells suppress immune cells through CD45 intracellular signals. The objective of this study was to determine the potential association of Gal3 with TNBC-secreted EVs induction of immunosuppression via the CD45 signaling pathway. EVs were isolated from MDA-MB-231 cells and the plasma of patients with TNBC. Mass spectrometry revealed the presence of Gal3 binding protein (Gal3BP) in the isolated small EVs, which interacted with TNBC secreted Gal3. Gal3BP and Gal3 form a complex that induces a significant increase in T-regulatory cells in peripheral blood mononuclear cells (PBMCs). This increase correlates with a significant increase in suppressive interleukins 10 and 35. Blocking the CD45 receptor in PBMCs cultured with tumor-derived EVs impeded the immunosuppression exerted by the Gal3BP/Gal3 complex. This led to an increase in IFN-γ and the activation of CD4, CD8 and CD56 effector cells. This study suggests a tumor escape mechanism that may contribute to the development of a different immunotherapy strategy that complements current therapies used for TNBC.

Equine β-defensin 1 regulates cytokine expression and phagocytosis in S. aureus-infected mouse monocyte macrophages via the Paxillin-FAK-PI3K pathway

β-defensin-1 (BD-1) is a rich source of disulfide bonds and antibacterial peptides that exhibit direct bactericidal function. The expression of BD-1 is primarily induced by external stimulation and is known to correlate with TLR-mediated inflammation, suggesting its association with innate immune responses. Equine β-defensin-1 (eBD-1) belongs to the BD-1 family. Our previous study demonstrated that eBD-1 enhances cytokine expression and promotes macrophage phagocytosis of S. aureus, although the underlying mechanism remains unknown. In this study, we utilized a PI-3K inhibitor (PKI-402) to treat eBD-1 -treated S. aureus-infected macrophages in vitro. Our results revealed that PKI-402 decreased the expression of eBD-1-promoted TNF-α, IL-6, CXCL10, CD40, RANTES, and p65 mRNA. To further investigate the relationship between eBD-1 and phagocytosis, we examined the expression of paxillin and FcγRIII (CD16 receptor) using western blot and immunofluorescence techniques. Our findings demonstrated that eBD-1 enhanced CD16 and paxillin expression in S. aureus -infected macrophages. Considering the correlation between paxillin expression and focal adhesion kinase (FAK), we transfected FAK siRNA into macrophages and evaluated paxillin expression using western blot analysis. Additionally, we quantified the number of S. aureus phagocytosed by macrophages. The results indicated a reduction in both paxillin expression and the number of S. aureus phagocytosed by macrophages upon FAK siRNA treatment. Our study showed the eBD-1 promotes cytokine mRNA expression in S. aureus-infected macrophages regulated by PI-3K-NF-κB pathway, and it increases macrophage phagocytosis of S. aureus associated with the FAK-paxillin signaling pathway.

Tumor-derived interleukin-1 receptor antagonist exhibits immunosuppressive functions and promotes pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDA) is a pernicious disease characterized by an immunosuppressive milieu that is unresponsive to current immunotherapies. Interleukin-1 receptor antagonist (IL-1Ra) is a natural anti-inflammatory cytokine; however, its contribution to cancer pathogenesis and immunosuppression remains elusive. In this research, we investigated the role and mechanism of IL-1Ra in malignant progression of PDA.ResultsThrough analyzing clinical dataset and examining the pathological tumor tissues and serum samples, we have demonstrated that IL-1Ra expression is elevated in human PDA and positively associated with malignant progression of PDA. To study the biological function of IL-1Ra in tumors, we generated a set of mouse pancreatic cancer cell lines with a knockout (KO) of the Il1rn gene, encoding IL-1Ra, and compared the tumor growth rates in immune-competent and immune-deficient mice. We found that the Il1rn KO cells exhibited greater tumor inhibition in immune-competent mice, highlighting the crucial role of a functional immune system in Il1rn KO-mediated anti-tumor response. Consistently, we found an increase in CD8+ T cells and a decrease in CD11b+Ly6G− immunosuppressive mononuclear population in the tumor microenvironment of Il1rn KO-derived tumors. To monitor the inhibitory effects of IL-1Ra on immune cells, we utilized a luciferase-based reporter CD4+ T cell line and splenocytes, which were derived from transgenic mice expressing ovalbumin-specific T cell receptors in CD8+ T cells, and mice immunized with ovalbumin. We showed that IL-1Ra suppressed T cell receptor signaling and inhibited antigen-specific interferon-γ (IFN-γ) secretion and cytolytic activity in splenocytes.ConclusionsOur findings illustrate the immunosuppressive properties of the natural anti-inflammatory cytokine IL-1Ra, and provide a rationale for considering IL-1Ra-targeted therapies in the treatment of PDA.

A Shiga Toxin B-Subunit-Based Lectibody Boosts T Cell Cytotoxicity towards Gb3-Positive Cancer Cells.

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments.