Effective bridging strategies prior to infusion with tisagenlecleucel results in high response rates and long-term remission in relapsed/refractory large B-cell lymphoma: findings from a German monocentric study.

BACKGROUND: Incorporating chimeric antigen receptor (CAR)-T cell therapy into relapsed or refractory large B-cell lymphoma (rr LBCL) treatment algorithms has yielded remarkable response rates and durable remissions, yet a substantial portion of patients experience progression or relapse. Variations in outcomes across treatment centers may be attributed to different bridging strategies and remission statuses preceding CAR-T cell therapy. PATIENTS: Twenty-nine consecutive adult patients receiving tisagenlecleucel (tisa-cel) for rr LBCL from December 2019 to February 2023 at Jena University Hospital were analyzed. RESULTS: The median age was 63, with a median of 3 prior treatments. Twenty patients (69%) were refractory to any systemic therapy before CAR-T cell treatment. Following leukapheresis, 25 patients (86%) received bridging therapy with the majority undergoing chemotherapy (52%) or combined modality therapy (32%). Radiotherapy (RT) was part of the bridging strategy in 44%, with moderately hypofractionated involved site RT (30.0 Gy/2.5 Gy) being applied most frequently (64%). Post-CAR-T infusion, the objective response rate at 30 days was 83%, with 55% achieving complete response. Twelve-month progression-free (PFS) and overall survival (OS) were 60% and 74%, respectively, with a median follow up of 11.1 months for PFS and 17.9 months for OS. Factors significantly associated with PFS were chemotherapy sensitivity pre-leukapheresis and response to bridging. CONCLUSION: The study underscores the importance of minimal tumor burden at CAR-T initiation, emphasizing the need for suitable bridging regimens. The findings advocate for clinical trials and further real-world analyses to optimize CAR-T cell therapy outcomes by identifying the most effective bridging strategies.

Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase.

Arachidonic acid (AA) is metabolized to diverse bioactive lipid mediators. Whereas the 5-lipoxygenase-activating protein (FLAP) facilitates AA conversion by 5-lipoxygenase (5-LOX) to pro-inflammatory leukotrienes (LTs), the soluble epoxide hydrolase (sEH) degrades anti-inflammatory epoxyeicosatrienoic acids (EETs). Accordingly, dual FLAP/sEH inhibition might be advantageous drugs for intervention of inflammation. We present the in vivo pharmacological profile and efficiency of N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N'-(3,4-dichlorophenyl)urea (diflapolin) that dually targets FLAP and sEH. Diflapolin inhibited 5-LOX product formation in intact human monocytes and neutrophils with IC50 = 30 and 170 nM, respectively, and suppressed the activity of isolated sEH (IC50 = 20 nM). Characteristic for FLAP inhibitors, diflapolin (I) failed to inhibit isolated 5-LOX, (II) blocked 5-LOX product formation in HEK cells only when 5-LOX/FLAP was co-expressed, (III) lost potency in intact cells when exogenous AA was supplied, and (IV) prevented 5-LOX/FLAP complex assembly in leukocytes. Diflapolin showed target specificity, as other enzymes related to AA metabolism (i.e., COX1/2, 12/15-LOX, LTA4H, LTC4S, mPGES1, and cPLA2) were not inhibited. In the zymosan-induced mouse peritonitis model, diflapolin impaired vascular permeability, inhibited cysteinyl-LTs and LTB4 formation, and suppressed neutrophil infiltration. Diflapolin is a highly active dual FLAP/sEH inhibitor in vitro and in vivo with target specificity to treat inflammation-related diseases.

ABO-Incompatible Living Donor Liver Transplantation in Focus of Antibody Rebound.

BACKGROUND: Living donor liver transplantation (LDLT) is an option to expand the donor organ pool for patients with life-threatening diseases who cannot be supplied with a cadaver organ. Next to the donor risks, complications after ABO-incompatible LDLT (ABOi LDLT) in the recipient are subject to controversial discussion. Improvement in ABOi graft survival rates have been achieved with plasma treatment procedures (PTP) and immunosuppression but antibody-mediated rejection (AMR) and graft loss still occur. METHODS: Since 2008, we have prepared 10 patients for ABOi LDLT. Seven of the 10 patients for transplantation had hepatocellular carcinoma (HCC). RESULTS: All patients underwent PTP before and after ABOi LDLT as well as immunosuppression according to the treatment schedule. We did not use anti-CD20 monoclonal antibodies in the transplant setting. We transplanted 6 of 10 preconditioned patients. After 3 years, 5 of the 6 transplanted patients were still alive. CONCLUSION: Even if B-cell depletion with anti-CD 20 treatment in the setting of ABOi LDLT is commonly accepted, our center successfully administered only quadruple drug immunosuppression combined with PTP. Especially patients with HCC had a high titer increment also pre-transplantation and were at high risk for arterial thrombosis and graft loss.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase.

Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)α. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFα in lipopolysaccharide (LPS)- or LPS/interferon (INF)γ-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFα production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1ß, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFα secretion. Archazolid selectively increased TNFα mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFκB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFα production in classically-activated macrophages along with NFκB and SAPK/JNK activation. Such increased TNFα release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.

Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy.

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.

Development of smart cell-free and cell-based assay systems for investigation of leukotriene C4 synthase activity and evaluation of inhibitors.

Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A4 (LTA4) that is subsequently conjugated with glutathione (GSH) by LTC4 synthase (LTC4S). Cys-LT receptor antagonists and LTC4S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA4 have hampered the development of LTC4S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying LTC4S activity and investigating LTC4S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC4S with isolated 5-LOX efficiently converted exogenous AA to LTC4 (~1.3µg/200µg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca2+-ionophore A23187 and 20µM AA resulted in strong LTC4 formation (~250ng/106 cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC4S, consistently inhibited LTC4 formation in all assay types (IC50=3.1-3.5µM) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50=17 and 300nM, respectively). We demonstrated transcellular LTC4 biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4 from AA and HEK293 cells expressing LTC4S that transform LTA4 to LTC4. In conclusion, our assay approaches are advantageous as the substrate LTA4 is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations and evaluation of LTC4S inhibitors.

Humudifucol and Bioactive Prenylated Polyphenols from Hops (Humulus lupulus cv. "Cascade").

Humulus lupulus (hop plant) has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the phytoestrogenic activity of the plant extracts as well as to the anti-inflammatory and chemopreventive properties of the prenylated chalcones present. In this study, an Italian sample of H. lupulus cv. "Cascade" has been investigated and three new compounds [4-hydroxycolupulone (6), humudifucol (7) and cascadone (8)] have been purified and identified by means of NMR spectroscopy along with four known metabolites. Notably, humudifucol (7) is the first prenylated dimeric phlorotannin discovered in nature. Because structurally related phloroglucinols from natural sources were found previously to inhibit microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), the isolated compounds were evaluated for their bioactivity against these pro-inflammatory target proteins. The prenylated chalcone xanthohumol inhibited both enzymes at low µM concentrations.