CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.

The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.

Identification and functional characterization of transcriptional activators in human cells.

Transcription is orchestrated by thousands of transcription factors (TFs) and chromatin-associated proteins, but how these are causally connected to transcriptional activation is poorly understood. Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. By combining interaction proteomics and chemical inhibitors, we delineate the preference of these transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct cofactors. We also identify potent transactivation domains among the hits and use AlphaFold2 to predict and experimentally validate interaction interfaces of two activation domains with BRD4. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent p300-dependent activator. Our work provides a functional catalog of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale.

Virus and cell fusion mechanisms.

In biomembrane fusion pathways, membranes are destabilized through insertions of amphipathic protein segments, lipid reorganization via hemifusion, protein restructuring, and dimpling of the membranes. Four classes of membrane proteins are known in virus and cell fusion. Class I virus-cell fusion proteins (fusogens) are α-helix-rich prefusion trimers that form coiled-coil structures that insert hydrophobic fusion peptides or loops (FPs or FLs) into membranes and refold into postfusion trimers. Class II virus-cell fusogens are ß-sheet-rich prefusion homo- or heterodimers that insert FLs into membranes, ending in postfusion trimers. Class III virus-cell fusogens are trimers with both α-helices and ß-sheets that dissociate into monomers, insert FLs into membranes, and oligomerize into postfusion trimers. Class IV reoviral cell-cell fusogens are small proteins with FLs that oligomerize to fuse membranes. Class I cell-cell fusogens (Syncytins) were captured by mammals from retroviruses, and class II cell-cell fusogens (EFF-1/AFF-1) fuse membranes via homotypic zippering. Mechanisms and fusogens for most cell fusion events are unknown.

The in vitro replication phenotype of hepatitis B virus (HBV) splice variants Sp3 and Sp9 and their impact on wild-type HBV replication.

Prior to nuclear export, the hepatitis B virus (HBV) pregenomic RNA may be spliced by the host cell spliceosome to form shorter RNA sequences known as splice variants. Due to deletions in the open reading frames, splice variants may encode novel fusion proteins. Although not essential for HBV replication, the role of splice variants and their novel fusion proteins largely remains unknown. Some splice variants and their encoded novel fusion proteins have been shown to impair or promote wild-type HBV replication in vitro, and although splice variants Sp3 and Sp9 are two of the most common splice variants identified to date, their in vitro replication phenotype and their impact on wild-type HBV replication are unclear. Here, we utilize greater than genome-length Sp3 and Sp9 constructs to investigate their replication phenotype in vitro, and their impact on wild-type HBV replication. We show that Sp3 and Sp9 were incapable of autonomous replication, which was rescued by providing the polymerase and core proteins in trans. Furthermore, we showed that Sp3 had no impact on wild-type HBV replication, whereas Sp9 strongly reduced wild-type HBV replication in co-transfection experiments. Knocking out Sp9 novel precore-surface and core-surface fusion protein partially restored replication, suggesting that these proteins contributed to suppression of wild-type HBV replication, providing further insights into factors regulating HBV replication in vitro. IMPORTANCE: The role of hepatitis B virus (HBV) splice variants in HBV replication and pathogenesis currently remains largely unknown. However, HBV splice variants have been associated with the development of hepatocellular carcinoma, suggesting a role in HBV pathogenesis. Several in vitro co-transfection studies have shown that different splice variants have varying impacts on wild-type HBV replication, perhaps contributing to viral persistence. Furthermore, all splice variants are predicted to produce novel fusion proteins. Sp1 hepatitis B splice protein contributes to liver disease progression and apoptosis; however, the function of other HBV splice variant novel fusion proteins remains largely unknown. We show that Sp9 markedly impairs HBV replication in a cell culture co-transfection model, mediated by expression of Sp9 novel fusion proteins. In contrast, Sp3 had no effect on wild-type HBV replication. Together, these studies provide further insights into viral factors contributing to regulation of HBV replication.

Endothelium as a Source of Cardiovascular Toxicity From Antitumor Kinase Inhibitors.

Kinase inhibitors (KIs) targeting oncogenic molecular pathways have revolutionized cancer therapy. By directly targeting specific tumor-driving kinases, targeted therapies have fewer side effects compared with chemotherapy. Despite the enhanced specificity, cardiovascular side effects have emerged with many targeted cancer therapies that limit long-term outcomes in patients with cancer. Endothelial cells lining all blood vessels are critical to cardiovascular health and are also exposed to circulating levels of systemic anticancer therapies. Both on- and off-target perturbation of signaling pathways from KIs can cause endothelial dysfunction, resulting in cardiovascular toxicity. As such, the endothelium is a potential source, and also a therapeutic target for prevention, of cardiovascular toxicity. In this review, we examine the evidence for KI-induced endothelial cell dysfunction as a mechanism for the cardiovascular toxicities of vascular endothelial growth factor inhibitors, BCR-Abl KIs, Bruton tyrosine inhibitors, and emerging information regarding endothelial toxicity of newer classes of KIs.

RAF1 gene fusions are recurrent driver events in infantile fibrosarcoma-like mesenchymal tumors.

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.

Safety, Immunogenicity, and Regimen Selection of Ad26.RSV.preF-Based Vaccine Combinations: A Randomized, Double-blind, Placebo-Controlled, Phase 1/2a Study.

BACKGROUND: Ad26.RSV.preF is an adenovirus serotype 26 vector-based respiratory syncytial virus (RSV) vaccine encoding a prefusion conformation-stabilized RSV fusion protein (preF) that demonstrated robust humoral and cellular immunogenicity and showed promising efficacy in a human challenge study in younger adults. Addition of recombinant RSV preF protein might enhance RSV-specific humoral immune responses, especially in older populations. METHODS: This randomized, double-blind, placebo-controlled, phase 1/2a study compared the safety and immunogenicity of Ad26.RSV.preF alone and varying doses of Ad26.RSV.preF-RSV preF protein combinations in adults aged ≥60 years. This report includes data from cohort 1 (initial safety, n = 64) and cohort 2 (regimen selection, n = 288). Primary immunogenicity and safety analyses were performed 28 days postvaccination (cohort 2) for regimen selection. RESULTS: All vaccine regimens were well tolerated, with similar reactogenicity profiles among them. Combination regimens induced greater humoral immune responses (virus-neutralizing and preF-specific binding antibodies) and similar cellular ones (RSV-F-specific T cells) as compared with Ad26.RSV.preF alone. Vaccine-induced immune responses remained above baseline up to 1.5 years postvaccination. CONCLUSIONS: All Ad26.RSV.preF-based regimens were well tolerated. A combination regimen comprising Ad26.RSV.preF, which elicits strong humoral and cellular responses, and RSV preF protein, which increases humoral responses, was selected for further development. Clinical Trials Registration. NCT03502707.

Unveiling success determinants for AMB-assisted phase expansion of fusion proteins in ARP/wARP.

Fusion proteins (FPs) are frequently utilized as a biotechnological tool in the determination of macromolecular structures using X-ray methods. Here, we explore the use of different protein tags in various FP, to obtain initial phases by using them in a partial molecular replacement (MR) and constructing the remaining FP structure with ARP/wARP. Usually, the tag is removed prior to crystallization, however leaving the tag on may facilitate crystal formation, and structural determination by expanding phases from known to unknown segments of the complex. In this study, the Protein Data Bank was mined for an up-to-date list of FPs with the most used protein tags, Maltose Binding Protein (MBP), Green Fluorescent Protein (GFP), Thioredoxin (TRX), Glutathione transferase (GST) and the Small Ubiquitin-like Modifier Protein (SUMO). Partial MR using the protein tag, followed by automatic model building, was tested on a subset of 116 FP. The efficiency of this method was analyzed and factors that influence the coordinate construction of a substantial portions of the fused protein were identified. Using MBP, GFP, and SUMO as phase generators it was possible to build at least 75 % of the protein of interest in 36 of the 116 cases tested. Our results reveal that tag selection has a significant impact; tags with greater structural stability, such as GFP, increase the success rate. Further statistical analysis identifies that resolution, Wilson B factor, solvent percentage, completeness, multiplicity, protein tag percentage in the FP (considering amino acids), and the linker length play pivotal roles using our approach. In cases where a structural homologous is absent, this method merits inclusion in the toolkit of protein crystallographers.

Respiratory syncytial virus entry mechanism in host cells: A general overview.

Respiratory syncytial virus (RSV) is a virus that causes acute respiratory infections in neonates and older adults. To infect host cells, the attachment glycoprotein (G) interacts with a cell surface receptor. This interaction determines the specific cell types that are susceptible to infection. RSV possesses a type I fusion protein F. Type I fusion proteins are metastable when rearrangement of the prefusion F occurs; the fusion peptide is exposed transforming the protein into postfusion form. The transition between the prefusion form and its postfusion form facilitates the viral envelope and the host cell membrane to fuse, enabling the virus to enter the host cell. Understanding the entry mechanism employed by RSV is crucial for developing effective antiviral therapies. In this review, we will discuss the various types of viral fusion proteins and explore the potential entry mechanisms utilized by RSV. A deeper understanding of these mechanisms will provide valuable insights for the development of novel approaches to treat RSV infections.

An Active and Versatile Electron Transport System for Cytochrome P450 Monooxygenases from the Alkane Degrading Organism Acinetobacter sp. OC4.

Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobacter sp. OC4 are investigated. Upon heterologous production of both proteins independently in Escherichia coli, spectral analysis showed their reduction capability towards reporter electron acceptors, e. g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25 U mg-1. Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK]n, flexible glycine linkers [GGGGS]n or rigid proline linkers [EPPPP]n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19 U mg-1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily.

89Zr-immunoPET-guided selection of a CD33xIL15 fusion protein optimized for antitumor immune cell activation and in vivo tumour retention in acute myeloid leukaemia.

PURPOSE: Immune cells are capable of eliminating leukemic cells, as evidenced by outcomes in hematopoietic cell transplantation (HCT). However, patients who fail induction therapy will not benefit from HCT due to their minimal residual disease (MRD) status. Thus, we aimed to develop an immunomodulatory agent to reduce MRD by activating immune effector cells in the presence of leukaemia cells via a novel fusion protein that chimerises two clinically tolerated biologics: a CD33 antibody and the IL15Ra/IL15 complex (CD33xIL15). METHODS: We generated a set of CD33xIL15 fusion protein constructs with varying configurations and identified those with the best in vitro AML-binding, T cell activation, and NK cell potentiation. Using 89Zr-immunoPET imaging we then evaluated the biodistribution and in vivo tumour retention of the most favourable CD33xIL15 constructs in an AML xenograft model. Ex vivo biodistribution studies were used to confirm the pharmacokinetics of the constructs. RESULTS: Two of the generated fusion proteins, CD33xIL15 (N72D) and CD33xIL15wt, demonstrated optimal in vitro behaviour and were further evaluated in vivo. These studies revealed that the CD33xIL15wt candidate was capable of being retained in the tumour for as long as its parental CD33 antibody, Lintuzumab (13.9 ± 3.1%ID/g vs 18.6 ± 1.1%ID/g at 120 h). CONCLUSION: This work demonstrates that CD33xIL15 fusion proteins are capable of targeting leukemic cells and stimulating local T cells in vitro and of concentrating in the tumour in AML xenografts. It also highlights the importance of 89Zr-immunoPET to guide the development and selection of tumour-targeted antibody-cytokine fusion proteins.

Electrostatically Mediated Attractive Self-Interactions and Reversible Self-Association of Fc-Fusion Proteins.

Attractive self-interactions and reversible self-association are implicated in many problematic solution behaviors for therapeutic proteins, such as irreversible aggregation, elevated viscosity, phase separation, and opalescence. Protein self-interactions and reversible oligomerization of two Fc-fusion proteins (monovalent and bivalent) and the corresponding fusion partner protein were characterized experimentally with static and dynamic light scattering as a function of pH (5 and 6.5) and ionic strength (10 mM to at least 300 mM). The fusion partner protein and monovalent Fc-fusion each displayed net attractive electrostatic self-interactions at pH 6.5 and net repulsive electrostatic self-interactions at pH 5. Solutions of the bivalent Fc-fusion contained higher molecular weight species that prevented quantification of typical interaction parameters (B22 and kD). All three of the proteins displayed reversible self-association at pH 6.5, where oligomers dissociated with increased ionic strength. Coarse-grained molecular simulations were used to model the self-interactions measured experimentally, assess net self-interactions for the bivalent Fc-fusion, and probe the specific electrostatic interactions between charged amino acids that were involved in attractive electrostatic self-interactions. Mayer-weighted pairwise electrostatic energies from the simulations suggested that attractive electrostatic self-interactions at pH 6.5 for the two Fc-fusion proteins were due to cross-domain interactions between the fusion partner domain(s) and the Fc domain.

Induction therapy in heart transplantation: A systematic review and network meta-analysis for developing evidence-based recommendations.

INTRODUCTION: Induction therapy (IT) utility in heart transplantation (HT) remains contested. Commissioned by a clinical-practice guidelines panel to evaluate the effectiveness and safety of IT in adult HT patients, we conducted this systematic review and network meta-analysis (NMA). METHODS: We searched for studies from January 2000 to October 2022, reporting on the use of any IT agent in adult HT patients. Based on patient-important outcomes, we performed frequentist NMAs separately for RCTs and observational studies with adjusted analyses, and assessed the certainty of evidence using the GRADE framework. RESULTS: From 5156 publications identified, we included 7 RCTs and 12 observational studies, and report on two contemporarily-used IT agents-basiliximab and rATG. The RCTs provide only very low certainty evidence and was uninformative of the effect of the two agents versus no IT or one another. With low certainty in the evidence from observational studies, basiliximab may increase 30-day (OR 1.13; 95% CI 1.06-1.20) and 1-year (OR 1.11; 95% CI 1.02-1.22) mortality compared to no IT. With low certainty from observational studies, rATG may decrease 5-year cardiac allograft vasculopathy (OR .82; 95% CI .74-.90) compared to no IT, as well as 30-day (OR .85; 95% CI .80-.92), 1-year (OR .87; 95% CI .79-.96), and overall (HR .84; 95% CI .76-.93) mortality compared to basiliximab. CONCLUSION: With low and very low certainty in the synthetized evidence, these NMAs suggest possible superiority of rATG compared to basiliximab, but do not provide compelling evidence for the routine use of these agents in HT recipients.

LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides.

An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) Escherichia coli strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against Staphylococcus aureus, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae bacteria clinical isolates at a lower minimum inhibition concentration level (10-33 µM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50-100 µM). KEY POINTS: • The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker. • The peptide was expressed with the carrier proteins SmbP and CusF3H+. • The hybrid peptide shows antibacterial potency against clinical bacterial isolates.

Fusion proteins development strategies and their role as cancer therapeutic agents.

Cancer continues to be leading cause of morbidity and mortality despite decades of research and advancement in chemotherapy. Most tumors can be reduced via standard oncology treatments, such as chemotherapy, radiotherapy, and surgical resection, and they frequently recur. Significant progress has been made since targeted cancer therapy inception in creation of medications that exhibit improved tumor-selective action. Particularly in preclinical and clinical investigations, fusion proteins have shown strong activity and improved treatment outcomes for a number of human cancers. Synergistically combining many proteins into one complex allows the creation of synthetic fusion proteins with enhanced characteristics or new capabilities. Signal transduction pathways are important for onset, development, and spread of cancer. As result, signaling molecules are desirable targets for cancer therapies, and significant effort has been made into developing fusion proteins that would act as inhibitors of these pathways. A wide range of biotechnological and medicinal applications are made possible by fusion of protein domains that improves bioactivities or creates new functional combinations. Such proteins may function as immune effectors cell recruiters to tumors or as decoy receptors for various ligands. In this review article, we have outlined the standard methods for creating fusion proteins and covered the applications of fusion proteins in treatment of cancer. This article also highlights the role of fusion proteins in targeting the signaling pathways involved in cancer for effective treatment.

From antibiotic to antiviral: computational screening reveals a multi-targeting antibiotic from Streptomyces spp. against Nipah virus fusion proteins.

Nipah Virus is a re-emerging zoonotic paramyxovirus that poses a significant threat to both swine industry and human health. The pursuit of potential antiviral agents with both preventive and therapeutic properties holds promise for targeting such viruses. To expedite this search, leveraging computational biology is essential. Streptomyces is renowned for its capacity to produce large and diverse metabolites with promising bioactivities. In the current study, we conducted a comprehensive structure-based virtual screening of 6524 Streptomyces spp. metabolites sourced from the StreptomeDB database to evaluate their potential inhibitory effects on three Nipah virus fusion (NiVF) protein conformations: NiVF pre-fusion 1-mer (NiVF-1mer), pre-fusion 3-mer (NiVF-3mer), and NiVF post-fusion (NiVF-PoF). Prior to virtual screening, the drug-likeness of Streptomyces spp. compounds was profiled using ADMET properties. From the 913 ADMET-filtered compounds, the subsequent targeted and confirmatory blind docking analysis revealed that S896 or virginiamycin M1, a known macrolide antibiotic, showed a maximum binding affinity with the NiVF proteins, suggesting a multi-targeting inhibitory property. In addition, the 200-ns molecular dynamics simulation and MM/PBSA analyses revealed stable and strong binding affinity between the NiVF-S896 complexes, indicating favorable interactions between S896 and the target proteins. These findings suggest the potential of virginiamycin M1, an antibiotic, as a promising multi-targeting antiviral drug. However, in vitro and in vivo experimental validations are necessary to assess their safety and efficacy.

The Molecular and Biological Function of MEF2D in Leukemia.

Myocyte enhancer factor 2 (MEF2) is a key transcription factor (TF) in skeletal, cardiac, and neural tissue development and includes four isoforms: MEF2A, MEF2B, MEF2C, and MEF2D. These isoforms significantly affect embryonic development, nervous system regulation, muscle cell differentiation, B- and T-cell development, thymocyte selection, and effects on tumorigenesis and leukemia. This chapter describes the multifaceted roles of MEF2 family proteins, covering embryonic development, nervous system regulation, and muscle cell differentiation. It further elucidates the contribution of MEF2 to various blood and immune cell functions. Specifically, in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), MEF2D is aberrantly expressed and forms a fusion protein with BCL9, CSF1R, DAZAP1, HNRNPUL1, and SS18. These fusion proteins are closely related to the pathogenesis of leukemia. In addition, it specifically introduces the regulatory effect of MEF2D fusion protein on the proliferation and growth of B-cell acute lymphoblastic leukemia (B-ALL) cells. Finally, we detail the positive feedback loop between MEF2D and IRF8 that significantly promotes the progression of acute myeloid leukemia (AML) and the importance of the ZMYND8-BRD4 interaction in regulating the IRF8 and MYC transcriptional programs. The MEF2D-CEBPE axis is highlighted as a key transcriptional mechanism controlling the block of leukemic cell self-renewal and differentiation in AML. This chapter starts with the structure and function of MEF2 family proteins, specifically summarizing and analyzing the role of MEF2D in B-ALL and AML, mediating the complex molecular mechanisms of transcriptional regulation and exploring their implications for human health and disease.

Optimum timing of antithymocyte globulin in relation to adoptive regulatory T cell therapy.

Reducing the recipient's T cell repertoire is considered to increase the efficacy of regulatory T cell (Treg) therapy. This necessitates timing the administration of antithymocyte globulin (ATG) early enough before adoptive cell therapy (ACT) so that residual serum ATG does not deplete the transferred Tregs. The optimum time point in this regard has not been defined. Herein, we report the effects of residual serum ATG on the viability of an in vitro expanded Treg cell product used in a clinical trial of ACT in kidney transplant recipients (NCT03867617). Patients received ATG monotherapy (either 6 or 3 mg/kg body weight) without concomitant immunosuppression 2 to 3 weeks before transplantation and Treg transfer. An anti-ATG immunoglobulin G (IgG) immune response was elicited in all patients within 14 days. In turn, the elimination of total and Treg-specific ATG was accelerated substantially over control patients receiving the same dose of ATG with concomitant immunosuppression. However, ATG serum concentrations of <1 µg/mL, which had previously been reported as subtherapeutic threshold, triggered apoptosis of Tregs in vitro. Therefore, ATG levels need to decline to lower levels than those previously thought for efficacious Treg transfer. In 5 of 6 patients, such low levels of serum ATG considered safe for Treg transfer were reached within 2 weeks after ATG administration.

Anti-HLA antibodies in recipients of CD19 versus BCMA-targeted CAR T-cell therapy.

Antibodies against foreign human leukocyte antigen (HLA) molecules are barriers to successful organ transplantation. B cell-depleting treatments are used to reduce anti-HLA antibodies but have limited efficacy. We hypothesized that the primary source for anti-HLA antibodies is long-lived plasma cells, which are ineffectively targeted by B cell depletion. To study this, we screened for anti-HLA antibodies in a prospectively enrolled cohort of 49 patients who received chimeric antigen receptor T-cell therapy (CARTx), targeting naïve and memory B cells (CD19-targeted, n = 21) or plasma cells (BCMA-targeted, n = 28) for hematologic malignancies. Longitudinal samples were collected before and up to 1 year after CARTx. All individuals were in sustained remission. We identified 4 participants with anti-HLA antibodies before CD19-CARTx. Despite B cell depletion, anti-HLA antibodies and calculated panel reactive antibody scores were stable for 1 year after CD19-CARTx. Only 1 BCMA-CARTx recipient had pre-CARTx low-level anti-HLA antibodies, with no follow-up samples available. These data implicate CD19neg long-lived plasma cells as an important source for anti-HLA antibodies, a model supported by infrequent HLA sensitization in BCMA-CARTx subjects receiving previous plasma cell-targeted therapies. Thus, plasma cell-targeted therapies may be more effective against HLA antibodies, thereby enabling improved access to organ transplantation and rejection management.

CD20/TNFR1 dual-targeting antibody enhances lysosome rupture-mediated cell death in B cell lymphoma.

Obinutuzumab is a therapeutic antibody for B cell non-Hodgkin's Lymphoma (BNHL), which is a glyco-engineered anti-CD20 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and causes binding-induced direct cell death (DCD) through lysosome membrane permeabilization (LMP). Tumour necrosis factor receptor 1 (TNFR1), a pro-inflammatory death receptor, also evokes cell death, partly through lysosomal rupture. As both obinutuzumab- and TNFR1-induced cell deaths are mediated by LMP and combining TNFR1 and obinutuzumab can amplify LMP-mediated cell death, we made dual-targeting antibody for CD20 and TNFR1 to enhance DCD of obinutuzumab.Obinutuzumab treatment-induced CD20 and TNFR1 colocalisation, and TNFR1-overexpressing cells showed increased obinutuzumab-induced DCD. Two targeting modes, anti-CD20/TNFR1 bispecific antibodies (bsAbs), and obinutuzumab-TNFα fusion proteins (OBI-TNFαWT and OBI-TNFαMUT), were designed to cluster CD20 and TNFR1 on the plasma membrane. OBI-TNFαWT and OBI-TNFαMUT showed significantly enhanced LMP, DCD, and ADCC compared with that induced by obinutuzumab. TNFR1 expression is upregulated in many BNHL subtypes compared to that in normal B cells; OBI-TNFαMUT specifically increased DCD and ADCC in a B cell lymphoma cell line overexpressing TNFR1. Further, OBI-TNFαMUT blocked NF-κB activation in the presence of TNF-α, implying that it can antagonise the proliferative role of TNF-α in cancers.Our study suggests that dual targeting of CD20 and TNFR1 can be a new therapeutic strategy for improving BNHL treatment. The OBI-TNFαMUT fusion protein enhances DCD and ADCC and prevents the proliferating effect of TNFα signalling; therefore, it may provide precision treatment for patients with BNHL, especially those with upregulated TNFR1 expression.