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Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell–cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ β = 13.5 h) than soluble ACE2 (t½ β = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. Statement of significance: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-β, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment., Deutsche Krebshilfe, Projekt DEAL

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with Endometrial Stromal Sarcomas (ESS) and Ossifying FibroMyxoid Tumors (OFMT) leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a co-activator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a mega-complex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome. These are linked to aberrant gene expression, in particular over an entire topologically-associated domain including part of the HOXD cluster. Furthermore, we show that JAZF1, implicated with PRC2 components in the most frequent translocations in ESS, is a potent transcription activator that physically associates with NuA4/TIP60. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas employ the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes leading to mislocalization of histone marks and aberrant polycomb target gene expression. HighlightsO_LIRecurrent oncogenic chromosomal translocations fuse Polycomb-like-1 (PHF1) with different subunits of the NuA4/TIP60 complex C_LIO_LITranslocation produces a mega-complex containing NuA4/TIP60 and PRC2 activities C_LIO_LITranslocation leads to mistargeting of histone marks throughout the genome and changes in gene expression C_LIO_LILoss of H3K27me3 and gain of H4ac on HOXD cluster are restricted to a topologically-associated domain (TAD) C_LIO_LIA highly recurrent JAZF1-SUZ12 translocation uses the same mechanism merging NuA4/TIP60 with PRC2 C_LI

Purpose Hemophilia B is a bleeding disorder, caused by a factor IX (FIX) deficiency. Recently, FIX concentrates with extended half-life (EHL) have become available. Prophylactic dosing of EHL-FIX concentrates can be optimized by assessment of individual pharmacokinetic (PK) parameters. To determine these parameters, limited sampling strategies (LSSs) may be applied. The study aims to establish adequate LSSs for estimating individual PK parameters of EHL-FIX concentrates using in silico evaluation. Methods Monte Carlo simulations were performed to obtain FIX activity versus time profiles using published population PK models for N9-GP (Refixia), rFIXFc (Alprolix), and rIX-FP (Idelvion). Fourteen LSSs, containing three or four samples taken within 8 days after administration, were formulated. Bayesian analysis was applied to obtain estimates for clearance (CL), half-life (t1/2), time to 1% (Time1%), and calculated weekly dose (Dose1%). Bias and precision of these estimates were assessed to determine which LSS was adequate. Results For all PK parameters of N9-GP, rFIXFc and rIX-FP bias was generally acceptable (range: −5% to 5%). For N9-GP, precision of all parameters for all LSSs was acceptable (1%, except for t1/2 (range: 27.1% to 44.7%) and Dose1% (range: 12% to 29.4%). For rIX-FP, all LSSs showed acceptable bias and precision, except for Dose1% using LSS with the last sample taken on day 3 (LSS 6 and 10). Conclusion Best performing LSSs were LSS with samples taken at days 1, 5, 7, and 8 (N9-GP and rFIXFc) and at days 1, 4, 6, and 8 (rIX-FP), respectively.

The development of safe and efficacious enzyme-based human therapies has increased greatly in the last decades, thanks to remarkable advances in the understanding of the molecular mechanisms responsible for different diseases, and the characterization of the catalytic activity of relevant exogenous enzymes that may play a remedial effect in the treatment of such pathologies. Several enzyme-based biotherapeutics have been approved by FDA (the U.S. Food and Drug Administration) and EMA (the European Medicines Agency) and many are undergoing clinical trials. Apart from enzyme replacement therapy in human genetic diseases, which is not discussed in this review, approved enzymes for human therapy find applications in several fields, from cancer therapy to thrombolysis and the treatment, e.g., of clotting disorders, cystic fibrosis, lactose intolerance and collagen-based disorders. The majority of therapeutic enzymes are of microbial origin, the most convenient source due to fast, simple and cost-effective production and manipulation. The use of microbial recombinant enzymes has broadened prospects for human therapy but some hurdles such as high immunogenicity, protein instability, short half-life and low substrate affinity, still need to be tackled. Alternative sources of enzymes, with reduced side effects and improved activity, as well as genetic modification of the enzymes and novel delivery systems are constantly searched. Chemical modification strategies, targeted- and/or nanocarrier-mediated delivery, directed evolution and site-specific mutagenesis, fusion proteins generated by genetic manipulation are the most explored tools to reduce toxicity and improve bioavailability and cellular targeting. This review provides a description of exogenous enzymes that are presently employed for the therapeutic management of human diseases with their current FDA/EMA-approved status, along with those already experimented at the clinical level and potential promising candidates.

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline–threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540–588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

Over the past 20 years, the rapid evolution in the diagnosis and treatment of primary immunodeficiencies (PI) and the recognition of immune dysregulation as a feature in some have prompted the use of "inborn errors of immunity" (IEI) as a more encompassing term used to describe these disorders [1, 2] . This article aims to review the future of therapy of PI/IEI (referred to IEI throughout this paper). Historically, immune deficiencies have been characterized as monogenic disorders resulting in immune deficiencies affecting T cells, B cells, combination of T and B cells, or innate immune disorders. More recently, immunologists are also recognizing a variety of phenotypes associated with one genotype or similar phenotypes across genotypes and a role for incomplete penetrance or variable expressivity of some genes causing inborn errors of immunity [3]. The IUIS classification of immune deficiencies (IEIs) has evolved over time to include 10 categories, with disorders of immune dysregulation accounting for a new subset, some treatable with small molecule inhibitors or biologics. [1] Until recently, management options were limited to prompt treatment of infections, gammaglobulin replacement, and possibly bone marrow transplant depending on the defect. Available therapies have expanded to include small molecule inhibitors, biologics, gene therapy, and the use of adoptive transfer of virus-specific T cells to fight viral infections in immunocompromised patients. Several significant contributions to the field of clinical immunology have fueled the rapid advancement of therapies over the past two decades. Among these are educational efforts to recruit young immunologists to the field resulting in the growth of a world-wide community of clinicians and investigators interested in rare diseases, efforts to increase awareness of IEI globally contributing to international collaborations, along with advancements in diagnostic genetic testing, newborn screening, molecular biology techniques, gene correction, use of immune modulators, and ex vivo expansion of engineered T cells for therapeutic use. The development and widespread use of newborn screening have helped to identify severe combined immune deficiency (SCID) earlier resulting in better outcomes [4]. Continual improvements and accessibility of genetic sequencing have helped to identify new IEI diseases at an accelerated pace [5]. Advances in gene therapy and bone marrow transplant have made treatments possible in otherwise fatal diseases. Furthermore, the increased awareness of IEI across the world has driven networks of immunologists working together to improve the diagnosis and treatment of these rare diseases. These improvements in the diagnosis and treatment of IEI noted over the past 20 years bring hope for a better future for the IEI community. This paper will review future directions in a few of the newer therapies emerging for IEI. For easy reference, most of the diseases discussed in this paper are briefly described in a summary table, in the order mentioned within the paper (Appendix).

Biological drugs or biopharmaceuticals off patent open a large market for biosimilars and biobetters, follow-on biologics. Biobetters, in particular, are new drugs designed from existing ones with improved properties such as higher selectivity, stability, half-life and/or lower toxicity/immunogenicity. Glycosylation is one of the most used strategies to improve biological drugs, nonetheless bioconjugation is an additional alternative and refers to the covalent attachment of polymers to biological drugs. Extensive research on novel polymers is underway, nonetheless PEGylation is still the best alternative with the longest clinical track record. Innovative trends based on genetic engineering techniques such as fusion proteins and PASylation are also promising. In this review, all these alternatives were explored as well as current market trends, legislation and future perspectives.

Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high β-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the β-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a β-strand and that the subsequent parallel β-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.

Clostridium perfringens is a potent producer of a variety of toxins. Well studied from these are five toxins (alpha, Beta (CPB), epsilon, iota and CPE) that are produced by seven toxinotype strains (A–G) of C. perfringens. Besides these toxins, C. perfringens produces also another toxin that causes necrotizing enterocolitis in piglets. This toxin termed consensus Beta2 toxin (cCPB2) has a molecular mass of 27,620 Da and shows only little homology to CPB and no one to the other toxins of C. perfringens. Its primary action on cells remained unknown to date. cCPB2 was heterogeneously expressed as fusion protein with GST in Escherichia coli and purified to homogeneity. Although cCPB2 does not exhibit the typical structure of beta-stranded pore-forming proteins and contains no indication for the presence of amphipathic alpha-helices we could demonstrate that cCPB2 is a pore-forming component with an extremely high activity in lipid bilayers. The channels have a single-channel conductance of about 700 pS in 1 M KCl and are highly cation-selective as judged from selectivity measurements in the presence of salt gradients. The high cation selectivity is caused by the presence of net negative charges in or near the channel that allowed an estimate of the channel size being about 1.4 nm wide. Our measurements suggest that the primary effect of cCPB2 is the formation of cation-selective channels followed by necrotic enteritis in humans and animals. We searched in databases for homologs of cCPB2 and constructed a cladogram representing the phylogenetic relationship to the next relatives of cCPB2.

Since the FDA approval of the first therapeutic antibody 35 years ago, antibody-based products have gained prominence in the pharmaceutical market. Building on the early successes of monoclonal antibodies, more recent efforts have capitalized on the exquisite specificity and/or favorable pharmacokinetic properties of antibodies by developing fusion proteins that enable targeted delivery of therapeutic payloads which are otherwise ineffective when administered systemically. This review focuses on recent engineering and translational advances for therapeutics that genetically fuse antibodies to disease-relevant payloads, including cytokines, toxins, enzymes, neuroprotective agents, and soluble factor traps. With numerous antibody fusion proteins in the clinic and other innovative molecules poised to follow suit, these potent, multifunctional drug candidates promise to be a major player in the therapeutic development landscape for years to come.

Preeclampsia is characterized by the development of elevated blood pressure during the second and third trimesters of pregnancy that is accompanied by end organ dysfunction. The pathogenesis of preeclampsia is multifactorial but is commonly characterized by endothelial dysfunction and the overproduction of antiangiogenic factors, including the soluble VEGF (vascular endothelial growth factor) receptor sFlt-1 (soluble Fms-like tyrosine kinase receptor 1). Previously, administration of exogenous VEGF-A, bound to a carrier protein called ELP (elastin-like polypeptide), significantly reduced free sFlt-1 levels and attenuated the hypertensive response in a rodent model of preeclampsia. However, VEGF-A administration induces multifactorial effects mediated through its direct activation of the Flk-1 receptor. In response to this, we developed a therapeutic chimera using ELP bound to VEGF-B, a VEGF isoform that binds to sFlt-1 but not to Flk-1. The purpose of this study was to evaluate the in vitro activity and pharmacological properties of ELP-VEGF-B and to test its efficacy in the reduced uterine perfusion pressure rat model of placental ischemia. ELP-VEGF-B was less potent than ELP-VEGF-A in stimulation of endothelial cell proliferation and matrix invasion, indicating that it is a weaker angiogenic driver. However, after repeated subcutaneous administration in pregnant rats, ELP-VEGF-B was maternally sequestered and reduced blood pressure when compared with saline treated animals following induction of placental ischemia (123.38±11.4 versus 139.98±10.56 mm Hg, P =0.0129). Blood pressure reduction was associated with a restoration of the angiogenic capacity of plasma from rats treated with ELP-VEGF-B. ELP-VEGF-B is a nonangiogenic, maternally sequestered protein with potential efficacy for treatment of preeclampsia.

In our previous study, we reported the design and recombinant production of the p28-apoptin as a novel chimeric protein for breast cancer (BC) treatment. This study aimed to evaluate the inhibitory activity of the chimeric protein against BC cells in vitro and in vivo. We developed a novel multifunctional protein, consisting of p28, as a tumor-homing killer peptide fused to apoptin as a tumor-selective killer. The chimeric protein showed significantly higher toxicity in BC cell lines dose-dependently than in non-cancerous control cell lines. IC50 values were 1.41 μM, 1.38 μM, 6.13 μM, and 264.49 μM for 4T1, MDA-MB-468, Vero, and HEK293 cells, respectively. The protein showed significantly enhanced uptake in 4T1 cancer cells compared with non-cancerous Vero cells. We also showed that the p28-apoptin chimeric protein binds significantly higher to human breast cancer tumor sections than the normal human breast tissue section. Also, significant apoptosis induction and tumor growth inhibition were observed in established tumor-bearing mice accompanied by a decreased frequency of metastases. Our results support that the chimeric protein has inhibitory activity in vitro and in vivo, making it a promising choice in targeted cancer therapy. This article is protected by copyright. All rights reserved.

A new and efficient purification process for recombinant human insulin production was developed by exploring new resins and optimizing purification steps from E. coli inclusion body washing to insulin polishing. A combined additives inclusion body wash protocol drastically improved efficiency in clarifying ZZ-proinsulin samples. ZZ-proinsulin recovery increased three-fold under optimized solubilization and sulfitolysis incubation temperature and duration. Desalting with Bio-Gel P4 and P6 resulted in higher sample loading and product recovery compared to conventional resins. A higher recovery (96%) and purity (81%) of ZZ-proinsulin were achievable with the Nuvia S cation exchanger for proinsulin purification compared to a reported process using expensive affinity chromatography resin. As the first step for insulin purification, process scale-up is more economical and practical when Nuvia HR-S cation exchanger was used instead of commonly used reversed-phase chromatography. Nuvia HR-S was highly effective in removing ZZ fusion protein (90% removal) after enzymatic cleavage, although ZZ fusion protein has a very close theoretical pI to human insulin, which was supposedly challenging to be removed by cation exchange chromatography. Also, insulin can be eluted at a lower ethanol % using Nuvia HR-S compared to other reported processes and is thus more environmentally sustainable. Recombinant human insulin was obtained with over 98% purity in just a single reversed-phase polishing step, which is comparable to the reference standard. The process workflow presented here can be potentially applied for the development of purification workflow for insulin analogs or other peptide products derived from E. coli inclusion body.Key points• Drastic efficiency improvement for inclusion body wash with combined additives.• High recovery of proinsulin purification with high capacity cation exchange resin.• Effective removal of fusion tag at lower ethanol % with high-resolution resin.

Ligand inducible proteins that enable precise and reversible control of nuclear translocation of passenger proteins have broad applications ranging from genetic studies in mammals to therapeutics that target diseases such as cancer and diabetes. One of the drawbacks of the current translocation systems is that the ligands used to control nuclear localization are either toxic or prone to crosstalk with endogenous protein cascades within live animals. We sought to take advantage of salicylic acid (SA), a small molecule that has been extensively used in humans. In plants, SA functions as a hormone that can mediate immunity and is sensed by the non-expressor of pathogenesis-related (NPR) proteins. Although it is well recognized that nuclear translocation of NPR1 is essential to promoting immunity in plants, the exact subdomain of Arabidopsis thaliana NPR1 (AtNPR1) essential for SA mediated nuclear translocation is controversial. Here, we utilized the fluorescent protein mCherry as the reporter to investigate the ability of SA to induce nuclear translocation of the full-length NPR1 protein or its C-terminal transactivation (TAD) domain using HEK293 cells as a heterologous system. HEK293 cells lack accessory plant proteins including NPR3/NPR4 and are thus ideally suited for studying the impact of SA-induced changes in NPR1. Our results obtained using a stable expression system show that the TAD of AtNPR1 is sufficient to enable the reversible SA mediated nuclear translocation of mCherry. Our studies advance a basic understanding of nuclear translocation mediated by the TAD of AtNPR1 and uncover a biotechnological tool for SA mediated nuclear localization. This article is protected by copyright. All rights reserved.

Mixed lineage leukemia (MLL) gene rearrangements are associated with acute leukemia. The protein menin is regarded as a critical oncogenic cofactor of the resulting MLL fusion proteins in acute leukemia. A direct interaction between menin and the MLL amino terminal sequences is necessary for MLL fusion protein-mediated leukemogenesis. Thus, inhibition of the interaction between menin and MLL has emerged as a novel therapeutic strategy. Recent improvements in structural biology and chemical reactivity have promoted the design and development of selective and potent menin-MLL interaction inhibitors. In this Perspective, different classes of menin-MLL interaction inhibitors are comprehensively summarized. Further research potential, challenges, and opportunities in the field are also discussed.

Pomalidomide and lenalidomide are immunomodulatory agents that were derived from thalidomide. Cereblon (CRBN) is a common direct target of thalidomide and related compounds and works as a Cullin Ring 4 E3 ubiquitin ligase (CRL4) with DDB1, CUL4, and ROC1. The substrate specificity of CRL4CRBN is modulated by thalidomide-related compounds. While lenalidomide is approved for the treatment of several diseases including multiple myeloma, 5q- syndrome, mantle cell lymphoma, and follicular lymphoma, pomalidomide is approved only for the treatment of lenalidomide-resistant multiple myeloma. Here we show that PLZF/ZBTB16 and its fusion proteins are pomalidomide-dependent neosubstrates of CRL4CRBN. PLZF joins to RARα or potentially other partner genes, and the translocation causes leukemias, such as acute promyelocytic leukemia and T-cell acute lymphoblastic leukemia. We demonstrate that pomalidomide treatment induces PLZF-RARα degradation, resulting in antiproliferation of leukemic cells expressing PLZF-RARα. This study highlights a potential therapeutic role of pomalidomide as a degrader of leukemogenic fusion proteins., Shimizu et al. identify PLZF/ZBTB16 and its fusion proteins as pomalidomide-dependent neosubstrates of the cereblon E3 ligase. The authors also report that pomalidomide induces their degradation and thereby exerts its anti-cancer effect in leukemic cells expressing the fusion proteins.

Chromosomal rearrangements can generate genetic fusions composed of two distinct gene sequences, many of which have been implicated in tumorigenesis and progression. Our study proposes a model whereby oncogenic gene fusions frequently alter the protein stability of the resulting fusion products, via exchanging protein degradation signal (degron) between gene sequences. Computational analyses of The Cancer Genome Atlas (TCGA) identify 2,406 cases of degron exchange events and reveal an enrichment of oncogene stabilization due to loss of degrons from fusion. Furthermore, we identify and experimentally validate that some recurrent fusions, such as BCR-ABL, CCDC6-RET and PML-RARA fusions, perturb protein stability by exchanging internal degrons. Likewise, we also validate that EGFR or RAF1 fusions can be stabilized by losing a computationally-predicted C-terminal degron. Thus, complementary to enhanced oncogene transcription via promoter swapping, our model of degron loss illustrates another general mechanism for recurrent fusion proteins in driving tumorigenesis., The impact of genetic fusions on degrons, which are motifs for ubiquitin-mediated protein degradation, has not been fully explored. Here, the authors analyse fusion genes affecting degrons in pan-cancer genomics data, validate their functional impact and find enrichment for both internal and C-terminal degron losses.

Domain swapping is a mechanism of protein oligomerization by which two or more subunits exchange structural elements to generate an intertwined complex. Numerous studies support a diversity of swapping mechanisms in which structural elements can be exchanged at different stages of the folding pathway of a subunit. Here, we used single-molecule optical tweezers technique to analyze the swapping mechanism of the forkhead DNA-binding domain of human transcription factor FoxP1. FoxP1 populates folded monomers in equilibrium with a swapped dimer. We generated a fusion protein linking two FoxP1 domains in tandem to obtain repetitive mechanical folding and unfolding trajectories. Thus, by stretching the same molecule several times, we detected either the independent folding of each domain or the elusive swapping step between domains. We found that a swapped dimer can be formed directly from fully or mostly folded monomer. In this situation, the interaction between the monomers in route to the domain-swapped dimer is the rate-limiting step. This approach is a useful strategy to test the different proposed domain swapping mechanisms for proteins with relevant physiological functions.

Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.

Laminins are heterotrimeric ECM proteins composed of α, β, and γ chains. The γ2 chain (Lm‐γ2) is a frequently expressed monomer and its expression is closely associated with cancer progression. Laminin‐γ2 contains an epidermal growth factor (EGF)‐like domain in its domain III (DIII or LEb). Matrix metalloproteinases can cleave off the DIII region of Lm‐γ2 that retains the ligand activity for EGF receptor (EGFR). Herein, we show that a novel short form of Lm‐γ2 (Lm‐γ2F) containing DIII is generated without requiring MMPs and chromosomal translocation between LAMC2 on chromosome 1 and NR6A1 gene locus on chromosome 9 in human ovarian cancer SKOV3 cells. Laminin‐γ2F is expressed as a truncated form lacking domains I and II, which are essential for its association with Lm‐α3 and ‐β3 chains of Lm‐332. Secreted Lm‐γ2F can act as an EGFR ligand activating the EGFR/AKT pathways more effectively than does the Lm‐γ2 chain, which in turn promotes proliferation, survival, and motility of ovarian cancer cells. LAMC2‐NR6A1 translocation was detected using in situ hybridization, and fusion transcripts were expressed in ovarian cancer cell tissues. Overexpression and suppression of fusion transcripts significantly increased and decreased the tumorigenic growth of cells in mouse models, respectively. To the best of our knowledge, this is the first report regarding a fusion gene of ECM showing that translocation of LAMC2 plays a crucial role in the malignant growth and progression of ovarian cancer cells and that the consequent product is a promising therapeutic target against ovarian cancers., Expression of LAMC2‐NR6A1 fusion transcripts in human ovarian carcinomas.

The tumor suppressor p16 protein is an endogenous CDK4/6 inhibitor. Inactivation of its encoding gene is found in nearly half of human cancers. Restoration of p16 function via adenovirus-based gene delivery has been shown to be effective in suppressing aberrant cell growth in many types of cancer, however, the potential risk of insertional mutagenesis in genomic DNA remains a major concern. Thus, there has been great interest in developing efficient strategies to directly deliver proteins into cells as an alternative that can avoid such safety concerns while achieving a comparable therapeutic effect. Nevertheless, intracellular delivery of protein therapeutics remains a challenge. Our group has recently developed a protein delivery platform based on an engineered Pos3Aa protein that forms sub-micrometer-sized crystals in Bacillus thuringiensis cells. In this report, we describe the further development of this platform (Pos3AaTM) via rationally designed site-directed mutagenesis, and its resultant potency for the delivery of cargo proteins into cells. Pos3AaTM-based fusion protein crystals are shown to exhibit improved release of their cargo proteins as demonstrated using a model mCherry protein. Importantly, this Pos3AaTM platform is able to mediate the efficient intracellular delivery of p16 protein with significant endosomal escape, resulting in p16-mediated inhibition of CDK4/6 kinase activity and Rb phosphorylation, and as a consequence, significant cell cycle arrest and cell growth inhibition. These results validate the ability of these improved Pos3AaTM crystals to mediate enhanced cytosolic protein delivery and highlight the potential of using protein therapeutics as selective CDK4/6 inhibitors for cancer therapy. Statement of significance Cytosolic delivery of bioactive therapeutic proteins capable of eliciting therapeutic benefit remains a significant challenge. We have previously developed a protein delivery platform based on engineered Pos3Aa protein crystals with excellent cell-permeability and endosomal escape properties. In this report, we describe the rational design of an improved Pos3Aa triple mutant (Pos3AaTM) with enhanced cargo release. We demonstrate that Pos3AaTM-mCherry-p16 fusion crystals can efficiently deliver p16 protein, a CDK4/6 inhibitor frequently inactivated in human cancers, into p16-deficient UM-SCC-22A cells, where it promotes significant G1 cell cycle arrest and cell growth inhibition. These results highlight the ability of the Pos3AaTM platform to promote potent cytosolic delivery of protein therapeutics, and the efficacy of p16 protein delivery as an effective strategy for treating cancer.

There is an increasing demand for monoclonal antibody (mAb) therapies to confer passive immunity against viral diseases. Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis, lower respiratory tract infections, and hospitalization in infants. Currently, there is no RSV vaccine but a humanized mAb available for high risk infants. MK-1654 is a fully human mAb with YTE mutation in the fragment crystallizable (Fc) region to extend the half-life in circulation. It binds to a highly conserved epitope of RSV Fusion protein with high affinity and neutralizes RSV infection. A functional cell-based assay is a regulatory requirement for clinical development, commercial release, and stability testing of MK-1654. In this study, we have evaluated three RSV neutralization assays to test the potency of MK-1654, including an imaging-based virus reduction neutralization test (VRNT) and two reporter virus-based assays (RSV-GFP and RSV-NLucP). All three methods showed good dose response curves of MK-1654 with similar EC50 values. RSV-NLucP method was chosen for further development because it is simple and can be easily adapted to quality control testing laboratories. After optimization, the RSV-NLucP assay was pre-qualified with good linearity, relative accuracy, intermediate precision, and specificity, therefore suitable for a cell-based potency assay.

Genomic analysis has recently identified multiple ESR1 gene translocations in estrogen receptor alpha–positive (ERα+) metastatic breast cancer (MBC) that encode chimeric proteins whereby the ESR1 ligand binding domain (LBD) is replaced by C-terminal sequences from many different gene partners. Here we functionally screened 15 ESR1 fusions and identified 10 that promoted estradiol-independent cell growth, motility, invasion, epithelial-to-mesenchymal transition, and resistance to fulvestrant. RNA sequencing identified a gene expression pattern specific to functionally active ESR1 gene fusions that was subsequently reduced to a diagnostic 24-gene signature. This signature was further examined in 20 ERα+ patient-derived xenografts and in 55 ERα+ MBC samples. The 24-gene signature successfully identified cases harboring ESR1 gene fusions and also accurately diagnosed the presence of activating ESR1 LBD point mutations. Therefore, the 24-gene signature represents an efficient approach to screening samples for the presence of diverse somatic ESR1 mutations and translocations that drive endocrine treatment failure in MBC. Significance: This study identifies a gene signature diagnostic for functional ESR1 fusions that drive poor outcome in advanced breast cancer, which could also help guide precision medicine approaches in patients harboring ESR1 mutations.

Undergraduate laboratory courses are essential to teaching core principles in STEM. This course, Quantitative Biological Methods, provides a unique approach to teaching molecular biology research techniques to students, in a laboratory that is delivered in a sequence that parallels standard biomedical research laboratory protocols. Students attend a lecture where they are taught the essential principles of biomedical research, and a lab where they learn to use laboratory equipment, perform experiments, and purify and quantify DNA and proteins. The course begins with an introduction to laboratory safety, pipetting, centrifugation, spectrophotometry, and other basic laboratory techniques. Next, the lab focuses on the purification and analysis of glutathione S-transferase (GST) fused to green fluorescent protein (GFP) from an Escherichia coli lysate. Students study this GST-GFP fusion protein and perform protein quantification, enzyme assays, chromatography, fluorescent detection, normalization, SDS-PAGE, and western blotting. Students then learn recombinant DNA technology using the GST-GFP vector that was the source of the fusion protein in the prior labs, and perform ligation, transformation of E. coli cells, blue/white screening, DNA purification via a miniprep, PCR, DNA quantification, restriction enzyme digestion, and agarose gel electrophoresis. Students write laboratory reports to demonstrate an understanding of the principles of the laboratory methods, and they must present and critically analyze their data. The lab methods described herein aim to emphasize the core molecular biology principles and techniques, prepare students for work in a biomedical research laboratory, and introduce students to both GST and GFP, two versatile laboratory proteins.

CD47, a transmembrane protein, acts as a "do not eat me" signal that is overexpressed in many tumor cell types, thereby forming a signaling axis with its ligand signal regulatory protein alpha (SIRPα) and enabling the tumor cells to escape from macrophage-mediated phagocytosis. Several clinical trials with CD47 targeting agents are underway and have achieved impressive results preliminarily. However, hematotoxicity (particularly anemia) has emerged as the most common side effect that cannot be neglected. In the development of CD47 targeting agents, various methods have been used to mitigate this toxicity. In this review, we summarized five strategies used to alleviate CD47 blockade-induced hematotoxicity, as follows: change in the mode of administration; dual targeting bispecific antibodies of CD47; CD47 antibodies/SIRPα fusion proteins with negligible red blood cell binding; anti-SIRPα antibodies; and glutaminyl-peptide cyclotransferase like inhibitors. With these strategies, the development of CD47 targeting agents can be improved.

Background Biosimilars have been used for 15 years in the European Union (EU), and have been shown to reduce costs and increase access to important biological medicines. In spite of their considerable exposure and excellent safety record, many prescribers still have doubts on the safety and interchangeability of biosimilars, especially monoclonal antibodies (mAbs) and fusion proteins. Objectives The aim of this study was to analyse the short- and long-term safety and interchangeability data of biosimilar mAbs and fusion proteins to provide unbiased information to prescribers and policy makers. Methods Data on the safety, immunogenicity and interchangeability of EU-licensed mAbs and fusion proteins were examined using European Public Assessment Reports (EPARs) and postmarketing safety surveillance reports from the European Medicines Agency (EMA). As recent biosimilar approvals allow self-administration by patients by the subcutaneous route, the administration devices were also analyzed. Results Prelicensing data of EPARs (six different biosimilar adalimumabs, three infliximabs, three etanercepts, three rituximabs, two bevacizumabs, and six trastuzumabs) revealed that the frequency of fatal treatment-emergent adverse events (TEAEs), TEAEs leading to discontinuation of treatment, serious adverse events (SAEs), and main immune-mediated adverse events (AEs) were comparable between the biosimilars and their reference products. The availability of new biosimilar presentations and administration devices may add to patient choice and be an emerging factor in the decision to switch patients. Analysis of postmarketing surveillance data covering up to 7 years of follow-up did not reveal any biosimilar-specific adverse effects. No product was withdrawn for safety reasons. This is in spite of considerable exposure to biosimilars in treatment-naïve patients and in patients switched from the reference medicinal product to the biosimilar. Analysis of data from switching studies provided in regulatory submissions showed that single or multiple switches between the originator and its biosimilar versions had no negative impact on efficacy, safety or immunogenicity. Conclusions In line with previous reports of prelicensing studies of biosimilar mAbs and etanercepts, this study demonstrated comparable efficacy, safety, and immunogenicity compared with the reference products. This is the first study to comprehensively analyze postmarketing surveillance data of the biosimilar mAbs and etanercept. An analysis of more than 1 million patient-treatment years of safety data raised no safety concerns. Based on these data, we argue that biosimilars approved in the EU are highly similar to and interchangeable with their reference products. Thus, additional systematic switch studies are not required to support the switching of patients. Supplementary Information The online version contains supplementary material available at 10.1007/s40265-021-01601-2.

The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1 þ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1–IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1–IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1–IL15m preferentially targeted CD8 þ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1–IL15m treatment induced the expansion of an exhausted CD8 þ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1–IL15m was dependent on CD8 þ T cells, as depletion of CD8 þ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1–IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1–IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8 þ and CD4 þ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1–IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1 þ TILs. See related Spotlight by Felices and Miller, p. 1110.

Interleukin-24 (IL-24) can specifically induce apoptosis in a broad range of cancer cells without harming normal cells. The interaction of contortrostatin (CN) with integrins on angiogenic vascular endothelial and tumor cells is modulated by the RGD motifs that can significantly inhibit metastasis and angiogenesis. To achieve superior therapeutic efficacy by combining anti-metastasis with tumor-selective apoptosis activity, CN was fused at the C-terminus of IL-24 with a flexible linker (G4S)2, and the recombinant IL-24-CN was expressed in Escherichia coli as a Thioredoxin (Trx)/IL-24-CN fusion protein. The target protein was purified using nickel affinity chromatography. Furthermore, we simplified the purification process by purifying Trx-IL-24-CN and cleaving the Trx tag in one step. The final yield of IL-24-CN was 27.6 mg/L based on flask fermentation. In vitro activity assay demonstrated that the recombinant IL-24-CN could more effectively suppress tumor growth and induce apoptosis of melanoma cells. Scratch and transwell assays suggested that IL-24-CN strongly reduced the migration and invasion behavior of melanoma cells. Immunofluorescence analysis and cell adhesion assay showed that CN could evidently improve the tumor inhibition capability of IL-24 by enhancing the affinity of recombinant protein toward cancer cells. In summary, a highly efficient strategy was developed for producing the bioactive IL-24-CN from prokaryotic cells, supporting IL-24-CN in melanoma therapy. Key points • Efficient heterologous production of recombinant IL-24-CN in E. coli using Trx fusion strategy. • Improved tumor growth suppression and apoptosis induction potency of IL-24-CN. • Enhanced cell adhesion ability of IL-24-CN in cancer cells.

Chromosome rearrangement is one of the hallmarks of human malignancies. Gene fusion is one of the consequences of chromosome rearrangements. In this report, we show that gene fusion between solute carrier family 45 member 2 (SLC45A2) and alpha-methylacyl-coenzyme A racemase (AMACR) occurs in eight different types of human malignancies, with frequencies ranging from 45% to 97%. The chimeric protein is translocated to the lysosomal membrane and activates the extracellular signal-regulated kinase signaling cascade. The fusion protein promotes cell growth, accelerates migration, resists serum starvation-induced cell death, and is essential for cancer growth in mouse xenograft cancer models. Introduction of SLC45A2-AMACR into the mouse liver using a sleeping beauty transposon system and somatic knockout of phosphatase and TENsin homolog (Pten) generated spontaneous liver cancers within a short period. Conclusion: The gene fusion between SLC45A2 and AMACR may be a driving event for human liver cancer development.

Leukemia patients bearing the t(4;11)(q21;q23) translocations can be divided into two subgroups: those expressing both reciprocal fusion genes, and those that have only the MLL-AF4 fusion gene. Moreover, a recent study has demonstrated that patients expressing both fusion genes have a better outcome than patients that are expressing the MLL-AF4 fusion protein alone. All this may point to a clonal process where the reciprocal fusion gene AF4-MLL could be lost during disease progression, as this loss may select for a more aggressive type of leukemia. Therefore, we were interested in unraveling the decisive role of the AF4-MLL fusion protein at an early timepoint of disease development. We designed an experimental model system where the MLL-AF4 fusion protein was constitutively expressed, while an inducible AF4-MLL fusion gene was induced for only 48 h. Subsequently, we investigated genome-wide changes by RNA- and ATAC-Seq experiments at distinct timepoints. These analyses revealed that the expression of AF4-MLL for only 48 h was sufficient to significantly change the genomic landscape (transcription and chromatin) even on a longer time scale. Thus, we have to conclude that the AF4-MLL fusion protein works through a hit-and-run mechanism, probably necessary to set up pre-leukemic conditions, but being dispensable for later disease progression.

Background: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). Objective: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. Methods: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. Results: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. Conclusion: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

Telitacicept (Tai'ai®) is fusion protein comprising a recombinant transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) receptor fused to the fragment crystallizable (Fc) domain of human immunoglobulin G (IgG). Telitacicept is being developed by Yantai Rongchang Pharmaceutical through its subsidiary RemeGen for the treatment of B cell-mediated autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and multiple sclerosis (MS). Telitacicept binds to and neutralizes the activity of two cell-signalling molecules, B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), thereby suppressing the development and survival of plasma cells and mature B cells. In March 2021, telitacicept received its first approval in China for the treatment of patients with active SLE. Clinical studies of telitacicept in several other indications, including IgA nephropathy, MS, myasthenia gravis, neuromyelitis optica spectrum disorders, RA and Sjogren's syndrome are underway in China. This article summarizes the milestones in the development of telitacicept leading to this first approval for SLE.

An approach comprising a novel fusion protein and inactivated virus, as a more efficacious vaccine against invading viruses is presented, using SARS-CoV-2 as a most prominent example. The fusion protein consists of the Hepatitis B surface antigen (HBsAg) conjugated to the N-terminal helix (NTH) of Angiotensin-Converting Enzyme 2 (ACE2), which is the receptor for SARS-CoV-2. For vaccination, this fusion protein is to be administered together with the whole killed virus. The NTH would bind to the Receptor Binding Domain (RBD) of the Spike protein of the killed virus. Due to HBsAg acting as a decoy, immune responses would be mounted. Neutralizing antibodies (NAbs) pre-existing in people already vaccinated with the recombinant Hepatitis B vaccine, fresh production of NAbs, and NAbs produced by memory B cells would bind to the HBsAg. This would lead to "presentation" of the killed virus to elements of the immune system at close range. Also, there would be enhanced opsonization and effective antigen presentation. This two-component vaccine could be a platform strategy, wherein HBsAg could be linked to the part of the cellular receptor that any new intractable virus binds to, and is administered together with whole inactivated virus. Now, the same fusion protein, administered by itself to persons with infection, would have therapeutic action, yet by harnessing elements of the immune system. NAbs would bind to the fusion protein as above, the NTH of which would bind to the RBDs of the infecting virus, which, in effect would be neutralized.

Bimolecular Fluorescence Complementation (BiFC) is a versatile approach for intracellular analysis of protein-protein interactions (PPIs), but the tendency of the split fluorescent protein (FP) fragments to self-assemble when brought into close proximity of each other by random collision can lead to generation of false-positive signals that hamper high-definition imaging of PPIs occurring on the nanoscopic level. While it is thought that expressing the fusion proteins at a low level can remove false positives without impacting specific signals, there has been no effective strategy to test this possibility. Here, we present a system capable of assessing and removing BiFC false positives, termed Background Assessable and Correctable-BiFC (BAC-BiFC), in which one of the split FP fragments is fused with an optically distinct FP that serves as a reference marker, and the single-cell fluorescence ratio of the BiFC signal to the reference signal is used to gauge an optimal transfection condition. We showed that when BAC-BiFC is designed to image PPIs regulating Human Immunodeficiency Virus type 1 (HIV-1) assembly, the fluorescence ratio could decrease with decreasing probe quantity, and ratios approaching the limit of detection could allow physiologically relevant characterization of the assembly process on the nanoscale by single-molecule localization microscopy (SMLM). With much improved clarity, previously undescribed features of HIV-1 assembly were revealed.

Recent studies have indicated the antitumor activity and reduced allogeneic response of universal chimeric antigen receptor-modified T (UCAR T) cells lacking endogenous T cell receptors and beta-2 microglobulin (B2M) generated using gene-editing technologies. However, these cells are vulnerable to lysis by allogeneic natural killer (NK) cells due to their lack of human leukocyte antigen (HLA) class I molecule expression. Here, constitutive expression of mutant B2M-HLA-E (mBE) and B2M-HLA-G (mBG) fusion proteins in anti-CD19 UCAR T (UCAR T-19) cells was conducted to protect against allogeneic NK cell-mediated lysis. The ability of cells expressing mBE or mBG to resist NK cell-mediated lysis was observed in gene-edited Jurkat CAR19 cells. UCAR T-19 cells constitutively expressing the mBE and mBG fusion proteins were manufactured and showed effective and specific anti-tumor activity. Constitutive expression of the mBE and mBG fusion proteins in UCAR T-19 cells prevented allogeneic NK cell-mediated lysis. In addition, these cells were not recognizable by allogeneic T cells. Additional experiments, including those in animal models and clinical trials, are required to evaluate the safety and efficacy of UCAR T-19 cells that constitutively express mBE and mBG.

Chromosomal translocations generate oncogenic fusion proteins in approximately one-third of sarcomas, but how these proteins promote tumorigenesis is not well understood. Interestingly, some translocation-driven cancers exhibit dramatic clinical responses to therapy, such as radiotherapy, although the precise mechanism has not been elucidated. Here we reveal a molecular mechanism by which the fusion oncoprotein FUS-CHOP promotes tumor maintenance that also explains the remarkable sensitivity of myxoid liposarcomas to radiation therapy. FUS-CHOP interacted with chromatin remodeling complexes to regulate sarcoma cell proliferation. One of these chromatin remodelers, SNF2H, colocalized with FUS-CHOP genome-wide at active enhancers. Following ionizing radiation, DNA damage response kinases phosphorylated the prion-like domain of FUS-CHOP to impede these protein–protein interactions, which are required for transformation. Therefore, the DNA damage response after irradiation disrupted oncogenic targeting of chromatin remodelers required for FUS-CHOP–driven sarcomagenesis. This mechanism of disruption links phosphorylation of the prion-like domain of an oncogenic fusion protein to DNA damage after ionizing radiation and reveals that a dependence on oncogenic chromatin remodeling underlies sensitivity to radiation therapy in myxoid liposarcoma. Significance: Prion-like domains, which are frequently translocated in cancers as oncogenic fusion proteins that drive global epigenetic changes, confer sensitivity to radiation via disruption of oncogenic interactions.

Set-nup214 is a recurrent fusion gene that is mainly observed in T-cell acute lymphoblastic leukemia (T-ALL). Dysregulation of homeobox (Hox) genes is frequently observed in patients with leukemia. Consistent with this, HoxA genes are upregulated in the set-nup214 + T-ALL cell line and patients. Although SET-Nup214 has been reported to be recruited to the promoter regions of HoxA genes, the detailed mechanisms of how SET-Nup214 specifically binds to HoxA gene promoters and regulates HoxA gene expression are not known. In this study, we demonstrated that SET-Nup214 interacts with MLL via the SET acidic region of SET-Nup214. SET-Nup214 and MLL cooperatively enhance the promoter activity of the HoxA10 gene. Neither the SET region alone nor the Nup214 region alone sufficiently enhanced the HoxA10 gene promoter. Our results indicated that the SET portion of the SET-Nup214 fusion protein is important for interactions with MLL and transcription enhancement of the HoxA10 gene. Thus, our study will contribute to the understanding of how SET-Nup214 and MLL disturb the expression of HoxA10 gene in leukemia.

Super-enhancers (SE) are clusters of transcription enhancers that drive gene expression. SEs are typically characterized by high levels of acetylation of histone H3 lysine 27 (H3K27ac), which is catalyzed by the histone lysine acetyltransferase CREB binding protein (CBP). Cancer cells frequently acquire tumor-specific SEs at key oncogenes, such as MYC, which induce several hallmarks of cancer. BRD4 is recruited to SEs and consequently functions as an epigenetic reader to promote transcription of SE-marked genes in cancer cells. miRNAs can be potent candidates for nucleic acid therapeutics for cancer. We previously identified miR-766-5p as a miRNA that downregulated MYC expression and inhibited cancer cell growth in vitro. In this study, we show that miR-766-5p directly targets CBP and BRD4. Concurrent suppression of CBP and BRD4 cooperatively downregulated MYC expression in cancer cells but not in normal cells. Chromatin immunoprecipitation analysis revealed that miR-766-5p reduced levels of H3K27ac at MYC SEs via CBP suppression. Moreover, miR-766-5p suppressed expression of a BRD4-NUT fusion protein that drives NUT midline carcinoma. In vivo administration of miR-766-5p suppressed tumor growth in two xenograft models. Collectively, these data suggest that targeting SEs using miR-766-5p–based therapeutics may serve as an effective strategy for the treatment of MYC-driven cancers. Significance: This study demonstrates that miR-766-5p targets CBP and BRD4, which can mitigate the protumorigenic consequences of SEs and oncogenic fusion proteins.

Activating TYK2-rearrangements have recently been identified and implicated in the leukemogenesis of high-risk acute lymphoblastic leukemia (HR-ALL) cases. Pre-clinical studies indicated the JAK/TYK2 inhibitor (JAKi), cerdulatinib, as a promising therapeutic against TYK2-rearranged ALL, attenuating the constitutive JAK/STAT signaling resulting from the TYK2 fusion protein. However, following a period of clinical efficacy, JAKi resistance often occurs resulting in relapse. In this study, we modeled potential mechanisms of JAKi resistance in TYK2-rearranged ALL cells in vitro in order to recapitulate possible clinical scenarios and provide a rationale for alternative therapies. Cerdulatinib resistant B-cells, driven by the MYB-TYK2 fusion oncogene, were generated by long-term exposure to the drug. Sustained treatment of MYB-TYK2-rearranged ALL cells with cerdulatinib led to enhanced and persistent JAK/STAT signaling, co-occurring with JAK1 overexpression. Hyperactivation of JAK/STAT signaling and JAK1 overexpression was reversible as cerdulatinib withdrawal resulted in re-sensitization to the drug. Importantly, histone deacetylase inhibitor (HDACi) therapies were efficacious against cerdulatinib-resistant cells demonstrating a potential alternative therapy for use in TYK2-rearranged B-ALL patients who have lost response to JAKi treatment regimens.

Teduglutide is the only drug approved for long-term treatment of short bowel syndrome (SBS). This drug exerts its pharmacological effects via binding to the GLP-2 receptors (ECD-GLP2R) located in intestinal tissue. The three dimensional (3D) structure of ECD-GLP2R hasn't been determined yet and hence its mode of interaction with agonists/antagonists is not clear. Therefore, it would be of great importance to develop a structural scaffold for investigation of ECD-GLP2R interactions with its binders. For this, the current study aimed to produce fusion protein of ECD-GLP2R-teduglutide. The ECD-GLP2R-teduglutide protein was expressed in bacterial expression system and purified using affinity and size exclusion chromatography techniques. Using circular dichroism the secondary structure content of purified protein was determined which was comparable to that of theoretical calculations. The low structural stability of purified protein (ΔG = 3.64 kJ.mol−1) was elucidated by monitoring its fluorescence emission at the presence of various concentrations of GdnHCl as a denaturant. Finally, a 3D model for ECD-GLP2R-teduglutide protein was generated and validated using molecular dynamics simulation whose information alongside the experimental studies can be useful for providing new insight into the mode of interaction of ECD-GLP2R with its specific ligands in order to design potent and specific GLP2R agonists.

Cytokine-induced killer cell immunotherapy is an ideal candidate for adoptive cell transfer therapy. However, therapeutic approaches to enhance the anti-tumor activity of cytokine-induced killer cells remain to be explored. Here, we described the successful development of a novel antibody-chemokine fusion protein containing the anti-human Endoglin antibody in the single-chain variable fragment format and human interferon-gamma-induced protein 10 (hENG scFv/hIP-10). Its anti-Endoglin immunoreactivity and chemotactic activity against the cytokine-induced killer cells were characterized in vitro. To evaluate the anti-tumor effect in vivo, cytokine-induced killer cells were intravenously injected into human hepatocellular carcinoma-bearing nude mice, together with intratumoral administration of the fusion protein hENG scFv/hIP-10 as an enhancer. The tumor volume and survival time of the mice were monitored, whilst the tumor-infiltrating cytokine-induced killer cells, serum levels of interferon-gamma, tumor cell proliferation, apoptosis, and angiogenesis were measured. The results demonstrated that hENG scFv/hIP-10 and cytokine-induced killer cells synergistically inhibited tumor growth and prolonged survival of tumor-bearing mice. Moreover, the number of tumor-infiltrating cytokine-induced killer cells, serum levels of interferon-gamma, and tumor cell apoptosis were increased, accompanied with decreased tumor proliferation and angiogenesis. Thus, our study suggests that hENG scFv/hIP-10 could enhance the anti-tumor activity of cytokine-induced killer cells against human hepatocellular carcinoma.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma. There are two main subtypes of RMS, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma. ARMS typically encompasses fusion‐positive rhabdomyosarcoma, which expresses either PAX3‐FOXO1 or PAX7‐FOXO1 fusion proteins. There are no targeted therapies for ARMS; however, recent studies have begun to illustrate the cooperation between epigenetic proteins and the PAX3‐FOXO1 fusion, indicating that epigenetic proteins may serve as targets in ARMS. Here, we investigate the contribution of BMI1, given the established role of this epigenetic regulator in sustaining aggression in cancer. We determined that BMI1 is expressed across ARMS tumors, patient‐derived xenografts, and cell lines. We depleted BMI1 using RNAi and inhibitors (PTC‐209 and PTC‐028) and found that this leads to a decrease in cell growth/increase in apoptosis in vitro, and delays tumor growth in vivo. Our data suggest that BMI1 inhibition activates the Hippo pathway via phosphorylation of LATS1/2 and subsequent reduction in YAP levels and YAP/TAZ target genes. These results identify BMI1 as a potential therapeutic vulnerability in ARMS and warrant further investigation of BMI1 in ARMS and other sarcomas., Treatment of alveolar rhabdomyosarcoma (ARMS) remains a major therapeutic challenge in pediatric oncology. Here, we demonstrate that genetic and pharmacologic inhibition of BMI1 reduces ARMS viability. We show that BMI1 inhibits the tumor suppressive Hippo pathway and, conversely, that BMI1 disruption upregulates Hippo signaling. Collectively, these findings provide an initial framework for targeting BMI1 in ARMS and additional sarcomas.

Targeted protein degradation is an emerging new strategy for the modulation of intracellular protein levels with applications in chemical biology and drug discovery. One approach to enable this strategy is to redirect the ubiquitin-proteasome system to mark and degrade target proteins of interest (POIs) through the use of proteolysis targeting chimeras (PROTACs). Although great progress has been made in enabling PROTACs as a platform, there are still a limited number of E3 ligases that have been employed for PROTAC design. Herein we report a novel phenotypic screening approach for the identification of E3 ligase binders. The key concept underlying this approach is the high-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ, which were then evaluated for their ability to degrade a GFP-HaloTag fusion protein in a cellular context. As proof of concept, we demonstrated that we could generate and detect functional HaloPROTACs in situ, using a validated Von Hippel-Lindau (VHL) binder that successfully degraded the GFP-HaloTag fusion protein in living cells. We then used this method to prepare and screen a library of approximately 2000 prospective E3 ligase-recruiting molecules.