Fc-Fc interactions and immune inhibitory effects of IgG4: implications for anti-PD-1 immunotherapies.

BACKGROUND: The majority of anti-programmed cell-death 1 (PD-1) monoclonal antibodies (mAbs) use S228P mutation IgG4 as the structural basis to avoid the activation of immune cells or complement. However, little attention has been paid to the Fc-Fc interactions between IgG4 and other IgG Fc fragments that could result in adverse effects. Fc-null IgG1 framework is a potential safer alternative to avoid the undesirable Fc-Fc interactions and Fc receptor binding derived effects observed with IgG4. This study provides a comprehensive evaluation of anti-PD-1 mAbs of these two frameworks. METHODS: Trastuzumab and rituximab (both IgG1), wildtype IgG1 and IgG4 were immobilized on nitrocellulose membranes, coated to microplates and biosensor chips, and bound to tumor cells as targets for Fc-Fc interactions. Wildtype IgG1 and IgG4, anti-PD-1 mAb nivolumab (IgG4 S228P), penpulimab (Fc-null IgG1), and tislelizumab (Fc-null IgG4 S228P-R409K) were assessed for their binding reactions to the immobilized IgG proteins and quantitative kinetic data were obtained. To evaluate the effects of the two anti-PD-1 mAbs on immune responses mediated by trastuzumab and rituximab in the context of combination therapy, we employed classic immune models for antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement dependent cytotoxicity. Tumor-bearing mouse models, both wildtype and humanized, were used for in vivo investigation. Furthermore, we also examined the effects of IgG1 and IgG4 on diverse immune cell populations RESULTS: Experiments demonstrated that wildtype IgG4 and nivolumab bound to immobilized IgG through Fc-Fc interactions, diminishing antibody-dependent cell-mediated cytotoxicity and phagocytosis reactions. Quantitative analysis of kinetic parameters suggests that nivolumab and wildtype IgG4 exhibit comparable binding affinities to immobilized IgG1 in both non-denatured and denatured states. IgG4 exerted inhibitory effects on various immune cell types. Wildtype IgG4 and nivolumab both promoted tumor growth in wildtype mouse models. Conversely, wildtype IgG1, penpulimab, and tislelizumab did not show similar adverse effects. CONCLUSIONS: Fc-null IgG1 represents a safer choice for anti-PD-1 immunotherapies by avoiding both the adverse Fc-Fc interactions and Fc-related immune inhibitory effects of IgG4. Fc-null IgG4 S228P-R409K and Fc-null IgG1 displayed similar structural properties and benefits. This study contributes to the understanding of immunotherapy resistance and the advancement of safer immune therapies for cancer.

Lectibodies as antivirals.

Growing concerns regarding the emergence of highly transmissible viral diseases highlight the urgent need to expand the repertoire of antiviral therapeutics. For this reason, new strategies for neutralizing and inhibiting these viruses are necessary. A promising approach involves targeting the glycans present on the surfaces of enveloped viruses. Lectins, known for their ability to recognize specific carbohydrate molecules, offer the potential for glycan-targeted antiviral strategies. Indeed, numerous studies have reported the antiviral effects of various lectins of both endogenous and exogenous origins. However, many lectins in their natural forms, are not suitable for use as antiviral therapeutics due to toxicity, other unfavorable pharmacological effects, and/or unreliable manufacturing sources. Therefore, improvements are crucial for employing lectins as effective antiviral therapeutics. A novel approach to enhance lectins' suitability as pharmaceuticals could be the generation of recombinant lectin-Fc fusion proteins, termed "lectibodies." In this review, we discuss the scientific rationale behind lectin-based antiviral strategies and explore how lectibodies could facilitate the development of new antiviral therapeutics. We will also share our perspective on the potential of these molecules to transcend their potential use as antiviral agents.

Potent synergistic effects of dulaglutide and food restriction in prevention of olanzapine-induced metabolic adverse effects in a rodent model.

BACKGROUND: Antipsychotics are indispensable in the treatment of severe mental illneses, however adverse metabolic effects including diabetes, weight gain, dyslipidemia, and related cardiovascular morbidity are common, and current pharmacological strategies for their management are unsatisfactory. Glucagon-like 1 peptide receptor agonists (GLP-1 RAs) are approved for the treatment of type 2 diabetes and obesity hold promise for the management of antipsychotic-associated adverse metabolic effects. METHODS: To characterize the molecular effects and identify biomarkers for GLP-1 RA preventive treatment, Sprague-Dawley female rats were treated with long-acting formulations of the antipsychotic olanzapine and the GLP-1 RA dulaglutide for 8 days. A pair-feeding protocol evaluated the combined effects of dulaglutide and food restriction on an olanzapine-induced metabolic phenotype. Body weight and food consumption were recorded. Biochemical analysis included a lipid profile, a spectrum of gastrointestinal and adipose tissue-derived hormones, and fibroblast growth factor 21 serum levels. RESULTS: Olanzapine induced hyperphagia, weight gain, increased serum triglycerides and HDL cholesterol. Food restriction affected the OLA-induced phenotype but not serum markers. Dulaglutide led to a modest decrease in food intake, with no effect on weight gain, and did not reverse the OLA-induced changes in serum lipid parameters. Concomitant dulaglutide and food restriction resulted in weight loss, decreased feed efficiency, and lower total and HDL cholesterol. CONCLUSIONS: A combined strategy of dulaglutide and food restriction manifested a massive synergistic benefit. GLP-1RAs represent a promising strategy and deserve thorough future research. Our findings underline the potential importance of lifestyle intervention in addition to GLP-1 RA treatment.

Dulaglutide restores endothelial progenitor cell levels in diabetic mice and mitigates high glucose-induced endothelial injury through SIRT1-mediated mitochondrial fission.

Type 2 diabetes mellitus (T2DM) significantly impairs the functionality and number of endothelial progenitor cells (EPCs) and resident endothelial cells, critical for vascular repair and regeneration, exacerbating the risk of vascular complications. GLP-1 receptor agonists, like dulaglutide, have emerged as promising therapeutic agents due to their multifaceted effects, including the enhancement of EPC activity and protection of endothelial cells. This study investigates dulaglutide's effects on peripheral blood levels of CD34+ and CD133+ cells in a mouse model of lower limb ischemia and its protective mechanisms against high-glucose-induced damage in endothelial cells. Results demonstrated that dulaglutide significantly improves blood flow, reduces tissue damage and inflammation in ischemic limbs, and enhances glycemic control. Furthermore, dulaglutide alleviated high-glucose-induced endothelial cell damage, evident from improved tube formation, reduced reactive oxygen species accumulation, and restored endothelial junction integrity. Mechanistically, dulaglutide mitigated mitochondrial fission in endothelial cells under high-glucose conditions, partly through maintaining SIRT1 expression, which is crucial for mitochondrial dynamics. This study reveals the potential of dulaglutide as a therapeutic option for vascular complications in T2DM patients, highlighting its role in improving endothelial function and mitochondrial integrity.

Comparative Analysis of the Anti-Inflammatory Effects of Liraglutide and Dulaglutide.

Inflammation plays a pathophysiological role in atherosclerosis and its clinical consequences. In addition to glycemic control, glucagon-like peptide-1 receptor agonists (GLP-1RAs) are of wide concern for cardioprotective effects. The structure, half-life, homology, and clinical efficacy of GLP-1RAs exhibit remarkable disparity. Several studies have compared the disparities in anti-inflammatory effects between daily and weekly GLP-1RAs. This study aimed to compare the similarities and differences between liraglutide and dulaglutide in terms of inhibiting atherosclerotic inflammation and improving co-cultured endothelial cell function. The expression of inflammation markers was examined by immunofluorescence, Western blotting, and real-time PCR. The tube-forming ability of endothelial cells was tested on Matrigel. The results verify that 10/50/100 nmol/L liraglutide and 100 nmol/L dulaglutide markedly suppressed the expression of inflammatory factors in LPS-induced atherosclerosis after 24 and 72 hours, respectively. Moreover, they promoted the polarization of M1 macrophages toward the M2 phenotype and improved the function of co-cultured endothelial cells. Both liraglutide and dulaglutide ameliorate atherosclerosis development. The difference between the two resided in the extended intervention duration required to observe the effect of dulaglutide, and liraglutide demonstrated a superior dose-dependent manner. We provide a potential strategy to understand the dynamics of drug action and possible timing administration.

Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation.

BACKGROUND: Elevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates. METHODS: The senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation. RESULTS: The high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG. CONCLUSIONS: FSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis ("FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients. Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

Bone marrow Tfr2 deletion improves the therapeutic efficacy of the activin-receptor ligand trap RAP-536 in ß-thalassemic mice.

ß-thalassemia is a disorder characterized by anemia, ineffective erythropoiesis (IE), and iron overload, whose treatment still requires improvement. The activin receptor-ligand trap Luspatercept, a novel therapeutic option for ß-thalassemia, stimulates erythroid differentiation inhibiting the transforming growth factor ß pathway. However, its exact mechanism of action and the possible connection with erythropoietin (Epo), the erythropoiesis governing cytokine, remain to be clarified. Moreover, Luspatercept does not correct all the features of the disease, calling for the identification of strategies that enhance its efficacy. Transferrin receptor 2 (TFR2) regulates systemic iron homeostasis in the liver and modulates the response to Epo of erythroid cells, thus balancing red blood cells production with iron availability. Stimulating Epo signaling, hematopoietic Tfr2 deletion ameliorates anemia and IE in Hbbth3/+ thalassemic mice. To investigate whether hematopoietic Tfr2 inactivation improves the efficacy of Luspatercept, we treated Hbbth3/+ mice with or without hematopoietic Tfr2 (Tfr2BMKO/Hbbth3/+) with RAP-536, the murine analog of Luspatercept. As expected, both hematopoietic Tfr2 deletion and RAP-536 significantly ameliorate IE and anemia, and the combined approach has an additive effect. Since RAP-536 has comparable efficacy in both Hbbth3/+ and Tfr2BMKO/Hbbth3/+ animals, we propose that the drug promotes erythroid differentiation independently of TFR2 and EPO stimulation. Notably, the lack of Tfr2, but not RAP-536, can also attenuate iron-overload and related complications. Overall, our results shed further light on the mechanism of action of Luspatercept and suggest that strategies aimed at inhibiting hematopoietic TFR2 might improve the therapeutic efficacy of activin receptor-ligand traps.

Combination of a TGF-ß ligand trap (RAP-GRL) and TMPRSS6-ASO is superior for correcting ß-thalassemia.

A recently approved drug that induces erythroid cell maturation (luspatercept) has been shown to improve anemia and reduce the need for blood transfusion in non-transfusion-dependent as well as transfusion-dependent ß-thalassemia (BT) patients. Although these results were predominantly positive, not all the patients showed the expected increase in hemoglobin (Hb) levels or transfusion burden reduction. Additional studies indicated that administration of luspatercept in transfusion-dependent BT was associated with increased erythropoietic markers, decreased hepcidin levels, and increased liver iron content. Altogether, these studies suggest that luspatercept may necessitate additional drugs for improved erythroid and iron management. As luspatercept does not appear to directly affect iron metabolism, we hypothesized that TMPRSS6-ASO could improve iron parameters and iron overload when co-administered with luspatercept. We used an agent analogous to murine luspatercept (RAP-GRL) and another novel therapeutic, IONIS TMPRSS6-LRx (TMPRSS6-ASO), a hepcidin inducer, to treat non-transfusion-dependent BT-intermedia mice. Our study shows that RAP-GRL alone improved red blood cell (RBC) production, with no or limited effect on splenomegaly and iron parameters. In contrast, TMPRSS6-ASO improved RBC measurements, ameliorated splenomegaly, and improved iron overload most effectively. Our results provide pre-clinical support for combining TMPRSS6-ASO and luspatercept in treating BT, as these drugs together show potential for simultaneously improving both erythroid and iron parameters in BT patients.

The effects of Fc fusion protein glucagon-like peptide-1 and glucagon dual receptor agonist with different receptor selectivity in vivo studies.

BACKGROUND: Glucagon-like peptide-1 (GLP-1)/glucagon (GCG) dual receptor agonists with different receptor selectivity are under investigation and have shown significant improvement in both weight loss and glycemic control, but the optimal potency ratio between the two receptors to balance efficacy and safety remains unclear. EXPERIMENTAL APPROACH: We designed and constructed several dual receptor agonists with different receptor potency ratios using Fc fusion protein technology. The long-term effects of the candidates on body weight and metabolic dysfunction-associated steatotic liver disease (MASLD) were evaluated in diet-induced obese (DIO) model mice, high-fat diet (HFD)-ob/ob mice and AMLN diet-induced MASLD mice. Repeat dose toxicity assays were performed to investigate the safety profile of the candidate (HEC-C070) in Sprague Dawley (SD) rats. KEY RESULTS: The high GCG receptor (GCGR) selectivity of HEC-C046 makes it more prominent than other compounds for weight loss and most MASLD parameters but may lead to safety concerns. The weight change of HEC-C052 with the lowest GCG agonism was inferior to that of selective GLP-1 receptor agonist (GLP-1RA) semaglutide in DIO model mice. The GLP-1R selectivity of HEC-C070 with moderate GCG agonism has a significant effect on weight loss and liver function in obese mice, and its lowest observed adverse effect level (LOAEL) was 30 nmol/kg in the repeat dose toxicity study. CONCLUSION: We compared the potential of the Fc fusion protein GLP-1/GCG dual receptor agonists with different receptor selectivity to provide the setting for future GLP-1/GCG dual receptor agonists to treat obesity and MASLD.

An engineered immunomodulatory IgG1 Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin.

Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell-produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.

A novel single domain bispecific antibody targeting VEGF and TNF-α ameliorates rheumatoid arthritis.

Anti-TNF-α therapy fails in 30% of patients, where TNF-α may not be the key causative factor in these patients. We developed a bispecific single-domain antibody block TNF-α and VEGF (V5-3).The experiments showed that V5-3 effectively activated proliferation and migration of RA-FLS and HUVEC, tube-forming role of HUVEC, and expression of inflammatory factors in vitro. Besides, the experiments indicated that the anti-RA activity of V5-3 was superior to Anbainuo in vivo. Application of V5-3 reduced the expression of inflammatory factors, extent of synovial inflammation and angiogenesis and attenuated the severity of autoimmune arthritis in collagen-induced arthritis (CIA) mice. Mechanistically, V5-3 suppressed p65, AKT and VEGFR2 phosphorylation, as well as production of TNF-α and VEGF in joint tissues. These results demonstrated that V5-3 displayed a superior effect of anti-RA, may be a new therapy to overcome the limitations of anti-TNF-α monoclonal antibody.

Pharmacokinetic similarity study comparing the biosimilar candidate, LY05008, with its reference product dulaglutide in healthy Chinese male subjects.

BACKGROUND: Dulaglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has been approved for improving glycemic control and reducing the risk of cardiovascular (CV) adverse events. This study compared the pharmacokinetic (PK) profiles, safety, and immunogenicity of LY05008, a biosimilar candidate, to a licensed product dulaglutide in healthy Chinese male subjects. RESEARCH DESIGN AND METHODS: In this double-blind, open-label, parallel-group study, healthy Chinese male subjects were randomized 1:1 to receive either LY05008 or dulaglutide subcutaneously. Primary study endpoints were PK parameters such as the area under the concentration-time curve (AUC) from time zero to infinity (AUC0 - ∞), AUC from time zero to the last quantifiable concentration (AUC0-t), and maximum serum concentration (Cmax). Safety and immunogenicity profiles were also included for data analysis. RESULTS: 82 subjects were randomized to receive LY05008 (n = 41) or dulaglutide (n = 41). The 90% confidence intervals (CIs) of the geometric mean ratios (GMRs) of AUC0 - ∞, AUC0-t and Cmax of LY05008 to dulaglutide were all within the bioequivalence limits of 80%-125%. Other PK parameters, safety, and immunogenicity profiles were comparable across the two treatment groups. CONCLUSION: This study demonstrated PK similarity of LY05008, a dulaglutide biosimilar, to dulaglutide in healthy Chinese male subjects, with comparable safety and immunogenicity data. TRIAL REGISTRATION: The trial is registered at the Chinese Clinical Trial Registry (Identifier No. ChiCTR2200066519).

Dulaglutide as a demethylating agent to improve the outcome of breast cancer.

Background: Dulaglutide emerged as a promising therapeutic option for diabetes mellitus Type 2 (DM2). Aims: Owing to epigenetic similarities between the pathophysiology of DM2 and breast cancer (BC), we investigated the antitumor effect of dulaglutide. Materials & methods: To investigate the effect of dulaglutide, we analyzed the expression of methylated gene promoter regions in BC (ESR1, CDH1 and ADAM33). Results: Dulaglutide increased the expression of ESR1, CDH1 and ADAM33 up to fourfold in the MDA-MB-231 lineage by demethylating the gene promoter regions. This effect was translated to in vivo antitumoral activity and revealed significant tumor inhibition by combining the half-dose of methotrexate with dulaglutide. Conclusion: This therapy may mitigate the severe side effects commonly associated with chemotherapy.

ICOSL Stimulation by ICOS-Fc Accelerates Cutaneous Wound Healing In Vivo.

BACKGROUND: ICOS and its ligand ICOSL are immune receptors whose interaction triggers bidirectional signals that modulate the immune response and tissue repair. AIM: The aim of this study was to assess the in vivo effects of ICOSL triggering by ICOS-Fc, a recombinant soluble form of ICOS, on skin wound healing. METHODS: The effect of human ICOS-Fc on wound healing was assessed, in vitro, and, in vivo, by skin wound healing assay using ICOS-/- and ICOSL-/- knockout (KO) mice and NOD-SCID-IL2R null (NSG) mice. RESULTS: We show that, in wild type mice, treatment with ICOS-Fc improves wound healing, promotes angiogenesis, preceded by upregulation of IL-6 and VEGF expression; increases the number of fibroblasts and T cells, whereas it reduces that of neutrophils; and increases the number of M2 vs. M1 macrophages. Fittingly, ICOS-Fc enhanced M2 macrophage migration, while it hampered that of M1 macrophages. ICOS-/- and ICOSL-/- KO, and NSG mice showed delayed wound healing, and treatment with ICOS-Fc improved wound closure in ICOS-/- and NSG mice. CONCLUSION: These data show that the ICOS/ICOSL network cooperates in tissue repair, and that triggering of ICOSL by ICOS-Fc improves cutaneous wound healing by increasing angiogenesis and recruitment of reparative macrophages.

Impact of glycoengineering and antidrug antibodies on the anticancer activity of a plant-made lectin-Fc fusion protein.

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core α1,3-fucose and ß1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFcΔXF , has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumour mouse model, we found that systemic administration of AvFcΔXF , but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumours, suggesting that Fc-mediated effector functions were integral. AvFcΔXF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of antidrug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFcΔXF before implanting tumours. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFcΔXF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFc's antitumor activity.

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants.

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Molecular Engineering of Efficacious Mono-Valent Ultra-Long Acting Two-Chain Insulin-Fc Conjugates.

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.

Glycaemic variability in patients with type 2 diabetes mellitus treated with dulaglutide, with and without concomitant insulin: Post hoc analyses of randomized clinical trials.

AIM: To investigate the association between treatment with dulaglutide and glycaemic variability (GV) in adult patients with type 2 diabetes mellitus (T2D). MATERIALS AND METHODS: Post hoc analyses of six randomized, phase 3 studies were conducted to investigate the association between treatment with dulaglutide 1.5 mg once weekly and GV in adult patients with T2D. Using data from seven- and eight-point self-monitored plasma glucose (SMPG) profiles over up to 28 weeks of treatment, GV in within- and between-day SMPG, and between-day fasting glucose from SMPG (FSMPG) was assessed according to standard deviation and coefficient of variation. RESULTS: Pooled data from five studies with dulaglutide as monotherapy or added to oral glucose-lowering medication, without concomitant insulin treatment, revealed clinically meaningful reductions in within- and between-day SMPG, and between-day FSMPG variability from baseline in the dulaglutide group. Comparisons between treatment groups in two studies demonstrated that reductions from baseline in within-day and between-day SMPG, and between-day FSMPG variability were greater for treatment with dulaglutide compared with insulin glargine, as well as for treatment with dulaglutide when added to insulin glargine compared with insulin glargine alone. CONCLUSIONS: In patients with T2D, treatment with dulaglutide as monotherapy or added to oral glucose-lowering medication, without concomitant insulin treatment, was potentially associated with a reduction in GV. Treatment with dulaglutide was associated with a reduction in GV to a greater degree than insulin glargine. When added to insulin glargine, treatment with dulaglutide was associated with greater decreases in GV compared with insulin glargine alone. As reduced GV may be associated with better outcomes, these findings may have clinical relevance.

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo.

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the "Y"- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.