Fc Fusion Protein Research Articles

Optimized Production of Fc Fusion Proteins by Sortase Enzymatic Ligation.

Fc fusions are a growing class of drugs comprising an antibody Fc domain covalently linked to a protein or peptide and can pose manufacturing challenges. In this study we evaluated three synthetic approaches to generate Fc fusions, using Fc-insulin as a model drug candidate. Engineered human IgG1 was digested with HRV3C to produce an Fc fragment with a C-terminal sortase tag (Fc-LPETGGH6). The synthesis of Fc-insulin2 from Fc-LPETGGH6 was evaluated with direct sortase-mediated ligation (SML) and two chemoenzymatic strategies. Direct SML was performed with triglycine-insulin, and chemoenzymatic strategies used to SML fuse either triglycine-azide or triglycine-DBCO prior to linking insulin with copper-catalyzed or strain-promoted azidealkyne cycloaddition. Reaction conditions were optimized by evaluating reagent concentrations, relative equivalents, temperature, and time. Direct SML provided the most effective reaction yields, converting 60-70% of Fc-LPETGGH6 to Fc-insulin2, whereas our optimized chemoenzymatic synthesis converted 30-40% of Fc-LPETGGH6 to Fc-insulin2. Here we show that SML is a practical and efficient method to synthesize Fc fusions and provide an optimized pathway for fusion drug synthesis.

IMMU-13. COMBINED MODULATION OF THE TGF-Β PATHWAY AND GITR IMMUNE CHECKPOINT SIGNALING PROMOTES ANTI-TUMOR IMMUNITY IN SYNGENEIC GLIOMA MODELS

Abstract The profound local immunosuppressive microenvironment is one hallmark of glioblastoma, which results in resistance to most immunotherapeutic strategies that have been explored so far. Reverting this condition in order to reinvigorate anti-glioma immunity might be a promising therapeutic approach. Transforming growth factor (TGF)-β signaling is deregulated in different cancer types and contributes to the malignant phenotype of glioma cells. Glioma-derived TGF-β is also a major immunosuppressive factor in the tumor microenvironment. Furthermore, intratumoral regulatory T (Treg) cells and activated T effector cells express high levels of the co-stimulatory immune checkpoint glucocorticoid-induced tumor necrosis factor receptor (GITR). Agonistic anti-GITR antibodies have been explored in preclinical tumor models and are under investigation in clinical trials for the treatment of solid tumors. We evaluated the effect of TGF-β and GITR targeting on anti-tumor immune responses in syngeneic mouse glioma models. In co-culture settings, GITR modulation with a GITR ligand (GITRL)-Fc fusion protein, given alone or in combination with a pharmacological TGF-β receptor inhibitor, led to increased T cell activation. Furthermore, the combined targeting of the two pathways resulted in significantly higher immune cell-mediated tumor cell killing than either treatment alone. In vivo, TGF-β inhibition and GITR signaling modulation resulted in a higher fraction of long-term surviving glioma-bearing mice than single-agent treatment. Surviving mice were resistant to tumor re-challenge, suggesting adaptive immunity as an underlying mechanism. These data support the assumption that combined immunotherapeutic strategies may represent a promising approach for the treatment of glioma.

RFIXFc prophylaxis improves pain and levels of physical activity in haemophilia B: Post hoc analysis of B-LONG using haemophilia-specific quality of life questionnaires.

Recurrent bleeding in severe haemophilia B causes painful hemarthroses and reduces capacity for physical activity. Recombinant factor IX Fc fusion protein (rFIXFc) prophylaxis results in low annualised bleeding rates, with the potential to improve patients' health-related quality of life (HRQoL). To present a post hoc analysis of data from B-LONG describing change over time in patient-reported outcomes associated with pain and physical activity. Patients (≥12 years) who received weekly dose-adjusted or interval-adjusted rFIXFc prophylaxis and completed the Haemophilia-Specific QoL questionnaire for adolescents (Haemo-QoL) or adults (Haem-A-QoL) at baseline (BL) and end of study (EoS). Individual level changes in items of the 'Physical Health' and 'Sports and Leisure' domains, categorised as 'never/rarely/seldom' or 'sometimes/often/all the time', were analysed using McNemar's test to compare distribution of responses at EoS versus BL. At EoS versus BL, a significantly greater proportion of patients did not experience painful swellings (64%vs. 44%; P=.004), painful joints (44%vs. 28%; P=.003) or pain when moving (54%vs. 41%; P=.026). Additionally, at EoS versus BL, patients were less likely to avoid participating in sports like football (30%vs. 8%; P=.002), avoid sports due to their haemophilia (47%vs. 27%; P=.007), or experience difficulty walking as far as they wanted (63%vs. 43%; P=.001). The proportion of patients who played sports as much as the general population was numerically increased (52%vs. 37%; P=.033) at EoS versus BL. Results of the analysis suggest that over time, rFIXFc prophylaxis is associated with significant improvements in pain and physical functioning. This contributes to previous evidence of overall HRQoL improvements in patients with haemophilia B treated with rFIXFc.

Isolation, physicochemical, and structure–function relationship of the hydrophobic variant of Fc-fusion proteins that bind to TNF-α receptor, HS002 and HS002A

HS002 is the recombinant human tumor necrosis factor-α receptor Ⅱ: IgG Fc fusion protein licensed in China to treat rheumatism and psoriasis. The aim of this study was to isolate and characterize the hydrophobic freeze-dried powder injection (HS002) and ampoule injection (HS002A) variants derived from proteins of the same sequence and then to explore the structure–function relationship. Extensive physicochemical and structural testing was performed during a side-by-side comparison of the monomer peak and variant. Then the TNF-α-related binding activity, cell biological activity and affinity with FcRn were analyzed. Finally, a transformation study of the hydrophobic variant was performed under serum-like redox conditions. This research revealed that HS002A has similar physicochemical and structure–function relationship profiles to those of HS002. The hydrophobic variant exhibited the presence of new incorrect disulfide bridging. At the same time, this novel disulfide scrambled species structure–function relationship was found to be the molecular basis for reduced TNF-α binding and cell biological activities. In addition, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring TNF-α binding and cell biological activities to almost normal levels, all of which indicate that the variant is probably irrelevant to clinical efficacy once the drug enters the bloodstream.

A Retrospective Observational Descriptive Study on the Effectiveness and Usage of Emicizumab and Antihemophilic Factor (recombinant), Fc Fusion Protein in Patients with Hemophilia A in the US

IntroductionHemophilia A is a rare bleeding disorder characterized by coagulation Factor VIII (FVIII) deficiency. Symptoms are primarily bleeding episodes that occur spontaneously or following injury, trauma, or surgical procedure. The treatment for hemophilia A involves replacement of FVIII for on-demand or prophylactic care. The aim of this analysis was to describe treatment outcomes and patterns between patients with hemophilia A who switch to emicizumab (Hemlibra®), Fc fusion protein (rFVIIIFc; Eloctate®), or other factor therapy using specialty pharmacy data from the US.MethodsThis was a retrospective observational descriptive study conducted between November 2014 and January 2021 using specialty pharmacy data including over 5,000 total patients with hemophilia A in the US. Patients included in the analysis were male, had a diagnosis of hemophilia A without a history of inhibitors, and had a minimum of 6 months baseline pre-index data and 6 months follow-up data post-index. Index date was defined as the first dispense of emicizumab after October 2018 or rFVIIIFc after May 2015. Patient reported and claims-based treatment outcomes assessed pre- and post-treatment included: annualized bleeding rate (ABR, calculated from patient bleed logs), any factor usage including on-demand FVIII use, prescribed and dispensed dosage of treatment, and frequency. Wastage as a percentage was a calculated as: (prophylaxis logged - prophylaxis prescribed)/prophylaxis dispensed.Results118 patients treated with emicizumab and 55 patients treated with rFVIIIFc were included in the analysis. Additionally, 26 patients switched from rFVIIIFc to emicizumab, and therefore met inclusion criteria for both treatment groups (these patients are described separately). Baseline characteristics are reported in Table 1; the majority of patients in all cohorts had severe hemophilia and prior prophylaxis therapy.The most frequently prescribed dosing patterns for patients in the emicizumab cohort was once every 2 weeks (Q2W) for 56 patients (47.5%) and once weekly (Q1W) for 51 patients (43.2%). Whereas the most frequently prescribed dosing patterns for the rFVIIIFc only cohort was twice per week (BIW) in 27 patients (49.1%), every 4 days (Q4D) in 15 patients (27.3%), and Q1W in 2 patients (3.6%).Total mean (95% confidence intervals [CI]) weekly prophylaxis consumption was 1.68 (1.65; 1.72) mg/kg in the emicizumab cohort and 83.17 (72.06; 94.29) IU/kg in the rFVIIIFc cohort post index. Mean (95% CI) weekly dispensed dosage for prophylaxis was 1.81 (1.76; 1.86) mg/kg emicizumab and 83.83 (72.55; 95.11) IU/kg rFVIIIFc post index. Mean (95% CI) total supplied FVIII for on-demand factor on hand was 5.14 (2.92; 7.35) IU/kg in the emicizumab cohort and 11.76 (9.06; 14.46) IU/kg in the rFVIIIFc cohort post index. Weekly percentage of supplied dosage wasted was 6.03% (4.50; 7.57; p<0.01) emicizumab for the overall cohort, with a mean (standard deviation [SD]) of 10.46% (12.21) wasted per patient within the emicizumab age <12 years cohort. Weekly percentage of supplied dosage wasted was 0.70 (-0.10; 1.50; p=0.09) rFVIIIFc for the overall cohort, with a mean (SD) of 2.63% (3.53) wasted per patient within the rFVIIIFc age <12 years cohort.Mean (95% CI) change in overall ABR pre- and post-treatment was -1.14 (-2.16; -0.18) in the rFVIIIFc cohort and -0.91 (-1.36; -0.50) in the emicizumab cohort (Table 2). Mean (95% CI) changes in spontaneous ABR and spontaneous joint ABR pre- and post-treatment were -0.36 (-1.12; 0.27) and -0.16 (-0.70; 0.31), respectively, in patients treated with rFVIIIFc, whereas within the emicizumab group they were -0.40 (-0.71; -0.13) and -0.37 (-0.66; -0.12).ConclusionPatients were prescribed emicizumab labeled dosing primarily on a Q1W or Q2W pattern, whereas patients prescribed rFVIIIFc had more individualized dosing primarily being prescribed Q4D or BIW. FVIII for on-demand treatment use was found post index in both the factor and non-factor treated cohorts. Overall product wastage with emicizumab is higher than previously reported. Product wastage was highest among emicizumab patients under 12 years, but wastage was still considerable in patients 12 or older. This descriptive analysis indicated that both treatments remained effective with lower bleed rates than prior FVIII therapies. [Display omitted] DisclosuresCockerham: Sanofi: Current Employment, Current equity holder in publicly-traded company. Wilson: Sanofi: Current Employment, Current equity holder in publicly-traded company; Alexion: Current equity holder in publicly-traded company. Frick: Trio Health, Inc.: Current Employment; Sanofi S.A.: Research Funding. Dasmahapatra: Sanofi: Current Employment, Current equity holder in publicly-traded company.

In Situ Vaccination with IL-12Fc and TLR Agonist - a Crucial Role for B Cells in Generating Anti-Tumor T Cell Immunity

Interleukin 12 (IL-12) is a potent proinflammatory cytokine that plays a central role in regulating both innate and adaptive immune responses. This cytokine has been tested as a cancer immunotherapy agent based on its impressive anti-tumor effects in animal models. Clinically, systemic delivery of IL-12 incurred dose-limiting toxicities because of its pharmaco-kinetic extremes of peaks and troughs, a result of its short serum half-life. Here, we evaluated mDF6006, a mouse IL-12 Fc-fusion protein which was designed to have a prolonged half-life with equivalent potency to recombinant mouse IL-12.We hypothesized that local intra-tumoral (IT) delivery of low doses IL-12Fc would lead to immune-therapeutic potential.To screen candidates for in situ vaccination, we have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting immune response is detected by the regression of the distant, untreated tumor.Using this assay for abscopal therapeutic effects we have tested a variety of immune stimulatory ligands and antibodies against immune checkpoints and T cell co-stimulatory targets, alone and in multiple combinations.Among these, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG), a TLR9 ligand, and IL-12Fc has provided the most impressive results. TLRs, expressed in antigen-presenting cells and are components of the innate immune system that recognize molecular patterns on bacterial, fungal, or viral pathogens.In a mouse model of lymphoma, IT injection of CpG and IL-12Fc, combined to cause tumor eradication at both the injected site and the untreated tumor. All these results were dependent on T cells in the animal.During this therapy we observed a significant increase in activated B cells within the treated tumor and especially in its draining lymph nodes (dLN). This B cell stimulation was contributed by both the CpG and the IL12-Fc. Surprisingly, depletion of the animals of B cells by an anti-CD20 antibody completely ablated the abscopal therapeutic effect.B cells function as antigen-presenting cells, secrete cytokines and are precursors of antibody producing plasma cells. To evaluate which of these roles B cells play in the therapeutic efficacy of CpG and IL-12Fc we isolated B cells from the LN of the treated mice and co-cultured them with T cells. These B cells activated T cells in an antigen-specific manner that was MHC dependent. No such T cell activation occurred when MHC molecules were blocked with anti-MHC antibodies or when the cells were physically separated by a semi-permeable membrane. Thus, local CpG and IL12-Fc directly activated B cells to present tumor antigens to specific T cells in the tumor microenvironment, resulting ultimately in an abscopal tumor response.IL-12Fc and CpG are both currently in phase-1/2 trials as single agents and in combinations with checkpoint antibodies. Our results provide strong rationale for using low doses of IL-12Fc with CpG as an in situ therapeutic vaccine for lymphoma. DisclosuresLevy: Virsti: Membership on an entity's Board of Directors or advisory committees; Khloris: Membership on an entity's Board of Directors or advisory committees; Abintus Bio: Membership on an entity's Board of Directors or advisory committees; Kira: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees.

Efmoroctocog Alfa: A Review in Haemophilia A.

Efmoroctocog alfa (Elocta®, Eloctate®, Eloctate™), an extended half-life (EHL) recombinant factor VIII (rFVIII)-Fc fusion protein, is approved for the treatment and prophylaxis of bleeding in patients with haemophilia A. The efficacy of efmoroctocog alfa in the prevention and treatment of bleeding in previously treated patients (PTPs) and previously untreated patients (PUPs) with severe haemophilia A has been demonstrated in phase III studies; this includes its use in the perioperative setting (in PTPs). Furthermore, the effectiveness of efmoroctocog alfa in clinical practice has been confirmed in numerous real-world studies; compared with conventional, standard half-life (SHL) FVIII products, prophylaxis with this EHL FVIII product achieved similar or reduced bleeding rates with fewer injections. Efmoroctocog alfa was generally well tolerated; inhibitors occurred in approximately one-third of PUPs in a phase III study. Efmoroctocog alfa is an established and effective EHL FVIII replacement therapy for the management of haemophilia A. Compared with SHL FVIII products, EHL FVIII products such as efmoroctocog alfa have the potential to optimise prophylactic outcomes by decreasing the burden of treatment or increasing the level of bleed protection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40265-021-01615-w.

380 GS-3583, a novel FLT3 agonist Fc fusion protein, expands conventional dendritic cells in healthy volunteers

BackgroundConventional dendritic cells subtype 1 (cDC1) play a vital role in the priming and expansion of tumor specific CD8+ T cells and their recruitment to tumor microenvironment (TME). However, cDC1s are often underrepresented in the TME. Systemic administration of Fms-like tyrosine kinase 3 ligand (FLT3L), a hematopoietic growth factor that binds to FLT3 on myeloid and lymphoid progenitor cells, leads to expansion of cDC1s in the periphery which can then be recruited into the TME. FLT3 pathway stimulation using GS-3583, a novel FLT3 agonistic Fc fusion protein, has the potential to promote T cell mediated anti-tumor activity. We sought to evaluate the pharmacodynamic (PD) effect of a single dose of GS-3583 in healthy volunteers alongside its safety. Herein, we present the updated results of the study.MethodsThis was a first-in-human, placebo-controlled study of GS-3583 in healthy volunteers to evaluate the safety, pharmacokinetics (PK), and PD of escalating single doses (ranging from 75 micrograms to 2000 micrograms) of GS-3583. The study was blinded to the subjects and the investigator. Each dose cohort enrolled 8–12 healthy subjects who received GS-3583 or placebo as single IV infusion at 3:1 ratio. Subjects were observed in the phase 1 unit for 15 days and then for 12 weeks as outpatients. As part of the PD evaluation, we investigated the changes in the number of cDC1 and cDC2 cells.ResultsAs of 2nd July 2021, selected safety, PK and PD data from all 4 cohorts were available. GS-3583 was well tolerated and all subjects had been discharged. To date, there have been no serious or grade 3 or higher adverse events. Preliminary PK analysis suggested dose-dependent increase in GS-3583 exposure (AUC and Cmax). Preliminary PD analysis shows that administration of GS-3583 resulted in temporary, dose-dependent increases in cDC1/cDC2 cells that peaked between days 5–11 (higher doses resulted in later peaks) and returned to baseline within 3 weeks of drug administration (table 1, figure 1).Abstract 380 Table 1Selected subject characteristics and pharmacodynamic resultsAbstract 380 Figure 1A) Comparison of cDC1 cell quantitative changes in cohorts 1–4; B) Comparison of cDC2 cell quantitative changes in cohorts 1–4ConclusionsGS-3583 infusion was well tolerated and induced dose dependent expansion of dendritic cells in the periphery in healthy volunteers. In patients with cancer, this increase in dendritic cells can be utilized to enhance anti-tumor therapeutic effects of immuno-oncology therapies.AcknowledgementsFunding provided by Gilead Sciences, Inc.Ethics ApprovalThe study received study site IRB/Ethics Committee approval prior to enrollment of subjects.

Development of a recombinant vaccine containing a spike S1-Fc fusion protein induced protection against MERS-CoV in human DPP4 knockin transgenic mice

The Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the family Coronaviridae and genus Betacoronavirus, has been recognized as a highly pathogenic virus. Due to the lack of therapeutic or preventive agents against MERS-CoV, developing an effective vaccine is essential for preventing a viral outbreak. To address this, we developed a recombinant S1 subunit of MERS-CoV spike protein fused with the human IgG4 Fc fragment (LV-MS1-Fc) in Chinese hamster ovary (CHO) cells. Thereafter, we identified the baculovirus gp64 signal peptide-directed secretion of LV-MS1-Fc protein in the extracellular fluid. To demonstrate the immunogenicity of the recombinant LV-MS1-Fc proteins, BALB/c mice were inoculated with 2.5 μg of LV-MS1-Fc. The inoculated mice demonstrated a significant humoral immune response, measured via total IgG and neutralizing antibodies. In addition, human dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with LV-MS1-Fc showed the protective capacity of LV-MS1-Fc against MERS-CoV with no inflammatory cell infiltration. These data showed that the S1 and Fc fusion protein induced potent humoral immunity and antigen-specific neutralizing antibodies in mice, and conferred protection against coronavirus viral challenge, indicating that LV-MS1-Fc is an effective vaccine candidate against MERS-CoV infection.

Protein engineering of a stable and potent anti-inflammatory IL-37-Fc fusion with enhanced therapeutic potential

Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics.

A survey of physicians’ treatment switching practice in people on long-term prophylaxis for hemophilia in five European countries

Objective Recombinant factor VIII and factor IX Fc fusion proteins (rFVIIIFc and rFIXFc) were developed with an extended half-life (EHL) to improve the management of people with hemophilia A (PwHA) and B (PwHB), respectively. Methods This survey gathered physician-reported treatment decisions and physician views on outcomes in PwHA or PwHB who switched to rFVIIIFc or rFIXFc in the 12 months prior to study completion. Results Physicians (N = 37) considered bleeds, pharmacokinetic parameters, joint health and adherence the most important factors to assess both in routine care and when deciding to switch to an EHL therapy. In the 12 months prior to study completion, 37 physicians switched 113 PwHA to rFVIIIFc and 25 physicians switched 36 PwHB to rFIXFc. Most PwH (>90%) had moderate or severe hemophilia and many (>60%) switched within 6 months of the survey. The main reason for switching PwHA to rFVIIIFc was to allow fewer injections (49%), while the main reason for switching PwHB to rFIXFc was the product becoming available for use (36%). Overall, 96% of PwHA and 89% of PwHB who were switched remained on these EHL products at the time of survey. Mean total weekly dose, injection frequency and annualized bleeding rate were reported to have reduced following switching. Conclusion This survey provides valuable insight into reasons for, and challenges to, the use of EHL products in clinical practice. Physicians perceived that switching to treatment with rFVIIIFc or rFIXFc can improve quality of life, treatment burden, disease control and adherence.

Antigen improves binding of IgGs to FcγRs in SPR analysis

FcγR binding characterization is one of the critical attributes during the development of therapeutic antibodies. Here, we report a novel assay format to characterize IgG-FcγR interaction in the presence of antigen using Surface plasmon resonance (SPR). The new assay format was developed by creating stable antigen/antibody immunocomplexes on a sensor chip surface before injection of FcγRs. In this assay format, binding activity of both huIgG1 (including IgG1 Fc fusion Protein) and huIgG2 increased significantly to most activating human FcγRs, especially to FcγRI, FcγRIIa-131H and FcγRIIIa-158F. To our knowledge, this study provides the first set of evidence using a biophysical method to demonstrate antigen binding facilitating IgG-FcγR interaction, especially for huIgG2 where previous studies did not indicate its binding to human FcγRI or FcγRIIIa-158F. Although further studies are needed to investigate the correlation of the binding data with effector function data in vivo, our results suggest that it may be useful to evaluate the IgG-FcγR interaction in the presence of antigen to help design safer and more effective biotherapeutics.

Collagen Fragments Produced in Cancer Mediate T Cell Suppression Through Leukocyte-Associated Immunoglobulin-Like Receptor 1.

The tumor microenvironment (TME) is a complex structure comprised of tumor, immune and stromal cells, vasculature, and extracellular matrix (ECM). During tumor development, ECM homeostasis is dysregulated. Collagen remodeling by matrix metalloproteinases (MMPs) generates specific collagen fragments, that can be detected in the circulation of cancer patients and correlate with poor disease outcome. Leukocyte-Associated Immunoglobulin-like Receptor-1 (LAIR-1) is an inhibitory collagen receptor expressed on immune cells in the TME and in the circulation. We hypothesized that in addition to ECM collagen, collagen fragments produced in cancer can mediate T cell immunosuppression through LAIR-1. Our analyses of TCGA datasets show that cancer patients with high tumor mRNA expression of MMPs, collagen I and LAIR-1 have worse overall survival. We show that in vitro generated MMP1 or MMP9 collagen I fragments bind to and trigger LAIR-1. Importantly, LAIR-1 triggering by collagen I fragments inhibits CD3 signaling and IFN-γ secretion in a T cell line. LAIR-2 is a soluble homologue of LAIR-1 with higher affinity for collagen and thereby acts as a decoy receptor. Fc fusion proteins of LAIR-2 have potential as cancer immunotherapeutic agents and are currently being tested in clinical trials. We demonstrate that collagen fragment-induced inhibition of T cell function could be reversed by LAIR-2 fusion proteins. Overall, we show that collagen fragments produced in cancer can mediate T cell suppression through LAIR-1, potentially contributing to systemic immune suppression. Blocking the interaction of LAIR-1 with collagen fragments could be an added benefit of LAIR-1-directed immunotherapy.

Evaluation of a Virus-like Nanoparticle Porcine Circovirus Type-2 (PCV2) Capsid Protein Fused with the Pig Immunoglobulin Fc Fragment as a Novel Vaccine Candidate against PCV2 in Mice.

Porcine circovirus Type 2 (PCV2) is a primary etiological pathogen of post-weaning multi-systemic wasting syndrome (PMWS). The capsid protein of PCV2 is the crucial immunogenic protein which can induce antibody generation and immune responses. However, there is still a lack of efficient PCV2 vaccines with high immunogenicity. In the current study, we developed a novel engineered PCV2 capsid (∆1-41aa)-pFc fusion protein (PCFP), which comprised a truncated capsid protein of PCV2 and a porcine IgG Fc fragment, fused to the capsid protein of PCV2 at the C-terminus. We found that this novel fusion protein could auto-assemble into virus-like nanoparticles with an estimated mean diameter of 22.6 nm, characterized by transmission electron microscopy. Immunization of BALB/c mice with this fusion protein significantly increased the production levels of anti-PCV2-capsid protein antibody in serum. Besides, the virus-like nanoparticles, PCFP was demonstrated to induce efficient cellular immune responses in mice, as evident by the high specific T cell reactivity to the PCFP fusion protein and the high production of the immune cytokines IFN-γ and IL-10 in an ex vivo re-stimulation system. Collectively, these findings demonstrate that the PCV2 truncated capsid subunit Fc-fusion protein can induce both cellular and humoral immune responses, and it displays great application potential.

Recombinant factor IX‐Fc fusion protein in severe hemophilia B: Patient‐reported outcomes and health‐related quality of life

IntroductionIn 2017, all people with severe hemophilia B in Ireland switched to recombinant factor IX Fc fusion protein concentrate (rFIXFc) prophylaxis. Patient‐reported outcomes (PROs) and health‐related quality of life (HRQoL) are important to evaluate with new treatments. AimsTo assess HRQoL in people with severe hemophilia B and their experience after switching to rFIXFc prophylaxis. MethodsParticipants completed a Patient Reported Outcomes Burden and Experience (PROBE) questionnaire on initiation and following two years of rFIXFc prophylaxis. The PROBE questionnaire has four domains: demographics, general health, haemophilia‐specific, and European Quality of Life 5‐Dimensions (EQ‐5D‐5L) questionnaire. ResultsTwenty‐three participants completed the questionnaire at both time points. The number of activities where chronic pain occurred and interfered with the activity was reduced by 25% and 33%, respectively (P< .001), following two years of rFIXFc prophylaxis. There was a 9% decrease in chronic pain during the second year of rFIXFc prophylaxis compared to baseline, but the rate remained high, at 74%. A 25% reduction in the number of affected activities of daily living (ADLs) was reported following 2 years of rFIXFc prophylaxis (P= .007). The most common health problems were arthritis, hypertension, anxiety/depression, and gingivitis. The median EQ‐5D‐5L score was similar following two years of rFIXFc prophylaxis, 0.76 (range, −0.01 to 0.95), compared to 0.77 (range, 0.36‐1) at baseline. ConclusionThis study of real‐world patient experience using PROs demonstrates a reduction in chronic pain and improvement in ADLs in participants after switching to rFIXFc prophylaxis. It provides important insights into patient‐identified health care needs and living with severe hemophilia B.

Development of an IgG-Fc fusion COVID-19 subunit vaccine, AKS-452

AKS-452 is a biologically-engineered vaccine comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain antigen (Ag) and human IgG1 Fc (SP/RBD-Fc) in clinical development for the induction and augmentation of neutralizing IgG titers against SARS-CoV-2 viral infection to address the COVID-19 pandemic. The Fc moiety is designed to enhance immunogenicity by increasing uptake via Fc-receptors (FcγR) on Ag-presenting cells (APCs) and prolonging exposure due to neonatal Fc receptor (FcRn) recycling. AKS-452 induced approximately 20-fold greater neutralizing IgG titers in mice relative to those induced by SP/RBD without the Fc moiety and induced comparable long-term neutralizing titers with a single dose vs. two doses. To further enhance immunogenicity, AKS-452 was evaluated in formulations containing a panel of adjuvants in which the water-in-oil adjuvant, Montanide™ ISA 720, enhanced neutralizing IgG titers by approximately 7-fold after one and two doses in mice, including the neutralization of live SARS-CoV-2 virus infection of VERO-E6 cells. Furthermore, ISA 720-adjuvanted AKS-452 was immunogenic in rabbits and non-human primates (NHPs) and protected from infection and clinical symptoms with live SARS-CoV-2 virus in NHPs (USA-WA1/2020 viral strain) and the K18 human ACE2-trangenic (K18-huACE2-Tg) mouse (South African B.1.351 viral variant). These preclinical studies support the initiation of Phase I clinical studies with adjuvanted AKS-452 with the expectation that this room-temperature stable, Fc-fusion subunit vaccine can be rapidly and inexpensively manufactured to provide billions of doses per year especially in regions where the cold-chain is difficult to maintain.

Monitoring of different factor VIII replacement products using a factor VIII one-stage clotting assay on cobas t 511/711 analysers.

IntroductionRecombinant coagulation factor VIII (FVIII) products are the standard of care for patients with haemophilia A. The development of modified FVIII products has provided benefit for patients but presented challenges for monitoring FVIII activity.AimThis single‐centre study evaluated the Roche FVIII one‐stage clotting assay (OSA) in measuring FVIII activity in plasma samples spiked with seven FVIII products at clinically relevant concentrations.MethodsFVIII‐deficient plasma samples were spiked with two batches of recombinant FVIII products (octocog alfa, moroctocog alfa, simoctocog alfa, efmoroctocog alfa, damoctocog alfa pegol, rurioctocog alfa pegol, lonoctocog alfa) at 1–120 IU/dL FVIII activity, according to their labelled potency. Measurement was conducted on the cobas t 511/711 analysers using the Roche FVIII OSA and the Technoclone TECHNOCHROM FVIII:C chromogenic substrate assay (CSA).ResultsUsing the OSA, FVIII activity was close to labelled potency for most analysed FVIII products including a recombinant FVIII Fc fusion protein. PEGylated FVIII product, damoctocog alfa pegol, was marginally above and single‐chain product, lonoctocog alfa, below the predefined acceptance criteria: for FVIII activity < 25 IU/dL: ± 5 IU/dL; for FVIII activity ≥ 25 IU/dL: ± 20% (relative). The different principles of OSA and CSA led to discrepancies in the estimation of all analysed FVIII products. Additionally, in vitro recovery was increased at lower levels of FVIII activity using the OSA, whereas recovery was more consistent using the CSA.ConclusionThese data allow the interpretation of FVIII activity results for different FVIII products using the Roche FVIII OSA on the cobas t 511/711 analysers.

BTN2A2 protein negatively regulates T cells to ameliorate collagen-induced arthritis in mice

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by persistent inflammatory responses in target tissues and organs, resulting in the destruction of joints. Collagen type II (CII)-induced arthritis (CIA) is the most used animal model for human RA. Although BTN2A2 protein has been previously shown to inhibit T cell functions in vitro, its effect on autoimmune arthritis has not been reported. In this study, we investigate the ability of a recombinant BTN2A2-IgG2a Fc (BTN2A2-Ig) fusion protein to treat CIA. We show here that administration of BTN2A2-Ig attenuates established CIA, as compared with control Ig protein treatment. This is associated with reduced activation, proliferation and Th1/Th17 cytokine production of T cells in BTN2A2-Ig-treated CIA mice. BTN2A2-Ig also inhibits CII-specific T cell proliferation and Th1/Th17 cytokine production. Although the percentage of effector T cells is decreased in BTN2A2-Ig-treated CIA mice, the proportions of naive T cells and regulatory T cells is increased. Furthermore, BTN2A2-Ig reduces the percentage of proinflammatory M1 macrophages but increases the percentage of anti-inflammatory M2 macrophages in the CIA mice. Our results suggest that BTN2A2-Ig protein has the potential to be used in the treatment of collagen-induced arthritis models.

Methods for Functional Characterization of FcRn Interactions with Therapeutic Antibodies and Fc-Fusion Proteins.

The neonatal Fc receptor (FcRn) plays a key role in determining the pharmacokinetic behavior of therapeutic monoclonal antibodies (mAbs). FcRn-mediated intracellular trafficking mechanisms extend the half-lives of mAbs by rescuing them from lysosomal degradation and contribute to their transportation from the vascular space to tissue compartments such as placenta and mucosal surfaces. It is important to characterize the FcRn interactions of therapeutic mAbs and Fc-fusion proteins due to its potential impact on their in vivo pharmacokinetic properties such as clearance and half-life. In this chapter, we describe protocols for two cell-based assays that measure the total function of FcRn which involves pH-dependent association and dissociation with IgG-Fc, as well as FcRn-mediated intracellular trafficking parameters. These assays are suitable for characterization of FcRn interactions with therapeutic mAbs and Fc-fusion proteins for the purpose of assessing lot-to-lot consistency and the structural and functional integrity of the Fc domain. In addition, they may serve as cost-effective screening tools for the evaluation of mAb-based drug candidates during lead selection and optimization for desired pharmacokinetic properties.

Circulating mature dendritic cells homing to the thymus promote thymic epithelial cells involution via the Jagged1/Notch3 axis

Multiple proinflammatory conditions, including chemotherapy, radiotherapy, transplant rejection, and microbial infections, have been identified to induce involution of the thymus. However, the underlying cellular and molecular mechanisms of these inflammatory conditions inducing apoptosis of thymic epithelial cells (TECs), the main components of the thymus, remain largely unknown. In the circulation, mature dendritic cells (mDCs), the predominant initiator of innate and adaptive immune response, can migrate into the thymus. Herein, we demonstrated that mDCs were able to directly inhibit TECs proliferation and induce their apoptosis by activating the Jagged1/Notch3 signaling pathway. Intrathymic injection of either mDCs or recombinant mouse Jagged1-human Fc fusion protein (rmJagged1-hFc) into mice resulted in acute atrophy of the thymus. Furthermore, DAPT, a γ-secretase inhibitor, reversed the effects induced by mDC or rmJagged1-hFc. These findings suggest that acute or aging-related thymus degeneration can be induced either by mass migration of circulating mDCs in a short period of time or by a few but constantly homing mDCs.