IgG Fc Fusion Protein Research Articles

Rhesus Macaque Killer Cell Ig-like Receptor Domain 0 Glycans Impact Surface Expression and Ligand Specificity.

Defining the MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as a model for infectious diseases and comparative immunogenetics. Several rhesus macaque KIRs belong to a phylogenetically distinct group with a three-amino acid deletion in domain 0 (D0). This deletion results in polymorphic differences in potential N-linked glycosylation (PNG) sites adjacent to a predicted KIR-MHC class I contact site. Whereas most KIRs have two tandem PNG sites in D0 (N36FTN39FT), the KIRs containing the deletion only have a single site in this region (N36FT). To discern the contribution of glycosylation to KIR expression and ligand recognition, we constructed PNG mutants for six lineage II KIR genes that eliminate or create sites for N-glycan addition at these locations. The impact of these mutations on total and surface expression was determined by immunoblotting and flow cytometry. Ligand engagement was assessed by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion proteins. We found that N36FT is glycosylated in KIR with a single site, and at least one site is glycosylated in KIRs with two tandem sites. In general, for rhesus KIRs with a single D0 glycosylation site, that site contributes to surface expression. For KIRs with two tandem sites, the first site can contribute to ligand specificity. This study establishes that D0 glycosylation of rhesus macaque KIRs modulates surface expression and contributes to ligand specificity.

Neurons upregulate PD-L1 via IFN/STAT1/IRF1 to alleviate damage by CD8+ T cells in cerebral malaria

BackgroundCerebral malaria (CM) is the most lethal complication of malaria, and survivors usually endure neurological sequelae. Notably, the cytotoxic effect of infiltrating Plasmodium-activated CD8+ T cells on cerebral microvasculature endothelial cells is a prominent feature of the experimental CM (ECM) model with blood–brain barrier disruption. However, the damage effect of CD8+ T cells infiltrating the brain parenchyma on neurons remains unclear. Based on the immunosuppressive effect of the PD-1/PD-L1 pathway on T cells, our previous study demonstrated that the systemic upregulation of PD-L1 to inhibit CD8+ T cell function could effectively alleviate the symptoms of ECM mice. However, it has not been reported whether neurons can suppress the pathogenic effect of CD8+ T cells through the PD-1/PD-L1 negative immunomodulatory pathway. As the important inflammatory factor of CM, interferons can induce the expression of PD-L1 via different molecular mechanisms according to the neuro-immune microenvironment. Therefore, this study aimed to investigate the direct interaction between CD8+ T cells and neurons, as well as the mechanism of neurons to alleviate the pathogenic effect of CD8+ T cells through up-regulating PD-L1 induced by IFNs.MethodsUsing the ECM model of C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), morphological observations were conducted in vivo by electron microscope and IF staining. The interaction between the ECM CD8+ T cells (immune magnetic bead sorting from spleen of ECM mice) and primary cultured cortical neurons in vitro was observed by IF staining and time-lapse photography. RNA-seq was performed to analyze the signaling pathway of PD-L1 upregulation in neurons induced by IFNβ or IFNγ, and verified through q-PCR, WB, IF staining, and flow cytometry both in vitro and in vivo using IFNAR or IFNGR gene knockout mice. The protective effect of adenovirus-mediated PD-L1 IgGFc fusion protein expression was verified in ECM mice with brain stereotaxic injection in vivo and in primary cultured neurons via viral infection in vitro.ResultsIn vivo, ECM mice showed infiltration of activated CD8+ T cells and neuronal injury in the brain parenchyma. In vitro, ECM CD8+ T cells were in direct contact with neurons and induced axonal damage, as an active behavior. The PD-L1 protein level was elevated in neurons of ECM mice and in primary cultured neurons induced by IFNβ, IFNγ, or ECM CD8+ T cells in vitro. Furthermore, the IFNβ or IFNγ induced neuronal expression of PD-L1 was mediated by increasing STAT1/IRF1 pathway via IFN receptors. The increase of PD-L1 expression in neurons during PbA infection was weakened after deleting the IFNAR or IFNGR. Increased PD-L1 expression by adenovirus partially protected neurons from CD8+ T cell-mediated damage both in vitro and in vivo.ConclusionOur study demonstrates that both type I and type II IFNs can induce neurons to upregulate PD-L1 via the STAT1/IRF1 pathway mediated by IFN receptors to protect against activated CD8+ T cell-mediated damage, providing a targeted pathway to alleviate neuroinflammation during ECM.Graphical

Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

Interim results from a phase I randomized, placebo-controlled trial of novel SARS-CoV-2 beta variant receptor-binding domain recombinant protein and mRNA vaccines as a 4th dose booster

SARS-CoV-2 booster vaccination should ideally enhance protection against variants and minimise immune imprinting. This Phase I trial evaluated two vaccines targeting SARS-CoV-2 beta-variant receptor-binding domain (RBD): a recombinant dimeric RBD-human IgG1 Fc-fusion protein, and an mRNA encoding a membrane-anchored RBD. 76 healthy adults aged 18-64y, previously triple vaccinated with licensed SARS-CoV-2 vaccines, were randomised to receive a 4th dose of either an adjuvanted (MF59®, CSL Seqirus) protein vaccine (5, 15 or 45μg, N=32), mRNA vaccine (10, 20, or 50μg, N=32), or placebo (saline, N=12) at least 90 days after a 3rd boost vaccination or SARS-CoV-2 infection. Bleeds occurred on days 1 (prior to vaccination), 8, and 29. govNCT05272605. No vaccine-related serious or medically-attended adverse events occurred. The protein vaccine reactogenicity was mild, whereas the mRNA vaccine was moderately reactogenic at higher dose levels. Best anti-RBD antibody responses resulted from the higher doses of each vaccine. A similar pattern was seen with live virus neutralisation and surrogate, and pseudovirus neutralisation assays. Breadth of immune response was demonstrated against BA.5 and more recent omicron subvariants (XBB, XBB.1.5 and BQ.1.1). Binding antibody titres for both vaccines were comparable to those of a licensed bivalent mRNA vaccine. Both vaccines enhanced CD4+ and CD8+ T cell activation. There were no safety concerns and the reactogenicity profile was mild and similar to licensed SARS-CoV-2 vaccines. Both vaccines showed strong immune boosting against beta, ancestral and omicron strains. Australian Government Medical Research Future Fund, and philanthropies Jack Ma Foundation and IFM investors.

Efficacy and safety of tripterygium wilfordii Hook F plus TNF inhibitor for active rheumatoid arthritis: A multicentre, randomized, double-blind, triple-dummy controlled trial

An investigator-initiated, multicentre, randomized, double-blind, triple-dummy, controlled trial was conducted at 14 tertiary rheumatology centers in China to evaluate the efficacy and safety of Tripterygium wilfordii Hook F (TwHF) with recombinant human TNF receptor IgGFc fusion protein (rhTNFR-Fc) in active Rheumatoid Arthritis (RA). Primary endpoint was the proportion of patients achieved a 50% improvement of American College of Rheumatology criteria (ACR50) in TwHF+rhTNFR-Fc vs. methotrexate (MTX) group at week 12. ACR50 was achieved in 57.1% (72/126), 41.3% (52/126), 23.0% (29/126), and 26.2% (33/126) patients receiving TwHF+rhTNFR-Fc, MTX + rhTNFR-Fc, TwHF and MTX monotherapy, respectively, at week 12 (TwHF+rhTNFR-Fc vs. other three groups, all p < 0.05). No statistical difference in serious adverse events or adverse events leading to discontinuation of study across all groups was documented. TwHF+rhTNFR-Fc was superior to MTX for active RA, and was more effective than MTX + rhTNFR-Fc on ACR50, with a similar safety profile.Trial registration:ClinicalTrials.govNCT03589833.

Dual phenotypic characteristics of P-selectin in a mouse model of hemorrhagic shock and hepatectomy

BackgroundMembrane-bound P-selectin induces endothelial adhesion of leucocytes and amplifies organ inflammations during major trauma, while soluble P-selectin (sP-sel) mediates survival rescue properties. This study characterized the differential effects of P-selectin in a “2-hit” model of hemorrhagic shock (HS) and partial hepatectomy (PH). Materials and methodsHS was induced by withdrawing blood (0.3 mL) directly from the mouse femoral arteries. 70% or 50% of liver volumes were resected after inducing HS. Time of survival in P-selectin deficient (Selp −/−) mice treated with and without intraperitoneal injections of recombinant P-sel IgG-Fc fusion proteins (rP-sel-Fc, 1.5 mg/kg) were recorded for up to 72h after injury. In addition, liver regeneration at 72h after HS and 50% PH was assessed in wild-type and Selp −/− mice. ResultsCompared to wild-types, Selp −/− mice had significantly higher mortality rates post HS and 70% PH, as none of these animals survived up to 48h postoperatively. The survival curve was restored in Selp −/− mice pre-treated with rP-sel-Fc. In the HS followed by 50% PH experimental arm, liver remnant growth ratios were significantly higher in Selp −/− mice (15.7 ± 3.1 vs 11.7 ± 4.9, P = 0.040). The elevated serum concentrations of alanine aminotransferase post-PH were significantly reduced in Selp −/− mice. Hepatocyte proliferation indices (CYP7a1 and PCNA) expression were enhanced and myeloperoxidase activity in the regenerated remnant liver was reduced in the Selp −/− mice. ConclusionIn critical conditions induced by HS and PH, P-selectin mediates two distinct phenotypic characteristics. Soluble-form circulating P-selectin improves survival in the acute stage of HS and extensive loss of liver parenchyma; membrane-bound P-selectin induces regional pro-inflammatory reactions in the remnant liver after the acute stage of two insults, thereby inhibiting hepatic regeneration. The results of this pre-clinical study may provide molecular mechanistic insight and clinical therapeutic applications of P-selectin in the acute and regenerative phases of traumatic hepatic injury.

A phase Ib trial of the VEGF fusion protein HB002.1T plus chemotherapy in patients with advanced solid tumors.

2530 Background: HB002.1T is a recombinant human vascular endothelial growth factor receptor (VEGF)-IgG Fc fusion protein, which functions as a decoy receptor to bind VEGF-A, VEGF-B and placental growth factor (PlGF). In phase Ia, HB002.1T monotherapy showed encouraging anti-tumor activity in gallbladder cancer. Methods: This study aimed to evaluate efficacy and safety of HB002.1T plus chemotherapy (mainly based on platinum, paclitaxel, gemcitabine, etc.) in advanced solid tumors. The tumor assessment was evaluated by investigator according to RECIST v1.1. The study included part 1 and part 2, part 1 study was dose escalation (12mg/kg, 16mg/kg Q3W) and dose expansion conducted in patients (pts) with advanced solid tumors, part 2 study was dose expansion (16mg/kgQ3W) in specified tumors (cohort 1: ovarian and cervical, cohort 2: pancreatic, gallbladder and bile duct cancer). Results: As of Dec 20,2022,68 (42F/26M) advanced solid tumor pts were enrolled in the study with 32 pts in part 1 and 36 pts in part 2. 33.3% pts failed at least 3 lines of therapy before enrollment. 63(92.6%) pts had stage IV disease. Median age was 58. It is to be note that 12 efficacy assessment evaluablepts with ovarian cancer (1 with 12mg/kg in part 1,11with 16mg/kg in part 2) were all stage IV, which included 3 complete response (CR),6 partial responses (PR) and 3 stable disease (SD). Median progression free survival (PFS) was 7.3 months and 6-month PFS rate was 58.3%. The ORR and DCR were 75%, 100% respectively. The most common treatment related adverse events (TRAEs) included neutropenia (9/12, 75.0%) and leukocytopenia (8/12, 66.7%). 8 (8/12, 66.7%) pts had grade ≥ 3 TRAEs, which neutropenia (4/12, 33.3%) and leukocytopenia (3/12, 25.0%) were commonly observed. Part 1: In 28 pts whose tumor assessment were available, including 1 gastric cancer patient achieved CR, 11 pts achieved PR (2 gastric, 2 ovarian, 2 cervical, 1 maxillary, 1 lung, 1 parotid, 1 nasopharynx, 1 pancreatic) and 15 achieved SD. Part 2: 32 pts were available for tumor assessment, including 4 achieved CR (3 ovarian, 1 cervical), 10 achieved PR (4 ovarian, 2 cervical, 3 bileduct, 1 pancreatic), and 16 achieved SD. Overall, in Part 1 and 2, TRAEs occurred in 64 (64/68, 94.1%) pts. The most common TRAEs were leukocytopenia (28/68, 41.2%), neutropenia (26/68, 38.2%) and proteinuria (25/68, 36.8%). 34 (34/68, 50.0%) pts had grade ≥ 3 TRAEs, which neutropenia (15/68, 22.1%) and leukocytopenia (11/68, 16.2%) were commonly observed. TRAE leading to discontinuation occurred in 3 pts. No death was reported related with HB002.1T. Conclusions: The Pts were well tolerated to 12mg/kg and 16mg/kg HB002.1T dose combined with chemotherapy, and HB002.1T showed acceptable safety profile. As well as, promising anti-tumor activity was observed in various tumors, especially in advanced ovarian cancer. More data is being accumulated, and confirmatory clinical trials are being prepared. Clinical trial information: NCT04802980 .

MHC Class I Ligands of Rhesus Macaque Killer Cell Ig-like Receptors.

Definition of MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as an animal model for infectious diseases, reproductive biology, and transplantation. To provide a more complete foundation for studying NK cell responses, rhesus macaque KIRs representing common allotypes of lineage II KIR genes were tested for interactions with MHC class I molecules representing diverse Macaca mulatta (Mamu)-A, -B, -E, -F, -I, and -AG alleles. KIR-MHC class I interactions were identified by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion proteins. Ligands for 12 KIRs of previously unknown specificity were identified that fell into three general categories: interactions with multiple Mamu-Bw4 molecules, interactions with Mamu-A-related molecules, including allotypes of Mamu-AG and the hybrid Mamu-B*045:03 molecule, or interactions with Mamu-A1*012:01. Whereas most KIRs found to interact with Mamu-Bw4 are inhibitory, most of the KIRs that interact with Mamu-AG are activating. The KIRs that recognize Mamu-A1*012:01 belong to a phylogenetically distinct group of macaque KIRs with a 3-aa deletion in the D0 domain that is also present in human KIR3DL1/S1 and KIR3DL2. This study more than doubles the number of rhesus macaque KIRs with defined MHC class I ligands and identifies interactions with Mamu-AG, -B*045, and -A1*012. These findings support overlapping, but nonredundant, patterns of ligand recognition that reflect extensive functional diversification of these receptors.

Abstract C035: Biotherapeutic strategies targeting the CXCR2 axis for depletion of myeloid-derived suppressor cells in pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) has a 5-year survival rate of only 10%, and limited treatment options exist. Immune checkpoint inhibitors are effective in a variety of cancer types; however, the complex immunosuppressive tumor microenvironment (TME) of PDAC is a significant barrier to immunotherapy. Myeloid-derived suppressor cells (MDSCs) are abundant in PDAC tumors from humans and mouse models. MDSCs suppress effector T cell function, leading us to hypothesize that targeting tumor MDSCs would be an effective therapeutic strategy for PDAC and enhance the efficacy of T cell-based immunotherapy. Our preliminary data and the work from others shows that CXCR2 ligands such as CXCL8/IL-8, CXCL5/ENA78, and CXCL1/GROα are the most abundant soluble factors secreted by PDAC cells. CXCR2 ligands are critical for the homing of myeloid cells to sites of injury in cutaneous wound healing, suggesting that dysregulated secretion of CXCR2 chemokines in PDAC may mimic a wound healing response that attracts myeloid cells and promotes the expansion of MDSCs and an immunosuppressive TME. To test this hypothesis, we have designed multiple biotherapeutic molecules that exploit CXCL5/ENA78 and CXCL1/GROa to target and eliminate MDSCs from PDAC tumors. Specifically, we have generated Pseudomonas aeruginosa exotoxin (ExoA) and IgG-Fc fusion proteins that will be used to deliver cytotoxic payloads to MDSCs. A variety of CXCR2 knockout and knock-in models will be used to demonstrate CXCR2 dependence and elimination of MDSCs in vitro. In vivo, we are utilizing the KPC (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) mouse model of PDAC, which we've shown in preliminary studies to, like human PDAC, support an abundance of MDSCs in tumors as well as secondary lymphoid organs such as the spleen. We anticipate that this study will demonstrate the therapeutic potential and actionability of targeting MDSCs and will provide novel biotherapeutic drug candidates that will ultimately improve immunotherapy outcomes in patients with PDAC. Citation Format: Ben N. Christopher, Reeder Robinson, Leticia Reyes, Lena Golick, Ashton Basar, Nathan Dolloff. Biotherapeutic strategies targeting the CXCR2 axis for depletion of myeloid-derived suppressor cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C035.

Efficacy and safety of etanercept biosimilar rhTNFR-Fc in Chinese patients with juvenile idiopathic arthritis: An open-label multicenter observational study.

BackgroundEtanercept biosimilar recombinant human TNF-α receptor II: IgG Fc fusion protein (rhTNFR-Fc) has showed its efficacy and safety in Chinese patients with rheumatoid arthritis. However, data on rhTNFR-Fc's application in juvenile idiopathic arthritis (JIA) is limited.MethodsA prospective, observational, multicenter study was performed at 6 institutes in China from July 2020 to December 2021. In a 24-week follow-up, patients with JIA including polyarticular JIA and enthesitis related arthritis received rhTNFR-Fc plus methotrexate (MTX) treatment. The primary outcome parameters were improvements of cJADAS-10 (clinical Juvenile Arthritis Disease Activity Score), and the secondary outcome parameter was an inactive disease.Results60 patients completed at least 12-week follow-up, and 57 completed 24-week follow-up. They had high C reactive protein values (11.6 mg/L) and cJADAS-10 (14.6) at baseline. Thirteen patients had morning stiffness. 33 patients showed synovial thickening, and 34 showed bone marrow edemas on MRI. Ultrasonography demonstrated significant joint effusions in 43 patients. The cJADAS-10 sharply decreased from 14.66 at the baseline to 2.4 at 24 weeks of rhTNFR-Fc therapy, respectively (P < 0.01). About half of patients achieved inactive disease at 24 weeks of therapy. Compared with the baseline, the number of patients with morning stiffness, joint effusions, bone marrow edema and synovial thickening on MRI significantly decreased at 24 weeks. Adverse events were consistent with known side effects of biologic agents.ConclusionsThe present study indicated that the combination of rhTNFR-Fc and MTX significantly improve symptoms and disease activity of children with JIA. This study suggests etanercept biosimilar rhTNFR-Fc as an effective and safe therapy for children with JIA.

Monoclonal Antibody Engineering and Design to Modulate FcRn Activities: A Comprehensive Review.

Understanding the biological mechanisms underlying the pH-dependent nature of FcRn binding, as well as the various factors influencing the affinity to FcRn, was concurrent with the arrival of the first recombinant IgG monoclonal antibodies (mAbs) and IgG Fc-fusion proteins in clinical practice. IgG Fc–FcRn became a central subject of interest for the development of these drugs for the comfort of patients and good clinical responses. In this review, we describe (i) mAb mutations close to and outside the FcRn binding site, increasing the affinity for FcRn at acidic pH and leading to enhanced mAb half-life and biodistribution, and (ii) mAb mutations increasing the affinity for FcRn at acidic and neutral pH, blocking FcRn binding and resulting, in vivo, in endogenous IgG degradation. Mutations modifying FcRn binding are discussed in association with pH-dependent modulation of antigen binding and (iii) anti-FcRn mAbs, two of the latest innovations in anti-FcRn mAbs leading to endogenous IgG depletion. We discuss the pharmacological effects, the biological consequences, and advantages of targeting IgG–FcRn interactions and their application in human therapeutics.

Synovitis, Acne, Pustulosis, Hyperostosis, and Osteitis (SAPHO) Syndrome with Cutis Verticis Gyrata: Case Report and Review of Literature

SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome is a rare disease clinically characterized by a wide range of cutaneous and osteoarticular manifestations. Here, we report a case of SAPHO syndrome with cutis verticis gyrata (CVG) and investigated the genetic causes in the four members of this pedigree. After failure of conventional treatments, a recombinant human TNF-α receptor II:IgG Fc fusion protein (rhTNFR:Fc, YISAIPU®) achieved good control of the disease at the 2-year follow-up. We did not identify any pathogenic mutation in this pedigree. We also summarized the clinical and therapeutic characteristics of 83 patients with SAPHO syndrome through the China National Knowledge Infrastructure (CKNI) database from 2016 to 2021. Patients with acne were young and predominantly male. About 45.8% patients were treated with biological therapies or traditional Chinese medicine (TCM), 84.2% of which showed positive effects against cutaneous and osteoarticular manifestations. We report a case of SAPHO syndrome with CVG that was successfully treated with rhTNFR:Fc. Our results reveal the genetic heterogeneity involved. Biologics and TCM are likely alternative options for the treatment of SAPHO syndrome.

802-P: Safety, Pharmacokinetics, and Pharmacodynamics of Once-Weekly Basal Insulin Fc (BIF) in Japanese Patients with Type 2 Diabetes: A Phase 1 Open-Label, Randomized, Active-Controlled Trial

The aim was to investigate the safety, pharmacokinetics (PK) , and pharmacodynamics (PD) of once-weekly Basal Insulin Fc (BIF) , a novel long-acting IgG Fc-fusion protein being developed for the treatment of diabetes. This multiple-site, open-label, randomized, active-controlled, multiple-dose Phase 1 study investigated individualized weekly subcutaneous injections of BIF compared to once-daily insulin degludec over 6 weeks in Japanese patients with T2D on stable doses of basal insulin, with or without antihyperglycemic medication. In a 5-week lead-in period to treatment, all patients titrated insulin degludec to a fasting glucose (FG) target of 100 to 139 mg/dL. Outcomes included safety (primary) and PK and PD (secondary) . ClinicalTrials.gov NCT04276428. Patients were randomized to receive BIF (n=18) or insulin degludec (n=10) . TEAE incidence was low for both BIF (n=4; 22.2%) and insulin degludec (n=2; 20.0%) . All TEAEs were mild, nonserious, and unrelated to BIF. A total of 6 (33.3%) and 2 (20.0%) patients reported hypoglycemic events during BIF and insulin degludec treatment, respectively, none of which were severe. Median tmax was 3 or 4 days. Geometric mean t1/2 of BIF was 15 days. The peak to trough ratio (Cmax to 168 hours postdose concentration after final dose at Week 6) was 1.13. Mean FG was statistically significantly increased from baseline from Weeks 3 to 7 following first dose of insulin degludec but was not increased with BIF (LS means [SE] change: Week 3 BIF, -6.6 [4.7]; degludec, 22.1 [5.9]; Week 7 BIF, -3.5 [7.1]; degludec, 25.3 [9.3] mg/dL) . The 7-point SMPG profile was unchanged with BIF over treatment. In Japanese patients with T2D, once-weekly BIF was well tolerated and associated with prolonged PK and improved glycemic control. PK/PD profiles in Japanese patients are consistent with prior studies and support participation in Phase 3 studies. Disclosure R. Nasu: None. T. Oura: Employee; Eli Lilly Japan K.K. Stock/Shareholder; Eli Lilly and Company. K. Ohwaki: None. M. Imori: Employee; Eli Lilly Japan K.K. K. Furihata: Other Relationship; Eli Lilly and Company, Pfizer Japan Inc. Funding Eli Lilly Japan K.K.

Possibility of RANKL as a pharmacological target

In bone tissue, RANKL acts as a positive regulator of bone turnover through both RANKL forward signaling, which induces osteoclast maturation, and RANKL reverse signaling, which promotes early osteoblast differentiation. Inhibition of osteoclast maturation by blocking RANKL forward signaling inevitably reduces the supply of osteoclast-derived coupling factors, leading to the suppression of bone formation. If we can design an agent that blocks RANKL forward signaling while simultaneously activating RANKL reverse signaling, the influence of reduced supply of osteoclast-derived coupling factors can be mitigated, which may be useful in the treatment of bone destructive diseases. Our previous study has shown that the cross-linking of RANKL molecules on the cell surface triggers the activation reverse signaling. Therefore, we screened various structures of IgG Fc-fusion protein constructs with multivalent binding sites for RANKL by arranging anti-RANKL single-chain antibody variable regions (scFv). Consequently, it was found that a structure in which the domain order inside the scFv was VH-VL order was advantageous for its ability to activate RANKL reverse signaling. Finally, the optimal structure was selected, and its pharmacological activity was evaluated using the ovariectomized mouse model. Results confirmed that the activation of RANKL reverse signaling could mitigate the decrease in bone formation associated with the suppression of osteoclast maturation.

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.

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.

Abstract 1703: Development of NAV-005, a humoral immuno-suppressor antagonist for the treatment of CA125-expressing cancers

Abstract Tumors employ a variety of mechanisms to avoid host cellular and humoral immune responses. Recent findings have found that the tumor-produced MUC16/CA125 protein elicits humoral immunosuppressive effects on antibody-mediated tumor cell killing. This effect is facilitated via direct binding to SIGLEC regulatory receptors on natural killer (NK) cells as well as through direct binding to therapeutic antibodies that blocks Fc-γ-receptor and C1q antibody engagement, leading to suppressed antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) activities, respectively. We have engineered a human IgG1 Fc fusion protein, called NAV-005, that binds to CA125 with high affinity and blocks its binding to SIGLEC receptors and affected antibodies, thus enabling efficient humoral immune mediated tumor cell killing. NAV-005 is being developed as a novel therapeutic agent to treat humoral immuno-suppressed CA125 expressing cancers. Citation Format: Nicholas C. Nicolaides, Brad Kline, Luigi Grasso. Development of NAV-005, a humoral immuno-suppressor antagonist for the treatment of CA125-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1703.

An ACE2 Triple Decoy that neutralizes SARS-CoV-2 shows enhanced affinity for virus variants

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.

Production of monoclonal shark-derived immunoglobulin new antigen receptor antibodies using Chinese hamster ovary cell expression system

Cartilaginous fishes such as sharks have adaptive immune systems based on immunoglobulins similar to those in mammals. During their evolution, cartilaginous fishes individually have acquired their adaptive immune system called immunoglobulin new antigen receptor (IgNARs). IgNARs maintain their functions in the harsh environment of shark serum, which contains a high concentration of urea to prevent water loss in seawater. Therefore, IgNARs have high structural stability, and are expected to be used as next-generation antibodies in applications different from those of conventional IgG antibodies. However, no recombinant expression system for IgNAR, which has a molecular weight of approximately 147kDa as a dimer and multiple N-glycosylation sites, has yet been constructed. This has stalled research into IgNAR development. Here, we constructed a recombinant expression system for IgNAR using Chinese hamster ovary (CHO) cells, widely used as hosts for IgG antibody production. Using this system, IgNAR was successfully expressed and purified as a human IgG Fc fusion protein and showed antigen-binding ability. After Protein A affinity purification, followed by specific cleavage and removal of the human Fc-region, the final yield of IgNAR was 1.07 mg/L-medium. Moreover, this CHO cell expression system modified IgNAR with various N-glycans, including high-mannose and complex types. This expression system will allow us to analyze the structure, physicochemical properties, and biological functions of IgNAR. This fundamental information will advance the development of IgNARs for industrial and biotechnological applications.

192-OR: Glycemic Control with Once-Weekly Basal Insulin Fc (BIF) in Persons with Type 2 Diabetes Mellitus (T2DM) Using Continuous Glucose Monitoring (CGM) in a Phase 2 Study

Basal insulin Fc (BIF; LY3209590) is a novel, once-weekly, long-acting IgG Fc-fusion protein assessed for the treatment of diabetes mellitus. A 32-week study evaluating the safety and efficacy of BIF vs. degludec in persons with T2DM previously treated with a basal insulin showed HbA1c non-inferiority of BIF vs. degludec with significantly fewer hypoglycemic events (≤70 mg/dL). Here we present CGM data derived by Dexcom G6, allowing a more detailed assessment of glycemic control of BIF vs. degludec. The study included 2 dosing algorithms for BIF with different fasting glucose (FG) targets: ≤140 mg/dL (BIF-A1) and ≤120 mg/dL (BIF-A2). Degludec was titrated to FG ≤100 mg/dL. Subjects were randomized to 1 of the 3 arms. Subject (N=399) mean age was 60.2 yrs and baseline HbA1c was 8.1%. For the entire 32 weeks, the percent of 24 hrs in range, hyperglycemia and hypoglycemia was similar for the 3 arms (Figure). At Week 32, total duration of hypoglycemia was similar across 7 days post-injection for BIF-A1 and A2, showing that duration of hypoglycemia is independent of day post-injection. CGM data confirm that BIF showed similar glycemic control vs. degludec despite higher FG targets and numerically lower time in hypoglycemia. The flat pharmacokinetic profile enables near peakless insulin concentrations without an increase in hypoglycemia risk at highest exposure. Disclosure C. M. Kazda: Employee; Self; Eli Lilly and Company. J. Chien: Stock/Shareholder; Self; Eli Lilly and Company. Q. Zhang: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. E. Chigutsa: Employee; Self; Eli Lilly and Company. W. Landschulz: Employee; Self; Lilly Diabetes, Employee; Spouse/Partner; Covance Inc., Stock/Shareholder; Self; Lilly Diabetes. P. Wullenweber: None. A. Haupt: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. J. P. Frias: Consultant; Self; 89bio, Inc., Altimmune, Axcella Health Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk, Pfizer Inc., Sanofi, Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Bristol-Myers Squibb Company, CymaBay Therapeutics, Eli Lilly and Company, Intercept Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Madrigal Pharmaceuticals, Inc., Merck & Co., Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk, Pfizer Inc., Sanofi, Speaker’s Bureau; Self; Merck & Co., Inc., Sanofi. Funding Eli Lilly and Company