An antigen-specific immunotherapeutic, AKS-107, deletes insulin-specific B cells and prevents murine autoimmune diabetes.

Introduction: The antigen-presenting cell function of insulin-reactive B cells promotes type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by stimulating pathogenic T cells leading to destruction of insulin-producing ß-cells of pancreatic islets. Methods/Results: To target insulin-reactive B cells, AKS-107, a human IgG1 Fc molecule fused with human insulin A and B chains, was engineered to retain conformational insulin epitopes that bound mouse and human B cell receptors but prevented binding to the insulin metabolic receptor. AKS-107 Fc-mediated deletion of insulin-reactive B cells was demonstrated via ex vivo and in vivo experiments with insulin-reactive B cell receptor transgenic mouse strains, VH125Tg/NOD and Tg125(H+L)/NOD. As an additional immune tolerance feature, the Y16A mutation of the insulin B(9-23) dominant T cell epitope was engineered into AKS-107 to suppress activation of insulin-specific T cells. In mice and non-human primates, AKS-107 was well-tolerated, non-immunogenic, did not cause hypoglycemia even at high doses, and showed an expectedly protracted pharmacokinetic profile. AKS-107 reproducibly prevented spontaneous diabetes from developing in NOD and VH125Tg/NOD mice that persisted for months after cessation of treatment, demonstrating durable immune tolerance. Discussion: These preclinical outcomes position AKS-107 for clinical development in T1D prevention settings.

Immunogenicity phase II study evaluating booster capacity of nonadjuvanted AKS-452 SARS-Cov-2 RBD Fc vaccine.

AKS-452, a subunit vaccine comprising an Fc fusion of the ancestral wild-type (WT) SARS-CoV-2 virus spike protein receptor binding domain (SP/RBD), was evaluated without adjuvant in a single cohort, non-randomized, open-labelled phase II study (NCT05124483) at a single site in The Netherlands for safety and immunogenicity. A single 90 µg subcutaneous booster dose of AKS-452 was administered to 71 adults previously primed with a registered mRNA- or adenovirus-based vaccine and evaluated for 273 days. All AEs were mild and no SAEs were attributable to AKS-452. While all subjects showed pre-existing SP/RBD binding and ACE2-inhibitory IgG titers, 60-68% responded to AKS-452 via ≥2-fold increase from days 28 to 90 and progressively decreased back to baseline by day 180 (days 28 and 90 mean fold-increases, 14.7 ± 6.3 and 8.0 ± 2.2). Similar response kinetics against RBD mutant proteins (including omicrons) were observed but with slightly reduced titers relative to WT. There was an expected strong inverse correlation between day-0 titers and the fold-increase in titers at day 28. AKS-452 enhanced neutralization potency against live virus, consistent with IgG titers. Nucleocapsid protein (Np) titers suggested infection occurred in 66% (46 of 70) of subjects, in which only 20 reported mild symptomatic COVID-19. These favorable safety and immunogenicity profiles support booster evaluation in a planned phase III universal booster study of this room-temperature stable vaccine that can be rapidly and inexpensively manufactured to serve vaccination at a global scale without the need of a complex distribution or cold chain.

A randomized phase I/II safety and immunogenicity study of the Montanide-adjuvanted SARS-CoV-2 spike protein-RBD-Fc vaccine, AKS-452.

BACKGROUND: Previous interim data from a phase I study of AKS-452, a subunit vaccine comprising an Fc fusion of the respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (SP/RBD) emulsified in the water-in-oil adjuvant, Montanide™ ISA 720, suggested a good safety and immunogenicity profile in healthy adults. This phase I study was completed and two dosing regimens were further evaluated in this phase II study. METHODS: This phase II randomized, open-labelled, parallel group study was conducted at a single site in The Netherlands with 52 healthy adults (18 - 72 years) receiving AKS-452 subcutaneously at one 90 µg dose (cohort 1, 26 subjects) or two 45 µg doses 28 days apart (cohort 2, 26 subjects). Serum samples were collected at the first dose (day 0) and at days 28, 56, 90, and 180. Safety and immunogenicity endpoints were assessed, along with induction of IgG isotypes, cross-reactive immunity against viral variants, and IFN-γ T cell responses. RESULTS: All AEs were mild/moderate (grades 1 or 2), and no SAEs were attributable to AKS-452. Seroconversion rates reached 100% in both cohorts, although cohort 2 showed greater geometric mean IgG titers that were stable through day 180 and associated with enhanced potencies of SP/RBD-ACE2 binding inhibition and live virus neutralization. AKS-452-induced IgG titers strongly bound mutant SP/RBD from several SARS-CoV-2 variants (including Omicrons) that were predominantly of the favorable IgG1/3 isotype and IFN-γ-producing T cell phenotype. CONCLUSION: These favorable safety and immunogenicity profiles of the candidate vaccine as demonstrated in this phase II study are consistent with those of the phase I study (ClinicalTrials.gov: NCT04681092) and suggest that a total of 90 µg received in 2 doses may offer a greater duration of cross-reactive neutralizing titers than when given in a single dose.

Ultra-long-acting recombinant insulin for the treatment of diabetes mellitus in dogs.

BACKGROUND: For the treatment of diabetes mellitus (DM) in dogs, novel insulins with decreased injection frequency while maintaining safety and efficacy are desirable. Insulin fused with immunoglobulin-fragment-crystallizable (Fc) has an ultra-long plasma half-life because it recycles through cells, protected from proteolysis. HYPOTHESIS: Glycemic control can be achieved in diabetic dogs with a recombinant fusion protein of a synthetic insulin and canine Fc (AKS-218d) administered subcutaneously once-weekly. ANIMALS: Five client-owned dogs with naturally occurring DM. METHODS: Prospective clinical trial in dogs with DM that were recruited from the UC Davis Veterinary Teaching Hospital and local veterinary clinics. Dogs previously controlled using intermediate-acting insulin q12h were transitioned to once-weekly injections of a preliminary construct identified as AKS-218d. The dose of AKS-218d was titrated weekly for 8 weeks based on clinical response and continuous interstitial glucose monitoring. Clinical signs, body weight, serum fructosamine concentrations, and mean interstitial glucose concentrations (IG) over the preceding week were compared between baseline (before AKS-218d) and during the last week of treatment. Data were compared using nonparametric paired tests. RESULTS: Once-weekly AKS-218d, compared to baseline twice-daily insulin therapy, resulted in no significant changes in clinical signs, median (range) body weight (+0.4 kg [-0.5-1.1]; P = .6), fructosamine concentration (-75 mmol/L [-215 to +126]; P = .4), or mean IG (+81 mg/dL [-282 to +144]; P = .8). No adverse reactions were reported. CONCLUSION: Control of clinical signs, body weight, and maintenance of glycemia was achieved with this once-weekly novel insulin construct in 4 of 5 dogs.

Phase I interim results of a phase I/II study of the IgG-Fc fusion COVID-19 subunit vaccine, AKS-452.

To address the coronavirus disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recombinant subunit vaccine, AKS-452, is being developed comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain (SP/RBD) antigen and human IgG1 Fc emulsified in the water-in-oil adjuvant, Montanide™ ISA 720. A single-center, open-label, phase I dose-finding and safety study was conducted with 60 healthy adults (18-65 years) receiving one or two doses 28 days apart of 22.5 µg, 45 µg, or 90 µg of AKS-452 (i.e., six cohorts, N = 10 subjects per cohort). Primary endpoints were safety and reactogenicity and secondary endpoints were immunogenicity assessments. No AEs ≥ 3, no SAEs attributable to AKS-452, and no SARS-CoV-2 viral infections occurred during the study. Seroconversion rates of anti-SARS-CoV-2 SP/RBD IgG titers in the 22.5, 45, and 90 µg cohorts at day 28 were 70%, 90%, and 100%, respectively, which all increased to 100% at day 56 (except 89% for the single-dose 22.5 µg cohort). All IgG titers were Th1-isotype skewed and efficiently bound mutant SP/RBD from several SARS-CoV-2 variants with strong neutralization potencies of live virus infection of cells (including alpha and delta variants). The favorable safety and immunogenicity profiles of this phase I study (ClinicalTrials.gov: NCT04681092) support phase II initiation of this room-temperature stable vaccine that can be rapidly and inexpensively manufactured to serve vaccination at a global scale without the need of a complex distribution or cold chain.

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.

An ultra-long-acting recombinant insulin for the treatment of diabetes mellitus in cats.

BACKGROUND: Treatment of diabetes mellitus (DM) in cats typically requires insulin injections q12h-q24h, posing a major compliance barrier for caregivers. Novel treatments enabling decreased injection frequency while maintaining safety are highly desirable. Insulin fused with feline immunoglobulin fragment crystallizable (Fc) has an ultra-long plasma half-life because it recycles through cells where it is protected from proteolysis. HYPOTHESIS: Glycemic control can be achieved in diabetic cats with a recombinant fusion protein of a synthetic insulin and feline Fc (AKS-267c) administered SC weekly. ANIMALS: Five cats with spontaneous DM. METHODS: Cats previously controlled using insulin glargine q12h were transitioned to once-weekly injection of AKS-267c. The dose of AKS-267c was titrated weekly for 7 weeks based on continuous glucose monitoring. Clinical signs, body weight, fructosamine concentrations, and mean interstitial glucose concentrations (IG) were compared between baseline (week 0, on insulin glargine) and the last week of treatment. Data were assessed for normality and compared using parametric or nonparametric paired tests (as appropriate). RESULTS: After 7 weeks of once-weekly injections, compared to baseline, there were no significant changes in clinical signs, body weight (median [range] gain, 0.1 kg [-0.1 to +0.7]; P = .5), fructosamine (-60 mmol/L [-338 to +206]; P = .6), and mean IG concentrations (change = -153 mmol/L [-179 to +29]; P = .3), and no adverse reactions were reported. CONCLUSION: Successful control of clinical signs and maintenance of glycemia was achieved with this once-weekly novel insulin treatment. The efficacy and safety of this novel formulation should be further assessed in a large clinical trial.

Neuregulin 1 expression is a predictive biomarker for response to AV-203, an ERBB3 inhibitory antibody, in human tumor models.

PURPOSE: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. EXPERIMENTAL DESIGN: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. RESULTS: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. CONCLUSIONS: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203.

Reliability of the mouse model of choroidal neovascularization induced by laser photocoagulation.

PURPOSE: We attempted to reproduce published studies that evaluated whether the following factors influence choroidal neovascularization (CNV) induced by laser photocoagulation in murine retinas: small interfering RNA (siRNA), cobra venom factor, complement factors C3 and C5, and complement receptor C5aR. In addition, we explored whether laser-induced CNV in mice was influenced by the vendor of origin of the animals. METHODS: Reagents or genotypes reported by others to influence CNV in this model were assessed using our standard procedures. Retrospective analyses of control or placebo mice in many experiments were done to evaluate whether the CNV area induced by laser photocoagulation varied according to vendor. RESULTS: Administration of the following agents did not have a substantial impact on the CNV induced by laser burns in mice: siRNA, low-molecular-weight inhibitor of the C5a receptor (PMX53), or cobra venom factor. Jackson Laboratory (JAX) mice lacking either C3 or C5 had increased neovascularization compared to non-littermate JAX wild-type controls. Taconic mice lacking C3 had reduced CNV compared to non-littermate Taconic wild-type control mice. A retrospective analysis of vehicle-treated wild-type C57BL/6 mice used as controls across 132 experiments conducted from 2007 to 2010 revealed that mice purchased from JAX or from Charles River produced less neovascularization than mice from Taconic. CONCLUSIONS: We present our recommended methods for conducting experiments with the mouse laser-induced CNV model to enhance reproducibility and minimize investigator bias.