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.

Suppression of cancer relapse and metastasis by inhibiting cancer stemness.

Partial or even complete cancer regression can be achieved in some patients with current cancer treatments. However, such initial responses are almost always followed by relapse, with the recurrent cancer being resistant to further treatments. The discovery of therapeutic approaches that counteract relapse is, therefore, essential for advancing cancer medicine. Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug sensitivity, and their potential to metastasize and cause relapse. Indeed, hypermalignant cancer cells, termed cancer stem cells or stemness-high cancer cells, that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types. Moreover, such stemness-high cancer cells are resistant to conventional chemotherapy and radiation. Here we show that BBI608, a small molecule identified by its ability to inhibit gene transcription driven by Stat3 and cancer stemness properties, can inhibit stemness gene expression and block spherogenesis of or kill stemness-high cancer cells isolated from a variety of cancer types. Moreover, cancer relapse and metastasis were effectively blocked by BBI608 in mice. These data demonstrate targeting cancer stemness as a novel approach to develop the next generation of cancer therapeutics to suppress cancer relapse and metastasis.

The effect of substrate modulus on the growth and function of matrix-embedded endothelial cells.

Endothelial cells (EC) are potent bioregulatory cells, modulating thrombosis, inflammation and control over mural smooth muscle cells and vascular health. The biochemical roles of EC are retained when cells are embedded within three-dimensional (3D) denatured collagen matrices. Though substrate mechanics have long been known to affect cellular morphology and function and 3D-EC systems are increasingly used as therapeutic modalities little is known about the effect of substrate mechanics on EC in these 3D systems. In this work, we examined the effect of isolated changes in modulus on EC growth and morphology, extracellular matrix gene expression, modulation of smooth muscle cell growth, and immunogenicity. EC growth, but not morphology was dependent on scaffold modulus. Increased scaffold modulus reduced secretion of smooth muscle cell growth inhibiting heparan sulfate proteoglycans (HSPGs), but had no effect on secreted growth factors, resulting in a loss of smooth muscle cell growth inhibition by EC on high modulus scaffolds. Expression of ICAM-1, VCAM-1 and induction of CD4(+) T cell proliferation was reduced by increased scaffold modulus, and correlated with changes in integrin α5 expression. Expression of several common ECM proteins by EC on stiffer substrates dropped, including collagen IV(α1), collagen IV(α5), fibronectin, HSPGs (perlecan and biglycan). In contrast, expression of elastin and TIMPs were increased. This work shows even modest changes in substrate modulus can have a significant impact on EC function in three-dimensional systems. The mechanism of these changes is not clear, but the data presented here within suggests a model wherein EC attempt to neutralize changes in environmental force balance by altering ECM and integrin expression, leading to changes in effects on downstream signaling and function.

Matrix-embedded endothelial cells are protected from the uremic milieu.

BACKGROUND: Endothelial cells (ECs) embedded in 3D matrices [matrix-embedded endothelial cells (MEECs)] of denatured collagen implanted around vascular access anastomoses preserve luminal patency. MEEC implant efficacy depends on embedded EC health. As the uremic milieu inhibits proliferation and induces apoptosis of ECs, we examined whether uremia might impact MEECs. METHODS: ECs grown on 2D gelatin-coated polystyrene tissue culture plates (gTCPS) or in MEEC were treated with sera pooled from 20 healthy control or uremic patients with end-stage renal disease. EC viability was examined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell counting and Trypan blue exclusion. Media conditioned (CM) with 2 and 3D-supported ECs were examined for its potential to inhibit vascular smooth muscle cell (vSMC) proliferation using (3)[H] thymidine incorporation and cyclin D1 expression. ECs grown on gTCPS were treated with uremic serum filtered through matrices to examine if matrices retain uremic toxins or whether EC effects were cell mediated. RESULTS: Uremic serum significantly reduced viability and number of live, and increased dead ECs when grown on gTCPS, but not in MEECs. EC survival correlated with vSMC inhibition. While CM from ECs grown in gTCPS with uremic serum inhibited vSMC proliferation no better than uremic serum alone (22 versus 27%), MEEC CM inhibited vSMC proliferation by 47% (P = 0.0004). Cyclin D1 expression tracked with indices of vSMC proliferation. There was no significant difference in EC viability between EC treated with matrix-filtered or unfiltered uremic serum. CONCLUSION: The viability, number and efficacy of MEECs were preserved in uremic serum compared to those of ECs on gTCPS. MEECs are protected from uremic toxicity, not from retention of uremic toxins by matrices, but likely from intrinsic changes in EC sensitivity to uremia. MEECs implanted at vascular access sites should inhibit neointimal hyperplasia in uremia. This study underscores the robustness of matrix embedding as a cell protectant, especially in hostile environments like uremia.