Development of a long-acting relaxin analogue, LY3540378, for treatment of chronic heart failure.

BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.

"What I Would Do to Take Away Your Pain": A Photovoice Project Conducted by Mothers of Children With Medical Complexity.

Research identifies that families of children with medical complexities in the United States have diverse and complex needs. Despite research emphasizing that families demonstrate higher needs that are not being met, limited research focuses solely on mothers of children with medical complexities. Specifically, how mothers understand and identify themselves, understand and define their role in coordinating care, and how they view their own mental and physical health. As such, this study provides a better understanding of how mothers in San Diego, CA, navigate the day-to-day psychological, social, and physical realities of having a child with medical complexities. Through the use of the participatory action research method photovoice, mothers conducted a critical analysis of their daily lives. Findings identify mothers' main concerns about the conditions of their lives as well as the strengths they employ to care for themselves and their children successfully. Implications provide recommendations for hospitals working with mothers of children with medical complexities.

Investigation of Immune Responses to Oxidation, Deamidation, And Isomerization in Therapeutic Antibodies using Preclinical Immunogenicity Risk Assessment Assays.

Product- and process- related critical quality attributes have the potential to impact pharmacokinetics, immunogenicity, potency, and safety of biotherapeutics. Among these critical quality attributes are chemical degradations, specifically oxidation, deamidation, and isomerization. These degradations can be induced by stressors such as light, pH, or temperature; they can also occur naturally under normal conditions. The immunogenicity risk of chemical degradations, particularly in the absence of aggregation, has not been thoroughly understood. In this study, model antibodies with known labile residues were stressed to induce each of the three chemical degradation classes. Aggregate-free and chemically modified antibody species were fractionalized and characterized, followed by testing in standardized and qualified preclinical immunogenicity risk assessment assays for dendritic cell internalization and presentation, monocyte activation, and pre-existing reactivity. Preclinical immunogenicity risk was assessed holistically in vitro based on changes in innate activation risk, CD4 T cell risk, and B cell risk compared to corresponding native antibody. The results of this study suggest an overall moderate increase in immune activation potential for the antibody with isomerization, with only slight increases observed in oxidized and deamidated antibodies. These findings could lend understanding to the immunogenicity risk of chemical degradations in therapeutic antibodies and therefore inform optimization engineering at particular labile residues and risk assessment under the Quality by Design framework.

Safety and Immunogenicity of LY3415244, a Bispecific Antibody Against TIM-3 and PD-L1, in Patients With Advanced Solid Tumors.

PURPOSE: Investigate the safety and efficacy of LY3415244, a TIM-3/PD-L1 bispecific antibody that blocks TIM-3 and PD-L1 in patients with advanced solid tumors. PATIENTS AND METHODS: A phase I, multicenter, open-label study was conducted in patients with advanced solid tumors. Patients were dosed every 2 weeks intravenously with flat doses of LY3415244 escalating from 3 to 70 mg. The primary endpoints were safety, tolerability, and identification of the recommended phase II dose. RESULTS: Between November 2018 and October 2019, 12 patients were enrolled into four cohorts and received at least one dose of LY3415244. Two patients (16.7%) developed clinically significant anaphylactic infusion-related reactions and all patients developed treatment-emergent antidrug antibodies (TE-ADA). ADA titers were sometimes very high and negatively impacted soluble TIM-3 target engagement in most patients. ADA epitope specificity was against both TIM-3 and PD-L1 arms of the bispecific antibody; most TE-ADAs initially targeted the TIM-3 arm after the first dose. Preexisting ADAs against LY3415244 were also detected in normal (unexposed) human serum samples. One patient with PD-1 refractory non-small cell lung cancer had a near partial response (-29.6%). CONCLUSIONS: This TIM-3 and PD-L1 bispecific format was associated with unexpected immunogenicity targeting both arms of the bispecific antibody, resulting in early study termination. Epitope specificity analysis revealed an initial response toward the TIM-3 arm and presence of preexisting ADAs to the bispecific molecule in the general population. This experience emphasizes the importance of thorough analyses for preexisting ADAs as part of immunogenicity risk assessment of novel antibodies.See related commentary by de Spéville and Moreno, p. 2669.

Inactivation of a GAL4-like transcription factor improves cell fitness and product yield in glycoengineered Pichia pastoris strains.

With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.

Protection provided by a herpes simplex virus 2 (HSV-2) glycoprotein C and D subunit antigen vaccine against genital HSV-2 infection in HSV-1-seropositive guinea pigs.

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.

Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris.

Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.

Sustained hemodynamic stress disrupts normal circadian rhythms in calcineurin-dependent signaling and protein phosphorylation in the heart.

RATIONALE: Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and disease, little is known regarding the circadian regulation of intracellular signaling pathways controlling cardiac function and remodeling. OBJECTIVE: To assess circadian changes in processes dependent on the protein phosphatase calcineurin, relative to changes in phosphorylation of cardiac proteins, in normal, hypertrophic, and failing hearts. METHODS AND RESULTS: We found evidence of large circadian oscillations in calcineurin-dependent activities in the left ventricle of healthy C57BL/6 mice. Calcineurin-dependent transcript levels and nuclear occupancy of the NFAT (nuclear factor of activated T cells) regularly fluctuated as much as 20-fold over the course of a day, peaking in the morning when mice enter a period of rest. Phosphorylation of the protein phosphatase 1 inhibitor 1 (I-1), a direct calcineurin substrate, and phospholamban, an indirect target, oscillated directly out of phase with calcineurin-dependent signaling. Using a surgical model of cardiac pressure overload, we found that although calcineurin-dependent activities were markedly elevated, the circadian pattern of activation was maintained, whereas, oscillations in phospholamban and I-1 phosphorylation were lost. Changes in the expression of fetal gene markers of heart failure did not mirror the rhythm in calcineurin/NFAT activation, suggesting that these may not be direct transcriptional target genes. Cardiac function in mice subjected to pressure overload was significantly lower in the morning than in the evening when assessed by echocardiography. CONCLUSIONS: Normal, opposing circadian oscillations in calcineurin-dependent activities and phosphorylation of proteins that regulate contractility are disrupted in heart failure.

Calcineurin is necessary for the maintenance but not embryonic development of slow muscle fibers.

Skeletal muscles are a mosaic of slow and fast twitch myofibers. During embryogenesis, patterns of fiber type composition are initiated that change postnatally to meet physiological demand. To examine the role of the protein phosphatase calcineurin in the initiation and maintenance of muscle fiber types, we used a "Flox-ON" approach to obtain muscle-specific overexpression of the modulatory calcineurin-interacting protein 1 (MCIP1/DSCR1), an inhibitor of calcineurin. Myo-Cre transgenic mice with early skeletal muscle-specific expression of Cre recombinase were used to activate the Flox-MCIP1 transgene. Contractile components unique to type 1 slow fibers were absent from skeletal muscle of adult Myo-Cre/Flox-MCIP1 mice, whereas oxidative capacity, myoglobin content, and mitochondrial abundance were unaltered. The soleus muscles of Myo-Cre/Flox-MCIP1 mice fatigued more rapidly than the wild type as a consequence of the replacement of the slow myosin heavy chain MyHC-1 with a fast isoform, MyHC-2A. MyHC-1 expression in Myo-Cre/Flox-MCIP1 embryos and early neonates was normal. These results demonstrate that developmental patterning of slow fibers is independent of calcineurin, while the maintenance of the slow-fiber phenotype in the adult requires calcineurin activity.