RESUMEN
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.
Asunto(s)
Cardiopatías , Insuficiencia Cardíaca , Relaxina , Animales , Femenino , Humanos , Ratones , Embarazo , Ratas , Cardiomegalia/tratamiento farmacológico , Cardiopatías/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Relaxina/farmacología , Relaxina/uso terapéutico , Relaxina/metabolismo , Volumen SistólicoRESUMEN
Development of biotherapeutics is hampered by the inherent risk of immunogenicity, which requires extensive clinical assessment and possible re-engineering efforts for mitigation. The focus in the pre-clinical phase is to determine the likelihood of developing treatment-emergent anti-drug antibodies (TE-ADA) and presence of pre-existing ADA in drug-naïve individuals as risk-profiling strategies. Pre-existing ADAs are routinely identified during clinical immunogenicity assessment, but their origin and impact on drug safety and efficacy have not been fully elucidated. One specific class of pre-existing ADAs has been described, which targets neoepitopes of antibody fragments, including Fabs, VH, or VHH domains in isolation from their IgG context. With the increasing number of antibody fragments and other small binding scaffolds entering the clinic, a widely applicable method to mitigate pre-existing reactivity against these molecules is desirable. Here is described a structure-based engineering approach to abrogate pre-existing ADA reactivity to the C-terminal neoepitope of VH(H)s. On the basis of 3D structures, small modifications applicable to any VH(H) are devised that would not impact developability or antigen binding. In-silico B cell epitope mapping algorithms were used to rank the modified VHH variants by antigenicity; however, the limited discriminating capacity of the computational methods prompted an experimental evaluation of the engineered molecules. The results identified numerous modifications capable of reducing pre-existing ADA binding. The most efficient consisted of the addition of two proline residues at the VHH C-terminus, which led to no detectable pre-existing ADA reactivity while maintaining favorable developability characteristics. The method described, and the modifications identified thereby, may provide a broadly applicable solution to mitigate immunogenicity risk of antibody-fragments in the clinic and increase safety and efficacy of this promising new class of biotherapeutics.
Asunto(s)
Factores Biológicos/inmunología , Simulación del Acoplamiento Molecular , Anticuerpos de Dominio Único/química , Linfocitos B/inmunología , Factores Biológicos/química , Epítopos/química , Epítopos/inmunología , Humanos , Unión Proteica , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Recombinant Pseudomonas fluorescens cells, expressing over 40% protein as bovine interferon-gamma (IFN-gamma), were chemically fixed to sterilize the culture and amend the bacterial cell wall. When killed and fixed recombinant cells, termed here amended-recombinant-cells (ARCs), were assayed for interferon activity, we obtained the following surprising results: 1) sterilization and fixation did not inactivate ARC-encapsulated IFN-gamma; 2) ARC-encapsulated IFN-gamma and soluble, recombinant IFN-gamma were equally active in vitro but proteolysis was required for release of the ARC cytokine; and 3) ARC-encapsulated IFN-gamma was active in vivo with optimal adjuvant activity at a dose about 1000-fold less than previously reported for soluble, recombinant IFN-gamma and 100-fold less than doses which induced adverse systemic effects. The mechanism by which ARC-encapsulation increased IFN-gamma activity in vivo remains uncertain. However, our in vitro results show that sustained release of soluble IFN-gamma is a likely factor. The ARC production and delivery system achieves enhanced adjuvant activity with reduced risk of systemic effects, and the low cost of IFN-gamma production offers new opportunities for the use of this important cytokine.