RESUMEN
Despite decades of development of treatments and the successful application of targeted therapies for multiple myeloma, clinical challenges remain for patients with relapsed/refractory disease. A drug designed for efficient delivery of an alkylating payload into tumor cells that yields a favorable therapeutic window can be an attractive choice. Herein we describe melphalan flufenamide (melflufen), a drug with a peptide carrier component conjugated to an alkylating payload, and its cellular metabolism. We further underline the fundamental role of enzymatic hydrolysis in the rapid and robust accumulation of alkylating metabolites in cancer cells and their importance for downstream effects. The formed alkylating metabolites were shown to cause DNA damage, both on purified DNA and on chromatin in cells, with both nuclear and mitochondrial DNA affected in the latter. Furthermore, the rapid intracellular enrichment of alkylating metabolites is shown to be essential for the rapid kinetics of the downstream intracellular effects such as DNA damage signaling and induction of apoptosis. To evaluate the importance of enzymatic hydrolysis for melflufen's efficacy, all four stereoisomers of the compound were studied in a systematic approach and shown to have a different pattern of metabolism. In comparison with melflufen, stereoisomers lacking intracellular accumulation of alkylating payloads showed cytotoxic activity only at significantly higher concentration, slower DNA damage kinetics, and different mechanisms of action to reach cellular apoptosis.
Asunto(s)
Melfalán , Mieloma Múltiple , Humanos , Melfalán/efectos adversos , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Fenilalanina/farmacologíaRESUMEN
We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM-3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15-23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.
RESUMEN
We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome-a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells.
RESUMEN
Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. Herein, we explore ancestral sequence reconstruction as an enzyme engineering tool to enhance the therapeutic potential of PAL/TALs. We reconstructed putative ancestors from fungi and compared their catalytic activity and stability to two modern fungal PAL/TALs. Surprisingly, most putative ancestors could be expressed as functional tetramers in Escherichia coli and thus retained their ability to oligomerize. All ancestral enzymes displayed increased thermostability compared to both modern enzymes, however, the increase in thermostability was accompanied by a loss in catalytic turnover. One reconstructed ancestral enzyme in particular could be interesting for further drug development, as its ratio of specific activities is more favourable towards tyrosine and it is more thermostable than both modern enzymes. Moreover, long-term stability assessment showed that this variant retained substantially more activity after prolonged incubation at 25 °C and 37 °C, as well as an increased resistance to incubation at 60 °C. Both of these factors are indicative of an extended shelf-life of biopharmaceuticals. We believe that ancestral sequence reconstruction has potential for enhancing the properties of enzyme therapeutics, especially with respect to stability. This work further illustrates that resurrection of putative ancestral oligomeric proteins is feasible and provides insight into the extent of conservation of a functional oligomerization surface area from ancestor to modern enzyme.
Asunto(s)
Suplementos Dietéticos , Terapia de Reemplazo Enzimático , Fenilanina Amoníaco-Liasa/uso terapéutico , Tirosinemias/terapia , Animales , Activación Enzimática , Terapia de Reemplazo Enzimático/métodos , Estabilidad de Enzimas , Hongos/clasificación , Hongos/enzimología , Hongos/genética , Humanos , Cinética , Modelos Moleculares , Fenilanina Amoníaco-Liasa/administración & dosificación , Fenilanina Amoníaco-Liasa/química , Fenilanina Amoníaco-Liasa/clasificación , Conformación Proteica , Proteínas Recombinantes , Relación Estructura-Actividad , Termodinámica , Tirosinemias/etiologíaRESUMEN
Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) characterized by severe central nervous system (CNS) degeneration. The disease is caused by mutations in the SGSH gene coding for the lysosomal enzyme sulfamidase. Sulfamidase deficiency leads to accumulation of heparan sulfate (HS), which triggers aberrant cellular function, inflammation and eventually cell death. There is currently no available treatment against MPS IIIA. In the present study, a chemically modified recombinant human sulfamidase (CM-rhSulfamidase) with disrupted glycans showed reduced glycan receptor mediated endocytosis, indicating a non-receptor mediated uptake in MPS IIIA patient fibroblasts. Intracellular enzymatic activity and stability was not affected by chemical modification. After intravenous (i.v.) administration in mice, CM-rhSulfamidase showed a prolonged exposure in plasma and distributed to the brain, present both in vascular profiles and in brain parenchyma. Repeated weekly i.v. administration resulted in a dose- and time-dependent reduction of HS in CNS compartments in a mouse model of MPS IIIA. The reduction in HS was paralleled by improvements in lysosomal pathology and neuroinflammation. Behavioral deficits in the MPS IIIA mouse model were apparent in the domains of exploratory behavior, neuromuscular function, social- and learning abilities. CM-rhSulfamidase treatment improved activity in the open field test, endurance in the wire hanging test, sociability in the three-chamber test, whereas other test parameters trended towards improvements. The unique properties of CM-rhSulfamidase described here strongly support the normalization of clinical symptoms, and this candidate drug is therefore currently undergoing clinical studies evaluating safety and efficacy in patients with MPS IIIA.
RESUMEN
CCL2 is known for its major role as a chemoattractant of monocytes for immunological surveillance and to site of inflammation. CCL2 acts mainly through the G-protein-coupled receptor CCR2 but has also been described to mediate its effects independently of this receptor in vitro and in vivo. Emerging pieces of evidence indicate that the CCL2/CCR2 axis is involved in fibrotic diseases, such as increased plasma levels of CCL2 and the presence of CCL2-hyperresponsive fibroblasts explanted from patients with systemic sclerosis and idiopathic pulmonary fibrosis. One of the profibrotic key mediators is the myofibroblast characterized by overexpression of α-smooth muscle actin and collagen I. However, the correlation between the CCL2/CCR2 axis and the activation of fibroblasts is not yet fully understood. We have screened human fibroblasts of various origins, human pulmonary fibroblasts (HPF), human fetal lung fibroblasts (HFL-1) and primary preadipocytes (SPF-1) in regard to CCL2 stimulated fibrotic responses. Surprisingly we found that CCL2 mediates anti-fibrotic effects independently of CCR2 in human fibroblasts of different origins.