RESUMO
Targeted degradation of pathological proteins is a promising approach to enhance the effectiveness of therapeutic monoclonal antibodies (mAbs) in cancer therapy. In this study, we demonstrate that this objective can be efficiently achieved by the grafting of mannose 6-phosphate analogues called AMFAs2 onto the therapeutic antibodies trastuzumab and cetuximab, both directed against membrane antigens. The grafting of AMFAs confers to these antibodies the novel property of being internalized via the mannose 6-phosphate receptor (M6PR) pathway. AMFA conjugation to these mAbs significantly increases their cellular uptake and leads to enhanced degradation of the target antigens in cancer cells. This results in a drastic inhibition of cancer cell proliferation compared to unconjugated mAbs, as demonstrated in various cancer cell lines, and an increased therapeutic efficacy in mouse and zebrafish xenografted models. These findings highlight the potential of this technology to improve therapeutic outcomes in cancer treatment.
Assuntos
Lisossomos , Proteínas de Membrana , Trastuzumab , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra , Animais , Humanos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Membrana/metabolismo , Trastuzumab/farmacologia , Cetuximab/farmacologia , Camundongos , Engenharia de Proteínas/métodos , Proliferação de Células/efeitos dos fármacos , Camundongos Nus , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Feminino , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
Inducing the degradation of pathological soluble antigens could be the key to greatly enhancing the efficacy of therapeutic monoclonal antibodies (mAbs), extensively used in the treatment of autoimmune and inflammatory disorders or cancer. Lysosomal targeting has gained increasing interest in recent years due to its pharmaceutical applications far beyond the treatment of lysosomal diseases, as a way to address proteins to the lysosome for eventual degradation. Mannose 6-phosphonate derivatives (M6Pn), called AMFA, are unique glycovectors that can significantly enhance the cellular internalization of the proteins conjugated to AMFA via the cation-independent mannose 6-phosphate receptor (M6PR) pathway. AMFA engineering of mAbs results in the generation of a bifunctional antibody that is designed to bind both the antigen and the M6PR. The improvement of the therapeutic potential by AMFA engineering was investigated using two antibodies directed against soluble antigens: infliximab (IFX), directed against tumor necrosis factor α (TNF-α), and bevacizumab (BVZ), directed against the vascular endothelial growth factor (VEGF). AMFA conjugations to the antibodies were performed either on the oligosaccharidic chains of the antibodies or on the lysine residues. Both conjugations were controlled and reproducible and provided a novel affinity for the M6PR without altering the affinity for the antigen. The grafting of AMFA to mAb increased their cellular uptake through an M6PR-dependent mechanism. The antigens were also 2.6 to 5.7 times more internalized by mAb-AMFA and rapidly degraded in the cells. Additional cell culture studies also proved the significantly higher efficacy of IFX-AMFA and BVZ-AMFA compared to their unconjugated counterparts in inhibiting TNF-α and VEGF activities. Finally, studies in a zebrafish embryo model of angiogenesis and in xenografted chick embryos showed that BVZ-AMFA was more effective than BVZ in reducing angiogenesis. These results demonstrate that AMFA grafting induces the degradation of soluble antigens and a significant increase in the therapeutic efficacy. Engineering with mannose 6-phosphate analogues has the potential to develop a new class of antibodies for autoimmune and inflammatory diseases.
Assuntos
Manose , Fator A de Crescimento do Endotélio Vascular , Embrião de Galinha , Animais , Fator de Necrose Tumoral alfa , Peixe-Zebra , Anticorpos Monoclonais , Bevacizumab , Infliximab , FosfatosRESUMO
The cation-independent mannose 6-phosphate receptor (CI-M6PR) is a ubiquitous transmembrane receptor whose main intracellular role is to direct enzymes carrying mannose 6-phosphate moieties to lysosomal compartments. Recently, the small membrane-bound portion of this receptor has appeared to be implicated in numerous pathophysiological processes. This review presents an overview of the main ligand partners and the roles of CI-M6PR in lysosomal storage diseases, neurology, immunology and cancer fields. Moreover, this membrane receptor has already been noted for its strong potential in therapeutic applications thanks to its cellular internalization activity and its ability to address pathogenic factors to lysosomes for degradation. A number of therapeutic delivery approaches using CI-M6PR, in particular with enzymes, antibodies or nanoparticles, are currently being proposed.
Assuntos
Lisossomos , Manose , Lisossomos/metabolismo , Proteínas de Transporte/metabolismo , Cátions , Fosfatos/metabolismoRESUMO
The concept of grafting mannose 6-phosphonate derivatives (M6Pn), named AMFA, on therapeutic proteins was first developed for the improvement of enzyme delivery in lysosomal storage disorders. This glycoengineering increases the cellular uptake of the protein via the cation-independent mannose 6-phosphate receptor (M6PR) which further allows their targeting to the lysosomes. In the present study, we investigated the extent to which the direct grafting of AMFA onto a drug, here a monoclonal antibody (mAb), affects the cell uptake and recycling of the antibody. The antibodies infliximab (IFX) and adalimumab (ADA), directed against the tumor necrosis factor α (TNFα), grafted with AMFA acquired an affinity for the M6PR, resulting in a >3-fold increase in drug release in cells. Subsequently, the impact of AMFA grafting to the Fc portion of mAb on its affinity for the neonatal Fc receptor (FcRn), which is the key receptor for antibody recycling, was evaluated. Whether one to three AMFA moieties were grafted, FcRn-mediated recycling of mAb was not affected. AMFA grafting did not impair the pharmacokinetics of both ADA and IFX and presented a high stability since AMFA were still bound to mAb in the plasma of mice 21 days after the treatment. In conclusion, this type of antibody engineering with a reduced number of AMFA confers M6PR targeting property and increases endocytosis, and yet appears fully compatible with FcRn binding and with antibody recycling and transcytosis.
Assuntos
Manose , Receptores Fc , Camundongos , Animais , Receptores Fc/metabolismo , Anticorpos Monoclonais/farmacocinética , Fator de Necrose Tumoral alfa , Antígenos de Histocompatibilidade Classe I/metabolismo , FosfatosRESUMO
5,15-Diazaporphyrin appended with D-mannose moieties was prepared through Suzuki-Miyaura cross-coupling reaction and SN2 alkylation. The resultant diazaporphyrin was hydrophilic enough to exhibit sufficient solubility in aqueous media. Because of the photosensitizing ability of diazaporphyrins, the in vitro activity of the D-mannose-appended diazaporphyrin in photodynamic therapy (PDT) was investigated. The specific internalization of the functionalized diazaporphyrin into human breast adenocarcinoma (MDA-MB-231) cells through mannose receptors was confirmed by confocal microscopy imaging. We also demonstrated the strong PDT activity of the functionalized diazaporphyrin at a nanomolar level with short light irradiation time.
Assuntos
Neoplasias da Mama , Fotoquimioterapia , Humanos , Feminino , Fotoquimioterapia/métodos , Manose , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular TumoralRESUMO
Porphyrin-based periodic mesoporous organosilica nanoparticles (PMO) synthesized from a large functional octatriethoxysilylated porphyrin precursor and allowing two-photon excitation photodynamic therapy (TPE-PDT) and NIR imaging were synthesized. These PMO were grafted with polyethylene glycol (PEG) moieties and an analogue of mannose 6-phosphate functionalized at the anomeric position (AMFA). AMFAs are known to efficiently target mannose 6-phosphate receptors (M6PRs) which are over-expressed in various cancers. Here, we demonstrated for the first time that M6PRs were over-expressed in rhabdomyosarcoma (RMS) cells and could be efficiently targeted with PMO-AMFA allowing TPE imaging and TPE-PDT of RMS cells. The comparison with healthy myoblasts demonstrated an absence of biological effects, suggesting a cancer cell specificity in the biomedical action observed.
Assuntos
Antineoplásicos/farmacologia , Materiais Biocompatíveis/farmacologia , Compostos de Organossilício/farmacologia , Receptor IGF Tipo 2/antagonistas & inibidores , Rabdomiossarcoma/tratamento farmacológico , Nanomedicina Teranóstica , Antineoplásicos/síntese química , Antineoplásicos/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Humanos , Nanopartículas/química , Imagem Óptica , Compostos de Organossilício/síntese química , Compostos de Organossilício/química , Tamanho da Partícula , Fotoquimioterapia , Porosidade , Porfirinas/química , Porfirinas/farmacologia , Proteômica , Receptor IGF Tipo 2/genética , Rabdomiossarcoma/diagnóstico por imagem , Rabdomiossarcoma/genética , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
The 300 kDa cation-independent M6P receptor (CI-MPR) mediates ligand internalization and trafficking to the endolysosomal compartments. Because of its endocytotic nature, it has been recognized as a promising class of receptors for target component delivery. Its cellular uptake involves the simultaneous binding of two protein units resulting in the formation of receptor dimers. While many multivalent glycoconjugates have been reported to date, little is known about the topological requests to induce an effective recruitment of CI-MPRs. We herein describe the synthesis and cell uptake ability of a set of highly organized glycoclusters bearing one to three saccharide units. The spatial arrangement of carbohydrate ligands is ensured by a heterocyclic γ-peptide central core.
Assuntos
Receptor IGF Tipo 2/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Conformação Proteica , Receptor IGF Tipo 2/químicaRESUMO
In the search of a better enzyme therapy in Pompe disease, the conjugation of mannose 6-phosphonates to the recombinant enzyme appeared as an enhancer of its efficacy. Here, we demonstrated that the increased efficacy of the conjugated enzyme is partly due to a higher intracellular maturation because of its insensitiveness to acid phosphatases during the routing to lysosomes.
Assuntos
Doença de Depósito de Glicogênio Tipo II/metabolismo , Manose/metabolismo , Organofosfonatos/metabolismo , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Lisossomos/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Mioblastos/metabolismoRESUMO
The aim of the present work is the development of highly efficient targeting molecules to specifically address mesoporous silica nanoparticles (MSNs) designed for the photodynamic therapy (PDT) of prostate cancer. We chose the strategy to develop a novel compound that allows the improvement of the targeting of the cation-independent mannose 6-phosphate receptor, which is overexpressed in prostate cancer. This original sugar, a dimannoside-carboxylate (M6C-Man) grafted on the surface of MSN for PDT applications, leads to a higher endocytosis and thus increases the efficacy of MSNs.
Assuntos
Fotoquimioterapia/métodos , Neoplasias da Próstata/metabolismo , Receptor IGF Tipo 2/metabolismo , Linhagem Celular Tumoral , Endocitose , Humanos , Masculino , Manosefosfatos/administração & dosagem , Manosefosfatos/química , Manosefosfatos/farmacologia , Nanopartículas/administração & dosagem , Nanopartículas/química , Nanopartículas/metabolismo , Dióxido de Silício/químicaRESUMO
BACKGROUND: Bridged silsesquioxane nanoparticles (BSNs) recently described represent a new class of nanoparticles exhibiting versatile applications and particularly a strong potential for nanomedicine. AIMS: In this work, we describe the synthesis of BSNs from an octasilylated functional porphyrin precursor (PORBSNs) efficiently obtained through a click reaction. These innovative and very small-sized nanoparticles were functionalized with PEG and mannose (PORBSNs-mannose) in order to target breast tumors in vivo. METHODS AND RESULTS: The structure of these nanoparticles is constituted of porphyrins J aggregates that allow two-photon spatiotemporal excitation of the nanoparticles. The therapeutic potential of such photoactivable nanoparticles was first studied in vitro, in human breast cancer cells in culture and then in vivo on zebrafish embryos bearing human tumors. These animal models were intravenously injected with 5 nL of a solution containing PORBSNs-mannose. An hour and half after the injection of photoactivable and targeted nanoparticles, the tumor areas were excited for few seconds with a two-photon beam induced focused laser. We observed strong tumor size decrease, with the involvement of apoptosis pathway activation. CONCLUSION: We demonstrated the high targeting, imaging, and therapeutic potential of PORBSNs-mannose injected in the blood stream of zebrafish xenografted with human tumors.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Nanopartículas/administração & dosagem , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Nanomedicina Teranóstica/métodos , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Injeções Intravenosas , Lasers , Microscopia de Fluorescência por Excitação Multifotônica , Nanopartículas/química , Nanopartículas/efeitos da radiação , Fotoquimioterapia/instrumentação , Fármacos Fotossensibilizantes/química , Porfirinas/administração & dosagem , Porfirinas/química , Silanos/administração & dosagem , Silanos/química , Nanomedicina Teranóstica/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Sugars play key roles in many molecular and cellular communication processes involving a family of proteins named lectins. The low affinity associated with sugar recognition is generally counterbalanced by the multivalent nature of the interaction. While many polyglycosylated architectures have been described, only a few studies focused on the impact of topology variations of the multivalent structures on the interaction with lectin proteins. One major interest of our group concerns the design of new highly predictable and stable molecular pseudo-peptide architectures for therapeutic applications. In such a context, we described a class of constrained heterocyclic γ-amino acids built around a thiazole ring, named ATCs. ATC oligomers are helical molecules resulting from the formation of a highly stable C9 hydrogen-bonding pattern. Following our program, we herein address the potential of ATC oligomers as tunable scaffolds for the development of original multivalent glycoclusters.
RESUMO
Pompe disease is a rare disorder due to deficiency of the acid α-glucosidase (GAA) treated by enzyme replacement therapy. The present authorized treatment with rhGAA, the recombinant human enzyme, provides an important benefit in the infantile onset; however, the juvenile and adult forms of the disease corresponding to >80% of the patients are less responsive to this treatment. This resistance has been mainly attributed to an insufficiency of mannose 6-phosphate residues in rhGAA to address lysosomes through the cation-independent mannose 6-phosphate receptor (CI-M6PR). As yet, several attempts to improve the enzyme delivery by increasing the number of mannose 6-phosphate on the enzyme were poorly effective on the late onset form of the disease. Here, we show that chemical conjugation of a synthetic analogue of the mannose 6-phosphate, named AMFA, onto rhGAA improves the affinity for CI-M6PR and the uptake of the enzyme in fibroblasts and myoblasts of adult Pompe patients. More importantly, only the conjugated rhGAA-AMFA was effective in aged Pompe mice when compared to rhGAA. Weekly treatment with 5-20mg·kg-1 rhGAA-AMFA provided major improvements of the motor function and of the myofiber structure, whereas rhGAA was inactive. Finally, AMFA addition did not induce supplementary immune response to the enzyme. This modified enzyme, displaying a muscle recovery in aged Pompe mice that was never attained before, could be considered as a potential therapy for the late onset Pompe disease.
Assuntos
Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Manosefosfatos/administração & dosagem , alfa-Glucosidases/administração & dosagem , Adulto , Animais , Células Cultivadas , Fibroblastos/metabolismo , Glicogênio/metabolismo , Doença de Depósito de Glicogênio Tipo II/metabolismo , Humanos , Manosefosfatos/química , Camundongos Knockout , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismoRESUMO
A challenge regarding the design of nanocarriers for drug delivery is to prevent their recognition by the immune system. To improve the blood residence time and prevent their capture by organs, nanoparticles can be designed with stealth properties using polymeric coating. In this study, we focused on the influence of surface modification with polyethylene glycol and/or mannose on the stealth behavior of porous silicon nanoparticles (pSiNP, ~200 nm). In vivo biodistribution of pSiNPs formulations were evaluated in mice 5 h after intravenous injection. Results indicated that the distribution in the organs was surface functionalization-dependent. Pristine pSiNPs and PEGylated pSiNPs were distributed mainly in the liver and spleen, while mannose-functionalized pSiNPs escaped capture by the spleen, and had higher blood retention. The most efficient stealth behavior was observed with PEGylated pSiNPs anchored with mannose that were the most excreted in urine at 5 h. The biodegradation kinetics evaluated in vitro were in agreement with these in vivo observations. The biocompatibility of the pristine and functionalized pSiNPs was confirmed in vitro on human cell lines and in vivo by cytotoxic and systemic inflammation investigations, respectively. With their biocompatibility, biodegradability, and stealth properties, the pSiNPs functionalized with mannose and PEG show promising potential for biomedical applications.
RESUMO
Porphyrins fused to imidazolium salts across two neighboring ß-pyrrolic positions were used as N-heterocyclic carbene (NHC) precursors to anchor AuI -Cl complexes at their periphery. Synthesis of several thiolato-AuI complexes was then achieved by substituting chloride for thiolates. Photodynamic properties of these complexes were investigated: the data obtained show that the Au-S bonds could be cleaved upon irradiation. The proposed mechanism to explain the release of thiolate moiety involves the S atom oxidation by singlet oxygen generated in the course of irradiation. In view of photodynamic therapy (PDT) applications, these porphyrins fused to NHC-AuI complexes were tested as photosensitizers to kill MCF-7 breast cancer cells. Results show the important role played by the ancillary ligands (chloride versus thiolates) on the photodynamic effect.
RESUMO
Improving therapeutics delivery in enzyme replacement therapy (ERT) for lysosomal storage disorders is a challenge. Herein, we present the synthesis of novel analogues of mannose 6-phosphate (M6P), known as AMFAs and functionalized at the anomeric position for enzyme grafting. AMFAs are non-phosphate serum-resistant derivatives that efficiently bind the cation-independent mannose 6-phosphate receptor (CI-M6PR), which is the main pathway to address enzymes to lysosomes. One of the AMFAs was used to improve the treatment of the lysosomal myopathy Pompe disease, in which acid α-glucosidase (GAA) is defective. AMFA grafting on a M6P-free recombinant GAA led to a higher uptake of the GAA in adult Pompe fibroblasts in culture as compared to Myozyme, the M6P recombinant GAA. Moreover, the treatment of Pompe adult mice with the AMFA-grafted recombinant enzyme led to a remarkable improvement, even at low doses, in muscle functionality and regeneration, whereas Myozyme had limited efficacy.
Assuntos
Terapia de Reposição de Enzimas , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Lisossomos/enzimologia , Manosefosfatos/farmacologia , alfa-Glucosidases/metabolismo , Animais , Configuração de Carboidratos , Desenho de Fármacos , Doença de Depósito de Glicogênio Tipo II/metabolismo , Humanos , Lisossomos/metabolismo , Manosefosfatos/síntese química , Manosefosfatos/química , CamundongosRESUMO
A novel non-toxic porous silicon nanoparticle grafted with a mannose-6-phosphate analogue and applicable in 2-photon imaging and photodynamic therapy was specifically designed for targeting prostate cancer cells.
RESUMO
The development of personalized and non-invasive cancer therapies based on new targets combined with nanodevices is a major challenge in nanomedicine. In this work, the over-expression of a membrane lectin, the cation-independent mannose 6-phosphate receptor (M6PR), was specifically demonstrated in prostate cancer cell lines and tissues. To efficiently target this lectin a mannose-6-phosphate analogue was synthesized in six steps and grafted onto the surface of functionalized mesoporous silica nanoparticles (MSNs). These MSNs were used for inâ vitro and exâ vivo photodynamic therapy to treat prostate cancer cell lines and primary cell cultures prepared from patient biopsies. The results demonstrated the efficiency of M6PR targeting for prostate cancer theranostic.
Assuntos
Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/tratamento farmacológico , Receptor IGF Tipo 2/antagonistas & inibidores , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Humanos , Masculino , Manosefosfatos/síntese química , Manosefosfatos/química , Nanopartículas/química , Nanopartículas/uso terapêutico , Tamanho da Partícula , Fotoquimioterapia , Porosidade , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptor IGF Tipo 2/genética , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
Porous silicon nanoparticles (pSiNPs) act as a sensitizer for the 2-photon excitation of a pendant porphyrin using NIR laser light, for imaging and photodynamic therapy. Mannose-functionalized pSiNPs can be vectorized to MCF-7 human breast cancer cells through a mannose receptor-mediated endocytosis mechanism to provide a 3-fold enhancement of the 2-photon PDT effect.
Assuntos
Nanopartículas/uso terapêutico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/uso terapêutico , Silício/uso terapêutico , Materiais Biocompatíveis/química , Materiais Biocompatíveis/uso terapêutico , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Endocitose/efeitos dos fármacos , Endocitose/efeitos da radiação , Humanos , Raios Infravermelhos , Células MCF-7 , Manose/química , Manose/uso terapêutico , Microscopia Confocal , Microscopia de Fluorescência , Nanopartículas/química , Fótons , Fármacos Fotossensibilizantes/química , Porosidade , Porfirinas/química , Silício/químicaRESUMO
The sulfamide analogue of the antiepileptic drug topiramate is a 210 times less potent inhibitor of isozyme II of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) compared to topiramate but effectively inhibits isozymes CA VA, VB, VII, XIII, and XIV (KI in the range of 21-35 nM). Its weak binding to CA II is due to a clash between one methyl group of the inhibitor and Ala65 and may be exploited for the drug design of compounds with lower affinity for this ubiquitous isozyme, as Ala65 is unique to CA II. As shown by X-ray crystallography, the sulfamide analogue binds to CA II with the deprotonated sulfamide moiety coordinated to Zn(II) and with the organic scaffold making an extended network of hydrogen bonds with Thr199, Gln92, His94, Asn62, and Thr200. Its binding to this isozyme is more similar to that of topiramate and quite different from that of the topiramate cyclic sulfate analogue RWJ-37947.