RESUMO
Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation.
Assuntos
Receptor de Asialoglicoproteína/metabolismo , Lisossomos/metabolismo , Acetilgalactosamina/metabolismo , Humanos , Células Tumorais CultivadasRESUMO
Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.
Assuntos
Imunoterapia/métodos , Melanoma Experimental/terapia , Neuraminidase/imunologia , Polissacarídeos/química , Receptor ErbB-2/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Aloenxertos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Humanos , Hidrólise , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Terapia de Alvo Molecular , Neuraminidase/química , Neuraminidase/genética , Polissacarídeos/imunologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Análise de Sobrevida , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.