RESUMEN
Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.
Asunto(s)
Antígenos de Diferenciación , Conformación Proteica , Humanos , Antígenos de Diferenciación/metabolismo , Antígenos de Diferenciación/química , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Antivirales/farmacología , Antivirales/química , Antivirales/metabolismoRESUMEN
CD47 on the surface of tumor cells has become a research hot spot in immunotherapy and anticancer therapy, as it can bind to SIRPα protein on the surface of macrophages, which ultimately leads to immune escape of tumor cells. In the present study, molecular interactions between CD47 and human SIRPα proteins (including variant 1, V1 and variant 2, V2) were analyzed through molecular dynamics (MD) simulation and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Hydrophobic interactions were found as the main driving force for the binding of CD47 on SIRPα. The residues including pyroglutamate acid (Z)1, L2, E35, Y37, E97, L101, and T102 of CD47 were identified with a significant favorable contribution to the binding of CD47 on SIRPα (both V1 and V2). Based on this, a peptide inhibitor library with the sequence ZLXRTLXEXY was designed (X represents the arbitrary residue of 20 standard amino acids) and then screened using molecular docking, MD simulations, and experimental validation. Finally, a peptide ZLIRTLHEWY was determined with high affinity with SIRPα from 8000 candidates, containing 6/10 residues favorable for the binding on SIRPα V1 and 8/10 residues favorable for the binding on SIRPα V2, which was thus considered to have potential anticancer function.
Asunto(s)
Antígeno CD47 , Neoplasias , Humanos , Antígeno CD47/genética , Antígeno CD47/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Simulación del Acoplamiento Molecular , Biomimética , Antígenos de Diferenciación/química , Antígenos de Diferenciación/metabolismo , Péptidos/farmacología , Biblioteca de Péptidos , FagocitosisRESUMEN
The intricate complex system of the differentiation 47 (CD47) and the signal-regulatory protein alpha (SIRPα) cluster is a crucial target for cancer immunotherapy. Although the conformational state of the CD47-SIRPα complex has been revealed through crystallographic studies, further characterization is needed to fully understand the binding mechanism and to identify the hot spot residues involved. In this study, molecular dynamics (MD) simulations were carried out for the complexes of CD47 with two SIRPα variants (SIRPαv1, SIRPαv2) and the commercially available anti-CD47 monoclonal antibody (B6H12.2). The calculated binding free energy of CD47-B6H12.2 is lower than that of CD47-SIRPαv1 and CD47-SIRPαv2 in all the three simulations, indicating that CD47-B6H12.2 has a higher binding affinity than the other two complexes. Moreover, the dynamical cross-correlation matrix reveals that the CD47 protein shows more correlated motions when it binds to B6H12.2. Significant effects were observed in the energy and structural analyses of the residues (Glu35, Tyr37, Leu101, Thr102, Arg103) in the C strand and FG region of CD47 when it binds to the SIRPα variants. The critical residues (Leu30, Val33, Gln52, Lys53, Thr67, Arg69, Arg95, and Lys96) were identified in SIRPαv1 and SIRPαv2, which surround the distinctive groove regions formed by the B2C, C'D, DE, and FG loops. Moreover, the crucial groove structures of the SIRPα variants shape into obvious druggable sites. The C'D loops on the binding interfaces undergo notable dynamical changes throughout the simulation. For B6H12.2, the residues Tyr32LC, His92LC, Arg96LC, Tyr32HC, Thr52HC, Ser53HC, Ala101HC, and Gly102HC in its initial half of the light and heavy chains exhibit obvious energetic and structural impacts upon binding with CD47. The elucidation of the binding mechanism of SIRPαv1, SIRPαv2, and B6H12.2 with CD47 could provide novel perspectives for the development of inhibitors targeting CD47-SIRPα.
Asunto(s)
Simulación de Dinámica Molecular , Neoplasias , Humanos , Receptores Inmunológicos/química , Antígenos de Diferenciación/química , Antígeno CD47/genética , Antígeno CD47/química , Anticuerpos Monoclonales , Inmunoterapia , Fagocitosis , Neoplasias/metabolismoRESUMEN
Cancer cells can evade immune surveillance through binding of its transmembrane receptor CD47 to CD172a on myeloid cells. CD47 is recognized as a promising immune checkpoint for cancer immunotherapy inhibiting macrophage phagocytosis. N-terminal post-translated modification (PTM) via glutaminyl cyclase is a landmark event in CD47 function maturation, but the molecular mechanism underlying the mechano-chemical regulation of the modification on CD47/CD172a remains unclear. Here, we performed so-called "ramp-clamp" steered molecular dynamics (SMD) simulations, and found that the N-terminal PTM enhanced interaction of CD172a with CD47 by inducing a dynamics-driven contraction of the binding pocket of the bound CD172a, an additional constraint on CYS15 on CD47 significantly improved the tensile strength of the complex with or without PTM, and a catch bond phenomenon would occur in complex dissociation under tensile force of 25 pN in a PTM-independent manner too. The residues GLN52 and SER66 on CD172a reinforced the H-bonding with their partners on CD47 in responding to PTM, while ARG69 on CD172 with its partner on CD47 might be crucial in the structural stability of the complex. This work might serve as molecular basis for the PTM-induced function improvement of CD47, should be helpful for deeply understanding CD47-relevant immune response and cancer development, and provides a novel insight in developing of new strategies of immunotherapy targeting this molecule interaction.
Asunto(s)
Antígeno CD47 , Neoplasias , Humanos , Antígeno CD47/metabolismo , Antígenos de Diferenciación/química , Macrófagos/metabolismo , Fagocitosis , Neoplasias/metabolismoRESUMEN
CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.
Asunto(s)
Inmunoconjugados , Neoplasias , Humanos , Antígenos CD28/genética , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Antígenos CD/genética , Antígenos de Diferenciación/química , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Abatacept/metabolismo , Inmunoconjugados/metabolismo , Neoplasias/genética , Neoplasias/terapia , Antígeno B7-1/genética , Antígeno B7-1/química , Antígeno B7-1/metabolismo , Inmunoterapia , Proteínas Portadoras , Antígeno B7-2/genética , Activación de LinfocitosRESUMEN
Background: Targeting the CD47/SIRPα signaling pathway represents a novel approach to enhance anti-tumor immunity. However, the crystal structure of the CD47/SIRPα has not been fully studied. This study aims to analyze the structure interface of the complex of CD47 and IMM01, a novel recombinant SIRPα-Fc fusion protein. Methods: IMM01-Fab/CD47 complex was crystalized, and diffraction images were collected. The complex structure was determined by molecular replacement using the program PHASER with the CD47-SIRPαv2 structure (PDB code 2JJT) as a search model. The model was manually built using the COOT program and refined using TLS parameters in REFMAC from the CCP4 program suite. Results: Crystallization and structure determination analysis of the interface of IMM01/CD47 structure demonstrated CD47 surface buried by IMM01. Comparison with the literature structure (PDB ID 2JJT) showed that the interactions of IMM01/CD47 structure are the same. All the hydrogen bonds that appear in the literature structure are also present in the IMM01/CD47 structure. These common hydrogen bonds are stable under different crystal packing styles, suggesting that these hydrogen bonds are important for protein binding. In the structure of human CD47 in complex with human SIRPα, except SER66, the amino acids that form hydrogen bonds are all conserved. Furthermore, comparing with the structure of PDB ID 2JJT, the salt bridge interaction from IMM01/CD47 structure are very similar, except the salt bridge bond between LYS53 in IMM01 and GLU106 in CD47, which only occurs between the B and D chains. However, as the side chain conformation of LYS53 in chain A is slightly different, the salt bridge bond is absent between the A and C chains. At this site between chain A and chain C, there are a salt bridge bond between LYS53 (A) and GLU104 (C) and a salt bridge bond between HIS56 (A) and GLU106 (C) instead. According to the sequence alignment results of SIRPα, SIRPß and SIRPγ in the literature of PDB ID 2JJT, except ASP100, the amino acids that form common salt bridge bonds are all conserved. Conclusion: Our data demonstrated crystal structure of the IMM01/CD47 complex and provides a structural basis for the structural binding interface and future clinical applications.
Asunto(s)
Aminoácidos , Antígenos de Diferenciación , Antígeno CD47 , Receptores Inmunológicos , Aminoácidos/química , Antígenos de Diferenciación/química , Antígeno CD47/química , Humanos , Fagocitosis , Unión Proteica , Receptores Inmunológicos/química , Proteínas Recombinantes de Fusión/químicaRESUMEN
Immunotherapy with anti-GD2 monoclonal antibodies (mAbs) provides some benefits for patients with neuroblastoma (NB). However, the therapeutic efficacy remains limited, and treatment is associated with significant neuropathic pain. Targeting O-acetylated GD2 (OAcGD2) by 8B6 mAb has been proposed to avoid pain by more selective tumor cell targeting. Thorough understanding of its mode of action is necessary to optimize this treatment strategy. Here, we found that 8B6-mediated antibody-dependent cellular phagocytosis (ADCP) performed by macrophages is a key effector mechanism. But efficacy is limited by upregulation of CD47 expression on neuroblastoma cells in response to OAcGD2 mAb targeting, inhibiting 8B6-mediated ADCP. Antibody specific for the CD47 receptor SIRPα on macrophages restored 8B6-induced ADCP of CD47-expressing NB cells and improved the antitumor activity of 8B6 mAb therapy. These results identify ADCP as a critical mechanism for tumor cytolysis by anti-disialoganglioside mAb and support a combination with SIRPα blocking agents for effective neuroblastoma therapy.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos de Diferenciación/química , Neuroblastoma/inmunología , Fagocitosis , Receptores Inmunológicos/química , Animales , Anticuerpos/química , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos Inmunológicos/farmacología , Antígeno CD47/biosíntesis , Línea Celular Tumoral , Citometría de Flujo , Gangliósidos/química , Humanos , Inmunoterapia/métodos , Macrófagos/metabolismo , Ratones , Microscopía Fluorescente , Neuroblastoma/metabolismo , Regulación hacia ArribaRESUMEN
Curcumin and curcuminoids have been discussed frequently due to their promising functional groups (such as scaffolds of α,ß-unsaturated ß-diketone, α,ß-unsaturated ketone and ß'-hydroxy-α,ß-unsaturated ketone connected with aromatic rings on both sides) that play an important role in various bioactivities, including antioxidant, anti-inflammatory, anti-proliferation and anticancer activity. A series of novel curcuminoid derivatives (a total of 55 new compounds) and three reference compounds were synthesized with good yields using three-step organic synthesis. The anti-proliferative activities of curcumin derivatives were examined for six human cancer cell lines: HeLaS3, KBvin, MCF-7, HepG2, NCI-H460 and NCI-H460/MX20. Compared to the IC50 values of all the synthesized derivatives, most α,ß-unsaturated ketones displayed potent anti-proliferative effects against all six human cancer cell lines, whereas ß'-hydroxy-α,ß-unsaturated ketones and α,ß-unsaturated ß-diketones presented moderate anti-proliferative effects. Two potent curcuminoid derivatives were found among all the novel derivatives and reference compounds: (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a). These were selected for further analysis after the evaluation of their anti-proliferative effects against all human cancer cell lines. The results of apoptosis assays revealed that the number of dead cells was increased in early apoptosis and late apoptosis, while cell proliferation was also decreased after applying various concentrations of (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) to MCF-7 and HpeG2 cancer cells. Analysis of the gene expression arrays showed that three genes (GADD45B, SESN2 and BBC3) were correlated with the p53 pathway. From the quantitative PCR analysis, it was seen that (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) effectively induced the up-regulated expression of GADD45B, leading to the suppression of MCF-7 cancer cell formation and cell death. Molecular docking analysis was used to predict and sketch the interactions of the GADD45B-α,ß-unsaturated ketone complex for help in drug design.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Diarilheptanoides/química , Diarilheptanoides/farmacología , Diseño de Fármacos , Antígenos de Diferenciación/química , Antígenos de Diferenciación/metabolismo , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diarilheptanoides/síntesis química , Ensayos de Selección de Medicamentos Antitumorales , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes p53/efectos de los fármacos , Humanos , Cetonas/química , Cetonas/farmacología , Simulación del Acoplamiento Molecular , Análisis de Componente Principal , Transducción de Señal/efectos de los fármacosRESUMEN
Antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), play a crucial role in bridging innate and adaptive immunity; thereby, innate immune checkpoint blockade-based therapy is an attractive approach for the induction of sustainable tumor-specific immunity. The interaction between the cluster of differentiation 47 (CD47) on tumor and signal-regulatory protein alpha (SIRPα) on phagocytic cells inhibits the phagocytic function of APCs, acting as a "don't eat me" signal. Accordingly, CD47 blockade is known to increase tumor cell phagocytosis, eliciting tumor-specific CD8+ T-cell immunity. Here, we introduced a nature-derived nanocage to deliver SIRPγ for blocking of antiphagocytic signaling through binding to CD47 and combined it with prophagocytic stimuli using a metabolic reprogramming reagent for APCs (CpG-oligodeoxynucleotides). Upon delivering the clustered SIRPγ variant, the nanocage showed enhanced CD47 binding profiles on tumor cells, thereby promoting active engulfment by phagocytes. Moreover, combination with CpG potentiated the prophagocytic ability, leading to the establishment of antitumorigenic surroundings. This combination treatment could competently inhibit tumor growth by invigorating APCs and CD8+ T-cells in TMEs in B16F10 orthotopic tumor models, known to be resistant to CD47-targeting therapeutics. Collectively, enhanced delivery of an innate immune checkpoint antagonist with metabolic modulation stimuli of immune cells could be a promising strategy for arousing immune responses against cancer.
Asunto(s)
Antígenos de Diferenciación/administración & dosificación , Antígenos de Diferenciación/inmunología , Ferritinas/administración & dosificación , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Nanoestructuras/uso terapéutico , Oxidorreductasas/administración & dosificación , Receptores Inmunológicos/administración & dosificación , Receptores Inmunológicos/inmunología , Animales , Antígenos de Diferenciación/química , Antígenos de Diferenciación/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Ferritinas/química , Ferritinas/genética , Humanos , Inmunoterapia/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Nanoestructuras/química , Células Neoplásicas Circulantes/inmunología , Oxidorreductasas/química , Oxidorreductasas/genética , Fagocitosis/inmunología , Receptores Inmunológicos/química , Receptores Inmunológicos/genéticaRESUMEN
Phagocytic resistance plays a key role in tumor-mediated immune escape, so phagocytosis immune checkpoints are a potential target for cancer immunotherapy. CD47 is one of the important phagocytosis immune checkpoints; thus, blocking the interaction between CD47 and signal regulatory protein α (SIRPα) may provide new options for cancer treatment. Using computer-aided targeted epitope mammalian cell-displayed antibody library, we screened and obtained an engineered SIRPα variant fragment crystallizable fusion protein, FD164, with higher CD47-binding activity than wild-type SIRPα Compared with wild-type SIRPα, FD164 has approximately 3-fold higher affinity for binding to CD47, which further enhanced its phagocytic effect in vitro and tumor suppressor activity in vivo. FD164 maintains the similar antitumor activity of the clinical research drug Hu5F9 in the mouse xenograft model. Furthermore, FD164 combined with rituximab can significantly improve the effect of single-agent therapy. On the other hand, compared with Hu5F9, FD164 does not cause hemagglutination, and its ability to bind to red blood cells or white blood cells is weaker at the same concentration. Finally, it was confirmed by computer structure prediction and alanine scanning experiments that the N45, E47, 52TEVYVK58, K60, 115EVTELTRE122, and E124 residues of CD47 are important for SIRPα or FD164 recognition. Briefly, we obtained a high-affinity SIRPα variant FD164 with balanced safety and effectiveness. SIGNIFICANCE STATEMENT: Up to now, few clinically marketed drugs targeting CD47 have been determined to be effective and safe. FD164, a potential signal regulatory protein α variant fragment crystallizable protein with balanced safety and effectiveness, could provide a reference for the development of antitumor drugs.
Asunto(s)
Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Antígeno CD47/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Animales , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos de Diferenciación/efectos adversos , Antígenos de Diferenciación/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Antígeno CD47/química , Células CHO , Línea Celular , Cricetulus , Diseño de Fármacos , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Hemaglutinación/efectos de los fármacos , Inmunoterapia , Ratones SCID , Modelos Moleculares , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Receptores Inmunológicos/química , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Rituximab/uso terapéutico , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interferon-induced transmembrane proteins (IFITMs) are a family of interferon-inducible proteins that inhibit a broad range of viruses by interfering with viral-to-cellular membrane fusion. The antiviral activity of IFITMs is highly regulated by several posttranslational modifications and by a number of protein domains that modulate steady-state protein levels, trafficking, and antiviral effectiveness. Taking advantage of the natural diversity existing among IFITMs of different animal species, we have compared 21 IFITMs for their ability to inhibit HIV-1 at two steps, during virus entry into cells (target cell protection) and during the production of novel virion particles (negative imprinting of virion particles' infectivity). We found a high functional heterogeneity among IFITM homologs with respect to both antiviral modalities, with IFITM members that exhibit enhanced viral inhibition, while others have no ability to block HIV-1. These differences could not be ascribed to known regulatory domains and could only be partially explained through differential protein stability, implying the existence of additional mechanisms. Through the use of chimeras between active and inactive IFITMs, we demonstrate that the cross talk between distinct domains of IFITMs is an important contributor of their antiviral potency. Finally, we identified murine IFITMs as natural variants competent for target cell protection, but not for negative imprinting of virion particles' infectivity, suggesting that the two properties may, at least in principle, be uncoupled. Overall, our results shed new light on the complex relationship between IFITMs and viral infection and point to the cross talk between IFITM domains as a novel layer of regulation of their activity. IMPORTANCE IFITMs are broad viral inhibitors capable of interfering with both early and late phases of the replicative cycle of many different viruses. By comparing 21 IFITM proteins issued from different animal species for their ability to inhibit HIV-1, we have identified several that exhibit either enhanced or impaired antiviral behavior. This functional diversity is not driven by differences in known domains and can only be partly explained through differential protein stability. Chimeras between active and inactive IFITMs point to the cross talk between individual IFITM domains as important for optimal antiviral activity. Finally, we show that murine IFITMs are not capable of decreasing the infectivity of newly produced HIV-1 virion particles, although they retain target cell protection abilities, suggesting that these properties may be, in principle, disconnected. Overall, our results shed new light on the complex layers of regulation of IFITM proteins and enrich our current understanding of these broad antiviral factors.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Antivirales/farmacología , Infecciones por VIH/prevención & control , VIH-1/fisiología , Interacciones Huésped-Patógeno , Ensamble de Virus , Internalización del Virus , Secuencia de Aminoácidos , Antígenos de Diferenciación/química , Antígenos de Diferenciación/genética , Células HEK293 , Infecciones por VIH/patología , Infecciones por VIH/virología , Humanos , Estabilidad Proteica , Homología de SecuenciaRESUMEN
Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a "don't eat me" signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N-terminal pyro-glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell-mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab-mediated direct growth inhibition, complement-dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα-Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose-dependent manner, suggesting that pyro-glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab-dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte-mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell-mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.
Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Antígenos de Diferenciación/química , Antineoplásicos Inmunológicos/administración & dosificación , Antígeno CD47/metabolismo , Neoplasias/tratamiento farmacológico , Receptores Inmunológicos/química , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Animales , Antígenos de Diferenciación/metabolismo , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cetuximab/administración & dosificación , Cetuximab/farmacología , Sinergismo Farmacológico , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Neoplasias/metabolismo , Panitumumab/administración & dosificación , Panitumumab/farmacología , Unión Proteica/efectos de los fármacos , Receptores Inmunológicos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The CD225 superfamily regulates vesicular membrane fusion events essential to neurotransmission, immunity, development, and metabolism. Its importance to physiology is reinforced by the identification of polymorphisms associated with disease. This article highlights the shared features that drive the function of CD225 proteins such as interferon-inducible transmembrane proteins 3 (IFITM3) and proline-rich transmembrane protein 2 (PRRT2) and is intended to catalyze efforts towards characterizing the lesser-known family members.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Antígenos de Diferenciación/química , Antígenos de Diferenciación/genética , Exocitosis/fisiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Fusión de Membrana/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Familia de Multigenes , Internalización del VirusAsunto(s)
Antígenos de Diferenciación/química , Endopeptidasas/fisiología , Leucemia Mieloide/patología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Regulación hacia Abajo , Leucemia Mieloide/etiología , Leucemia Mieloide/metabolismo , Ratones , Ratones NoqueadosRESUMEN
CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRPα receptors, that block the SIRPα-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRPα and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRPα variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRPα binding. The SIRPα-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRPα-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.
Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Antígenos de Diferenciación/genética , Antígeno CD47/genética , Neoplasias/tratamiento farmacológico , Receptores Inmunológicos/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Inmunidad Adaptativa/genética , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/química , Antígenos de Diferenciación/química , Antígeno CD47/química , Desarrollo de Medicamentos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Inmunoterapia/métodos , Células Jurkat , Citometría de Barrido por Láser , Ligandos , Oncología Médica/tendencias , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Fagocitosis/efectos de los fármacos , Mapas de Interacción de Proteínas/genética , Receptores Inmunológicos/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
CD47 is an immunoglobulin that is overexpressed on the surface of many types of cancer cells. CD47 forms a signaling complex with signal-regulatory protein α (SIRPα), enabling the escape of these cancer cells from macrophage-mediated phagocytosis. In recent years, CD47 has been shown to be highly expressed by various types of solid tumors and to be associated with poor patient prognosis in various types of cancer. A growing number of studies have since demonstrated that inhibiting the CD47-SIRPα signaling pathway promotes the adaptive immune response and enhances the phagocytosis of tumor cells by macrophages. Improved understanding in this field of research could lead to the development of novel and effective anti-tumor treatments that act through the inhibition of CD47 signaling in cancer cells. In this review, we describe the structure and function of CD47, provide an overview of studies that have aimed to inhibit CD47-dependent avoidance of macrophage-mediated phagocytosis by tumor cells, and assess the potential and challenges for targeting the CD47-SIRPα signaling pathway in anti-cancer therapy.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos de Diferenciación/metabolismo , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno CD47/inmunología , Antígeno CD47/metabolismo , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Receptores Inmunológicos/metabolismo , Transducción de Señal/efectos de los fármacos , Inmunidad Adaptativa/efectos de los fármacos , Animales , Antígenos de Diferenciación/química , Antígeno CD47/antagonistas & inhibidores , Antígeno CD47/química , Humanos , Macrófagos/inmunología , Fagocitosis/efectos de los fármacos , Receptores Inmunológicos/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lectins mediate adhesion of pathogens to host tissues, filling in a key role in the first steps of infection. Belonging to the opportunistic pathogen Burkholderia cenocepacia, BC2L-C is a superlectin with dual carbohydrate specificity, believed to mediate cross-linking between bacteria and host cells. Its C-terminal domain binds to bacterial mannosides while its N-terminal domain (BCL2-CN) recognizes fucosylated human epitopes. BC2L-CN presents a tumor necrosis factor alpha (TNF-ï¡) fold previously unseen in lectins with a novel fucose binding mode. We report, here, the production of a novel recombinant form of BC2L-CN (rBC2L-CN2), which allowed better protein stability and unprecedented co-crystallization with oligosaccharides. Isothermal calorimetry measurements showed no detrimental effect on ligand binding and data were obtained on the binding of Globo H hexasaccharide and l-galactose. Crystal structures of rBC2L-CN2 were solved in complex with two blood group antigens: H-type 1 and H-type 3 (Globo H) by X-ray crystallography. They provide new structural information on the binding site, of importance for the structural-based design of glycodrugs as new antimicrobials with antiadhesive properties.
Asunto(s)
Antígenos de Grupos Sanguíneos/química , Burkholderia cenocepacia/química , Lectinas/química , Oligosacáridos/química , Antígenos de Diferenciación/química , Antígenos de Carbohidratos Asociados a Tumores/química , Sitios de Unión , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Cristalografía por Rayos X , Epítopos/química , Fucosa/química , Expresión Génica , Humanos , Manósidos/química , Modelos Moleculares , Unión Proteica , Proteínas Recombinantes/genética , Factor de Necrosis Tumoral alfa/químicaRESUMEN
Type I interferon (IFN) induced by virus infections during pregnancy can cause placental damage, but the mechanisms and identities of IFN-stimulated genes that are involved in this damage remain under investigation. The IFN-induced transmembrane proteins (IFITMs) inhibit virus infections by preventing virus membrane fusion with cells and by inhibiting fusion of infected cells (syncytialization). Fusion of placental trophoblasts via expression of endogenous retroviral fusogens known as syncytins forms the syncytiotrophoblast, a multinucleated cell structure essential for fetal development. We found here that IFN blocks fusion of BeWo human placental trophoblasts. Stably expressed IFITM1, -2, and -3 also blocked fusion of these trophoblasts while making them more resistant to virus infections. Conversely, stable IFITM knockdowns in BeWo trophoblasts increased their spontaneous fusion and allowed fusion in the presence of IFN while also making the cells more susceptible to virus infection. We additionally found that exogenous expression of IFITMs in HEK293T cells blocked fusion with cells expressing syncytin-1 or syncytin-2, confirming the ability of IFITMs to block individual syncytin-mediated fusion. Overall, our data indicate that IFITMs inhibit trophoblast fusion and suggest that there may be a critical balance between these antifusogenic effects and the beneficial antiviral effects of IFITMs in virus infections during pregnancy.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Productos del Gen env/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Gestacionales/metabolismo , Proteínas de Unión al ARN/metabolismo , Antígenos de Diferenciación/química , Antígenos de Diferenciación/genética , Antivirales/farmacología , Fusión Celular , Femenino , Células HEK293 , Humanos , Interferón Tipo I/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Placenta/citología , Embarazo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Trofoblastos/citología , Trofoblastos/metabolismo , Internalización del Virus/efectos de los fármacos , Virus Zika/fisiologíaRESUMEN
Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies. Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; CFSE: carboxyfluorescein succinimidyl ester; Fab: fragment antigen binding; Fc: fragment crystallizable; FcγR: Fcγ receptor; Ig: immunoglobulin; IND: investigational new drug; MDMâ: monocyte-derived macrophage; NOD: non-obese diabetic; scFv: single chain fragment variable; SCID: severe combined immunodeficiency; SIRP: signal-regulatory protein.
Asunto(s)
Anticuerpos Monoclonales , Especificidad de Anticuerpos , Antígenos de Diferenciación , Receptores Inmunológicos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación/química , Antígenos de Diferenciación/inmunología , Antígeno CD47/inmunología , Pollos , Cristalografía por Rayos X , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Femenino , Humanos , Inmunoterapia , Masculino , Neoplasias/inmunología , Neoplasias/terapia , Dominios Proteicos , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/química , Receptores Inmunológicos/inmunologíaRESUMEN
CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.