RESUMO
Two types of engineered T cells have been successfully used to treat patients with cancer, one with an antigen recognition domain derived from antibodies [chimeric antigen receptors (CARs)] and the other derived from T cell receptors (TCRs). CARs use high-affinity antigen-binding domains and costimulatory domains to induce T cell activation but can only react against target cells with relatively high amounts of antigen. TCRs have a much lower affinity for their antigens but can react against target cells displaying only a few antigen molecules. Here, we describe a new type of receptor, called a Co-STAR (for costimulatory synthetic TCR and antigen receptor), that combines aspects of both CARs and TCRs. In Co-STARs, the antigen-recognizing components of TCRs are replaced by high-affinity antibody fragments, and costimulation is provided by two modules that drive NF-κB signaling (MyD88 and CD40). Using a TCR-mimic antibody fragment that targets a recurrent p53 neoantigen presented in a common human leukocyte antigen (HLA) allele, we demonstrate that T cells equipped with Co-STARs can kill cancer cells bearing low densities of antigen better than T cells engineered with conventional CARs and patient-derived TCRs in vitro. In mouse models, we show that Co-STARs mediate more robust T cell expansion and more durable tumor regressions than TCRs similarly modified with MyD88 and CD40 costimulation. Our data suggest that Co-STARs may have utility for other peptide-HLA antigens in cancer and other targets where antigen density may limit the efficacy of engineered T cells.
Assuntos
Neoplasias , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos , Humanos , Animais , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Transdução de SinaisRESUMO
BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.
Assuntos
Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Humanos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Animais , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidoresRESUMO
The B-cell lymphoma 2 (BCL2) family members, BCL2-associated protein X (BAX) and BCL2 homologous antagonist killer (BAK), are required for programmed cell death via the mitochondrial pathway. When cells are stressed, damaged or redundant, the balance of power between the BCL2 family of proteins shifts towards BAX and BAK, allowing their transition from an inactive, monomeric state to a membrane-active oligomeric form that releases cytochrome c from the mitochondrial intermembrane space. That oligomeric state has an essential intermediate, a symmetric homodimer of BAX or BAK. Here we describe crystal structures of dimers of the core domain of BAX, comprising its helices α2-α5. These structures provide an atomic resolution description of the interactions that drive BAX homo-dimerisation and insights into potential interaction between core domain dimers and membrane lipids. The previously identified BAK lipid-interacting sites are not conserved with BAX and are likely to determine the differences between them in their interactions with lipids. We also describe structures of heterodimers of BAK/BAX core domains, yielding further insight into the differences in lipid binding between BAX and BAK.
RESUMO
Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRASG12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.
Assuntos
Anticorpos , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Proto-Oncogênicas p21(ras)/genética , Reconhecimento Psicológico , Interações Hidrofóbicas e Hidrofílicas , Antígenos HLA-A/genéticaRESUMO
Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.
Assuntos
Apoptose , Proteína Killer-Antagonista Homóloga a bcl-2 , Anticorpos , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Membranas Mitocondriais/metabolismo , Estrutura Secundária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genéticaRESUMO
Chimeric antigen receptor (CAR) T cells have emerged as a promising class of therapeutic agents, generating remarkable responses in the clinic for a subset of human cancers. One major challenge precluding the wider implementation of CAR therapy is the paucity of tumor-specific antigens. Here, we describe the development of a CAR targeting the tumor-specific isocitrate dehydrogenase 2 (IDH2) with R140Q mutation presented on the cell surface in complex with a common human leukocyte antigen allele, HLA-B*07:02. Engineering of the hinge domain of the CAR, as well as crystal structure-guided optimization of the IDH2R140Q-HLA-B*07:02-targeting moiety, enhances the sensitivity and specificity of CARs to enable targeting of this HLA-restricted neoantigen. This approach thus holds promise for the development and optimization of immunotherapies specific to other cancer driver mutations that are difficult to target by conventional means.
Assuntos
Antígeno HLA-B7/química , Isocitrato Desidrogenase/metabolismo , Engenharia de Proteínas/métodos , Receptores de Antígenos Quiméricos/química , Animais , Antígenos de Neoplasias/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Epitopos , Antígeno HLA-B7/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/imunologia , Mutação , Biblioteca de Peptídeos , Conformação Proteica , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/fisiologiaRESUMO
A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2-α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6-α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far.
Assuntos
Detergentes/química , Multimerização Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/química , Sequência de Aminoácidos , Animais , Lipossomos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Estrutura Secundária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias , Biomarcadores Tumorais/antagonistas & inibidores , Proteínas Mutantes/antagonistas & inibidores , Proteínas ras/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Linhagem Celular , Reações Cruzadas , Antígenos HLA/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Proteínas Mutantes/química , Proteínas Mutantes/imunologia , Mutação , Fragmentos de Peptídeos , Ligação Proteica/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/imunologiaRESUMO
TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Antígeno HLA-A2/imunologia , Neoplasias/terapia , Proteína Supressora de Tumor p53/imunologia , Alelos , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/uso terapêutico , Arginina/genética , Células COS , Chlorocebus aethiops , Feminino , Células HEK293 , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Histidina/genética , Humanos , Imunização Passiva , Células Jurkat , Ativação Linfocitária , Camundongos Endogâmicos NOD , Mutação , Linfócitos T/imunologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mutations in CTNNB1, the gene encoding ß-catenin, are common in colon and liver cancers, the most frequent mutation affecting Ser-45 in ß-catenin. Peptides derived from WT ß-catenin have previously been shown to be presented on the cell surface as part of major histocompatibility complex (MHC) class I, suggesting an opportunity for targeting this common driver gene mutation with antibody-based therapies. Here, crystal structures of both the WT and S45F mutant peptide bound to HLA-A*03:01 at 2.20 and 2.45 Å resolutions, respectively, confirmed the accessibility of the phenylalanine residue for antibody recognition. Phage display was then used to identify single-chain variable fragment clones that selectively bind the S45F mutant peptide presented in HLA-A*03:01 and have minimal WT or other off-target binding. Following the initial characterization of five clones, we selected a single clone, E10, for further investigation. We developed a computational model of the binding of E10 to the mutant peptide-bound HLA-A3, incorporating data from affinity maturation as initial validation. In the future, our model may be used to design clones with maintained specificity and higher affinity. Such derivatives could be adapted into either cell-based (CAR-T) or protein-based (bispecific T-cell engagers) therapies to target cancer cells harboring the S45F mutation in CTNNB1.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Fragmentos de Imunoglobulinas/química , Engenharia de Proteínas , beta Catenina/genética , Linhagem Celular , Antígenos de Histocompatibilidade Classe I/química , Humanos , Modelos Moleculares , Mutação , beta Catenina/químicaRESUMO
We present a patient with Phelan-McDermid syndrome, a rare neurodevelopmental disorder caused by a 22q13 deletion, with the previously undescribed finding of progressive dilation of the great arteries. While congenital heart defects have been identified in patients previously, dilation of the great arteries has not been described to our knowledge.
RESUMO
Phosphatidylinositol 3-kinases (PI3Ks) are important therapeutic targets for the treatment of cancer, thrombosis, and inflammatory and immune diseases. The four highly homologous Class I isoforms, PI3K, PI3K, PI3K and PI3K have unique, non-redundant physiological roles and as such, isoform selectivity has been a key consideration driving inhibitor design and development. In this review, we discuss the structural biology of PI3Ks and how our growing knowledge of structure has influenced the medicinal chemistry of PI3K inhibitors. We present an analysis of the available structure-selectivity-activity relationship data to highlight key insights into how the various regions of the PI3K binding site influence isoform selectivity. The picture that emerges is one that is far from simple and emphasizes the complex nature of protein-inhibitor binding, involving protein flexibility, energetics, water networks and interactions with non-conserved residues.
Assuntos
Indazóis/química , Indazóis/farmacologia , Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Química Farmacêutica , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Modelos Moleculares , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).
Assuntos
Cristalografia por Raios X , Modelos Moleculares , Proteínas/química , Cristalografia por Raios X/métodos , Fosfatidilinositol 3-Quinases/química , Conformação Proteica , Pirofosfatases/químicaRESUMO
Acquired diaphragmatic hernia is a rare complication of pediatric intervention or surgery. In this study, we report an infant with iatrogenic diaphragmatic hernia following neonatal complex congenital cardiac surgery, and then we review the associated literature.
Assuntos
Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Cardiopatias Congênitas/cirurgia , Hérnia Diafragmática/diagnóstico , Síndrome do Desconforto Respiratório do Recém-Nascido/etiologia , Feminino , Seguimentos , Hérnia Diafragmática/complicações , Hérnia Diafragmática/cirurgia , Herniorrafia/métodos , Humanos , Doença Iatrogênica , Recém-Nascido , Radiografia Torácica , Reoperação , Síndrome do Desconforto Respiratório do Recém-Nascido/diagnósticoRESUMO
Glycosaminoglycans (GAGs) play critical roles in diverse processes ranging from viral infection to neuroregeneration. Their regiospecific sulfation patterns, which are generated by sulfotransferases, are key structural determinants that underlie their biological activity. Small-molecule modulators of these sulfotransferases could serve as powerful tools for understanding the physiological functions of GAGs, as well as potential therapeutic leads for human diseases. Here, we report the development of the first cell-permeable, small-molecule inhibitor selective for GAG sulfotransferases, which was obtained using a high-throughput screen targeted against Chst15, the sulfotransferase responsible for biosynthesis of chondroitin sulfate-E (CS-E). We demonstrate that the molecule specifically inhibits GAG sulfotransferases in vitro, decreases CS-E and overall sulfation levels on cell-surface and secreted chondroitin sulfate proteoglycans (CSPGs), and reverses CSPG-mediated inhibition of axonal growth. These studies pave the way toward a new set of pharmacological tools for interrogating GAG sulfation-dependent processes and may represent a novel therapeutic approach for neuroregeneration.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Glicoproteínas de Membrana/metabolismo , Sulfotransferases/metabolismo , Animais , Clonagem Molecular , Ensaios de Triagem em Larga Escala , Glicoproteínas de Membrana/genética , Camundongos , Microssomos/metabolismo , Células NIH 3T3 , Ratos , Sulfotransferases/genéticaRESUMO
Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that activate signaling cascades controlling cell survival, proliferation, protein synthesis, and vesicle trafficking. PI3Ks have dual kinase specificity: a lipid kinase activity that phosphorylates the 3'-hydroxyl of phosphoinositides and a protein-kinase activity that includes autophosphorylation. Despite the wealth of biochemical and structural information on PI3Kα, little is known about the identity and roles of individual active-site residues in catalysis. To close this gap, we explored the roles of residues of the catalytic domain and the regulatory subunit of human PI3Kα in lipid and protein phosphorylation. Using site-directed mutagenesis, kinetic assays, and quantitative mass spectrometry, we precisely mapped key residues involved in substrate recognition and catalysis by PI3Kα. Our results revealed that Lys-776, located in the P-loop of PI3Kα, is essential for the recognition of lipid and ATP substrates and also plays an important role in PI3Kα autophosphorylation. Replacement of the residues His-936 and His-917 in the activation and catalytic loops, respectively, with alanine dramatically changed PI3Kα kinetics. Although H936A inactivated the lipid kinase activity without affecting autophosphorylation, H917A abolished both the lipid and protein kinase activities of PI3Kα. On the basis of these kinetic and structural analyses, we propose possible mechanistic roles of these critical residues in PI3Kα catalysis.
Assuntos
Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Processamento de Proteína Pós-Traducional , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Sítios de Ligação , Biocatálise , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase , Histidina/química , Histidina/metabolismo , Humanos , Cinética , Lisina/química , Lisina/metabolismo , Conformação Molecular , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 4,5-Difosfato/química , Fosforilação , Mutação Puntual , Conformação Proteica , Multimerização ProteicaRESUMO
PIK3CA, the gene that encodes the catalytic subunit of phosphatidylinositol 3-kinase α (PI3Kα), is frequently mutated in breast and other types of cancer. A specific inhibitor that targets the mutant forms of PI3Kα could maximize treatment efficiency while minimizing side-effects. Herein we describe the identification of novel binding pockets that may provide an opportunity for the design of mutant selective inhibitors. Using a fragment-based approach, we screened a library of 352 fragments (MW<300Da) for binding to PI3Kα by X-ray crystallography. Five novel binding pockets were identified, each providing potential opportunities for inhibitor design. Of particular interest was a binding pocket near Glu542, which is located in one of the two most frequently mutated domains.
Assuntos
Sítio Alostérico , Desenho de Fármacos , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/genética , Classe I de Fosfatidilinositol 3-Quinases , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Proteínas Mutantes/genética , Mutação , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Molecular imaging can report on the status of the tumor immune microenvironment and guide immunotherapeutic strategies to enhance the efficacy of immune modulation therapies. Imaging agents that can rapidly report on targets of immunomodulatory therapies are few. The programmed death ligand 1 (PD-L1) is an immune checkpoint protein over-expressed in several cancers and contributes to tumor immune suppression. Tumor PD-L1 expression is indicative of tumor response to PD-1 and PD-L1 targeted therapies. Herein, we report a highly specific peptide-based positron emission tomography (PET) imaging agent for PD-L1. We assessed the binding modes of the peptide WL12 to PD-L1 by docking studies, developed a copper-64 labeled WL12 ([64Cu]WL12), and performed its evaluation in vitro, and in vivo by PET imaging, biodistribution and blocking studies. Our results show that [64Cu]WL12 can be used to detect tumor PD-L1 expression specifically and soon after injection of the radiotracer, to fit within the standard clinical workflow of imaging within 60 min of administration.
Assuntos
Antígeno B7-H1/análise , Neoplasias/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Animais , Antígeno B7-H1/metabolismo , Células CHO , Radioisótopos de Cobre/administração & dosagem , Radioisótopos de Cobre/farmacocinética , Cricetulus , Usos Diagnósticos de Compostos Químicos , Feminino , Humanos , Camundongos SCID , Simulação de Acoplamento Molecular , Neoplasias/diagnóstico por imagem , Peptídeos/administração & dosagem , Receptor de Morte Celular Programada 1/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of PI3Kδ inhibitors derived from the pan-PI3K inhibitor ZSTK474 was prepared that target a non-conserved region of the catalytic site. Dependent upon the substituents present, these analogues show different levels of isoform selectivity and sensitivity to the mutation N836D in PI3Kδ. As a marker of 'on-target' activity and permeability, a selection of the most potent PI3Kδ inhibitors were shown to inhibit pAkt production in the Nawalma Burkitt lymphoma cell line.