RESUMO
The unique class of heavy chain-only antibodies, present in Camelidae, can be shrunk to just the variable region of the heavy chain to yield VHHs, also called nanobodies. About one-tenth the size of their full-size counterparts, nanobodies can serve in applications similar to those for conventional antibodies, but they come with a number of signature advantages that find increasing application in biology. They not only function as crystallization chaperones but also can be expressed inside cells as such, or fused to other proteins to perturb the function of their targets, for example, by enforcing their localization or degradation. Their small size also affords advantages when applied in vivo, for example, in imaging applications. Here we review such applications, with particular emphasis on those areas where conventional antibodies would face a more challenging environment.
Assuntos
Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Animais , Formação de Anticorpos , Técnicas de Visualização da Superfície Celular , Engenharia Genética , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Molecular biologists and chemists alike have long sought to modify proteins with substituents that cannot be installed by standard or even advanced genetic approaches. We here describe the use of transpeptidases to achieve these goals. Living systems encode a variety of transpeptidases and peptide ligases that allow for the enzyme-catalyzed formation of peptide bonds, and protein engineers have used directed evolution to enhance these enzymes for biological applications. We focus primarily on the transpeptidase sortase A, which has become popular over the past few years for its ability to perform a remarkably wide variety of protein modifications, both in vitro and in living cells.
Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Peptídeos/genética , Peptidil Transferases/genética , Sequência de Aminoácidos/genética , Aminoaciltransferases/química , Proteínas de Bactérias/química , Catálise , Cisteína Endopeptidases/química , Humanos , Peptídeos/química , Peptidil Transferases/química , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Extracellular matrix (ECM) deposition is a hallmark of many diseases, including cancer and fibroses. To exploit the ECM as an imaging and therapeutic target, we developed alpaca-derived libraries of "nanobodies" against disease-associated ECM proteins. We describe here one such nanobody, NJB2, specific for an alternatively spliced domain of fibronectin expressed in disease ECM and neovasculature. We showed by noninvasive in vivo immuno-PET/CT imaging that NJB2 detects primary tumors and metastatic sites with excellent specificity in multiple models of breast cancer, including human and mouse triple-negative breast cancer, and in melanoma. We also imaged mice with pancreatic ductal adenocarcinoma (PDAC) in which NJB2 was able to detect not only PDAC tumors but also early pancreatic lesions called pancreatic intraepithelial neoplasias, which are challenging to detect by any current imaging modalities, with excellent clarity and signal-to-noise ratios that outperformed conventional 2-fluorodeoxyglucose PET/CT imaging. NJB2 also detected pulmonary fibrosis in a bleomycin-induced fibrosis model. We propose NJB2 and similar anti-ECM nanobodies as powerful tools for noninvasive detection of tumors, metastatic lesions, and fibroses. Furthermore, the selective recognition of disease tissues makes NJB2 a promising candidate for nanobody-based therapeutic applications.
Assuntos
Carcinogênese/genética , Carcinoma Ductal Pancreático/diagnóstico por imagem , Matriz Extracelular/efeitos dos fármacos , Neoplasias Pancreáticas/diagnóstico por imagem , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibrose/patologia , Humanos , Masculino , Camundongos , Neoplasias Pancreáticas/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/farmacologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Neoplasias PancreáticasRESUMO
Ipilimumab, a monoclonal antibody that recognizes cytotoxic T lymphocyte antigen (CTLA)-4, was the first approved "checkpoint"-blocking anticancer therapy. In mouse tumor models, the response to antibodies against CTLA-4 depends entirely on expression of the Fcγ receptor (FcγR), which may facilitate antibody-dependent cellular phagocytosis, but the contribution of simple CTLA-4 blockade remains unknown. To understand the role of CTLA-4 blockade in the complete absence of Fc-dependent functions, we developed H11, a high-affinity alpaca heavy chain-only antibody fragment (VHH) against CTLA-4. The VHH H11 lacks an Fc portion, binds monovalently to CTLA-4, and inhibits interactions between CTLA-4 and its ligand by occluding the ligand-binding motif on CTLA-4 as shown crystallographically. We used H11 to visualize CTLA-4 expression in vivo using whole-animal immuno-PET, finding that surface-accessible CTLA-4 is largely confined to the tumor microenvironment. Despite this, H11-mediated CTLA-4 blockade has minimal effects on antitumor responses. Installation of the murine IgG2a constant region on H11 dramatically enhances its antitumor response. Coadministration of the monovalent H11 VHH blocks the efficacy of a full-sized therapeutic antibody. We were thus able to demonstrate that CTLA-4-binding antibodies require an Fc domain for antitumor effect.
Assuntos
Antígeno CTLA-4/imunologia , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos de Imunoglobulinas/administração & dosagem , Neoplasias/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígeno CTLA-4/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Domínios ProteicosRESUMO
CD47 is an antiphagocytic ligand broadly expressed on normal and malignant tissues that delivers an inhibitory signal through the receptor signal regulatory protein alpha (SIRPα). Inhibitors of the CD47-SIRPα interaction improve antitumor antibody responses by enhancing antibody-dependent cellular phagocytosis (ADCP) in xenograft models. Endogenous expression of CD47 on a variety of cell types, including erythrocytes, creates a formidable antigen sink that may limit the efficacy of CD47-targeting therapies. We generated a nanobody, A4, that blocks the CD47-SIRPα interaction. A4 synergizes with anti-PD-L1, but not anti-CTLA4, therapy in the syngeneic B16F10 melanoma model. Neither increased dosing nor half-life extension by fusion of A4 to IgG2a Fc (A4Fc) overcame the issue of an antigen sink or, in the case of A4Fc, systemic toxicity. Generation of a B16F10 cell line that secretes the A4 nanobody showed that an enhanced response to several immune therapies requires near-complete blockade of CD47 in the tumor microenvironment. Thus, strategies to localize CD47 blockade to tumors may be particularly valuable for immune therapy.
Assuntos
Antígeno CD47/antagonistas & inibidores , Imunoterapia/métodos , Melanoma Experimental/terapia , Anticorpos de Domínio Único/uso terapêutico , Anemia/induzido quimicamente , Animais , Antígeno CD47/imunologia , Avaliação Pré-Clínica de Medicamentos , Camundongos Endogâmicos C57BL , Fagocitose , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/farmacologia , Microambiente TumoralRESUMO
Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response.
Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Orthomyxoviridae/fisiologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Especificidade de Anticorpos/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Morte Celular , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Pulmão/citologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/virologia , Linfonodos/citologia , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Testes de Neutralização , Técnicas de Transferência Nuclear , Orthomyxoviridae/patogenicidade , Receptores de Antígenos de Linfócitos B/metabolismo , Replicação ViralRESUMO
Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47-SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.
Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Antígeno CD47/efeitos dos fármacos , Antígeno CD47/imunologia , Vacinas Anticâncer/administração & dosagem , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/imunologia , Vacinas Anticâncer/imunologia , Feminino , Imunização/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Neoplasias Experimentais/patologia , Resultado do TratamentoRESUMO
mAbs specific for surface proteins on APCs can serve as Ag-delivery vehicles that enhance immunogenicity. The practical use of such constructs is limited by the challenge of expressing and modifying full-sized mAbs. We generated single-domain Ab fragments (VHHs) specific for class II MHC (MHCII), CD11b, and CD36. VHH sequences were modified by inclusion of a C-terminal sortase motif to allow site-specific conjugation with various Ag payloads. We tested T cell activation using VHHs that target distinct APC populations; anti-MHCII adducts elicited strong activation of CD4+ T cells, whereas anti-CD11b showed CD8+ T cell activation superior to targeting via MHCII and CD36. Differences in Ag presentation among constructs were unrelated to dendritic cell subtype or routing to acidic compartments. When coupled to antigenic payloads, anti-MHCII VHH primed Ab responses against GFP, ubiquitin, an OVA peptide, and the α-helix of influenza hemagglutinin's stem; the last afforded protection against influenza infection. The versatility of the VHH scaffold and sortase-mediated covalent attachment of Ags suggests their broader application to generate desirable immune responses.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Células Dendríticas/fisiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Anticorpos de Domínio Único/metabolismo , Animais , Apresentação de Antígeno , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Camelídeos Americanos , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Influenza Humana/prevenção & controle , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/prevenção & controle , Anticorpos de Domínio Único/imunologiaRESUMO
Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7-kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.
Assuntos
Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Toxoplasma/enzimologia , Regulação Alostérica , Animais , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/metabolismo , Anticorpos Antiprotozoários/farmacologia , Biocatálise/efeitos dos fármacos , Western Blotting , Cálcio/metabolismo , Camelídeos Americanos , Células Cultivadas , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/farmacologia , Toxoplasma/genéticaRESUMO
Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.
RESUMO
Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Macrófagos , Mycobacterium tuberculosis , Fagossomos , Anticorpos de Domínio Único , Humanos , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Anticorpos de Domínio Único/metabolismoRESUMO
ABSTRACT: Quality measurement across healthcare is undertaken with a goal of improving care and outcomes for patients; however, the relationship between quality measurement and patient outcomes remains largely untested, particularly in inpatient behavioral health. Using a retrospective quantitative design, we assessed 142 behavioral health organizations' quality data submitted to the Hospital-Based Inpatient Psychiatric Services and Inpatient Psychiatric Facility Quality Reporting programs from 2017 to 2018 and tested relationships between compliance on 16 quality measures and symptom improvement on patient self-report outcomes (SROs) at the facility level. Performance on many quality measures was negatively skewed (at least four have almost no room for improvement on average), and there was high interrelatedness between most quality measures. Nine of the assessed measures correlated with patient SROs but not in clear groupings. Findings indicate that an underlying organizational construct may be driving compliance rates on quality measures, but the measures are not linked to treatment outcomes as expected. We encourage an expansion of the current framework of behavioral health quality measurement beyond process and organization and suggest the addition of patient outcomes such as SROs as quality measures to directly assess patient improvement.
Assuntos
Atenção à Saúde , Pacientes Internados , Humanos , Estudos RetrospectivosRESUMO
Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Anticorpos de Domínio Único/química , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação , Camelídeos Americanos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos de Domínio Único/metabolismoRESUMO
Chimeric antigen receptor (CAR) T-cell therapy is effective in the treatment of cancers of hematopoietic origin. In the immunosuppressive solid tumor environment, CAR T cells encounter obstacles that compromise their efficacy. We developed a strategy to address these barriers by having CAR T cells secrete single-domain antibody fragments [variable heavy domain of heavy chain antibodies (VHH) or nanobodies] that can modify the intratumoral immune landscape and thus support CAR T-cell function in immunocompetent animals. VHHs are small in size and able to avoid domain swapping when multiple nanobodies are expressed simultaneously-features that can endow CAR T cells with desirable properties. The secretion of an anti-CD47 VHH by CAR T cells improves engagement of the innate immune system, enables epitope spreading, and can enhance the antitumor response. CAR T cells that secrete anti-PD-L1 or anti-CTLA-4 nanobodies show improved persistence and demonstrate the versatility of this approach. Furthermore, local delivery of secreted anti-CD47 VHH-Fc fusions by CAR T cells at the tumor site limits their systemic toxicity. CAR T cells can be further engineered to simultaneously secrete multiple modalities, allowing for even greater tailoring of the antitumor immune response.
Assuntos
Antígeno CD47/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Melanoma Experimental/imunologia , Receptores de Antígenos Quiméricos/imunologia , Proteínas Recombinantes/genética , Anticorpos de Domínio Único/imunologia , Animais , Linhagem Celular Tumoral , Granzimas/antagonistas & inibidores , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores de Antígenos Quiméricos/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Microambiente TumoralRESUMO
Cancer-specific mutations can lead to peptides of unique sequence presented on MHC class I to CD8 T cells. These neoantigens can be potent tumour-rejection antigens, appear to be the driving force behind responsiveness to anti-CTLA-4 and anti-PD1/L1-based therapies and have been used to develop personalized vaccines. The platform for delivering neoantigen-based vaccines has varied, and further optimization of both platform and adjuvant will be necessary to achieve scalable vaccine products that are therapeutically effective at a reasonable cost. Here, we developed a platform for testing potential CD8 T cell tumour vaccine candidates. We used a high-affinity alpaca-derived VHH against MHC class II to deliver peptides to professional antigen-presenting cells. We show in vitro and in vivo that peptides derived from the model antigen ovalbumin are better able to activate naive ovalbumin-specific CD8 T cells when conjugated to an MHC class II-specific VHH when compared with an irrelevant control VHH. We then used the VHH-peptide platform to evaluate a panel of candidate neoantigens in vivo in a mouse model of pancreatic cancer. None of the candidate neoantigens tested led to protection from tumour challenge; however, we were able to show vaccine-induced CD8 T cell responses to a melanoma self-antigen that was augmented by combination therapy with the synthetic cytokine mimetic Neo2/15.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-2/administração & dosagem , Melanoma/tratamento farmacológico , Peptídeos/administração & dosagem , Anticorpos de Domínio Único/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Camelídeos Americanos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Interleucina-2/imunologia , Melanoma/imunologia , Camundongos , Peptídeos/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Vacinas de Subunidades Antigênicas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human cytomegalovirus has hijacked and evolved a human G-protein-coupled receptor into US28, which functions as a promiscuous chemokine 'sink' to facilitate evasion of host immune responses. To probe the molecular basis of US28's unique ligand cross-reactivity, we deep-sequenced CX3CL1 chemokine libraries selected on 'molecular casts' of the US28 active-state and find that US28 can engage thousands of distinct chemokine sequences, many of which elicit diverse signaling outcomes. The structure of a G-protein-biased CX3CL1-variant in complex with US28 revealed an entirely unique chemokine amino terminal peptide conformation and remodeled constellation of receptor-ligand interactions. Receptor signaling, however, is remarkably robust to mutational disruption of these interactions. Thus, US28 accommodates and functionally discriminates amongst highly degenerate chemokine sequences by sensing the steric bulk of the ligands, which distort both receptor extracellular loops and the walls of the ligand binding pocket to varying degrees, rather than requiring sequence-specific bonding chemistries for recognition and signaling.
Assuntos
Quimiocina CX3CL1/química , Receptores de Quimiocinas/química , Receptores Acoplados a Proteínas G/química , Transdução de Sinais , Proteínas Virais/química , Animais , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/farmacologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/agonistas , Proteínas Virais/metabolismoRESUMO
Cytokine-based therapies for cancer have not achieved widespread clinical success because of inherent toxicities. Treatment for pancreatic cancer is limited by the dense stroma that surrounds tumors and by an immunosuppressive tumor microenvironment. To overcome these barriers, we developed constructs of single-domain antibodies (VHHs) against PD-L1 fused with IL-2 and IFNγ. Targeting cytokine delivery in this manner reduced pancreatic tumor burden by 50%, whereas cytokines fused to an irrelevant VHH, or blockade of PD-L1 alone, showed little effect. Targeted delivery of IL-2 increased the number of intratumoral CD8+ T cells, whereas IFNγ reduced the number of CD11b+ cells and skewed intratumoral macrophages toward the display of M1-like characteristics. Imaging of fluorescent VHH-IFNγ constructs, as well as transcriptional profiling, demonstrated targeting of IFNγ to the tumor microenvironment. Many tumors and tumor-infiltrating myeloid cells express PD-L1, rendering them potentially susceptible to this form of targeted immunotherapy. Cancer Immunol Res; 6(4); 389-401. ©2018 AACR.
Assuntos
Antígeno B7-H1/metabolismo , Citocinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Anticorpos de Domínio Único/farmacologia , Microambiente Tumoral , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Citocinas/genética , Modelos Animais de Doenças , Humanos , Melanoma Experimental , Camundongos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/terapia , Anticorpos de Domínio Único/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Sortase A, a calcium-dependent transpeptidase derived from Staphylococcus aureus, is used in a broad range of applications, such as the conjugation of fluorescent dyes and other moieties to proteins or to the surface of eukaryotic cells. In vivo and cell-based applications of sortase have been somewhat limited by the large range of calcium concentrations, as well as by the often transient nature of protein-protein interactions in living systems. In order to use sortase A for cell labeling applications, we generated a new sortase A variant by combining multiple mutations to yield an enzyme that was both calcium-independent and highly active. This variant has enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions.
Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Cálcio/metabolismo , Cisteína Endopeptidases/genética , Peptidil Transferases/genética , Coloração e Rotulagem/métodos , Staphylococcus aureus/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Cisteína Endopeptidases/metabolismo , Mutação , Peptidil Transferases/metabolismo , Staphylococcus aureus/enzimologiaRESUMO
Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non-small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno-positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8+ T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Antígeno CTLA-4/antagonistas & inibidores , Neoplasias Mamárias Experimentais/terapia , Animais , Antígeno CTLA-4/fisiologia , Feminino , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/uso terapêutico , Imunoterapia/métodos , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Transplante de Neoplasias , Tomografia por Emissão de Pósitrons/métodos , Resultado do TratamentoRESUMO
Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or ß-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state.Current approaches to visualise brown adipose tissue (BAT) rely primarily on markers that reflect its metabolic activity. Here, the authors show that PD-L1 is expressed on brown adipocytes, does not change upon BAT activation, and that BAT volume in mice can be measured by PET-CT with a radiolabeled anti-PD-L1 antibody.