RESUMO
The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent phagocytic activity. Although SIRP-1 and SIRP-2 bind SIRPγ with varying affinity, they show no adverse effects on T cell proliferation. Finally, both Abs also enhance phagocytosis when combined with tumor-opsonizing Abs, including a highly differentiated anti-CD47 Ab, AO-176, currently being evaluated in phase 1 clinical trials, NCT03834948 and NCT04445701 SIRP-1 and SIRP-2 are novel, differentiated SIRP Abs that induce in vitro single-agent and combination phagocytosis and show no adverse effects on T cell functionality. These data support their future development, both as single agents and in combination with other anticancer drugs.
Assuntos
Apresentação de Antígeno , Antígenos de Diferenciação/imunologia , Antineoplásicos Imunológicos/imunologia , Macrófagos/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Fagocitose , Receptores Imunológicos/imunologia , Linfócitos T/imunologia , Humanos , Células Jurkat , Células THP-1 , Células U937RESUMO
We investigated whether blockade of the CD47 signaling pathway could reduce ischemia-reperfusion injury (IRI) of renal allografts donated after cardiac death (DCD) in a porcine animal model of transplantation. Renal allografts were subjected to 30 minutes of warm ischemia, 3.5 hours of cold ischemia, and then perfused with a humanized anti-CD47 monoclonal antibody (CD47mAb) in the treatment group or HTK solution in the control group (n = 4/group). The animals were euthanized five days after transplantation. At the time of reperfusion, indocyanine green-based in vivo imaging showed that CD47mAb-treated organs had greater and more uniform reperfusion. On post-transplant days 3-5, the treatment group had lower values compared to the control for creatinine and blood urea nitrogen. Histological examination of allograft tissues showed a significant decrease of acute tubular injury in the CD47mAb-treated group compared to control. Compared to the control group, CD47mAb treatment significantly decreased genes expression related to oxidative stress (sod-1, gpx-1, and txn), the inflammatory response (il-2, il-6, inf-g, and tgf-b), as well as reduced protein levels of BAX, Caspase-3, MMP2, and MMP9. These data demonstrate that CD47mAb blockade decreases IRI and subsequent tissue injury in DCD renal allografts in a large animal transplant model.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/antagonistas & inibidores , Morte , Rejeição de Enxerto/prevenção & controle , Falência Renal Crônica/cirurgia , Transplante de Rim/métodos , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose , Antígeno CD47/imunologia , Modelos Animais de Doenças , Feminino , Taxa de Filtração Glomerular , Sobrevivência de Enxerto , Inflamação/prevenção & controle , Testes de Função Renal , Estresse Oxidativo , Transdução de Sinais , SuínosRESUMO
Orthotopic liver transplantation (OLT) remains the standard treatment option for nonresponsive liver failure. Because ischemia/reperfusion injury (IRI) is an important impediment to the success of OLT, new therapeutic strategies are needed to reduce IRI. We investigated whether blocking the CD47/thrombospondin-1 inhibitory action on nitric oxide signaling with a monoclonal antibody specific to CD47 (CD47mAb400) would reduce IRI in liver grafts. Syngeneic OLT was performed with Lewis rats. Control immunoglobulin G or CD47mAb400 was administered to the donor organ at procurement or to both the organ and the recipient at the time of transplant. Serum transaminases, histological changes of the liver, and animal survival were assessed. Oxidative stress, inflammatory responses, and hepatocellular damage were also quantified. A significant survival benefit was not achieved when CD47mAb400 was administered to the donor alone. However, CD47mAb400 administration to both the donor and the recipient increased animal survival afterward. The CD47mAb400-treated group showed lower serum transaminases, bilirubin, oxidative stress, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, caspase-3 activity, and proinflammatory cytokine expression of tumor necrosis factor α, interleukin-1ß, and interleukin-6. Thus, CD47 blockade with CD47mAb400 administered both to the donor and the recipient reduced liver graft IRI in a rat liver transplantation model. This may translate to decreased liver dysfunction and increased survival of liver transplant recipients.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/metabolismo , Isquemia Fria/efeitos adversos , Transplante de Fígado/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/cirurgia , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Antígeno CD47/imunologia , Citoproteção , Modelos Animais de Doenças , Mediadores da Inflamação/sangue , Fígado/irrigação sanguínea , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Masculino , Necrose , Estresse Oxidativo/efeitos dos fármacos , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Antagonists of the cannabinoid receptor 1 (CB1) impart anti-inflammatory activity even though, paradoxically, CB2 receptors are more predominant on cells of the immune system. We attempted to understand the mechanism of this activity by using an acute model of lipopolysaccharide-induced inflammation/stress in both rat and mouse, with selective antagonists to CB1 receptors. We demonstrate that the ability of a CB1 antagonist to inhibit release of proinflammatory cytokines is not dependent on either adrenal-derived catecholamines or corticosteroids or input from the pituitary or thymus glands but does involve the spleen. Furthermore, we show that the anti-inflammatory activity is retained without communication from the central nervous system following ganglionic blockade, suggesting a peripheral site of action. Finally, we show that the anti-inflammatory activity can be inhibited with the use of a selective beta2-adrenoceptor antagonist.
Assuntos
Anti-Inflamatórios/farmacologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptores Adrenérgicos beta 2/fisiologia , Adrenalectomia , Antagonistas de Receptores Adrenérgicos beta 2 , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Artrite/fisiopatologia , Benzoxazinas/farmacologia , Feminino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Morfolinas/farmacologia , Naftalenos/farmacologia , Ratos , Ratos Endogâmicos Lew , Receptor CB1 de Canabinoide/fisiologia , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Efforts to refine the SAR of the piperazinyl-glutamate-pyridines for more potent analogs with improved pharmacokinetic profiles are described. Exploring substituted piperidines and other ring systems at the 4-pyridyl position led to compounds with improved potency and pharmacokinetic properties over candidate I. In particular, compounds 4t and 5t were discovered with a 10-fold improvement over potency and improved pharmacokinetic profiles in both the rat and dog.
Assuntos
Fibrinolíticos/farmacologia , Ácido Glutâmico/síntese química , Piperidinas/síntese química , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Piridinas/síntese química , Piridinas/farmacologia , Administração Oral , Animais , Células CHO , Cricetinae , Cricetulus , Cães , Fibrinolíticos/síntese química , Fibrinolíticos/química , Ácido Glutâmico/química , Ácido Glutâmico/farmacologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Piperidinas/química , Piperidinas/farmacologia , Piridinas/química , Ratos , Receptores Purinérgicos P2Y12 , Relação Estrutura-AtividadeRESUMO
Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and overexpressed on many tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next-generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPα interaction to induce tumor cell phagocytosis, but also induces tumor cytotoxicity in hematologic and solid human tumor cell lines, but not normal noncancerous cells, by a cell autonomous mechanism (not ADCC). AO-176 also binds preferentially to tumor versus many normal cell types. In particular, AO-176 binds negligibly to RBCs in contrast to tumor cells, even at high concentrations up to 200 µg/mL and does not agglutinate RBCs up to 1 mg/mL in vitro These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. When tested in cynomolgus monkeys, AO-176 was well tolerated with no adverse effects. Finally, we show that AO-176 demonstrates dose-dependent antitumor activity in tumor xenograft models. Taken together, the unique properties and antitumor activity of our next-generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPα axis targeting agents in clinical development.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígeno CD47/antagonistas & inibidores , Eritrócitos/metabolismo , Imunidade Inata/imunologia , Neoplasias/tratamento farmacológico , Fagocitose , Receptores Imunológicos/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Antígenos de Diferenciação/imunologia , Apoptose , Antígeno CD47/imunologia , Proliferação de Células , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/imunologia , Neoplasias/patologia , Receptores Imunológicos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Caenorhabditis elegans/isolamento & purificação , Membrana Celular/metabolismo , Endopeptidases/metabolismo , Proteínas de Homeodomínio/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Clonagem Molecular , Proteínas de Drosophila , Drosophila melanogaster , Elementos Facilitadores Genéticos/genética , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Proteínas de Helminto/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , Precursores de Proteínas/metabolismo , Receptores Notch , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/genéticaRESUMO
Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl-glutamate-pyridine as a P2Y(12) antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 22J with good human PRP potency, selectivity, in vivo efficacy and oral bioavailability.
Assuntos
Ácido Glutâmico/química , Piperazinas/química , Inibidores da Agregação Plaquetária/química , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Piridinas/química , Administração Oral , Animais , Disponibilidade Biológica , Humanos , Masculino , Inibidores da Agregação Plaquetária/farmacocinética , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the 4-position of the pyrimidine leading to highly potent P2Y12 antagonists. In particular, 4-substituted piperidine-4-pyrimidines provided compounds with exceptional potency. Pharmacokinetic and physicochemical properties were fine-tuned through modifications at the 4-position of the piperidine ring leading to compounds with good human PRP potency, selectivity, clearance and oral bioavailability.
Assuntos
Fibrinolíticos/química , Ácido Glutâmico/química , Piperidinas/química , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Pirimidinas/química , Animais , Fibrinolíticos/síntese química , Fibrinolíticos/farmacocinética , Humanos , Masculino , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Despite the efficacy of orthotopic liver transplantation in the treatment of end-stage liver diseases, its therapeutic utility is severely limited by the availability of donor organs. The ability to rehabilitate marginal organs, such as steatotic allografts, has the potential to address some of the supply limitations of available organs for transplantation. Steatotic livers are more susceptible to ischemia-reperfusion injury (IRI), which is exacerbated by the thrombospondin-1/CD47 pathway through inhibition of nitric oxide signaling. We postulated that CD47 blockade with a monoclonal antibody specific to CD47, clone 400 (CD47mAb400) may reduce the extent of IRI in steatotic liver allografts. METHODS: Orthotopic liver transplantation was performed using steatotic liver grafts from Zucker rats transplanted into lean recipients. Control IgG or the CD47mAb400 was administered to the donor livers at procurement. Serum transaminases, histological changes, and animal survival were assessed. Hepatocellular damage, oxidative and nitrosative stress, and inflammation were also quantified. RESULTS: Administration of CD47mAb400 to donor livers increased recipient survival and resulted in significant reduction of serum transaminases, bilirubin, triphosphate nick-end labeling staining, caspase-3 activity, oxidative and nitrosative stresses, and proinflammatory cytokine expression of TNF-α, IL-6 and IL-1ß. CONCLUSIONS: We conclude that administration of CD47mAb400 to donor grafts may reduce IRI through CD47 blockade to result in improved function of steatotic liver allografts and increased survival of recipients and represent a novel strategy to allow the use of livers with higher levels of steatosis.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/imunologia , Fígado Gorduroso/imunologia , Transplante de Fígado , Traumatismo por Reperfusão/tratamento farmacológico , Aloenxertos , Animais , Movimento Celular , Ensaio de Imunoadsorção Enzimática , Fígado Gorduroso/cirurgia , Imunoglobulina G/imunologia , Inflamação , Fígado/patologia , Fígado/cirurgia , Óxido Nítrico/metabolismo , Estresse Oxidativo , Ratos , Ratos Zucker , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Transaminases/sangue , Resultado do TratamentoRESUMO
Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/imunologia , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígeno CD47/biossíntese , Carcinoma Hepatocelular/imunologia , Movimento Celular/imunologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos SCID , Fagocitose/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Ischemia-reperfusion injury (IRI) significantly contributes to delayed graft function and inflammation, leading to graft loss. Ischemia-reperfusion injury is exacerbated by the thrombospondin-1-CD47 system through inhibition of nitric oxide signaling. We postulate that CD47 blockade and prevention of nitric oxide inhibition reduce IRI in organ transplantation. METHODS: We used a syngeneic rat renal transplantation model of IRI with bilaterally nephrectomized recipients to evaluate the effect of a CD47 monoclonal antibody (CD47mAb) on IRI. Donor kidneys were flushed with CD47mAb OX101 or an isotype-matched control immunoglobulin and stored at 4°C in University of Wisconsin solution for 6 hr before transplantation. RESULTS: CD47mAb perfusion of donor kidneys resulted in marked improvement in posttransplant survival, lower levels of serum creatinine, blood urea nitrogen, phosphorus and magnesium, and less histological evidence of injury. In contrast, control groups did not survive more than 5 days, had increased biochemical indicators of renal injury, and exhibited severe pathological injury with tubular atrophy and necrosis. Recipients of CD47mAb-treated kidneys showed decreased levels of plasma biomarkers of renal injury including Cystatin C, Osteopontin, Tissue Inhibitor of Metalloproteinases-1 (TIMP1), ß2-Microglobulin, Vascular Endothelial Growth Factor A (VEGF-A), and clusterin compared to the control group. Furthermore, laser Doppler assessment showed higher renal blood flow in the CD47mAb-treated kidneys. CONCLUSION: These results provide strong evidence for the use of CD47 antibody-mediated blockade to reduce IRI and improve organ preservation for renal transplantation.
Assuntos
Antígeno CD47/fisiologia , Transplante de Rim , Rim/irrigação sanguínea , Traumatismo por Reperfusão/prevenção & controle , Animais , Anticorpos Monoclonais/uso terapêutico , Antígeno CD47/imunologia , Sobrevivência de Enxerto , Rim/patologia , Masculino , Ratos , Ratos Endogâmicos Lew , Resultado do TratamentoRESUMO
Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl glutamate pyridine as a P2Y(12) antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized through modifications at the 4-position of the pyridine ring and the terminal nitrogen of the piperazine ring, leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 47s with good human PRP potency, selectivity, in vivo efficacy, and oral bioavailability. Compound 47s was selected for further preclinical evaluations.