RESUMEN
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.
Asunto(s)
Presentación de Antígeno , Antígenos de Diferenciación/inmunología , Antineoplásicos Inmunológicos/inmunología , Macrófagos/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Fagocitosis , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Humanos , Células Jurkat , Células THP-1 , Células U937RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno CD47/metabolismo , Isquemia Fría/efectos adversos , Trasplante de Hígado/efectos adversos , Hígado/efectos de los fármacos , Hígado/cirugía , Daño por Reperfusión/prevención & control , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/sangre , Antígeno CD47/inmunología , Citoprotección , Modelos Animales de Enfermedad , Mediadores de Inflamación/sangre , Hígado/irrigación sanguínea , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Masculino , Necrosis , Estrés Oxidativo/efectos de los fármacos , Ratas Endogámicas Lew , Daño por Reperfusión/sangre , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptores Adrenérgicos beta 2/fisiología , Adrenalectomía , Antagonistas de Receptores Adrenérgicos beta 2 , Animales , Artritis/inducido químicamente , Artritis/tratamiento farmacológico , Artritis/fisiopatología , Benzoxazinas/farmacología , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos DBA , Morfolinas/farmacología , Naftalenos/farmacología , Ratas , Ratas Endogámicas Lew , Receptor Cannabinoide CB1/fisiología , Receptores Adrenérgicos beta 2/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.
Asunto(s)
Fibrinolíticos/farmacología , Ácido Glutámico/síntesis química , Piperidinas/síntesis química , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2 , Piridinas/síntesis química , Piridinas/farmacología , Administración Oral , Animales , Células CHO , Cricetinae , Cricetulus , Perros , Fibrinolíticos/síntesis química , Fibrinolíticos/química , Ácido Glutámico/química , Ácido Glutámico/farmacología , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Piperidinas/química , Piperidinas/farmacología , Piridinas/química , Ratas , Receptores Purinérgicos P2Y12 , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antígeno CD47/antagonistas & inhibidores , Eritrocitos/metabolismo , Inmunidad Innata/inmunología , Neoplasias/tratamiento farmacológico , Fagocitosis , Receptores Inmunológicos/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/inmunología , Antígenos de Diferenciación/inmunología , Apoptosis , Antígeno CD47/inmunología , Proliferación Celular , Femenino , Humanos , Inmunidad Innata/efectos de los fármacos , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/inmunología , Neoplasias/patología , Receptores Inmunológicos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Fibrinolíticos/química , Ácido Glutámico/química , Piperidinas/química , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2 , Pirimidinas/química , Animales , Fibrinolíticos/síntesis química , Fibrinolíticos/farmacocinética , Humanos , Masculino , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Ácido Glutámico/química , Piperazinas/química , Inhibidores de Agregación Plaquetaria/química , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2 , Piridinas/química , Administración Oral , Animales , Disponibilidad Biológica , Humanos , Masculino , Inhibidores de Agregación Plaquetaria/farmacocinética , Inhibidores de Agregación Plaquetaria/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno CD47/inmunología , Hígado Graso/inmunología , Trasplante de Hígado , Daño por Reperfusión/tratamiento farmacológico , Aloinjertos , Animales , Movimiento Celular , Ensayo de Inmunoadsorción Enzimática , Hígado Graso/cirugía , Inmunoglobulina G/inmunología , Inflamación , Hígado/patología , Hígado/cirugía , Óxido Nítrico/metabolismo , Estrés Oxidativo , Ratas , Ratas Zucker , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/patología , Transducción de Señal , Transaminasas/sangre , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno CD47/inmunología , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígeno CD47/biosíntesis , Carcinoma Hepatocelular/inmunología , Movimiento Celular/inmunología , Células Hep G2 , Humanos , Neoplasias Hepáticas/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones SCID , Fagocitosis/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antígeno CD47/fisiología , Trasplante de Riñón , Riñón/irrigación sanguínea , Daño por Reperfusión/prevención & control , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígeno CD47/inmunología , Supervivencia de Injerto , Riñón/patología , Masculino , Ratas , Ratas Endogámicas Lew , Resultado del TratamientoRESUMEN
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.