RESUMO
BACKGROUND: SGN-B7H4V is a novel investigational vedotin antibody-drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline (mc-vc) linker. This vedotin linker-payload system has been clinically validated in multiple Food and Drug Administration approved agents including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumors, including breast, ovarian, and endometrial tumors, and limited normal tissue expression. SGN-B7H4V is designed to induce direct cytotoxicity against target cells by binding to B7-H4 on the surface of target cells and releasing the cytotoxic payload MMAE upon internalization of the B7-H4/ADC complex. METHODS: B7-H4 expression was characterized by immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the antitumor activity of SGN-B7H4V as monotherapy and in combination with an anti-programmed cell death-1 (PD-1) agent was evaluated using an immunocompetent murine B7-H4-expressing Renca tumor model. RESULTS: Immunohistochemistry confirmed B7-H4 expression across multiple solid tumors, with the highest prevalence in breast, endometrial, and ovarian tumors. In vitro, SGN-B7H4V killed B7-H4-expressing tumor cells by MMAE-mediated direct cytotoxicity and antibody-mediated effector functions including antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In vivo, SGN-B7H4V demonstrated strong antitumor activity in multiple xenograft models of breast and ovarian cancer, including xenograft tumors with heterogeneous B7-H4 expression, consistent with the ability of vedotin ADCs to elicit a bystander effect. In an immunocompetent murine B7-H4-expressing tumor model, SGN-B7H4V drove robust antitumor activity as a monotherapy that was enhanced when combined with an anti-PD-1 agent. CONCLUSION: The immune checkpoint ligand B7-H4 is a promising molecular target expressed by multiple solid tumors. SGN-B7H4V demonstrates robust antitumor activity in preclinical models through multiple potential mechanisms. Altogether, these preclinical data support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.
Assuntos
Antineoplásicos , Imunoconjugados , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoconjugados/química , Imuno-Histoquímica , LigantesRESUMO
The oncogenic receptor HER2 is overexpressed in many cancers, including up to 20% of breast cancers. Despite the availability of HER2-targeted treatments, patients' disease often progresses during therapy, underscoring the need for novel treatment strategies. The addition of tucatinib, a reversible, highly selective HER2 tyrosine kinase inhibitor (TKI), to treatment with trastuzumab and capecitabine significantly improved survival outcomes of patients with HER2-positive metastatic breast cancer, including those with active brain metastases. We rationalized that combining tucatinib with other HER2-targeting agents with complementary mechanisms of action would further increase efficacy against tumors. We characterized the activity of tucatinib with the antibodydrug conjugate T-DM1 in preclinical models of breast cancer, including HER2-positive breast cancer cells and patient-derived xenograft (PDX) models. Mechanistic details on tucatinib activity were obtained in internalization and catabolism studies. In combination, tucatinib and T-DM1 showed an enhanced, often synergistic, cytotoxic response and demonstrated improved antitumor activity in vivo, including in PDX models refractory to T-DM1 single-agent activity. Mechanistically, tucatinib mediated an increase in inactive HER2 molecules at the cell surface through inhibition of HER2 ubiquitination, resulting in increased internalization and catabolism of T-DM1. The combination was correlated with enhanced HER2 pathway inhibition, decreased proliferation, and increased apoptosis. In a xenograft model of brain metastasis, tucatinib penetrated intracranial tumor tissues, inhibiting tumor growth and improving survival. These results suggest that tucatinib may be the optimal TKI partner for HER2-targeted therapies and support clinical studies of its combination with T-DM1, including in patients with brain metastases. SIGNIFICANCE: The preclinical findings in breast cancer models presented here demonstrate that combining tucatinib with T-DM1 enhances the antitumor activity of either agent alone, supporting clinical studies of the combination in HER2-positive breast cancer, including in patients with brain metastases, which remains an important unmet medical need.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Trastuzumab , Receptor ErbB-2 , Ado-Trastuzumab EmtansinaRESUMO
SGN-CD228A is an investigational antibody-drug conjugate (ADC) directed to melanotransferrin (CD228, MELTF, MFI2, p97), a cell-surface protein first identified in melanoma. SGN-CD228A consists of a humanized antibody, hL49, with high specificity and affinity for CD228 that is stably conjugated to 8 molecules of the clinically validated microtubule-disrupting agent monomethyl auristatin E (MMAE) via a novel glucuronide linker. We performed comprehensive IHC studies, which corroborated published RNA sequencing data and confirmed low CD228 expression in normal tissues and high expression in several cancers, including melanoma, squamous non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), colorectal cancer, and pancreatic cancer. SGN-CD228A was efficiently internalized in various tumor cell types, and its cytotoxic activity was dependent on CD228 expression and internalization and intrinsic sensitivity to the MMAE payload. Compared with the valine-citrulline dipeptide linker, the novel glucuronide linker increased the cellular retention of MMAE in vitro and conferred improved antitumor activity against melanoma cell lines in vitro and in vivo. In addition, SGN-CD228A was active across melanoma, TNBC, and NSCLC cell line- and patient-derived xenograft models with heterogeneous antigen expression. In vivo, CD228 expression was important for response to SGN-CD228A but was not well correlated across all tumor types, suggesting that other factors associated with ADC activity are important. Overall, SGN-CD228A is a CD228-directed, investigational ADC that employs innovative technology and has compelling preclinical antitumor activity. SGN-CD228A is investigated in a Phase I clinical trial (NCT04042480) in patients with advanced solid tumors.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Imunoconjugados , Neoplasias Pulmonares , Melanoma , Neoplasias de Mama Triplo Negativas , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Glucuronídeos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoconjugados/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Feline McDonough Sarcoma-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell-mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T-cell-mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with 2 bispecific T-cell engager molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells, whereas only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development.
Assuntos
Leucemia Mieloide Aguda , Receptores de Antígenos Quiméricos , Adulto , Animais , Gatos , Linhagem Celular Tumoral , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Macaca fascicularisRESUMO
There is an unmet need in severe asthma where approximately 40% of patients exhibit poor ß-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca2+-activated Cl- channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of â¼580,000 compounds. The anthelmintics niclosamide, nitazoxanide, and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting ß-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the ß-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.
RESUMO
BACKGROUND: To further understand the role of pituitary adenylate cyclase-activating polypeptide 1 (PAC1) receptors in headache disorders, we mapped their expression in tissues of the trigemino-autonomic system by immunohistochemistry and in situ hybridization. METHODS: To optimize screening for monoclonal antibodies suitable for immunohistochemistry on formalin-fixed, paraffin-embedded tissues, we developed a new enzyme-linked immunosorbent assay using formalin-fixed, paraffin-embedded cells overexpressing human PAC1 receptors. 169G4.1 was selected from these studies for analysis of rat and human tissues and chimerized onto a mouse backbone to avoid human-on-human cross-reactivity. Immunoreactivity was compared to PAC1 receptor mRNA by in situ hybridization in both species. RESULTS: 169G4.1 immunoreactivity delineated neuronal cell bodies in the sphenopalatine ganglion in both rat and human, whereas no staining was detected in the trigeminal ganglion. The spinal trigeminal nucleus in both species showed immunoreactivity as especially strong in the upper laminae with both cell bodies and neuropil being labelled. No immunoreactivity was seen in either rat or human dura mater vessels. In situ hybridization in both species revealed mRNA in sphenopalatine ganglion neurons and the spinal trigeminal nucleus, a weak signal in the trigeminal nucleus and no signal in dural vessels. CONCLUSION: Taken together, these data support a role for PAC1 receptors in the trigemino-autonomic system as it relates to headache pathophysiology.
Assuntos
Gânglios Parassimpáticos/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Gânglio Trigeminal/metabolismo , Núcleo Espinal do Trigêmeo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Cefaleia/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/análiseRESUMO
Etelcalcetide, a novel peptide agonist of the calcium-sensing receptor, prevents vascular calcification in a rat model of renal insufficiency with secondary hyperparathyroidism. Vascular calcification occurs frequently in patients with chronic kidney disease (CKD) and is a consequence of impaired mineral homeostasis and secondary hyperparathyroidism (SHPT). Etelcalcetide substantially lowers parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) levels in SHPT patients on hemodialysis. This study compared the effects of etelcalcetide and paricalcitol on vascular calcification in rats with adenine-induced CKD and SHPT. Uremia and SHPT were induced in male Wistar rats fed a diet supplemented with 0.75% adenine for 4 weeks. Rats were injected with vehicle, etelcalcetide, or paricalcitol for 4 weeks from the beginning of adenine diet. Rats fed an adenine-free diet were included as nonuremic controls. Similar reductions in plasma PTH and parathyroid chief cell proliferation were observed in both etelcalcetide- and paricalcitol-treated rats. Serum calcium and phosphorus were significantly lower in etelcalcetide-treated uremic rats and was unchanged in paricalcitol-treated rats. Both serum FGF23 and aortic calcium content were significantly lower in etelcalcetide-treated uremic rats compared with either vehicle- or paricalcitol-treated uremic rats. The degree of aortic calcium content for etelcalcetide-treated rats was similar to that in nonuremic controls and corroborated findings of lack of histologic aortic mineralization in those groups. In conclusion, etelcalcetide and paricalcitol similarly attenuated progression of SHPT in an adenine rat model of CKD. However, etelcalcetide differentially prevented vascular calcification, at least in part, due to reductions in serum FGF23, calcium, and phosphorus levels.
Assuntos
Hiperparatireoidismo Secundário/complicações , Peptídeos/farmacologia , Insuficiência Renal/complicações , Calcificação Vascular/etiologia , Animais , Modelos Animais de Doenças , Ergocalciferóis/farmacologia , Masculino , Ratos , Ratos WistarRESUMO
Sustained elevation of parathyroid hormone (PTH) is catabolic to cortical bone, as evidenced by deterioration in bone structure (cortical porosity), and is a major factor for increased fracture risk in chronic kidney disease (CKD). Etelcalcetide (AMG 416), a novel peptide agonist of the calcium-sensing receptor, reduces PTH levels in subtotal nephrectomized (Nx) rats and in hemodialysis patients with secondary hyperparathyroidism (SHPT) in clinical studies; however, effects of etelcalcetide on bone have not been determined. In a rat model of established SHPT with renal osteodystrophy, etelcalcetide or vehicle was administered by subcutaneous (s.c.) injection to subtotal Nx rats with elevated PTH (>750pg/mL) once per day for 6weeks. Sham-operated rats receiving vehicle (s.c.) served as non-SHPT controls. Prior to treatment, significant increases in serum creatinine (2-fold), blood urea nitrogen (BUN, 3-fold), PTH (5-fold), fibroblast growth factor-23 (FGF23; 13-fold) and osteocalcin (12-fold) were observed in SHPT rats compared to non-SHPT controls. Elevations in serum creatinine and BUN were unaffected by treatment with vehicle or etelcalcetide. In contrast, etelcalcetide significantly decreased PTH, FGF23 and osteocalcin, whereas vehicle treatment did not. Cortical bone porosity increased and bone strength decreased in vehicle-treated SHPT rats compared to non-SHPT controls. Cortical bone structure improved and energy to failure was significantly greater in SHPT rats treated with etelcalcetide compared to vehicle. Mineralization lag time and marrow fibrosis were significantly reduced by etelcalcetide. In conclusion, etelcalcetide reduced bone turnover, attenuated mineralization defect and marrow fibrosis, and preserved cortical bone structure and bone strength by lowering PTH in subtotal Nx rats with established SHPT.
Assuntos
Osso Cortical/fisiopatologia , Hiperparatireoidismo Secundário/tratamento farmacológico , Hiperparatireoidismo Secundário/fisiopatologia , Nefrectomia , Peptídeos/uso terapêutico , Receptores de Detecção de Cálcio/agonistas , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Cálcio/sangue , Osso Cortical/efeitos dos fármacos , Creatinina/sangue , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Hiperparatireoidismo Secundário/sangue , Hiperplasia , Testes de Função Renal , Masculino , Osteocalcina/sangue , Glândulas Paratireoides/patologia , Hormônio Paratireóideo/sangue , Peptídeos/farmacologia , Fósforo/sangue , Ratos Sprague-Dawley , Fosfatase Ácida Resistente a Tartarato/sangueRESUMO
The discovery of brown adipose tissue (BAT) as a key regulator of energy expenditure has sparked interest in identifying novel soluble factors capable of activating inducible BAT (iBAT) to combat obesity. Using a high content cell-based screen, we identified fibroblast growth factor 16 (FGF16) as a potent inducer of several physical and transcriptional characteristics analogous to those of both "classical" BAT and iBAT. Overexpression of Fgf16 in vivo recapitulated several of our in vitro findings, specifically the significant induction of the Ucp1 gene and UCP1 protein expression in inguinal white adipose tissue (iWAT), a common site for emergent active iBAT. Despite significant UCP1 up-regulation in iWAT and dramatic weight loss, the metabolic improvements observed due to Fgf16 overexpression in vivo were not the result of increased energy expenditure, as measured by indirect calorimetric assessment. Instead, a pattern of reduced food and water intake, combined with feces replete with lipid and bile acid, indicated a phenotype more akin to that of starvation and intestinal malabsorption. Gene expression analysis of the liver and ileum indicated alterations in several steps of bile acid metabolism, including hepatic synthesis and reabsorption. Histological analysis of intestinal tissue revealed profound abnormalities in support of this conclusion. The in vivo data, together with FGF receptor binding analysis, indicate that the in vivo outcome observed is the likely result of both direct and indirect mechanisms and probably involves multiple receptors. These results highlight the complexity of FGF signaling in the regulation of various metabolic processes.
Assuntos
Tecido Adiposo Branco/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Termogênese , Proteases Específicas de Ubiquitina/biossíntese , Tecido Adiposo Branco/patologia , Animais , Linhagem Celular , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Fatores de Crescimento de Fibroblastos/genética , Humanos , Camundongos , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/metabolismo , Proteases Específicas de Ubiquitina/genéticaRESUMO
AMG X, a human neutralizing monoclonal antibody (mAb) against a soluble human protein, caused thrombocytopenia, platelet activation, reduced mean arterial pressure, and transient loss of consciousness in cynomolgus monkeys after first intravenous administration. In vitro, AMG X induced activation in platelets from macaque species but not from humans or baboons. Other similar mAbs against the same pharmacological target failed to induce these in vivo and in vitro effects. In addition, the target protein was known to not be expressed on platelets, suggesting that platelet activation occurred through an off-target mechanism. AMG X bound directly to cynomolgus platelets and required both the Fab and Fc portion of the mAb for platelet activation. Binding to platelets was inhibited by preincubation of AMG X with its pharmacological target or with anti-human Fc antibodies or by preincubation of platelets with AMG X F(ab')(2) or human immunoglobulin (IVIG). AMG X F(ab')(2) did not activate platelets. Thus, platelet activation required both recognition/binding of a platelet ligand with the Fab domain and interaction of platelet Fc receptors (i.e., FcγRIIa) with the Fc domain. These findings reflect the complexity of the mechanism of action of mAbs and the increasing awareness of potential for unintended effects in preclinical species.
Assuntos
Anticorpos Monoclonais/toxicidade , Plaquetas/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Administração Intravenosa , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Plaquetas/metabolismo , Humanos , Hipotensão/sangue , Hipotensão/induzido quimicamente , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Macaca fascicularis , Masculino , Papio , Agregação Plaquetária/efeitos dos fármacos , Ligação Proteica , Serotonina/metabolismo , Síncope/sangue , Síncope/induzido quimicamente , Trombocitopenia/sangue , Trombocitopenia/induzido quimicamente , Tromboxano B2/metabolismoRESUMO
Interleukin-1 (IL-1) has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified an isoform of the IL-1 receptor (IL-1R) accessory protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1R but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild-type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild-type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest that it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival.
Assuntos
Processamento Alternativo , Sistema Nervoso Central/imunologia , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucina-1/metabolismo , Neurônios/imunologia , Sequência de Aminoácidos , Animais , Astrócitos/imunologia , Sequência de Bases , Encéfalo/citologia , Encéfalo/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Inflamação/imunologia , Proteína Acessória do Receptor de Interleucina-1/química , Proteína Acessória do Receptor de Interleucina-1/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais/imunologiaRESUMO
CD11c, a member of the leukointegrin family, is expressed prominently on tissue macrophages and dendritic cells and binds to complement fragment (iC3b), provisional matrix molecules (fibrinogen), and the Ig superfamily cell adhesion molecule, ICAM-1. CD11c has been proposed to function in phagocytosis, cell migration, and cytokine production by monocytes/macrophages as well as induction of T cell proliferation by Langerhans cells. Using assays to quantify CD11c-mediated cell adhesion, we demonstrate that CD11c recognizes ICAM-2 and VCAM-1. The CD11c-binding site on VCAM-1 appears to be different from that used by the integrin alpha4. CD11c and alpha4beta1 contributed to monocyte capture and transmigration on inflamed human aortic endothelial cells. We discovered that the anti-mouse CD11c mAb N418 blocks CD11c binding to iC3b, ICAM-1, and VCAM-1. Treatment of mice with N418 reduced SRBC-induced delayed-type hypersensitivity significantly. CD11c appeared to contribute predominantly to the sensitization phase and somewhat less to the response to SRBC challenge. This suggests a novel role for CD11c during leukocyte recruitment, antigen uptake, and the survival of APC.