RESUMO
BACKGROUND & AIMS: BiTE (bispecific T-cell engager) immune therapy has demonstrated clinical activity in multiple tumor indications, but its influence in the tumor microenvironment remains unclear. CLDN18.2 is overexpressed in solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC), both of which are characterized by the presence of immunosuppressive cells, including regulatory T cells (Tregs) and few effector T cells (Teffs). METHODS: We evaluated the activity of AMG 910, a CLDN18.2-targeted half-life extended (HLE) BiTE molecule, in GC and PDAC preclinical models and cocultured Tregs and Teffs in the presence of CLDN18.2-HLE-BiTE. RESULTS: AMG 910 induced potent, specific cytotoxicity in GC and PDAC cell lines. In GSU and SNU-620 GC xenograft models, AMG 910 engaged human CD3+ T cells with tumor cells, resulting in significant antitumor activity. AMG 910 monotherapy, in combination with a programmed death-1 (PD-1) inhibitor, suppressed tumor growth and enhanced survival in an orthotopic Panc4.14 PDAC model. Moreover, Treg infusion enhanced the antitumor efficacy of AMG 910 in the Panc4.14 model. In syngeneic KPC models of PDAC, treatment with a mouse surrogate CLDN18.2-HLE-BiTE (muCLDN18.2-HLE-BiTE) or the combination with an anti-PD-1 antibody significantly inhibited tumor growth. Tregs isolated from mice bearing KPC tumors that were treated with muCLDN18.2-HLE-BiTE showed decreased T cell suppressive activity and enhanced Teff cytotoxic activity, associated with increased production of type I cytokines and expression of Teff gene signatures. CONCLUSIONS: Our data suggest that BiTE molecule treatment converts Treg function from immunosuppressive to immune enhancing, leading to antitumor activity in immunologically "cold" tumors.
Assuntos
Anticorpos Biespecíficos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Linfócitos T Reguladores/metabolismo , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Moléculas de Adesão Celular , Carcinoma Ductal Pancreático/tratamento farmacológico , Imunidade , Microambiente Tumoral , ClaudinasRESUMO
Cancers with microsatellite instability (MSI), which include ≤20% of solid tumors, are characterized by resistance to chemotherapy due to deficiency in the DNA mismatch repair (MMR) pathway. Rhodium metalloinsertors make up a class of compounds that bind DNA mismatches with high specificity and show selective cytotoxicity in MSI cancer cells. We determined that rhodium complexes with an Nâ§O coordination showed significantly increased cell potency compared with that of Nâ§N-coordinated compounds, and we identified [Rh(chrysi)(phen)(PPO)]2+ (RhPPO) as the most potent, selective compound in this class. Using matched cell lines that are MMR-deficient (HCT116O) and MMR-proficient (HCT116N), we demonstrated that RhPPO preferentially activates the DNA damage response and inhibits DNA replication and cell proliferation in HCT116O cells, leading to cell death by necrosis. Using a fluorescent conjugate of RhPPO, we established that the metalloinsertor localizes to DNA mismatches in the cell nucleus and causes DNA double-strand breaks at or near the mismatch sites. Evaluation of RhPPO across MMR-deficient and MMR-proficient cell lines confirmed the broad potential for RhPPO to target MSI cancers, with cell potency significantly higher than that of platinum complexes used broadly as chemotherapeutics. Moreover, in a mouse xenograft model of MSI cancer, RhPPO shows promising antitumor activity and increased survival. Thus, our studies indicate that RhPPO is a novel DNA-targeted therapy with improved potency and selectivity over standard-of-care platinum-based chemotherapy and, importantly, that DNA mismatches offer a critical new target in the design of chemotherapeutics for MSI cancers.
Assuntos
Antineoplásicos/uso terapêutico , Complexos de Coordenação/uso terapêutico , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , DNA/metabolismo , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Humanos , Estrutura Molecular , Necrose/induzido quimicamente , Ródio/químicaRESUMO
Transthyretin (TTR) is an abundant homotetrameric serum protein and was selected here for engineering higher-valency molecules because of its compact size, simple structure, and natural propensity to tetramerize. To demonstrate this utility, we fused TTR to the C terminus of conatumumab, an antibody that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2, as heavy chains to form antibody dimers and Fab heavy chains to form Fab tetramers. Moreover, we used constant heavy domain 3 heterodimerization substitutions to create TTR-mediated conatumumab tetramers. The conatumumab-TTR fusions displayed substantially enhanced potency in cell-based assays, as well as in murine tumor xenograft models. We conclude that antibody-TTR fusions may provide a powerful platform for multimerizing antibody and Fab fragments to enhance the capabilities of human therapeutics that benefit from target clustering and higher-order antigen-binding valency.
Assuntos
Anticorpos Monoclonais , Antineoplásicos Imunológicos , Fragmentos Fab das Imunoglobulinas , Neoplasias Experimentais , Pré-Albumina , Multimerização Proteica , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/farmacologia , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Pré-Albumina/genética , Pré-Albumina/farmacocinética , Pré-Albumina/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Up to 20% of solid tumors are characterized by DNA mismatch repair (MMR) deficiency and microsatellite instability that confer resistance to standard of care chemotherapy. MMR-deficient cancers have an increased mutation rate, and DNA mismatches accumulate as part of these cancers. We previously described a class of compounds, rhodium metalloinsertors, that bind DNA mismatches with high specificity and selectivity and have potential as targeted therapy. [Rh(chrysi)(phen)(PPO)]2+ (RhPPO) is the most potent, selective compound in this class and acts by targeting DNA mismatches, resulting in preferential cytotoxicity to MMR-deficient cancers. To explore further the cellular mechanism of action of RhPPO, we conjugated the metal complex to a fluorescent probe, cyanine 3 (Cy3). RhPPO-Cy3 binds DNA mismatches and retains the selectivity and potent cytotoxic activity of RhPPO for MMR-deficient cell lines. RhPPO-Cy3 forms discrete foci in the cell nucleus that overlap with sites of DNA damage, suggesting that the lesions occur at or near DNA mismatch sites. RhPPO-Cy3 foci persist over time, despite initial processing of the lesion and recruitment of repair proteins, consistent with the idea that the complex binding to a mismatch prevents repair. RhPPO-Cy3 binding does not lead to activation of p53 and the apoptotic pathway. Together, these findings support the idea that RhPPO-Cy3 binding leads to irreversible DNA damage at DNA mismatches that enables selective cytotoxicity to MMR-deficient cells.
Assuntos
Antineoplásicos/farmacologia , Carbocianinas/farmacologia , Complexos de Coordenação/farmacologia , Dano ao DNA , Corantes Fluorescentes/farmacologia , Ródio/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Carbocianinas/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HCT116 , Humanos , Estrutura Molecular , Imagem Óptica , Ródio/químicaRESUMO
The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)]2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.
Assuntos
Ciclo Celular/efeitos dos fármacos , Complexos de Coordenação/toxicidade , Citotoxinas/toxicidade , Dano ao DNA , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , DNA/metabolismo , Ródio/toxicidade , Linhagem Celular Tumoral , Complexos de Coordenação/química , Citotoxinas/química , Humanos , Ródio/químicaRESUMO
The tumour-suppressor gene ATM, mutations in which cause the human genetic disease ataxia telangiectasia (A-T), encodes a key protein kinase that controls the cellular response to DNA double-strand breaks (DSBs). DNA DSBs caused by ionizing radiation or chemicals result in rapid ATM autophosphorylation, leading to checkpoint activation and phosphorylation of substrates that regulate cell-cycle progression, DNA repair, transcription and cell death. However, the precise mechanism by which damaged DNA induces ATM and checkpoint activation remains unclear. Here, we demonstrate that linear DNA fragments added to Xenopus egg extracts mimic DSBs in genomic DNA and provide a platform for ATM autophosphorylation and activation. ATM autophosphorylation and phosphorylation of its substrate NBS1 are dependent on DNA fragment length and the concentration of DNA ends. The minimal DNA length required for efficient ATM autophosphorylation is approximately 200 base pairs, with cooperative autophosphorylation induced by DNA fragments of at least 400 base pairs. Importantly, full ATM activation requires it to bind to DNA regions flanking DSB ends. These findings reveal a direct role for DNA flanking DSB ends in ATM activation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/efeitos dos fármacos , Extratos Celulares/química , DNA/farmacologia , DNA/fisiologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Morfolinas/farmacologia , Óvulo/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Pironas/farmacologia , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/efeitos dos fármacos , Regulação para Cima , XenopusRESUMO
The tumor-associated antigen STEAP1 is a potential therapeutic target that is expressed in most prostate tumors and at increased levels in metastatic castration-resistant prostate cancer (mCRPC). We developed a STEAP1-targeted XmAb 2+1 T-cell engager (TCE) molecule, AMG 509 (also designated xaluritamig), that is designed to redirect T cells to kill prostate cancer cells that express STEAP1. AMG 509 mediates potent T cell-dependent cytotoxicity of prostate cancer cell lines in vitro and promotes tumor regression in xenograft and syngeneic mouse models of prostate cancer in vivo. The avidity-driven activity of AMG 509 enables selectivity for tumor cells with high STEAP1 expression compared with normal cells. AMG 509 is the first STEAP1 TCE to advance to clinical testing, and we report a case study of a patient with mCRPC who achieved an objective response on AMG 509 treatment. SIGNIFICANCE: Immunotherapy in prostate cancer has met with limited success due to the immunosuppressive microenvironment and lack of tumor-specific targets. AMG 509 provides a targeted immunotherapy approach to engage a patient's T cells to kill STEAP1-expressing tumor cells and represents a new treatment option for mCRPC and potentially more broadly for prostate cancer. See related commentary by Hage Chehade et al., p. 20. See related article by Kelly et al., p. 76. This article is featured in Selected Articles from This Issue, p. 5.
Assuntos
Anticorpos Biespecíficos , Neoplasias de Próstata Resistentes à Castração , Masculino , Camundongos , Animais , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Linfócitos T , Imunoterapia , Anticorpos Biespecíficos/uso terapêutico , Microambiente Tumoral , Antígenos de Neoplasias , Oxirredutases/uso terapêuticoRESUMO
Cdc7 kinase is responsible for the initiation and regulation of DNA replication and has been proposed as a target for cancer therapy. We have identified a class of Cdc7 inhibitors based on a substituted indole core. Synthesis of focused indole and azaindole analogs yielded potent and selective 5-azaindole Cdc7 inhibitors with improved intrinsic metabolic stability (ie 36). In parallel, quantum mechanical conformational analysis helped to rationalize SAR observations, led to a proposal of the preferred binding conformation in the absence of co-crystallography data, and allowed the design of 7-azaindole 37 as a second lead in this series.
Assuntos
Compostos Aza/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Compostos Aza/síntese química , Compostos Aza/química , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from modest cell potency (cellular IC50=0.71 µM measuring pMCM2), low selectivity against structurally related kinases, and high IV clearance in rats (CL=18 L/h/kg). Extensive optimization resulted in azaindole 26 (Cdc7 IC50=1.1 nM, pMCM2 IC50=32 nM) that demonstrated robust lowering of pMCM2 in a mouse pharmacodynamic (PD) model when dosed orally. Modifications to improve the pharmacokinetic profile of this series were guided by trapping experiments with glutathione in rat hepatocytes.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Oxidiazóis/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Nus , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma with a poor prognosis. Initial responses to standard-of-care chemo-immunotherapy are, unfortunately, followed by rapid disease recurrence in most patients. Current treatment options are limited, with no therapies specifically approved as third-line or beyond. Delta-like ligand 3 (DLL3), a Notch inhibitory ligand, is an attractive therapeutic target because it is overexpressed on the surface of SCLC cells with minimal to no expression on normal cells. Several DLL3-targeted therapies are being developed for the treatment of SCLC and other neuroendocrine carcinomas, including antibody-drug conjugates (ADCs), T-cell engager (TCE) molecules, and chimeric antigen receptor (CAR) therapies. First, we discuss the clinical experience with rovalpituzumab tesirine (Rova-T), a DLL3-targeting ADC, the development of which was halted due to a lack of efficacy in phase 3 studies, with a view to understanding the lessons that can be garnered for the rapidly evolving therapeutic landscape in SCLC. We then review preclinical and clinical data for several DLL3-targeting agents that are currently in development, including the TCE molecules-tarlatamab (formerly known as AMG 757), BI 764532, and HPN328-and the CAR T-cell therapy AMG 119. We conclude with a discussion of the future challenges and opportunities for DLL3-targeting therapies, including the utility of DLL3 as a biomarker for patient selection and disease progression, and the potential of rational combinatorial approaches that can enhance efficacy.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Ligantes , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas de Membrana , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Effective treatments for de novo and treatment-emergent small-cell/neuroendocrine (t-SCNC) prostate cancer represent an unmet need for this disease. Using metastatic biopsies from patients with advanced cancer, we demonstrate that delta-like ligand 3 (DLL3) is expressed in de novo and t-SCNC and is associated with reduced survival. We develop a PET agent, [89Zr]-DFO-DLL3-scFv, that detects DLL3 levels in mouse SCNC models. In multiple patient-derived xenograft models, AMG 757 (tarlatamab), a half-life-extended bispecific T-cell engager (BiTE) immunotherapy that redirects CD3-positive T cells to kill DLL3-expressing cells, exhibited potent and durable antitumor activity. Late relapsing tumors after AMG 757 treatment exhibited lower DLL3 levels, suggesting antigen loss as a resistance mechanism, particularly in tumors with heterogeneous DLL3 expression. These findings have been translated into an ongoing clinical trial of AMG 757 in de novo and t-SCNC, with a confirmed objective partial response in a patient with histologically confirmed SCNC. Overall, these results identify DLL3 as a therapeutic target in SCNC and demonstrate that DLL3-targeted BiTE immunotherapy has significant antitumor activity in this aggressive prostate cancer subtype. SIGNIFICANCE: The preclinical and clinical evaluation of DLL3-directed immunotherapy, AMG 757, and development of a PET radiotracer for noninvasive DLL3 detection demonstrate the potential of targeting DLL3 in SCNC prostate cancer.
Assuntos
Proteínas de Membrana , Neoplasias da Próstata , Animais , Humanos , Masculino , Camundongos , Anticorpos Monoclonais , Imunoterapia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/terapia , Neoplasias da Próstata/patologia , Zircônio , Tumores Neuroendócrinos/diagnóstico por imagem , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/terapiaRESUMO
T-cell engager (TCE) molecules provide a targeted immunotherapy approach to treat hematologic malignancies and solid tumors. Since the approval of the CD19-targeted BiTE® (bispecific T-cell engager) molecule blinatumomab, multiple TCE molecules against different targets have been developed in several tumor types, with the approval of three additional TCE molecules in 2022. Some of the initial challenges, such as the need for continuous intravenous administration and low productivity, have been addressed in subsequent iterations of the platform by advancing half-life extended, Fc-based molecules. As clinical data from these molecules emerge, additional optimization of formats and manufacturability will be necessary. Ongoing efforts are focused on further improving TCE efficacy, safety, and convenience of administration.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Neoplasias , Humanos , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD19/uso terapêutico , Imunoterapia , Linfócitos T , Neoplasias/tratamento farmacológicoRESUMO
Heterochromatin performs a central role in chromosome segregation and stability by promoting cohesion at centromeres. Establishment of both heterochromatin-mediated silencing and cohesion requires passage through S phase, although the mechanism is unknown. Here we demonstrate that Schizosaccharomyces pombe Hsk1 (CDC7), a conserved Dbf4-dependent protein kinase (DDK) that regulates replication initiation, interacts with and phosphorylates the heterochromatin protein 1 (HP1) equivalent Swi6 (ref. 6). Hsk1 and its regulatory subunit Dfp1 function downstream of Swi6 localization to promote heterochromatin function and cohesion specifically at centromeres. This role for Hsk1-Dfp1 is separable from its replication initiation activity, providing a temporal link between S phase and centromere cohesion that is mediated by heterochromatin.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Centrômero/genética , Segregação de Cromossomos/genética , Heterocromatina/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/genética , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fase S/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
BiTE ® (bispecific T-cell engager) molecules exert antitumor activity by binding one arm to CD3 on cytotoxic T-cells and the other arm to a tumor-associated antigen. We generated a fully mouse cross-reactive mesothelin (MSLN)-targeted BiTE molecule that is genetically fused to a Fc-domain for half-life extension, and evaluated biodistribution and tumor targeting of a zirconium-89 (89Zr)-labeled MSLN HLE BiTE molecule in 4T1 breast cancer bearing syngeneic mice with positron emission tomography (PET). Biodistribution of 50 µg 89Zr-MLSN HLE BiTE was studied over time by PET imaging in BALB/c mice and revealed uptake in tumor and lymphoid tissues with an elimination half-life of 63.4 hours. Compared to a non-targeting 89Zr-control HLE BiTE, the 89Zr-MLSN HLE BiTE showed a 2-fold higher tumor uptake and higher uptake in lymphoid tissues. Uptake in the tumor colocalized with mesothelin expression, while uptake in the spleen colocalized with CD3 expression. Evaluation of the effect of protein doses on the biodistribution and tumor targeting of 89Zr-MSLN HLE BiTE revealed for all dose groups that uptake in the spleen was faster than in the tumor (day 1 vs day 5). The lowest dose of 10 µg 89Zr-MSLN HLE BiTE had higher spleen uptake and faster blood clearance compared to higher doses of 50 µg and 200 µg. 89Zr-MSLN HLE BiTE tumor uptake was similar at all doses. Conclusion: The MSLN HLE BiTE showed specific tumor uptake and both arms contributed to the biodistribution profile. These findings support the potential for clinical translation of HLE BiTE molecules.
RESUMO
MUC12 is a transmembrane mucin that is highly expressed in >50% of primary and metastatic colorectal tumors. MUC12 is also expressed by normal epithelial cells of the colon and small intestine. Although MUC12 localization in normal epithelial cells is restricted to the apical membrane, expression in tumors is depolarized and shows broad membrane localization. The differential localization of MUC12 in tumor cells as compared with normal cells makes it a potential therapeutic target. Here, we evaluated targeting of MUC12 with a BiTE (bispecific T-cell engager) molecule. We generated a panel of proof-of-concept half-life extended (HLE) BiTE molecules that bind MUC12 on tumor cells and CD3 on T cells. We prioritized one molecule based on in vitro activity for further characterization in vivo In vitro, the MUC12 HLE BiTE molecule mediated T-cell-redirected lysis of MUC12-expressing cells with half-maximal lysis of 4.4 ± 0.9 to 117 ± 78 pmol/L. In an exploratory cynomolgus monkey toxicology study, the MUC12 HLE BiTE molecule administered at 200 µg/kg with a step dose to 1,000 µg/kg was tolerated with minimal clinical observations. However, higher doses were not tolerated, and there was evidence of damage in the gastrointestinal tract, suggesting dose levels projected to be required for antitumor activity may be associated with on-target toxicity. Together, these data demonstrate that the apically restricted expression of MUC12 in normal tissues is accessible to BiTE molecule target engagement and highlight the difficult challenge of identifying tumor-selective antigens for solid tumor T-cell engagers.
Assuntos
Anticorpos Biespecíficos/farmacologia , Biomarcadores Tumorais/metabolismo , Complexo CD3/imunologia , Neoplasias Colorretais/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Mucinas/antagonistas & inibidores , Linfócitos T/imunologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citotoxicidade Imunológica/imunologia , Humanos , Imunoterapia , Macaca fascicularis , Masculino , Mucinas/imunologia , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AMG 596 is a bispecific T-cell engager (BiTE) immuno-oncology therapy in clinical development for treatment of glioblastoma multiforme (GBM), the most common primary brain tumor in adults with limited therapeutic options. AMG 596 is composed of two single-chain variable fragments that simultaneously bind to the tumor-specific antigen, EGFR variant III (EGFRvIII), on GBM cells and to CD3 on T cells, thereby activating T cells to proliferate and secrete cytotoxic substances that induce lysis of the bound tumor cell. T-cell-redirected lysis by AMG 596 is very potent; in vitro studies revealed EC50 values in the low picomolar range, and in vivo studies showed that AMG 596 treatment significantly increased the overall survival of mice bearing EGFRvIII-expressing orthotopic tumors. In addition, AMG 596 activity is highly specific; no AMG 596-induced T-cell activity can be observed in assays with EGFRvIII-negative GBM cells, and no signs of toxicity and activity were observed in cynomolgus monkeys, which lack expression of EGFRvIII on normal tissues. With EGFRvIII-expressing GBM cells, we showed shedding of EGFRvIII-containing membrane vesicles, followed by vesicle uptake and EGFRvIII cell surface presentation by EGFRvIII noncoding GBM cells. Cell membrane presentation of EGFRvIII following microvesicle transfer allows engagement by AMG 596, resulting in T-cell activation and T-cell-dependent lysis of GBM cells. Together, these data show a compelling preclinical efficacy and safety profile of AMG 596, supporting its development as a novel immunotherapy for treatment of GBM.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Imunoterapia/métodos , Animais , Anticorpos Biespecíficos/farmacologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Receptores ErbB , Glioblastoma/patologia , Humanos , CamundongosRESUMO
PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) remains a disease with high unmet medical need, as most patients do not achieve durable response with available treatments. Prostate-specific membrane antigen (PSMA) is a compelling target for mCRPC. It is highly expressed by primary and metastatic prostate cancer cells, with increased expression after progression on androgen deprivation therapy. EXPERIMENTAL DESIGN: We developed AMG 160, a half-life extended, bispecific T-cell engager immuno-oncology therapy that binds PSMA on prostate cancer cells and cluster of differentiation 3 on T cells for treatment of mCRPC. AMG 160 was evaluated in vitro and in mCRPC xenograft models. AMG 160 tolerability was assessed in nonhuman primates (NHP). AMG 160 activity as monotherapy and in combination with a PSMA-imaging agent, novel hormonal therapy, and immune checkpoint blockade was evaluated. RESULTS: AMG 160 induces potent, specific killing of PSMA-expressing prostate cancer cell lines in vitro, with half-maximal lysis of 6-42 pmol/L. In vivo, AMG 160 administered weekly at 0.2 mg/kg engages T cells administered systemically and promotes regression of established 22Rv-1 mCRPC xenograft tumors. AMG 160 is compatible with the imaging agent gallium 68-labeled PSMA-11, and shows enhanced cytotoxic activity when combined with enzalutamide or an anti-programmed death-1 antibody. AMG 160 exhibits an extended half-life and has an acceptable safety profile in NHPs. CONCLUSIONS: The preclinical characterization of AMG 160 highlights its potent antitumor activity in vitro and in vivo, and its potential for use with known diagnostic or therapeutic agents in mCRPC. These data support the ongoing clinical evaluation of AMG 160 in patients with mCRPC.See related commentary by Kamat et al., p. 2675.
Assuntos
Transferência Adotiva/métodos , Antígenos de Superfície/imunologia , Glutamato Carboxipeptidase II/imunologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linfócitos T/imunologia , Animais , Complexo CD3/antagonistas & inibidores , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Glutamato Carboxipeptidase II/antagonistas & inibidores , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Neoplasias de Próstata Resistentes à Castração/patologia , Linfócitos T/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. SIGNIFICANCE: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.
Assuntos
Mutação , Síndromes Mielodisplásicas/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Fator de Processamento U2AF/genética , Ciclo Celular , Linhagem Celular Tumoral , Instabilidade Cromossômica , Corantes Fluorescentes , Regulação da Expressão Gênica , Genes Reporter , Estudo de Associação Genômica Ampla , Humanos , Células K562 , Proteínas de Ligação a RNA , RNA-Seq , Ribonuclease H/metabolismo , SpliceossomosRESUMO
PURPOSE: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. EXPERIMENTAL DESIGN: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). RESULTS: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients. CONCLUSIONS: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.
Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares , Proteínas de Membrana , Carcinoma de Pequenas Células do Pulmão , Linfócitos T , Animais , Feminino , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/antagonistas & inibidores , Camundongos Endogâmicos NOD , Camundongos SCID , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers. Despite high rates of response to first-line chemotherapy and radiotherapy, patients with extensive-stage disease eventually relapse, and very few patients survive more than 5 years from diagnosis. Treatment options for recurrent or refractory disease are limited, and the treatments that do exist are associated with significant treatment-related toxicities. Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is highly expressed in SCLC and other neuroendocrine tumors but minimally expressed in normal tissues. It is therefore being explored as a potential therapeutic target in SCLC. Here, we review the preclinical and clinical evidence for targeting DLL3 in SCLC and discuss several DLL3-specific therapies being developed for the treatment of SCLC: the antibody-drug conjugate rovalpituzumab tesirine, the bispecific T cell engager immuno-oncology therapy AMG 757, and the chimeric antigen receptor T cell therapy AMG 119.