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The field of chemical modification of proteins has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently, we have developed oxaziridine chemistry for highly selective modification of methionine called redox-activated chemical tagging (ReACT) but have not broadly tested the molecular parameters for efficient and stable protein modification. Here we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, used for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and excellent reaction efficiencies with a panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than 10-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, presumably because of their reduced access to water. There was also a 10-fold variation in stability depending on the nature of the oxaziridine, which was determined to be inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs were sufficient to support their use as antibody drug conjugates and potent in a breast cancer mouse xenograft model over a month. These studies provide key parameters for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation to native or engineered methionines.
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Aziridinas/análisis , Inmunoconjugados/química , Metionina/análisis , Animales , Antineoplásicos/normas , Línea Celular Tumoral , Estabilidad de Medicamentos , Femenino , Humanos , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Ratones , Ratones Desnudos , Ingeniería de Proteínas/métodos , Estabilidad ProteicaRESUMEN
Alternative splicing of the androgen receptor (AR) is frequently observed in castration resistant prostate cancer (CRPC). One AR isoform, the AR-V7 splice variant, is a constitutively active transcription factor which lacks a ligand binding domain and is therefore undruggable. AR-V7 expression correlates with resistance to androgen receptor signaling inhibitors (ARSi) and poor clinical prognoses. The occurrence of the AR-V7 splice variant is driven by alternative splicing of AR pre-mRNA by the spliceosome, however the mechanistic details are poorly understood. We demonstrate that the splicing factor RBM39 is critical for alternative splicing of the AR-V7 splice variant mRNA transcripts from AR pre-mRNA, and that the anti-cancer drug, indisulam, reduces AR-V7 mRNA levels by degrading RBM39. We report that indisulam effectively reduces AR-V7 in in vitro and in vivo models.
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Antineoplásicos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/genética , Sulfonamidas/farmacología , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Androgénicos/metabolismo , Relación Estructura-Actividad , Sulfonamidas/química , Células Tumorales CultivadasRESUMEN
High-grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle-tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re-establishing endothelial barrier integrity. LIGHT-mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK-LIGHT with anti-vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T-cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide-binding activities in mouse and human primary brain tumour vessels. Thus, peptide-mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Inhibidores de la Angiogénesis/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos , Glioblastoma/tratamiento farmacológico , Neovascularización Patológica , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/farmacología , Remodelación Vascular/efectos de los fármacos , Vénulas/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/metabolismo , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular , Composición de Medicamentos , Femenino , Glioblastoma/sangre , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Micelas , Persona de Mediana Edad , Terapia Molecular Dirigida , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Pericitos/patología , Fenotipo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Vénulas/metabolismo , Vénulas/patologíaRESUMEN
RATIONALE: The dramatic upregulation of αvß3-integrin that occurs in the vasculature during tumor growth has long suggested that the endothelial expression of this molecule is an ideal target for antiangiogenic therapy to treat cancer. This discovery led to the development of small-molecule inhibitors directed against αvß3-integrin that are currently in clinical trials. In 2002, we reported that ß3-integrin-knockout mice exhibit enhanced tumor growth and angiogenesis. However, as ß3-integrin is expressed by a wide variety of cells, endothelial cell-specific contributions to tumor angiogenesis are muddied by the use of a global knockout of ß3-integrin function. OBJECTIVE: Our aim was to examine the endothelial-specific contribution ß3-integrin makes to tumor growth and angiogenesis. METHODS AND RESULTS: We have crossed ß3-integrin-floxed (ß3-floxed) mice to 2 endothelial-specific Cre models and examined angiogenic responses in vivo, ex vivo, and in vitro. We show that acute depletion of endothelial ß3-integrin inhibits tumor growth and angiogenesis preventatively, but not in already established tumors. However, the effects are transient, and long-term depletion of the molecule is ineffective. Furthermore, long-term depletion of the molecule correlates with many molecular changes, such as reduced levels of focal adhesion kinase expression and a misbalance in focal adhesion kinase phosphorylation, which may lead to a release from the inhibitory effects of decreased endothelial ß3-integrin expression. CONCLUSIONS: Our findings imply that timing and length of inhibition are critical factors that need to be considered when targeting the endothelial expression of ß3-integrin to inhibit tumor growth and angiogenesis.
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Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Integrina alfaVbeta3/genética , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Células Endoteliales/patología , Endotelio Vascular/patología , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrina alfaVbeta3/metabolismo , Pulmón/irrigación sanguínea , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neovascularización Patológica/patologíaRESUMEN
For nearly two decades now, the RGD (Arg-Gly-Asp)-binding αvß3-integrin has been a focus of anti-angiogenic drug design. These inhibitors are well-tolerated, but have shown only limited success in patients. Over the years, studies in ß3-integrin-knockout mice have shed some light on possible explanations for disappointing clinical outcomes. However, studying angiogenesis in ß3-integrin-knockout mice is a blunt tool to investigate ß3-integrin's role in pathological angiogenesis. Since establishing our laboratory at University of East Anglia (UEA), we have adopted more refined models of genetically manipulating the expression of the ß3-integrin subunit. The present review will highlight some of our findings from these models and describe how data from them have forced us to rethink how targeting αvß3-integrin expression affects tumour angiogenesis and cancer progression. Revisiting the fundamental biology behind how this integrin regulates tumour growth and angiogenesis, we believe, is the key not only to understanding how angiogenesis is normally co-ordinated, but also in success with drugs directed against it.
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Endotelio Vascular/metabolismo , Integrinas/fisiología , Humanos , Integrinas/metabolismoRESUMEN
New epitopes for immune recognition provide the basis of anticancer immunity. Due to the high concentration of extracellular adenosine triphosphate in the tumor microenvironment, we hypothesized that extracellular kinases (ectokinases) could have dysregulated activity and introduce aberrant phosphorylation sites on cell surface proteins. We engineered a cell-tethered version of the extracellular kinase CK2α, demonstrated it was active on cells under tumor-relevant conditions, and profiled its substrate scope using a chemoproteomic workflow. We then demonstrated that mice developed polyreactive antisera in response to syngeneic tumor cells that had been subjected to surface hyperphosphorylation with CK2α. Interestingly, these mice developed B cell and CD4+ T cell responses in response to these antigens but failed to develop a CD8+ T cell response. This work provides a workflow for probing the extracellular phosphoproteome and demonstrates that extracellular phosphoproteins are immunogenic even in a syngeneic system.
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PURPOSE: Multiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [89Zr]Zr-DFO-YS5 for imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [89Zr]Zr-DFO-YS5 for immunoPET imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models. EXPERIMENTAL DESIGN: In vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [89Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [225Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity. RESULTS: [89Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (Kd = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [89Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [225Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups. CONCLUSIONS: Our study showed that the CD46-targeted probe [89Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [225Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.
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Mieloma Múltiple , Masculino , Humanos , Animales , Ratones , Mieloma Múltiple/diagnóstico por imagen , Mieloma Múltiple/tratamiento farmacológico , Medicina de Precisión , Actinio , Radioisótopos , Radiofármacos , Circonio , Línea Celular Tumoral , Tomografía Computarizada por Tomografía de Emisión de Positrones , Anticuerpos , Proteína Cofactora de MembranaRESUMEN
Despite the success of BCMA-targeting CAR-Ts in multiple myeloma, patients with high-risk cytogenetic features still relapse most quickly and are in urgent need of additional therapeutic options. Here, we identify CD70, widely recognized as a favorable immunotherapy target in other cancers, as a specifically upregulated cell surface antigen in high risk myeloma tumors. We use a structure-guided design to define a CD27-based anti-CD70 CAR-T design that outperforms all tested scFv-based CARs, leading to >80-fold improved CAR-T expansion in vivo. Epigenetic analysis via machine learning predicts key transcription factors and transcriptional networks driving CD70 upregulation in high risk myeloma. Dual-targeting CAR-Ts against either CD70 or BCMA demonstrate a potential strategy to avoid antigen escape-mediated resistance. Together, these findings support the promise of targeting CD70 with optimized CAR-Ts in myeloma as well as future clinical translation of this approach.
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Targeted degradation of cell surface and extracellular proteins via lysosomal delivery is an important means to modulate extracellular biology. However, these approaches have limitations due to lack of modularity, ease of development, restricted tissue targeting and applicability to both cell surface and extracellular proteins. We describe a lysosomal degradation strategy, termed cytokine receptor-targeting chimeras (KineTACs), that addresses these limitations. KineTACs are fully genetically encoded bispecific antibodies consisting of a cytokine arm, which binds its cognate cytokine receptor, and a target-binding arm for the protein of interest. We show that KineTACs containing the cytokine CXCL12 can use the decoy recycling receptor, CXCR7, to target a variety of target proteins to the lysosome for degradation. Additional KineTACs were designed to harness other CXCR7-targeting cytokines, CXCL11 and vMIPII, and the interleukin-2 (IL-2) receptor-targeting cytokine IL-2. Thus, KineTACs represent a general, modular, selective and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and cell surface targets with broad or tissue-specific distribution.
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Quimera Dirigida a la Proteólisis , Receptores de Citocinas , Membrana Celular , Interleucina-2 , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Transducción de Señal , Proteolisis , Quimiocina CXCL12/químicaRESUMEN
Mutations in the receptor tyrosine kinases (RTKs) FLT3 and KIT are frequent and associated with poor outcomes in acute myeloid leukemia (AML). Although selective FLT3 inhibitors (FLT3i) are clinically effective, remissions are short-lived due to secondary resistance characterized by acquired mutations constitutively activating the RAS/MAPK pathway. Hereby, we report the pre-clinical efficacy of co-targeting SHP2, a critical node in MAPK signaling, and BCL2 in RTK-driven AML. The allosteric SHP2 inhibitor RMC-4550 suppresses proliferation of AML cell lines with FLT3 and KIT mutations, including cell lines with acquired resistance to FLT3i. We demonstrate that pharmacologic SHP2 inhibition unveils an Achilles' heel of RTK-driven AML, increasing apoptotic dependency on BCL2 via MAPK-dependent mechanisms, including upregulation of BMF and downregulation of MCL1. Consequently, RMC-4550 and venetoclax are synergistically lethal in AML cell lines and in clinically relevant xenograft models. Our results provide mechanistic rationale and pre-clinical evidence for co-targeting SHP2 and BCL2 in RTK-driven AML.
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Apoptosis , Leucemia Mieloide Aguda , Humanos , Línea Celular Tumoral , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/farmacologíaRESUMEN
BACKGROUND: Approximately 50% of patients who receive anti-CD19 CAR-T cells relapse, and new immunotherapeutic targets are urgently needed. We recently described CD72 as a promising target in B-cell malignancies and developed nanobody-based CAR-T cells (nanoCARs) against it. This cellular therapy design is understudied compared with scFv-based CAR-T cells, but has recently become of significant interest given the first regulatory approval of a nanoCAR in multiple myeloma. METHODS: We humanized our previous nanobody framework regions, derived from llama, to generate a series of humanized anti-CD72 nanobodies. These nanobody binders were inserted into second-generation CD72 CAR-T cells and were evaluated against preclinical models of B cell acute lymphoblastic leukemia and B cell non-Hodgkin's lymphoma in vitro and in vivo. Humanized CD72 nanoCARs were compared with parental ("NbD4") CD72 nanoCARs and the clinically approved CD19-directed CAR-T construct tisangenlecleucel. RNA-sequencing, flow cytometry, and cytokine secretion profiling were used to determine differences between the different CAR constructs. We then used affinity maturation on the parental NbD4 construct to generate high affinity binders against CD72 to test if higher affinity to CD72 improved antitumor potency. RESULTS: Toward clinical translation, here we humanize our previous nanobody framework regions, derived from llama, and surprisingly discover a clone ("H24") with enhanced potency against B-cell tumors, including patient-derived samples after CD19 CAR-T relapse. Potentially underpinning improved potency, H24 has moderately higher binding affinity to CD72 compared with a fully llama framework. However, further affinity maturation (KD<1 nM) did not lead to improvement in cytotoxicity. After treatment with H24 nanoCARs, in vivo relapse was accompanied by CD72 antigen downregulation which was partially reversible. The H24 nanobody clone was found to have no off-target binding and is therefore designated as a true clinical candidate. CONCLUSION: This work supports translation of H24 CD72 nanoCARs for refractory B-cell malignancies, reveals potential mechanisms of resistance, and unexpectedly demonstrates that nanoCAR potency can be improved by framework alterations alone. These findings may have implications for future engineering of nanobody-based cellular therapies.
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Linfoma de Burkitt , Camélidos del Nuevo Mundo , Receptores Quiméricos de Antígenos , Animales , Humanos , Inmunoterapia Adoptiva , Linfocitos T , Camélidos del Nuevo Mundo/metabolismo , Recurrencia , Antígenos de Diferenciación de Linfocitos B , Antígenos CDRESUMEN
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.
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Proteínas de la Membrana , Neoplasias de la Próstata , Animales , Humanos , Masculino , Ratones , Anticuerpos Monoclonales , Inmunoterapia , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligandos , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/terapia , Neoplasias de la Próstata/patología , Circonio , Tumores Neuroendocrinos/diagnóstico por imagen , Tumores Neuroendocrinos/patología , Tumores Neuroendocrinos/terapiaRESUMEN
Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.
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Leucemia Mieloide Aguda , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T , Integrinas/metabolismo , Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/genéticaRESUMEN
The androgen receptor (AR) is a central driver of aggressive prostate cancer. After initial treatment with androgen receptor signaling inhibitors (ARSi), reactivation of AR signaling leads to resistance. Alternative splicing of AR mRNA yields the AR-V7 splice variant, which is currently an undruggable mechanism of ARSi resistance: AR-V7 lacks a ligand binding domain, where hormones and anti-androgen antagonists act, but still activates AR signaling. We reveal PKCß as a druggable regulator of transcription and splicing at the AR genomic locus. We identify a clinical PKCß inhibitor in combination with an FDA-approved anti-androgen as an approach for repressing AR genomic locus expression, including expression of AR-V7, while antagonizing full-length AR. PKCß inhibition reduces total AR gene expression, thus reducing AR-V7 protein levels and sensitizing prostate cancer cells to current anti-androgen therapies. We demonstrate that this combination may be a viable therapeutic strategy for AR-V7-positive prostate cancer.
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Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Proteína Quinasa C beta/metabolismo , Antagonistas de Andrógenos/farmacología , Antagonistas de Andrógenos/uso terapéutico , Resistencia a Medicamentos , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Empalme del ARN/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismoRESUMEN
Objective.The Office of Radiological Security, U.S. Department of Energy's National Nuclear Security Administration, is implementing a radiological risk reduction program which seeks to minimize or eliminate the use of high activity radiological sources, including137Cs, by replacing them with non-radioisotopic technologies, such as x-ray irradiators. The main goal of this paper is to evaluate the equivalence of the dose delivered by gamma- and x-ray irradiators in mice using experimental measurements and Monte Carlo simulations. We also propose a novel biophantom as anin situdose calibration method.Approach.We irradiated mouse carcasses and 3D-printed mouse biophantoms in a137Cs irradiator (Mark I-68) and an x-ray irradiator (X-Rad320) at three voltages (160 kVp, 225 kVp and 320 kVp) and measured the delivered radiation dose. A Geant4-based Monte Carlo model was developed and validated to provide a comprehensive picture of gamma- and x-ray irradiation in mice.Main Results.Our Monte Carlo model predicts a uniform dose delivered in soft-tissue for all the explored irradiation programs and in agreement with the absolute dose measurements. Our Monte Carlo model shows an energy-dependent difference between dose in bone and in soft tissue that decreases as photon energy increases. Dose rate depends on irradiator and photon energy. We observed a deviation of the measured dose from the target value of up to -9% for the Mark I-68, and up to 35% for the X-Rad320. The dose measured in the 3D-printed phantoms are equivalent to that in the carcasses within 6% uncertainty.Significance.Our results suggest that 320 kVp irradiation is a good candidate to substitute137Cs irradiation barring a few caveats. There is a significant difference between measured and targeted doses for x-ray irradiation that suggests a strong need forin situcalibration, which can be achieved with 3D-printed mouse biophantoms. A dose correction is necessary for bone doses, which can be provided by a Monte Carlo calculation. Finally, the biological implications of the differences in dose rates and dose per photon for the different irradiation methods should be carefully assessed for each small-animal irradiation experiment.
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Calibración , Animales , Ratones , Método de Montecarlo , Fantasmas de Imagen , Rayos XRESUMEN
Surface-targeting biotherapeutic agents have been successful in treating HER2-amplified cancers through immunostimulation or chemodelivery but have failed to produce effective inhibitors of constitutive HER2-HER3 signaling. We report an extensive structure-function analysis of this tumor driver, revealing complete uncoupling of intracellular signaling and tumorigenic function from regulation or constraints from their extracellular domains (ECDs). The canonical HER3 ECD conformational changes and exposure of the dimerization interface are nonessential, and the entire ECDs of HER2 and HER3 are redundant for tumorigenic signaling. Restricting the proximation of partner ECDs with bulk and steric clash through extremely disruptive receptor engineering leaves tumorigenic signaling unperturbed. This is likely due to considerable conformational flexibilities across the span of these receptor molecules and substantial undulations in the plane of the plasma membrane, none of which had been foreseen as impediments to targeting strategies. The massive overexpression of HER2 functionally and physically uncouples intracellular signaling from extracellular constraints.
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Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Carcinogénesis/efectos de los fármacos , Receptor ErbB-2/efectos de los fármacos , Receptor ErbB-3/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Trastuzumab/farmacologíaRESUMEN
Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance.
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Antineoplásicos , Antígenos de Histocompatibilidad Clase I/inmunología , Neoplasias , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Anticuerpos , Antineoplásicos/farmacología , Humanos , Factores Inmunológicos , Inmunoterapia , Péptidos/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Effective inactivation of the HER2-HER3 tumor driver has remained elusive because of the challenging attributes of the pseudokinase HER3. We report a structure-function study of constitutive HER2-HER3 signaling to identify opportunities for targeting. The allosteric activation of the HER2 kinase domain (KD) by the HER3 KD is required for tumorigenic signaling and can potentially be targeted by allosteric inhibitors. ATP binding within the catalytically inactive HER3 KD provides structural rigidity that is important for signaling, but this is mimicked, not opposed, by small molecule ATP analogs, reported here in a bosutinib-bound crystal structure. Mutational disruption of ATP binding and molecular dynamics simulation of the apo KD of HER3 identify a conformational coupling of the ATP pocket with a hydrophobic AP-2 pocket, analogous to EGFR, that is critical for tumorigenic signaling and feasible for targeting. The value of these potential target sites is confirmed in tumor growth assays using gene replacement techniques.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinogénesis/efectos de los fármacos , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Compuestos de Anilina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Nitrilos/farmacología , Quinolinas/farmacología , Receptor ErbB-2/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.
Asunto(s)
Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/inmunología , Epítopos/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/inmunología , Neoplasias Pancreáticas/inmunología , Proteolisis , Animales , Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Epítopos/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Pancreáticas/genéticaRESUMEN
Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.