RESUMEN
Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG-binding protein 2 (MECP2). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), â¼50% of cells express mutant MECP2 and the other â¼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked MECP2 in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called "XCI factors" or "XCIFs"). Whether XCIF inhibitors in living animals can reactivate Xi-linked MECP2 in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked MECP2 in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked MECP2 corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked Mecp2 in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked Mecp2 in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked MECP2 as a therapeutic approach for RTT.
Asunto(s)
Corteza Cerebral/metabolismo , Proteína 2 de Unión a Metil-CpG , Mutación , Neuronas/metabolismo , Síndrome de Rett/metabolismo , Animales , Línea Celular , Corteza Cerebral/patología , Humanos , Proteína 2 de Unión a Metil-CpG/biosíntesis , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Ratones Noqueados , Neuronas/patología , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/patologíaRESUMEN
Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.
Asunto(s)
Neoplasias Ováricas , Receptores Quiméricos de Antígenos , Humanos , Femenino , Epítopos , Receptor fas/genética , Receptor fas/metabolismo , Proteína Ligando Fas , Linfocitos T , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapia , Apoptosis , Anticuerpos/farmacologíaRESUMEN
INTRODUCTION: There are more than two dozen bispecific antibodies (BsAbs) in development with a variety of designs which are relevant to breast cancer. The field of BsAbs for breast cancer includes agents that co-direct immune recognition of the cancer cell, target unique cancer antigens, and target the microenvironment. BsAbs are being developed for use as antibody-drug conjugates and as homing signals for engineered T-cells. AREAS COVERED: This review covers potential targets for bispecific antibodies, agents in pre-clinical development, agents in clinical trials, combinatorial therapies, and future directions. EXPERT OPINION: There is no BsAb approval expected for breast cancer in the near term, but late-stage trials are underway. Future BsAb roles in breast cancer are possible given unmet needs in estrogen receptor+ disease, residual disease, and de-escalating chemotherapy use. The HER2+ space shows hints of success for BsAbs, but is already crowded. Areas of unmet need still exist.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias de la Mama , Anticuerpos Biespecíficos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Humanos , Inmunoterapia , Linfocitos T , Microambiente TumoralRESUMEN
The COVID-19 causing coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human lung cells via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). Notably, the cleavage of the spike by the host cell protease furin in virus-producing cells is critical for subsequent spike-driven entry into lung cells. Thus, effective targeted therapies blocking the spike cleavage and activation in viral producing cells may provide an alternate strategy to break the viral transmission cycle and to overcome disease pathology. Here we engineered and described an antibody-based targeted strategy, which directly competes with the furin mediated proteolytic activation of the spike in virus-producing cells. The described approach involves engineering competitive furin substrate residues in the IgG1 Fc-extended flexible linker domain of SARS-CoV-2 spike targeting antibodies. Considering the site of spike furin cleavage and SARS-CoV-2 egress remains uncertain, the experimental strategy pursued here revealed novel mechanistic insights into proteolytic processing of the spike protein, which suggest that processing does not occur in the constitutive secretory pathway. Furthermore, our results show blockade of furin-mediated cleavage of the spike protein for membrane fusion activation and virus host-cell entry function. These findings provide an alternate insight of targeting applicability to SARS-CoV-2 and the future coronaviridae family members, exploiting the host protease system to gain cellular entry and subsequent chain of infections. IMPORTANCE Since its emergence in December 2019, COVID-19 has remained a global economic and health threat. Although RNA and DNA vector-based vaccines induced antibody response and immunological memory have proven highly effective against hospitalization and mortality, their long-term efficacy remains unknown against continuously evolving SARS-CoV-2 variants. As host cell-enriched furin-mediated cleavage of SARS-CoV-2 spike protein is critical for viral entry and chain of the infection cycle, the solution described here of an antibody Fc-conjugated furin competing peptide is significant. In a scenario where spike mutational drifts do not interfere with the Fc-conjugated antibody's epitope, the proposed furin competing strategy confers a broad-spectrum targeting design to impede the production of efficiently transmissible SARS-CoV-2 viral particles. In addition, the proposed approach is plug-and-play against other potentially deadly viruses that exploit secretory pathway independent host protease machinery to gain cellular entry and subsequent transmissions to host cells.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/enzimología , COVID-19/virología , Furina/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/inmunología , Secuencia de Aminoácidos , COVID-19/genética , Furina/genética , Interacciones Huésped-Patógeno , Humanos , Proteolisis , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del VirusRESUMEN
The long-held paradigm that tumor suppressors are un-targetable in cancer therapy is challenged by a study published in Science. This recent work elegantly describes and characterizes a p53 mutant peptide-selective TCR-mimic antibody and its co-targeting T cell-activating bispecific antibody to eliminate neoantigen-expressing tumors.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Humanos , Linfocitos TRESUMEN
Receptor clustering is the first and critical step to activate apoptosis by death receptor-5 (DR5). The recent discovery of the autoinhibitory DR5 ectodomain has challenged the long-standing view of its mechanistic activation by the natural ligand Apo2L. Because the autoinhibitory residues have remained unknown, here we characterize a crucial patch of positively charged residues (PPCR) in the highly variable domain of DR5. The PPCR electrostatically separates DR5 receptors to autoinhibit their clustering in the absence of ligand and antibody binding. Mutational substitution and antibody-mediated PPCR interference resulted in increased apoptotic cytotoxic function. A dually specific antibody that enables sustained tampering with PPCR function exceptionally enhanced DR5 clustering and apoptotic activation and distinctively improved the survival of animals bearing aggressive metastatic and recurrent tumors, whereas clinically tested DR5 antibodies without PPCR blockade function were largely ineffective. Our study provides mechanistic insights into DR5 activation and a therapeutic analytical design for potential clinical success.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Células A549 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Especificidad de Anticuerpos , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/metabolismo , Epítopos , Humanos , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lack of effective immune infiltration represents a significant barrier to immunotherapy in solid tumors. Thus, solid tumor-enriched death receptor-5 (DR5) activating antibodies, which generates tumor debulking by extrinsic apoptotic cytotoxicity, remains a crucial alternate therapeutic strategy. Over past few decades, many DR5 antibodies moved to clinical trials after successfully controlling tumors in immunodeficient tumor xenografts. However, DR5 antibodies failed to significantly improve survival in phase-II trials, leading in efforts to generate second generation of DR5 agonists to supersize apoptotic cytotoxicity in tumors. Here we have discovered that clinical DR5 antibodies activate an unexpected immunosuppressive PD-L1 stabilization pathway, which potentially had contributed to their limited success in clinics. The DR5 agonist stimulated caspase-8 signaling not only activates ROCK1 but also undermines proteasome function, both of which contributes to increased PD-L1 stability on tumor cell surface. Targeting DR5-ROCK1-PD-L1 axis markedly increases immune effector T-cell function, promotes tumor regression, and improves overall survival in animal models. These insights have identified a potential clinically viable combinatorial strategy to revive solid cancer immunotherapy using death receptor agonism.
Asunto(s)
Antígeno B7-H1 , Neoplasias de la Mama Triple Negativas , Animales , Anticuerpos Monoclonales , Humanos , Evasión Inmune , Inmunoterapia , Quinasas Asociadas a rhoRESUMEN
Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Neoplasias/terapia , Animales , Células CHO , Línea Celular Tumoral , Cricetulus , Humanos , Inmunoterapia , Ratones , Proteína Estafilocócica A , Distribución TisularRESUMEN
The majority of clinical deaths in patients with triple-negative breast cancer (TNBC) are due to chemoresistance and aggressive metastases, with high prevalence in younger women of African ethnicity. Although tumorigenic drivers are numerous and varied, the drivers of metastatic transition remain largely unknown. Here, we uncovered a molecular dependence of TNBC tumors on the TRIM37 network, which enables tumor cells to resist chemotherapeutic as well as metastatic stress. TRIM37-directed histone H2A monoubiquitination enforces changes in DNA repair that rendered TP53-mutant TNBC cells resistant to chemotherapy. Chemotherapeutic drugs triggered a positive feedback loop via ATM/E2F1/STAT signaling, amplifying the TRIM37 network in chemoresistant cancer cells. High expression of TRIM37 induced transcriptomic changes characteristic of a metastatic phenotype, and inhibition of TRIM37 substantially reduced the in vivo propensity of TNBC cells. Selective delivery of TRIM37-specific antisense oligonucleotides using antifolate receptor 1-conjugated nanoparticles in combination with chemotherapy suppressed lung metastasis in spontaneous metastatic murine models. Collectively, these findings establish TRIM37 as a clinically relevant target with opportunities for therapeutic intervention. SIGNIFICANCE: TRIM37 drives aggressive TNBC biology by promoting resistance to chemotherapy and inducing a prometastatic transcriptional program; inhibition of TRIM37 increases chemotherapy efficacy and reduces metastasis risk in patients with TNBC.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Proteínas de Motivos Tripartitos/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The structural basis of blocking human epidermal growth factor receptor-2 (HER2) dimerization remains of great interest to generate effective anti-cancer therapies. Despite clinically feasible outcome in mammary tumors, a fine consensus between efficacy and safety remains a critical challenge beyond breast cancer. Here we extrapolate on the balancing act using recently reported clinical findings in salivary ductal carcinomas.
RESUMEN
Therapeutic antibodies targeting ovarian cancer (OvCa)-enriched receptors have largely been disappointing due to limited tumor-specific antibody-dependent cellular cytotoxicity. Here we report a symbiotic approach that is highly selective and superior compared with investigational clinical antibodies. This bispecific-anchored cytotoxicity activator antibody is rationally designed to instigate "cis" and "trans" cytotoxicity by combining specificities against folate receptor alpha-1 (FOLR1) and death receptor 5 (DR5). Whereas the in vivo agonist DR5 signaling requires FcγRIIB interaction, the FOLR1 anchor functions as a primary clustering point to retain and maintain a high level of tumor-specific apoptosis. The presented proof of concept study strategically makes use of a tumor cell-enriched anchor receptor for agonist death receptor targeting to potentially generate a clinically viable strategy for OvCa.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Receptor 1 de Folato/antagonistas & inhibidores , Neoplasias Ováricas/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/patología , Receptores de IgG/fisiologíaRESUMEN
INTRODUCTION: Generating effective RNAi-based therapies with the potential to achieve leukemia remission remains critical unmet need. Despite a growing number of leukemia clinical trials, tissue specific delivery of therapeutic siRNA is a major roadblock in translating its clinical potential. The most recent reports in the antibody-siRNA-conjugates (ARCs) field add new dimensions to leukemic therapy, where a covalently ligated therapeutic antisense-RNA with the potential to repress the oncogenic transcript is selectively delivered into the cancer cells. Despite ARC localization to leukemic cells due to high affinity antigen-antibody interactions, multiple challenges exist to unlock the therapeutic potential of siRNA targeting. Areas covered: This review focuses on antibody and siRNA-based therapies for leukemia as well as potential antibody engineering-based strategies to generate an optimal ARC platform. Expert opinion: In vitro and clinical results have revealed that non-targeted delivery and inefficient cellular internalization of therapeutic siRNA are major contributing factors for the lack of efficacy in leukemia patients. Rational antibody design and selective protein engineering with the potential to neutralize siRNA charge, stabilize ARC complex, restrict off-targeted delivery, optimize endosomal escape, and extend serum half-life will generate clinically relevant leukemic therapies that are safe, selective, and effective.
Asunto(s)
Anticuerpos/uso terapéutico , Inmunoconjugados/uso terapéutico , Leucemia/tratamiento farmacológico , ARN Interferente Pequeño/uso terapéutico , Animales , Anticuerpos/inmunología , Humanos , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Leucemia/genética , Leucemia/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
In mammals, gene expression is largely controlled at the transcriptional level. In response to environmental or intrinsic signaling, gene expression is often fine-tuned by epigenetic modifications, including DNA methylation and histone modifications. One such histone modification is ubiquitination that predominately occurs in mono-ubiquitinated forms on histone H2A and H2B. We recently identified and characterized a novel E3 ligase called TRIM37 that ubiquitinates H2A. This study highlights the consequence of aberrant histone ubiquitination at the promoters of tumor suppressor genes in breast cancer. Regulatory mechanism by which TRIM37 and other auxiliary proteins are involved in the initiation and progression of breast cancer is of utmost importance toward generating effective therapeutics. Here, we describe a detailed step-by-step process of carrying out in vitro ubiquitination assay using purified histone proteins or reconstituted nucleosomes and affinity-purified recombinant E3 ligase like TRIM37. These experimental procedures are largely based on our studies in mammalian cells and will be a useful tool to identify substrate for E3 ubiquitin ligase as well as characterizing new E3 ligases.