RESUMEN
Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.
Asunto(s)
ARN Interferente Pequeño/administración & dosificación , Proteínas de Unión al ARN/administración & dosificación , Secuencia de Aminoácidos , Fenómenos Biofísicos , Línea Celular , Citosol/metabolismo , Sistemas de Liberación de Medicamentos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Marcación de Gen/métodos , Humanos , Modelos Moleculares , Conformación Proteica , Ingeniería de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/farmacocinética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Proteínas Virales/administración & dosificación , Proteínas Virales/genética , Proteínas Virales/farmacocinéticaRESUMEN
Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47-SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Antígeno CD47/efectos de los fármacos , Antígeno CD47/inmunología , Vacunas contra el Cáncer/administración & dosificación , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Inmunidad Adaptativa/efectos de los fármacos , Inmunidad Adaptativa/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/inmunología , Vacunas contra el Cáncer/inmunología , Femenino , Inmunización/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Terapia Molecular Dirigida/métodos , Neoplasias Experimentales/patología , Resultado del TratamientoRESUMEN
Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacologic efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Mol Cancer Ther; 17(8); 1773-80. ©2018 AACR.
Asunto(s)
Neoplasias/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias/patología , Transducción de SeñalRESUMEN
We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Fibronectinas/química , Melanoma Experimental/tratamiento farmacológico , Ingeniería de Proteínas/métodos , Receptores de IgG/inmunología , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Células HEK293 , Humanos , Melanoma Experimental/inmunología , Ratones , Modelos Moleculares , Fagocitosis , Receptores de IgG/química , Homología Estructural de Proteína , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ras is at the hub of signal transduction pathways controlling cell proliferation and survival. Its mutants, present in about 30% of human cancers, are major drivers of oncogenesis and render tumors unresponsive to standard therapies. Here we report the engineering of a protein scaffold for preferential binding to K-Ras G12D. This is the first reported inhibitor to achieve nanomolar affinity while exhibiting specificity for mutant over wild type (WT) K-Ras. Crystal structures of the protein R11.1.6 in complex with K-Ras WT and K-Ras G12D offer insight into the structural basis for specificity, highlighting differences in the switch I conformation as the major defining element in the higher affinity interaction. R11.1.6 directly blocks interaction with Raf and reduces signaling through the Raf/MEK/ERK pathway. Our results support greater consideration of the state of switch I and provide a novel tool to study Ras biology. Most importantly, this work makes an unprecedented contribution to Ras research in inhibitor development strategy by revealing details of a targetable binding surface. Unlike the polar interfaces found for Ras/effector interactions, the K-Ras/R11.1.6 complex reveals an extensive hydrophobic interface that can serve as a template to advance the development of high affinity, non-covalent inhibitors of K-Ras oncogenic mutants.
Asunto(s)
Ingeniería de Proteínas , Proteínas Recombinantes/farmacología , Proteínas ras/antagonistas & inhibidores , Secuencia de Aminoácidos , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas ras/metabolismoRESUMEN
Early stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few "false negatives" (assuming a complex cell extract as the "true" value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Proteínas de Choque Térmico/inmunología , Animales , Reacciones Cruzadas/inmunología , Humanos , RatonesRESUMEN
Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti-PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.
Asunto(s)
Inmunidad Adaptativa , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Inmunoterapia , Integrinas/metabolismo , Inmunidad Adaptativa/efectos de los fármacos , Animales , Formación de Anticuerpos/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Neoplasias del Colon/patología , Reacciones Cruzadas/efectos de los fármacos , Reacciones Cruzadas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Inflamación/patología , Interleucina-2/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos/metabolismo , Receptores de IgG/metabolismo , Albúmina Sérica/metabolismo , Especificidad de la Especie , Distribución Tisular/efectos de los fármacos , Resultado del TratamientoRESUMEN
Numerous synergistic cancer immunotherapy combinations have been identified, but the effects of relative dose timing are rarely considered. In established syngeneic mouse tumor models, we found that staggering interferon-α (IFNα) administration after, rather than before or simultaneously with, serum-persistent interleukin-2 (IL-2) and tumor-specific antibody significantly increased long-term survival. Successful combination therapy required IFNα-induced activation of cross-presenting CD8α+ dendritic cells (DCs) following the release of antigenic tumor debris by the IL-2- and antibody-mediated immune response. Due to decreased phagocytic ability post-maturation, DCs activated too early captured less antigen and could not effectively prime CD8+ T cells. Temporally programming DC activation to occur after tumoricidal activity enhanced tumor control by multiple distinct combination immunotherapies, highlighting dose schedule as an underappreciated factor that can profoundly affect the success of multi-component immunotherapies.
Asunto(s)
Interferón-alfa/inmunología , Interleucina-2/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Células Dendríticas/inmunología , Células Dendríticas/patología , Modelos Animales de Enfermedad , Humanos , Inmunoterapia , Interferón-alfa/administración & dosificación , Interleucina-2/administración & dosificación , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Melanoma Experimental/patología , RatonesRESUMEN
Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8+ T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.
Asunto(s)
Antineoplásicos/farmacología , Vacunas contra el Cáncer/farmacología , Citocinas/efectos de los fármacos , Inmunoterapia/métodos , Interleucina-2/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Inmunidad Adaptativa , Animales , Línea Celular Tumoral , Citocinas/inmunología , Quimioterapia Combinada , Citometría de Flujo , Técnicas de Inactivación de Genes , Inmunidad Innata , Immunoblotting , Oxidorreductasas Intramoleculares/genética , Ratones , Linfocitos T/inmunologíaRESUMEN
Cancer immunotherapies under development have generally focused on either stimulating T cell immunity or driving antibody-directed effector functions of the innate immune system such as antibody-dependent cell-mediated cytotoxicity (ADCC). We find that a combination of an anti-tumor antigen antibody and an untargeted IL-2 fusion protein with delayed systemic clearance induces significant tumor control in aggressive isogenic tumor models via a concerted innate and adaptive response involving neutrophils, NK cells, macrophages, and CD8(+) T cells. This combination therapy induces an intratumoral "cytokine storm" and extensive lymphocyte infiltration. Adoptive transfer of anti-tumor T cells together with this combination therapy leads to robust cures of established tumors and development of immunological memory.