RESUMEN

Lipid nanoparticle (LNP)-encapsulated mRNA has been used for in vivo production of several secreted protein classes, such as IgG, and has enabled the development of personalized vaccines in oncology. Establishing the feasibility of delivering complex multispecific modalities that require higher-order structures important for their function could help expand the use of mRNA/LNP biologic formulations. Here, we evaluated whether in vivo administration of mRNA/LNP formulations of SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT could achieve oligomerization and extend exposure, on-target activity, and antitumor responses comparable with that of the corresponding recombinant fusion proteins. Intravenous infusion of the formulated LNP-encapsulated mRNAs led to rapid and sustained production of functional hexameric proteins in vivo, which increased the overall exposure relative to the recombinant protein controls by ∼28 to 140 fold over 96 hours. High concentrations of the mRNA-encoded proteins were also observed in secondary lymphoid organs and within implanted tumors, with protein concentrations in tumors up to 134-fold greater than with the recombinant protein controls 24 hours after treatment. In addition, SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT mRNAs induced a greater increase in antigen-specific CD8+ T cells in the tumors. These mRNA/LNP formulations were well tolerated and led to a rapid increase in serum and intratumoral IL2, delayed tumor growth, extended survival, and outperformed the activities of benchmark mAb controls. Furthermore, the mRNA/LNPs demonstrated improved efficacy in combination with anti-PD-L1 relative to the recombinant fusion proteins. These data support the delivery of complex oligomeric biologics as mRNA/LNP formulations, where high therapeutic expression and exposure could translate into improved patient outcomes. SIGNIFICANCE: Lipid nanoparticle-encapsulated mRNA can efficiently encode complex fusion proteins encompassing immune checkpoint blockers and costimulators that functionally oligomerize in vivo with extended pharmacokinetics and durable exposure to induce potent antitumor immunity.

Asunto(s)

Nanopartículas , ARN Mensajero , Proteínas Recombinantes de Fusión , Animales , Ratones , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Nanopartículas/química , Humanos , Femenino , Ratones Endogámicos C57BL , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Lípidos/química , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Línea Celular Tumoral

RESUMEN

Coinhibition of TIGIT (T cell immunoreceptor with Ig and ITIM domains) and PD-1/PD-L1 (PD-1/L1) may improve response rates compared with monotherapy PD-1/L1 blockade in checkpoint naive non-small cell lung cancer with PD-L1 expression >50%. TIGIT mAbs with an effector-competent Fc can induce myeloid cell activation, and some have demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT Ab blockade translates to antitumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Furthermore, DNAM-1 is downregulated on tumor-infiltrating lymphocytes (TILs) in advanced and checkpoint inhibition-resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or checkpoint inhibition-resistant tumors, may be induced by immune costimulation that operates independently from PD-1/L1 inhibition. TNFSF14 (LIGHT) was identified through genomic screens, in vitro functional analysis, and immune profiling of TILs as a TNF ligand that could provide broad immune activation. Accordingly, murine and human bifunctional fusion proteins were engineered linking the extracellular domain of TIGIT to the extracellular domain of LIGHT, yielding TIGIT-Fc-LIGHT. TIGIT competitively inhibited binding to all PVR ligands. LIGHT directly activated myeloid cells through interactions with LTßR (lymphotoxin ß receptor), without the requirement for a competent Fc domain to engage Fcγ receptors. LIGHT costimulated CD8+ T and NK cells through HVEM (herpes virus entry mediator A). Importantly, HVEM was more widely expressed than DNAM-1 on T memory stem cells and TILs across a range of tumor types. Taken together, the mechanisms of TIGIT-Fc-LIGHT promoted strong antitumor activity in preclinical tumor models of primary and acquired resistance to PD-1 blockade, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Antígeno B7-H1/genética , Humanos , Ratones , Células Mieloides/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Inmunológicos , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética

RESUMEN

BACKGROUND: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DESIGN: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. RESULTS: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. CONCLUSIONS: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Cuello del Útero/inmunología , ADN-Topoisomerasas de Tipo II/inmunología , Proteínas de Unión al ADN/inmunología , Inmunohistoquímica , Componente 2 del Complejo de Mantenimiento de Minicromosoma/inmunología , Fase S , Análisis de Matrices Tisulares/métodos , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Biopsia , Western Blotting , Núcleo Celular/enzimología , Núcleo Celular/inmunología , Núcleo Celular/patología , Cuello del Útero/enzimología , Cuello del Útero/patología , Mapeo Epitopo/métodos , Epítopos , Femenino , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Valor Predictivo de las Pruebas , Índice de Severidad de la Enfermedad , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/inmunología , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/patología

RESUMEN

The chromosomal translocation t(8;21) often found in acute myeloid leukemia generates an oncogenic fusion protein AML1-ETO. This chimeric oncoprotein disrupts wild-type AML1 function and dysregulates genes important for normal myelopoiesis. Monoclonal antibodies that can capture and detect the AML1-ETO fusion protein would help with early diagnosis and treatment prognosis of acute myeloid leukemia. We report the development of murine monoclonal antibodies (MAbs) that specifically bind epitopes encoded by either AML1 or ETO. Since alignment to the human ETO protein indicated almost 100% homology to the mouse ortholog, a strategy was needed to instruct humoral immunity in mice to focus and respond to self-epitopes. Our strategy to develop capture/detector reagents involved producing MAbs that would bind to epitopes within the non-fused myelopic protein (i.e., either AML1 or ETO). This included a process to select antibodies for their ability to also recognize the translocated chromosomal AML1-ETO fusion protein and to identify complementary capture/detector antibody pairs. Construction of a peptide hapten-carrier complex and use of a rapid immunization protocol resulted in IgM-IgG ETO specific MAbs. These MAbs bound specifically to a recombinant form of AML1-ETO fusion protein expressed in HEK and to an endogenous AML1-ETO form of the fusion protein expressed in Kasumi-1. We report the development of murine hybridoma MAbs derived from immunizations with a peptide "self-epitope." Our findings provide a potential strategy to instruct humoral immunity in mice to focus and respond to self-epitopes. This strategy has been validated with the oncogenic fusion protein AML1-ETO involved in acute myeloid leukemia.

Asunto(s)

Anticuerpos Monoclonales de Origen Murino/biosíntesis , Subunidad alfa 2 del Factor de Unión al Sitio Principal/inmunología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Factores de Transcripción/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Clonación Molecular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Femenino , Células HEK293 , Humanos , Hibridomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteína 1 Compañera de Translocación de RUNX1 , Proteínas Recombinantes de Fusión/aislamiento & purificación , Homología de Secuencia de Aminoácido , Factores de Transcripción/aislamiento & purificación

RESUMEN

BACKGROUND: Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay. METHODS: Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples. RESULTS: Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4×10(-9) M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells. CONCLUSION: In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples.

Asunto(s)

Anticuerpos Monoclonales/análisis , Western Blotting/métodos , Proteínas de Ciclo Celular/inmunología , Proteínas de Unión al ADN/inmunología , Mapeo Epitopo/métodos , Inmunohistoquímica/métodos , Proteínas Nucleares/inmunología , Displasia del Cuello del Útero/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Biopsia , Proteínas de Ciclo Celular/análisis , Proteínas de Unión al ADN/análisis , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Epítopos/química , Epítopos/inmunología , Femenino , Humanos , Componente 6 del Complejo de Mantenimiento de Minicromosoma , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Displasia del Cuello del Útero/inmunología , Displasia del Cuello del Útero/patología

RESUMEN

BACKGROUND: The AMPLICOR HPV Test has been validated for use with cervical cells collected in liquid-based preservative fluids, such as BD SurePath. It is currently recommended, however, that residual BD SurePath samples be stored at 4 degrees C prior to testing. OBJECTIVES: The aim of this study was to demonstrate that DNA isolated from SurePath cervical cytology specimens and stored at ambient temperature was also compatible with the AMPLICOR HPV Test. STUDY DESIGN: DNA was extracted using the AmpliLute Media Sample Preparation Kit. Amplification and detection of HPV was performed both as directed by the manufacturer and with minor protocol modifications. RESULTS: Cervical specimens collected in SurePath preservative fluid remained stable for testing with the AMPLICOR HPV Test for at least 21 days. The performance of DNA extracted from specimens stored at room temperature was equivalent to DNA extracted from specimens stored at 4 degrees C. The beta-globin internal control was detected in all of the 146 residual SurePath cervical cytology specimens tested using the AMPLICOR HPV Test, and high-risk HPV was detected in 46.2% (18/39) of ASCUS cases, in 63.3% (19/30) of LSIL cytology specimens, and 92.3% (24/26) of HSIL cases. Concordance of AMPLICOR HPV Test results with Hybrid Capture II was 83.9%. CONCLUSIONS: The AMPLICOR HPV Test can be successfully and reproducibly performed from DNA isolated from residual SurePath cervical cytology specimens stored at ambient temperature for at least 21 days. This provides clinical laboratories flexible storage conditions for residual SurePath cytology specimens.

Asunto(s)

Cuello del Útero/virología , ADN Viral/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Papillomaviridae/genética , Manejo de Especímenes/métodos , Virología/métodos , Femenino , Humanos , Juego de Reactivos para Diagnóstico , Temperatura , Frotis Vaginal