RESUMEN
Worldwide, the incidence rate of breast cancer is the highest in women. Approximately 2.3 million people were newly diagnosed and 0.685 million were dead of breast cancer in 2020, which continues to grow. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with a higher risk of recurrence and metastasis, but disappointly, there are no effective and specific therapies clinically, especially for patients presenting with metastatic diseases. Therefore, it is urgent to develop a new type of cancer therapy for survival improvisation and adverse effects alleviation of breast cancers. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed, photochemistry-based cancer therapy. It was drive by an antibody-photoabsorber conjugate (APC) which is triggered by near-infrared light. The key part of APC is a cancer-targeting monoclonal antibody (mAb) that can bind to receptors or antigens on the surface of tumor cells. Because of this targeted conjugate accumulation, subsequent deployment of focal NIR-light results in functional damage on the targeted cell membranes without harming the immediately adjacent receptor-negative cells and evokes a kind of photochemical, speedy, and highly specific immunogenic cell death (ICD) of cancer cells with corresponding antigens. Subsequently, immature dendritic cells adjacent to dying cancer cells will become mature, further inducing a host-oriented anti-cancer immune response, complicatedly and comprehensively. Currently, NIR-PIT has progressed into phase 3 clinical trial for recurrent head and neck cancer. And preclinical studies have illustrated strong therapeutic efficacy of NIR-PIT targeting various molecular receptors overexpressed in breast cancer cells, including EGFR, HER2, CD44c, CD206, ICAM-1 and FAP-α. Thereby, NIR-PIT is in early trials, but appears to be a promising breast cancer therapy and moving into the future. Here, we present the specific advantages and discuss the most recent preclinical studies against several transmembrane proteins of NIR-PIT in breast cancers.
Asunto(s)
Inmunoconjugados , Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Inmunoconjugados/uso terapéutico , Inmunoconjugados/química , Línea Celular Tumoral , Recurrencia Local de Neoplasia/tratamiento farmacológico , Fototerapia/métodos , Inmunoterapia/métodos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
Drug-load (DL) characterization of antibody-drug conjugates (ADCs) is an important analytical task due to its designation as a critical quality attribute (CQA) affecting potency and stability. Intact and subunit liquid chromatography-mass spectrometry (LC-MS) analyses can determine global drug-to-antibody ratios (DARs) that correlate well with other orthogonal analytical methods; however, peptide mapping liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has struggled to provide complementary site-specific quantitation of drug conjugation sites. The peptide mapping method described herein utilizes stable isotope labeling to accurately quantitate the site-specific conjugation levels of a cysteine-conjugated ADC to provide "bottom-up" DAR characterization in parallel with protein sequence and post-translational modification (PTM) characterization in one multi-attribute analytical method (MAM).
Asunto(s)
Inmunoconjugados , Cromatografía Liquida/métodos , Cisteína/química , Inmunoconjugados/química , Marcaje Isotópico , Mapeo Peptídico , Espectrometría de Masas en TándemRESUMEN
Near-infrared photoimmunotherapy (NIR-PIT) is a new type of cancer treatment, which was recently approved in Japan for patients with inoperable head and neck cancer. NIR-PIT utilizes antibody-IRDye700DX (IR700) conjugates and NIR light at a wavelength of 690 nm. NIR light exposure leads to physicochemical changes in the antibody-IR700 conjugate cell receptor complex, inducing rapid necrotic cell death. Just as fluorescence guided surgery is useful for surgeons to resect tumors completely, real-time information of tumor locations would help clinicians irradiate NIR light more precisely. IR700 is a fluorescence dye that emits at 702 nm; however, there is no clinically available device optimized for detecting this fluorescence. On the other hand, many indocyanine green (ICG) fluorescence imaging devices have been approved for clinical use. Therefore, we investigated whether LIGHTVISION, one of the clinically available ICG cameras, could be employed for tumor detection. We hypothesized that irradiation with even low-power 690-nm laser light, attenuated by 99% with a neutral-density filter, could be detected with LIGHTVISION without fluorescence decay or therapeutic effect because of the long emission tail of IR700 beyond 800 nm (within the detection range of LIGHTVISION). We demonstrated that the LIGHTVISION camera, originally designed for ICG detection, can detect the tail of IR700 fluorescence in real time, thus enabling the visualization of target tumors.
Asunto(s)
Inmunoterapia/métodos , Neoplasias/diagnóstico por imagen , Imagen Óptica/instrumentación , Fototerapia/métodos , Animales , Línea Celular Tumoral , Terapia Combinada/métodos , Femenino , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Indoles/administración & dosificación , Indoles/química , Ratones , Neoplasias/terapia , Compuestos de Organosilicio/administración & dosificación , Compuestos de Organosilicio/química , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/química , Trastuzumab/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses antibody-IRDye700DX (IR700) conjugates and was recently approved in Japan for patients with inoperable head and neck cancer. Exposure of the tumor with NIR light at a wavelength of 690 nm leads to physicochemical changes in the antibody-IR700 conjugate-cell receptor complex, resulting in increased hydrophobicity and damage to the integrity of the cell membrane. However, it is important that the tumor be completely exposed to light during NIR-PIT, and thus, a method to provide real-time information on tumor location would help clinicians direct light more accurately. IR700 is a fluorophore that emits at 702 nm; however, there is no clinically available device optimized for detecting this fluorescence. On the other hand, many indocyanine green (ICG) fluorescence imaging devices have been approved for clinical use in operating rooms. Therefore, we investigated whether LIGHTVISION, one of the clinically available ICG cameras, could be employed for NIR-PIT target tumor detection. Due to the limited benefits of adding IR700 molecules, the additional conjugation of IRDye800CW (IR800) or ICG-EG4-Sulfo-OSu (ICG-EG4), which has an overlapping spectrum with ICG, to trastuzumab-IR700 conjugates was performed. Conjugation of second NIR dyes did not interfere the efficacy of NIR-PIT. The dual conjugation of IR800 and IR700 to trastuzumab clearly visualized target tumors with LIGHTVISION by detecting emission light of IR800. We demonstrated that the conjugation of second NIR dyes enables us to provide a real-time feedback of tumor locations prior to NIR-PIT.
Asunto(s)
Anticuerpos Monoclonales/química , Colorantes Fluorescentes/química , Inmunoconjugados/química , Verde de Indocianina/química , Imagen Óptica/métodos , Fototerapia/métodos , Células 3T3 , Animales , Línea Celular , Línea Celular Tumoral , Fluorescencia , Humanos , Inmunoterapia/métodos , Verde de Indocianina/análogos & derivados , Rayos Infrarrojos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fármacos Fotosensibilizantes/química , Trastuzumab/química , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells. Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody. Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.
Asunto(s)
Inmunoconjugados/química , Fragmentos Fab de Inmunoglobulinas/química , Transglutaminasas/inmunología , Trastuzumab/química , Secuencia de Aminoácidos , Neoplasias de la Mama/patología , Carcinoma/patología , Línea Celular Tumoral , Cistina/química , ADN Complementario/genética , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Escherichia coli , Femenino , Colorantes Fluorescentes , Humanos , Inmunoconjugados/inmunología , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Moleculares , Fragmentos de Péptidos/síntesis química , Conformación Proteica , Ingeniería de Proteínas , Multimerización de Proteína , Receptor ErbB-2/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Resonancia por Plasmón de Superficie , Trastuzumab/inmunologíaRESUMEN
B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg.
Asunto(s)
Antígenos B7/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoconjugados/farmacología , Neoplasias/tratamiento farmacológico , Animales , Antígenos B7/genética , Antígenos B7/metabolismo , Efecto Espectador , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas , Técnicas de Silenciamiento del Gen , Humanos , Inhibidores de Puntos de Control Inmunológico/química , Inhibidores de Puntos de Control Inmunológico/aislamiento & purificación , Inmunoconjugados/química , Inmunoconjugados/aislamiento & purificación , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody-drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti-CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid-erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Benzodiazepinas/química , Inmunoconjugados/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de Microfilamentos/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Pirroles/química , Animales , Antineoplásicos/química , Apoptosis , Proliferación Celular , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inmunoconjugados/química , Macaca fascicularis , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Microfilamentos/inmunología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein conjugates such as antibody-drugs conjugates (ADCs) represents the next generation of therapeutic proteins. They allow to combine the biological properties of the protein format with the characteristics of the conjugated ligands. The reaction implemented to couple ligands to the peptide backbone represents a crucial aspect of the production of protein conjugates, influencing the nature and the heterogeneity of the conjugates obtained. Here, we report the concomitant use of MALDI-TOF MS and LC-MS/MS analysis to investigate the chemical functionalization of human serum albumin (HSA) by the intermediate of lysine residues, previously used to generate biopharmaceutical agents for medical imaging. A kinetic was performed by collecting samples after different reaction times and analyzing them using the two techniques. MALDI-TOF MS analyses allowed estimating the number of conjugated ligands in a robust manner and assess the global functionalization kinetic on the intact protein level. Results demonstrated a maximum of 38 modified residues out of the 59 lysines available showing the limitation of the chemical functionalization. Consequently, LC-MS/MS analysis provided a site-specific characterization of the residues undergoing chemical modification. Data exhibited unique properties due to the presence of the ligands which allowed to identify without ambiguity the residues exhibiting different modification rate and enabled the identification of the unmodified lysine. Results were compared to the structure of HSA described from crystallography data. The comparison strongly suggested that accessibility is influencing the residues respective reactivity. The relevant complementarity of the different techniques could be emphasized in order to perform an extensive characterization concerning the evolution of the primary structure of the protein during the chemical reaction, providing an improved insight on the conjugation process and offering the potentiality to tune the reaction.
Asunto(s)
Inmunoconjugados/análisis , Lisina/análisis , Albúmina Sérica Humana/análisis , Secuencia de Aminoácidos , Química Farmacéutica/métodos , Cristalografía por Rayos X , Imidoésteres/química , Inmunoconjugados/química , Cinética , Proteolisis , Albúmina Sérica Humana/química , Albúmina Sérica Humana/ultraestructura , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Photoimmunotherapy involves targeted delivery of photosensitizers via an antibody conjugate (i.e., photoimmunoconjugate, PIC) followed by light activation for selective tumor killing. The trade-off between PIC selectivity and PIC uptake is a major drawback limiting the efficacy of photoimmunotherapy. Despite ample evidence showing that photoimmunotherapy is most effective when combined with chemotherapy, the design of nanocarriers to co-deliver PICs and chemotherapy drugs remains an unmet need. To overcome these challenges, we developed a novel photoimmunoconjugate-nanoliposome (PIC-Nal) comprising of three clinically used agents: anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody cetuximab (Cet), benzoporphyrin derivative (BPD) photosensitizer, and irinotecan (IRI) chemotherapy. RESULTS: The BPD photosensitizers were first tethered to Cet at a molar ratio of 6:1 using carbodiimide chemistry to form PICs. Conjugation of PICs onto nanoliposome irinotecan (Nal-IRI) was facilitated by copper-free click chemistry, which resulted in monodispersed PIC-Nal-IRI with an average size of 158.8 ± 15.6 nm. PIC-Nal-IRI is highly selective against EGFR-overexpressing epithelial ovarian cancer cells with 2- to 6-fold less accumulation in low EGFR expressing cells. Successful coupling of PIC onto Nal-IRI enhanced PIC uptake and photoimmunotherapy efficacy by up to 30% in OVCAR-5 cells. Furthermore, PIC-Nal-IRI synergistically reduced cancer viability via a unique three-way mechanism (i.e., EGFR downregulation, mitochondrial depolarization, and DNA damage). CONCLUSION: It is increasingly evident that the most effective therapies for cancer will involve combination treatments that target multiple non-overlapping pathways while minimizing side effects. Nanotechnology combined with photochemistry provides a unique opportunity to simultaneously deliver and activate multiple drugs that target all major regions of a cancer cell-plasma membrane, cytoplasm, and nucleus. PIC-Nal-IRI offers a promising strategy to overcome the selectivity-uptake trade-off, improve photoimmunotherapy efficacy, and enable multi-tier cancer targeting. Controllable drug compartmentalization, easy surface modification, and high clinical relevance collectively make PIC-Nal-IRI extremely valuable and merits further investigations in living animals.
Asunto(s)
Inmunoconjugados/uso terapéutico , Irinotecán/uso terapéutico , Nanopartículas/química , Neoplasias/terapia , Fototerapia , Línea Celular Tumoral , Terapia Combinada , Liberación de Fármacos , Estabilidad de Medicamentos , Humanos , Inmunoconjugados/química , Irinotecán/química , LiposomasRESUMEN
We produced a prometryn-specific monoclonal antibody and propose a strategy for convenient on-site detection of prometryn residues in herbs for the first time. This strategy has perfect applicability in a complex herbal medicine matrix. The strategy combines a semiquantitative immunochromatographic strip assay with a heterologous indirect competitive ELISA. When there was no matrix interference, the ELISA had a half-maximal inhibitory concentration of 2.6 ng·mL-1 and a limit of detection of 0.2 ng·mL-1. The immunochromatographic strip assay can be completed within 5 min with a visual limit of detection of 1 ng·mL-1. Although the sample matrix had different effects on the sensitivity of the antibody, excellent repeatability and accuracy were achieved. The method was successfully applied for the screening and determination of prometryn residue in multiple complex herb samples for the first time, and the results were in good agreement with those obtained by liquid chromatography-tandem mass spectrometry. The proposed strategy is rapid, of high-throughput, and of low cost, and may be a promising choice for on-site detection of prometryn in different kinds of herbs. Graphical abstract.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Herbicidas/análisis , Plantas Medicinales/química , Prometrina/análisis , Animales , Anticuerpos Monoclonales/química , Ensayo de Inmunoadsorción Enzimática/instrumentación , Diseño de Equipo , Femenino , Contaminación de Alimentos/análisis , Oro Coloide/química , Inmunoconjugados/química , Límite de Detección , Ratones Endogámicos BALB C , Tiras Reactivas/análisisRESUMEN
Cancer treatment has been founded traditionally on the three approaches of surgery, radiation, and chemotherapy with the latter recognized as the obvious systemic treatment approach applicable to disease that has spread. Although significant progress has been made over nearly 100 years of developing systemic treatments, it remains clear that use of the toxic agents involved is a two-edged sword with normal organ toxicities always needing to be balanced with and against administration of relevant therapeutic doses. With the advent of monoclonal antibodies targeted against tumor-associated antigens that could be used as carriers of potently toxic chemotherapy drugs, it was thought that such antibody-drug conjugates (ADCs) could engender the answer to the toxicity/therapeutic equation by shifting the equation more toward beneficial therapeutic efficacy. However, over 40 or so years, antibody-drug conjugates have not significantly affected the toxicity/therapy balance paradigm in most cancer indications, especially in solid tumors. Ideally, a further step may be required in that a non-tumor-targeted antibody-drug conjugate should be essentially nontoxic in its native administered form, with toxic effects unleashed only at the site of targeted tumors. A new approach that employs this principle is the use of an antibody-drug conjugate that is essentially nontoxic to normal tissues by virtue of requiring an extra step of light activation to become potent. We describe the preclinical data and first clinical results gained over the past few years by use of antibody-drug conjugates wherein the drug comprises a near-infrared photoactivatable dye delivered to tumors by a monoclonal antibody and is subsequently activated to a toxic entity solely at sites of tumors.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Inmunoterapia/métodos , Neoplasias/terapia , Animales , Antineoplásicos Inmunológicos/química , Humanos , Inmunoconjugados/química , Neoplasias/inmunología , Fototerapia/métodosRESUMEN
Closed system transfer devices (CSTD) are a supplemental engineering control designed to reduce occupational exposure of hazardous drugs and are currently implemented in accordance with evolving regulations. Owing to the novelty and complexity of these devices and their importance in clinical in-use testing, here we evaluated FDA-approved CSTD, assessing product quality through stability indicating assays to determine any drug product incompatibilities. Six devices were used in a simulated compounding and administration of a late-phase IgG1 antibody-drug conjugate (ADC) and the resulting samples were analyzed for visible and subvisible particle counts by light obscuration and micro-flow imaging, physical stability by size exclusion chromatography, and biological activities by relative potency. Potential challenges included improper fit of CSTD components, loss of product to void volume, and material incompatibility. Results showed compatibility of the ADC with the 6 CSTD evaluated. One CSTD introduced subvisible particles into the ADC during compounding that were identified through morphological assessment as silicone oil. This study highlights the importance of clinical in use testing with new devices and proposes strategies to mitigate the risk of drug product incompatibility with CSTD.
Asunto(s)
Composición de Medicamentos/instrumentación , Inmunoconjugados/química , Inmunoglobulina G/química , Exposición Profesional/prevención & control , Equipos de Seguridad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Estabilidad de Medicamentos , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/toxicidad , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/toxicidad , Ensayo de Materiales , Exposición Profesional/efectos adversos , Agregado de Proteínas , Estabilidad ProteicaRESUMEN
From a structural point of view, the complete characterization of ADCs is a challenging task due to their high complexity. ADCs combine the heterogeneity of the initial antibody to the variability associated with the conjugation strategy, the manufacturing process, and the storage. Given the inherent complexity of these biomolecules, online comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive technique to address the challenges associated with ADC characterization. Compared to conventional one-dimensional liquid chromatography techniques (1D-LC), LC × LC combines two different and complementary separation systems. In the context of ADC analysis, LC × LC has been proven to be a rapid and efficient analytical tool: (1) to provide a higher resolving power by increasing the overall peak capacity and thus allowing to gain more information within a single run and (2) to allow mass spectrometry (MS) coupling with some chromatographic techniques that are not MS-compatible and hence to facilitate the structural elucidation of ADCs. In this chapter, we present the coupling of different chromatographic techniques including hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophilic liquid chromatography (HILIC). The interest of HIC × SEC, SEC × SEC, HIC × RPLC, IEX × RPLC, RPLC × RPLC, and HILIC × RPLC, all hyphenated to high-resolution mass spectrometry (HRMS), is discussed in the context of the characterization of ADCs.
Asunto(s)
Cromatografía Liquida , Inmunoconjugados/análisis , Inmunoconjugados/química , Espectrometría de Masas , Aminoácidos/química , Anticuerpos Monoclonales/química , Cromatografía Líquida de Alta Presión , Cromatografía Liquida/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoconjugados/aislamiento & purificación , Espectrometría de Masas/métodosRESUMEN
Recently, the photothermal effect of nanomaterials has opened the door for new appealing strategy, which can generate a promising and powerful tool when combined with immunoassay. As a new kind of nanomaterial, black phosphorus (BP) has aroused widespread interest. In this study, a novel immunofiltration strip method with temperature as the readout signal based on the photothermal effect of BP nanosheets was established. The temperature was monitored by a portable temperature sensor. Using an indirect competitive strategy, it provides a simple, rapid, sensitive, and economic platform for the detection of 17ß-estradiol, a kind of endocrine disrupting compound that is frequently detected in environmental water or food samples. The higher the concentration of 17ß-estradiol in the sample, the less BP nanosheets are brought to bind to the strip surface, along with lower temperature variation when exposed to intensive laser irradiation. Under optimum conditions, a detection limit of 0.104 ng mL-1 was achieved. The feasibility of this assay was assessed by a standard addition method in water and milk samples, showing good performance and indicating potential application value for easy-to-use, inexpensive, and on-site monitoring of 17ß-estradiol.
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Disruptores Endocrinos/análisis , Estradiol/análisis , Inmunoensayo/métodos , Inmunoconjugados/inmunología , Fósforo/química , Animales , Agua Potable/análisis , Disruptores Endocrinos/química , Disruptores Endocrinos/inmunología , Estradiol/química , Estradiol/inmunología , Contaminación de Alimentos/análisis , Inmunoconjugados/química , Límite de Detección , Leche/química , Nanoestructuras/química , Nanoestructuras/efectos de la radiación , Ovalbúmina/química , Fósforo/efectos de la radiación , Temperatura , Rayos Ultravioleta , Contaminantes Químicos del Agua/análisisRESUMEN
Stimulation of Toll-like receptors (TLRs) and/or NOD-like receptors on immune cells initiates and directs immune responses that are essential for vaccine adjuvants. The small-molecule TLR7 agonist, imiquimod, has been approved by the FDA as an immune response modifier but is limited to topical application due to its poor pharmacokinetics that causes undesired adverse effects. Nanoparticles are increasingly used with innate immune stimulators to mitigate side effects and enhance adjuvant efficacy. In this study, a potent small-molecule TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), was conjugated to hollow silica nanoshells (NS). Proinflammatory cytokine (IL-6, IL-12) release by mouse bone-marrow-derived dendritic cells and human peripheral blood mononuclear cells revealed that the potency of silica nanoshells-TLR7 conjugates (NS-TLR) depends on nanoshell size and ligand coating density. Silica nanoshells of 100 nm diameter coated with a minimum of â¼6000 1V209 ligands/particle displayed 3-fold higher potency with no observed cytotoxicity when compared to an unconjugated TLR7 agonist. NS-TLR activated the TLR7-signaling pathway, triggered caspase activity, and stimulated IL-1ß release, while neither unconjugated TLR7 ligands nor silica shells alone produced IL-1ß. An in vivo murine immunization study, using the model antigen ovalbumin, demonstrated that NS-TLR increased antigen-specific IgG antibody induction by 1000× with a Th1-biased immune response, compared to unconjugated TLR7 agonists. The results show that the TLR7 ligand conjugated to silica nanoshells is capable of activating an inflammasome pathway to enhance both innate immune-stimulatory and adjuvant potencies of the TLR7 agonist, thereby broadening applications of innate immune stimulators.
Asunto(s)
Imiquimod/inmunología , Inmunidad Innata/efectos de los fármacos , Inmunoconjugados/inmunología , Receptor Toll-Like 7/inmunología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Humanos , Imiquimod/química , Imiquimod/uso terapéutico , Inmunidad Innata/genética , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Ratones , Nanocáscaras/química , Transducción de Señal/efectos de los fármacos , Dióxido de Silicio/química , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/genéticaRESUMEN
PURPOSE: Fibroblast growth factor receptor 2 (FGFR2) has been previously reported to be overexpressed in several types of cancer, whereas the expression in normal tissue is considered to be moderate to low. Thus, FGFR2 is regarded as an attractive tumor antigen for targeted alpha therapy. This study reports the evaluation of an FGFR2-targeted thorium-227 conjugate (FGFR2-TTC, BAY 2304058) comprising an anti-FGFR2 antibody, a chelator moiety covalently conjugated to the antibody, and the alpha particle-emitting radionuclide thorium-227. FGFR2-TTC was assessed as a monotherapy and in combination with the DNA damage response inhibitor ATRi BAY 1895344. METHODS AND MATERIALS: The in vitro cytotoxicity and mechanism of action were evaluated by determining cell viability, the DNA damage response marker γH2A.X, and cell cycle analyses. The in vivo efficacy was determined using human tumor xenograft models in nude mice. RESULTS: In vitro mechanistic assays demonstrated upregulation of γH2A.X and induction of cell cycle arrest in several FGFR2-expressing cancer cell lines after treatment with FGFR2-TTC. In vivo, FGFR2-TTC significantly inhibited tumor growth at a dose of 500 kBq/kg in the xenograft models NCI-H716, SNU-16, and MFM-223. By combining FGFR2-TTC with the ATR inhibitor BAY 1895344, an increased potency was observed in vitro, as were elevated levels of γH2A.X and inhibition of FGFR2-TTC-mediated cell cycle arrest. In the MFM-223 tumor xenograft model, combination of the ATRi BAY 1895344 with FGFR2-TTC resulted in significant tumor growth inhibition at doses at which the single agents had no effect. CONCLUSIONS: The data provide a mechanism-based rationale for combining the FGFR2-TTC with the ATRi BAY 1895344 as a new therapeutic approach for treatment of FGFR2-positive tumors from different cancer indications.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Neoplasias de la Mama/radioterapia , Inhibidores de Proteínas Quinasas/uso terapéutico , Radioinmunoterapia/métodos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/uso terapéutico , Torio/uso terapéutico , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quelantes/uso terapéutico , Daño del ADN , Combinación de Medicamentos , Sinergismo Farmacológico , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Histonas/metabolismo , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Ratones , Ratones Desnudos , Terapia Molecular Dirigida/métodos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Torio/farmacocinética , Compuestos de Torio/uso terapéutico , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Innovative protein engineering and chemical conjugation technologies have yielded an impressive number of drug candidates in clinical development including >80 antibody drug conjugates, >60 bispecific antibodies, >35 Fc-fusion proteins and >10 immuno-cytokines. Despite these innovations, technological advances are needed to address unmet medical needs with new pharmacological mechanisms. Age-related eye diseases are among the most common causes of blindness and poor vision in the world. Many such diseases affect the back of the eye, where the inaccessibility of the site of action necessitates therapeutic delivery via intravitreal (IVT) injection. Treatments administered via this route typically have vitreal half-lives <10 days in humans, requiring frequent administration. Since IVT injection is burdensome to patients, there exists a strong need to develop therapeutics with prolonged residence time in the eye. We report here a strategy to increase retention of a therapeutic fragment antibody (Fab) in the eye, using an anti-complement factor D Fab previously optimized for ocular delivery. Polyethylene glycol structures, varying in length, geometry and degree of branching, were coupled to the Fab via maleimide-activated termini. A screening strategy was developed to allow for key determinants of ocular half-life to be measured in vitro. After compound selection, a scalable process was established to enable tolerability and pharmacokinetic studies in cynomolgus monkeys, demonstrating an increase in vitreal half-life with no associated adverse events. Further, we show that the technique for compound selection, analytical characterization, and scalable production is general for a range of antibody fragments. The application of the technology has broad impact in across many therapeutic areas with the first major advancement in the treatment of an important ocular disease.
Asunto(s)
Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Ojo , Inmunoconjugados/química , Polietilenglicoles/química , Proteínas/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Evaluación Preclínica de Medicamentos , Ojo/efectos de los fármacos , Femenino , Haplorrinos , Humanos , Inmunoconjugados/aislamiento & purificación , Inmunoconjugados/farmacología , Fragmentos Fab de Inmunoglobulinas/química , Ingeniería de Proteínas , Proteínas/aislamiento & purificación , Proteínas/farmacologíaRESUMEN
PURPOSE: Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue. EXPERIMENTAL DESIGN: The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer. RESULTS: BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data. CONCLUSIONS: These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).
Asunto(s)
Partículas alfa/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/farmacología , Neoplasias/tratamiento farmacológico , Radiofármacos/farmacología , Torio/farmacología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Femenino , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/farmacocinética , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Mesotelina , Mesotelioma/tratamiento farmacológico , Mesotelioma/metabolismo , Mesotelioma/patología , Mesotelioma Maligno , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Radiofármacos/farmacocinética , Torio/administración & dosificación , Torio/química , Torio/farmacocinética , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic intranasal administration of antibodies to glutamate to aging C57Bl/6 mice improved passive avoidance conditioning, had no effect on horizontal and vertical locomotor activity, but slowed locomotion in the open-field test. Administration of antibodies to glutamate increased the content of dopamine and its metabolites in mouse hippocampus, but had no effect on the metabolism of neurotransmitter amino acids. In the frontal cortex, antibodies to glutamate did not affect neurotransmitter metabolism, but increased the level of both excitatory and inhibitory amino acids without changing their ratio.
Asunto(s)
Envejecimiento/fisiología , Anticuerpos/farmacología , Reacción de Prevención/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Memoria/efectos de los fármacos , Administración Intranasal , Animales , Anticuerpos/química , Ácido Aspártico/metabolismo , Reacción de Prevención/fisiología , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Dopamina/metabolismo , Antagonistas de Aminoácidos Excitadores/química , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/fisiología , Glicina/metabolismo , Haptenos/química , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Ácido Hidroxiindolacético/metabolismo , Inmunoconjugados/química , Locomoción/efectos de los fármacos , Locomoción/fisiología , Masculino , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Conejos , Serotonina/metabolismo , Albúmina Sérica Bovina/química , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The antibody-drug conjugate (ADC) field has seen a remarkable expansion in the number of entrants in clinical studies. Many of these agents employ newer conjugation technologies that have been developed over the last decade that confer various attributes to the ADCs prepared with them, including stability, potency, and homogeneity. In many cases, these new ADCs appear demonstrably superior to earlier technologies in preclinical models of activity and toxicology, but the degree to which these improvements will translate to the clinic is only starting to be seen. Many of these technologies are now competing head-to-head by targeting the same antigen in similar patient populations, allowing for a direct comparison of their clinical performance properties. As lessons from these experiences feed back into discovery research, future iterations of ADC design may be expected to bring improved therapeutics into the clinic.