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1.
Int J Mol Sci ; 25(3)2024 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-38339208

RESUMEN

Structure and function of therapeutic antibodies can be modulated by a variety of post-translational modifications (PTM). Tyrosine (Tyr) sulfation is a type of negatively charged PTM that occurs during protein trafficking through the Golgi. In this study, we discovered that an anti-interleukin (IL)-4 human IgG1, produced by transiently transfected HEK293 cells, contained a fraction of unusual negatively charged species. Interestingly, the isolated acidic species exhibited a two-fold higher affinity to IL-4 and a nearly four-fold higher potency compared to the main species. Mass spectrometry (MS) showed the isolated acidic species possessed an +80-Dalton from the expected mass, suggesting an occurrence of Tyr sulfation. Consistent with this hypothesis, we show the ability to control the acidic species during transient expression with the addition of Tyr sulfation inhibitor sodium chlorate or, conversely, enriched the acidic species from 30% to 92% of the total antibody protein when the IL-4 IgG was co-transfected with tyrosylprotein sulfotransferase genes. Further MS and mutagenesis analysis identified a Tyr residue at the light chain complementarity-determining region-1 (CDRL-1), which was sulfated specifically. These results together have demonstrated for the first time that Tyr sulfation at CDRL-1 could modulate antibody binding affinity and potency to a human immune cytokine.


Asunto(s)
Interleucina-4 , Tirosina , Humanos , Tirosina/metabolismo , Células HEK293 , Aparato de Golgi/metabolismo , Mutagénesis
2.
Immunology ; 143(3): 416-27, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24831554

RESUMEN

Interleukin-4 (IL-4) and IL-13 are critical drivers of immune activation and inflammation in ulcerative colitis, asthma and other diseases. Because these cytokines may have redundant function, dual targeting holds promise for achieving greater efficacy. We have recently described a bifunctional therapeutic targeting IL-4 and IL-13 developed on a novel protein scaffold, generated by combining specific binding domains in an optimal configuration using appropriate linker regions. In the current study, the bifunctional IL-4/IL-13 antagonist was evaluated in the murine oxazolone-induced colitis model, which produces disease with features of ulcerative colitis. The bifunctional IL-4/IL-13 antagonist reduced body weight loss throughout the 7-day course of the model, and ameliorated the increased colon weight and decreased colon length that accompany disease. Colon tissue gene expression was modulated in accordance with the treatment effect. Concentrations of serum amyloid P were elevated in proportion to disease severity, making it an effective biomarker. Serum concentrations of the bifunctional IL-4/IL-13 antagonist were inversely proportional to disease severity, colon tissue expression of pro-inflammatory genes, and serum amyloid P concentration. Taken together, these results define a panel of biomarkers signifying engagement of the IL-4/IL-13 pathway, confirm the T helper type 2 nature of disease in this model, and demonstrate the effectiveness of dual cytokine blockade.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Colitis Ulcerosa/metabolismo , Interleucina-13/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/farmacología , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Ratones , Oxazolona/efectos adversos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteína Amiloide A Sérica/metabolismo , Componente Amiloide P Sérico/metabolismo , Índice de Severidad de la Enfermedad
3.
Am J Respir Cell Mol Biol ; 49(1): 37-46, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23449738

RESUMEN

IL-4 and IL-13 comprise promising targets for therapeutic interventions in asthma and other Th2-associated diseases, but agents targeting either IL-4 or IL-13 alone have shown limited efficacy in human clinical studies. Because these cytokines may involve redundant function, dual targeting holds promise for achieving greater efficacy. We describe a bifunctional therapeutic targeting IL-4 and IL-13, developed by a combination of specific binding domains. IL-4-targeted and IL-13-targeted single chain variable fragments were joined in an optimal configuration, using appropriate linker regions on a novel protein scaffold. The bifunctional IL-4/IL-13 antagonist displayed high affinity for both cytokines. It was a potent and efficient neutralizer of both murine IL-4 and murine IL-13 bioactivity in cytokine-responsive Ba/F3 cells, and exhibited a half-life of approximately 4.7 days in mice. In a murine model of ovalbumin-induced ear swelling, the bifunctional molecule blocked both the IL-4/IL-13-dependent early-phase response and the IL-4-dependent late-phase response. In the ovalbumin-induced lung inflammation model, the bifunctional IL-4/IL-13 antagonist reduced the IL-4-dependent rise in serum IgE titers, and reduced IL-13-dependent airway hyperresponsiveness, lung inflammation, mucin gene expression, and serum chitinase responses. Taken together, these findings demonstrate the effective dual blockade of IL-4 and IL-13 with a single agent, which resulted in the modulation of a more extensive range of endpoints than could be achieved by targeting either cytokine alone.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inmunoglobulina E/inmunología , Interleucina-13/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Neumonía/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/inmunología , Sitios de Unión , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/inmunología , Células CHO , Cricetinae , Reactivos de Enlaces Cruzados/química , Oído/fisiopatología , Femenino , Semivida , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Ratones , Ratones Endogámicos C57BL , Conformación Molecular , Pruebas de Neutralización , Ovalbúmina/efectos adversos , Ovalbúmina/inmunología , Neumonía/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Anticuerpos de Cadena Única/metabolismo
4.
J Immunol ; 187(1): 561-9, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21622864

RESUMEN

Human studies using Abs to two different, nonoverlapping epitopes of IL-13 suggested that epitope specificity can have a clinically significant impact on clearance of IL-13. We propose that Ab modulation of IL-13 interaction with IL-13Rα2 underlies this effect. Two Abs were administered to healthy subjects and mild asthmatics in separate dose-ranging studies and allergen-challenge studies. IMA-638 allows IL-13 interaction with IL-13Rα1 or IL-13Rα2 but blocks recruitment of IL-4Rα to the IL-13/IL-13Rα1 complex, whereas IMA-026 competes with IL-13 interaction with IL-13Rα1 and IL-13Rα2. We found ∼10-fold higher circulating titer of captured IL-13 in subjects treated with IMA-026 compared with those administered IMA-638. To understand how this difference could be related to epitope, we asked whether either Ab affects IL-13 internalization through cell surface IL-13Rα2. Humans inducibly express cell surface IL-13Rα2 but lack the soluble form that regulates IL-13 responses in mice. Cells with high IL-13Rα2 expression rapidly and efficiently depleted extracellular IL-13, and this activity persisted in the presence of IMA-638 but not IMA-026. The potency and efficiency of this clearance pathway suggest that cell surface IL-13Rα2 acts as a scavenger for IL-13. These findings could have important implications for the design and characterization of IL-13 antagonists.


Asunto(s)
Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Interleucina-13/inmunología , Interleucina-13/metabolismo , Isoanticuerpos/fisiología , Receptores Depuradores/metabolismo , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Relación Dosis-Respuesta Inmunológica , Sistemas de Liberación de Medicamentos , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Células HT29 , Humanos , Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/biosíntesis , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Receptores Depuradores/antagonistas & inhibidores , Receptores Depuradores/fisiología
5.
Sci Rep ; 12(1): 7262, 2022 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-35508689

RESUMEN

Next-generation site-specific cysteine-based antibody-drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.


Asunto(s)
Antineoplásicos , Inmunoconjugados , Animales , Anticuerpos , Antineoplásicos/uso terapéutico , Células CHO , Cricetinae , Cricetulus , Cisteína , Disulfuros , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Ingeniería Metabólica
6.
Mol Cancer Ther ; 21(9): 1462-1472, 2022 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-35793468

RESUMEN

Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.


Asunto(s)
Neoplasias de la Mama , Inmunoconjugados , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Fibronectinas/metabolismo , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Ratones , Neovascularización Patológica/metabolismo , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto
7.
MAbs ; 13(1): 1850395, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33459147

RESUMEN

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Complejo CD3/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores de Enterotoxina/inmunología , Animales , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Biespecíficos/uso terapéutico , Línea Celular Tumoral , Femenino , Humanos , Hibridomas , Macaca fascicularis/inmunología , Macaca fascicularis/metabolismo , Ratones Endogámicos BALB C , Neoplasias/inmunología , Neoplasias/metabolismo , Ingeniería de Proteínas/métodos , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/farmacocinética , Anticuerpos de Cadena Única/uso terapéutico , Linfocitos T/inmunología , Linfocitos T/metabolismo
8.
Mol Cancer Ther ; 19(10): 2068-2078, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32747418

RESUMEN

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo-acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Inmunoconjugados/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Gástricas/tratamiento farmacológico , Animales , Neoplasias de la Mama/patología , Femenino , Humanos , Inmunoconjugados/farmacología , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Neoplasias Gástricas/patología
9.
J Pharmacol Exp Ther ; 325(3): 882-92, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18337474

RESUMEN

Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Ralpha1/IL-4Ralpha) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Ralpha, and Ab02 blocks IL-13 interaction with IL-13Ralpha1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Ralpha-binding epitope or the IL-13Ralpha1-binding epitope.


Asunto(s)
Antígenos Helmínticos/inmunología , Ascaris/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina G/inmunología , Inflamación/inmunología , Interleucina-13/inmunología , Pulmón/inmunología , Receptores de Interleucina-13/inmunología , Animales , Anticuerpos Antihelmínticos/inmunología , Basófilos/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Epítopos/inmunología , Liberación de Histamina/inmunología , Humanos , Macaca fascicularis , Masculino
10.
AAPS J ; 19(4): 1123-1135, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28439809

RESUMEN

As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build "design rules" that will enable more efficient prosecution of next-generation ADC discovery programs.


Asunto(s)
Cisteína/química , Inmunoconjugados/química , Secuencia de Aminoácidos , Simulación de Dinámica Molecular
11.
ACS Med Chem Lett ; 7(11): 977-982, 2016 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-27882194

RESUMEN

As part of our efforts to develop new classes of tubulin inhibitor payloads for antibody-drug conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated excellent in vitro activity but suffered from rapid metabolism of a critical acetate ester. A two-pronged strategy was employed to address this metabolism. First, the hydrolytically labile ester was replaced by a carbamate functional group resulting in a more stable ADC that retained potency in cellular assays. Second, site-specific conjugation was employed in order to design ADCs with reduced metabolic liabilities. Using the later approach, we were able to identify a conjugate at the 334C position of the heavy chain that resulted in an ADC with considerably reduced metabolism and improved efficacy. The examples discussed herein provide one of the clearest demonstrations to-date that site of conjugation can play a critical role in addressing metabolic and PK liabilities of an ADC. Moreover, a clear correlation was identified between the hydrophobicity of an ADC and its susceptibility to metabolic enzymes. Importantly, this study demonstrates that traditional medicinal chemistry strategies can be effectively applied to ADC programs.

12.
Mol Cancer Ther ; 14(8): 1868-76, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26089370

RESUMEN

Antibody-drug conjugates (ADC) represent a promising therapeutic modality for managing cancer. Here, we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1. Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts in nude mice, including non-small cell lung cancer and pancreas, prostate, and colon cancers. As v1.10 did not react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-LP2 together was more effective than either ADC alone. These data provide proof-of-concept that TM4SF1-targeting ADCs have potential as anticancer agents with dual action against tumor cells and the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further investigation. Mol Cancer Ther; 14(8); 1868-76. ©2015 AACR.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de la Angiogénesis/toxicidad , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antineoplásicos/toxicidad , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Expresión Génica , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica , Conejos , Distribución Tisular , Ensayos Antitumor por Modelo de Xenoinjerto
13.
PLoS One ; 8(5): e62616, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23700410

RESUMEN

Strong genetic data link the Tyrosine kinase receptor B (TrkB) and its major endogenous ligand brain-derived neurotrophic factor (BDNF) to the regulation of energy homeostasis, with loss-of-function mutations in either gene causing severe obesity in both mice and humans. It has previously been reported that peripheral administration of the endogenous TrkB agonist ligand neurotrophin-4 (NT-4) profoundly decreases food intake and body weight in rodents, while paradoxically increasing these same parameters in monkeys. We generated a humanized TrkB agonist antibody, TAM-163, and characterized its therapeutic potential in several models of type 2 diabetes and obesity. In vitro, TAM-163 bound to human and rodent TrkB with high affinity, activated all aspects of the TrkB signaling cascade and induced TrkB internalization and degradation in a manner similar to BDNF. In vivo, peripheral administration of TAM-163 decreased food intake and/or body weight in mice, rats, hamsters, and dogs, but increased food intake and body weight in monkeys. The magnitude of weight change was similar in rodents and non-human primates, occurred at doses where there was no appreciable penetration into deep structures of the brain, and could not be explained by differences in exposures between species. Rather, peripherally administered TAM-163 localized to areas in the hypothalamus and the brain stem located outside the blood-brain barrier in a similar manner between rodents and non-human primates, suggesting differences in neuroanatomy across species. Our data demonstrate that a TrkB agonist antibody, administered peripherally, causes species-dependent effects on body weight similar to the endogenous TrkB ligand NT-4. The possible clinical utility of TrkB agonism in treating weight regulatory disorder, such as obesity or cachexia, will require evaluation in man.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Depresores del Apetito/farmacología , Peso Corporal/efectos de los fármacos , Receptor trkB/agonistas , Administración Intravenosa , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Depresores del Apetito/administración & dosificación , Depresores del Apetito/farmacocinética , Cricetinae , Dieta Alta en Grasa/efectos adversos , Perros , Evaluación Preclínica de Medicamentos , Ingestión de Energía/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Hipotálamo/metabolismo , Macaca fascicularis , Macaca mulatta , Masculino , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/patología , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Receptor trkB/metabolismo , Distribución Tisular
14.
Mol Cancer Ther ; 12(1): 38-47, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23223830

RESUMEN

Antibody-drug conjugates (ADC) represent a promising therapeutic modality for the clinical management of cancer. We sought to develop a novel ADC that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells (TIC), which comprise the most aggressive cell population in the tumor. We optimized an anti-5T4 ADC (A1mcMMAF) by sulfydryl-based conjugation of the humanized A1 antibody to the tubulin inhibitor monomethylauristatin F (MMAF) via a maleimidocaproyl linker. A1mcMMAF exhibited potent in vivo antitumor activity in a variety of tumor models and induced long-term regressions for up to 100 days after the last dose. Strikingly, animals showed pathologic complete response in each model with doses as low as 3 mg antibody/kg dosed every 4 days. In a non-small cell lung cancer patient-derived xenograft model, in which 5T4 is preferentially expressed on the less differentiated tumor cells, A1mcMMAF treatment resulted in sustained tumor regressions and reduced TIC frequency. These results highlight the potential of ADCs that target the most aggressive cell populations within tumors, such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered to cynomolgus monkeys at doses up to 10 mg antibody/kg/cycle × 2 and displayed a half-life of 5 days. The preclinical efficacy and safety data established a promising therapeutic index that supports clinical testing of A1mcMMAF.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Glicoproteínas de Membrana/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/toxicidad , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Macaca fascicularis , Masculino , Dosis Máxima Tolerada , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Inducción de Remisión , Distribución Tisular , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Cancer Res ; 71(12): 4236-46, 2011 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-21540235

RESUMEN

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy.


Asunto(s)
Antígenos de Neoplasias/análisis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Inmunotoxinas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Glicoproteínas de Membrana/análisis , Células Madre Neoplásicas/inmunología , Animales , Antígeno CD24/análisis , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Humanos , Receptores de Hialuranos/análisis , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/fisiología , Ratones
16.
Am J Respir Cell Mol Biol ; 36(3): 368-76, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17023688

RESUMEN

IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.


Asunto(s)
Asma/tratamiento farmacológico , Asma/inmunología , Modelos Animales de Enfermedad , Interleucina-13/antagonistas & inhibidores , Interleucina-13/inmunología , Oveja Doméstica/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Ascaris suum/fisiología , Asma/inducido químicamente , Asma/fisiopatología , Secuencia de Bases , Hiperreactividad Bronquial/parasitología , Hiperreactividad Bronquial/patología , Broncoconstricción/efectos de los fármacos , Broncoconstricción/inmunología , Carbacol/farmacología , Femenino , Células HT29 , Humanos , Interleucina-13/química , Interleucina-13/genética , Cinética , Datos de Secuencia Molecular , Pruebas de Neutralización , Receptores de Interleucina-13/metabolismo , Oveja Doméstica/parasitología , Solubilidad/efectos de los fármacos , Resonancia por Plasmón de Superficie , Factores de Tiempo
17.
J Allergy Clin Immunol ; 119(5): 1251-7, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17379289

RESUMEN

BACKGROUND: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. OBJECTIVE: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. METHODS: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. RESULTS: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals. CONCLUSION: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. CLINICAL IMPLICATIONS: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Ascariasis/inmunología , Interleucina-13/antagonistas & inhibidores , Neumonía/inmunología , Neumonía/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/uso terapéutico , Antígenos Helmínticos/inmunología , Ascaris suum , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interleucina-13/genética , Interleucina-13/inmunología , Macaca fascicularis , Masculino , Datos de Secuencia Molecular , Neumonía/metabolismo , Homología de Secuencia de Aminoácido
18.
J Immunol ; 176(1): 616-24, 2006 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-16365457

RESUMEN

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial tethering of leukocytes to activated platelets and endothelium. We report molecular cloning and characterization of the rat PSGL-1 gene. A neutralizing Ab was generated, and its binding epitope was mapped to the N-terminal binding region of rat PSGL-1. We examined the effects of early PSGL-1 blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 Ab with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pretreatment with anti-PSGL-1 Ab improved portal venous flow, increased bile production, and decreased hepatocellular damage. Rat pretreatment with anti-PSGL-1 Ab prevented hepatic insult in a model of cold ischemia, followed by OLT, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-1, CD3, and OX-62 cells; 3) inhibited expression of proinflammatory cytokine genes (TNF-alpha, IL-1beta, IL-6, IFN-gamma, and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic Bcl-2/Bcl-xL protective genes. Thus, targeting PSGL-1 with a blocking Ab that has diminished Fc-mediated effector function is a simple and effective strategy that provides the rationale for novel therapeutic approaches to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.


Asunto(s)
Anticuerpos Bloqueadores/farmacología , Leucocitos/inmunología , Trasplante de Hígado/inmunología , Daño por Reperfusión/prevención & control , Secuencia de Aminoácidos , Animales , Apoptosis/inmunología , Secuencia de Bases , Sitios de Unión de Anticuerpos/genética , Western Blotting , Biblioteca Genómica , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Humanos , Etiquetado Corte-Fin in Situ , Leucocitos/metabolismo , Trasplante de Hígado/patología , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Daño por Reperfusión/inmunología , Daño por Reperfusión/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido
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