RESUMEN
GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.
Asunto(s)
Productos Biológicos/uso terapéutico , Encéfalo/metabolismo , Enfermedades por Almacenamiento Lisosomal/terapia , Progranulinas/uso terapéutico , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Endosomas/metabolismo , Femenino , Demencia Frontotemporal/sangre , Demencia Frontotemporal/líquido cefalorraquídeo , Gliosis/complicaciones , Gliosis/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/patología , Metabolismo de los Lípidos , Lipofuscina/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Degeneración Nerviosa/patología , Fenotipo , Progranulinas/deficiencia , Progranulinas/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Transferrina/metabolismo , Distribución TisularRESUMEN
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/terapia , Mastocitos/enzimología , Mastocitos/inmunología , Triptasas/antagonistas & inhibidores , Triptasas/inmunología , Adolescente , Regulación Alostérica/inmunología , Animales , Línea Celular , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , ConejosRESUMEN
Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos/uso terapéutico , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacocinética , Antineoplásicos/efectos adversos , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Macaca fascicularis , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Inmunoglobulina G/química , Animales , Formación de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Complemento C1q/inmunología , Cricetinae , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Ratones , Conformación Proteica , Receptores de IgG/metabolismo , TemperaturaRESUMEN
Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Regulación de la Expresión Génica , Inmunoglobulina G/inmunología , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Animales , Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Pulmón/inmunología , Pulmón/metabolismo , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Plásmidos/metabolismo , Ingeniería de Proteínas/métodos , Resonancia por Plasmón de SuperficieRESUMEN
Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.
Asunto(s)
Barrera Hematoencefálica , Oligonucleótidos Antisentido , ARN Largo no Codificante , Receptores de Transferrina , Animales , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/administración & dosificación , Barrera Hematoencefálica/metabolismo , Receptores de Transferrina/metabolismo , Humanos , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Ratones , Transporte Biológico , Macaca fascicularis , Técnicas de Silenciamiento del Gen , Distribución TisularRESUMEN
Brain exposure of systemically administered biotherapeutics is highly restricted by the blood-brain barrier (BBB). Here, we report the engineering and characterization of a BBB transport vehicle targeting the CD98 heavy chain (CD98hc or SLC3A2) of heterodimeric amino acid transporters (TVCD98hc). The pharmacokinetic and biodistribution properties of a CD98hc antibody transport vehicle (ATVCD98hc) are assessed in humanized CD98hc knock-in mice and cynomolgus monkeys. Compared to most existing BBB platforms targeting the transferrin receptor, peripherally administered ATVCD98hc demonstrates differentiated brain delivery with markedly slower and more prolonged kinetic properties. Specific biodistribution profiles within the brain parenchyma can be modulated by introducing Fc mutations on ATVCD98hc that impact FcγR engagement, changing the valency of CD98hc binding, and by altering the extent of target engagement with Fabs. Our study establishes TVCD98hc as a modular brain delivery platform with favorable kinetic, biodistribution, and safety properties distinct from previously reported BBB platforms.
Asunto(s)
Barrera Hematoencefálica , Encéfalo , Animales , Ratones , Distribución Tisular , Anticuerpos , Ingeniería , Macaca fascicularisRESUMEN
Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR-binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.
Asunto(s)
Iduronato Sulfatasa , Mucopolisacaridosis II , Receptores de Transferrina , Proteínas Recombinantes de Fusión , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Iduronato Sulfatasa/metabolismo , Iduronato Sulfatasa/farmacología , Lisosomas/metabolismo , Ratones , Mucopolisacaridosis II/metabolismo , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Distribución TisularRESUMEN
Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the ß-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits ß-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive ß-tryptase monomers, and may provide an alternative strategy for antibody engineering.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Inmunoglobulina G/metabolismo , Triptasas/metabolismo , Regulación Alostérica/efectos de los fármacos , Secuencia de Aminoácidos , Heparina/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Modelos Moleculares , Proteínas Mutantes/química , Unión Proteica/efectos de los fármacos , Multimerización de Proteína , Triptasas/químicaRESUMEN
Systemic cytokine release and on-target/off-tumor toxicity to normal tissues are the main adverse effects limiting the clinical utility of T cell-redirecting therapies. This study was designed to determine how binding affinity for CD3 and tumor target HER2 impact the efficacy and nonclinical safety of anti-HER2/CD3 T cell-dependent antibodies (TDBs). Affinity was found to be a major determinant for the overall tolerability. Higher affinity for CD3 associated with rapidly elevated peripheral cytokine concentrations, weight loss in mice, and poor tolerability in cynomolgus monkeys. A TDB with lower CD3 affinity was better tolerated in cynomolgus monkeys compared with a higher CD3-affinity TDB. In contrast to tolerability, T cell binding affinity had only limited impact on in vitro and in vivo antitumor activity. High affinity for HER2 was critical for the tumor-killing activity of anti-HER2/CD3 TDBs, but higher HER2 affinity also associated with a more severe toxicity profile, including cytokine release and damage to HER2-expressing tissues. The tolerability of the anti-HER2/CD3 was improved by implementing a dose-fractionation strategy. Fine-tuning the affinities for both the tumor target and CD3 is likely a valuable strategy for achieving maximal therapeutic index of CD3 bispecific antibodies.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Afinidad de Anticuerpos , Antineoplásicos Inmunológicos/inmunología , Receptor ErbB-2/inmunología , Animales , Anticuerpos Biespecíficos/química , Antineoplásicos Inmunológicos/química , Complejo CD3/química , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Humanos , Macaca fascicularis , Receptor ErbB-2/químicaRESUMEN
Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-ß-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.
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Secretasas de la Proteína Precursora del Amiloide , Barrera Hematoencefálica , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Haplorrinos/metabolismo , Fragmentos Fc de Inmunoglobulinas , Ratones , Receptores de Transferrina/metabolismoRESUMEN
Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.
Asunto(s)
Barrera Hematoencefálica , Iduronato Sulfatasa , Animales , Encéfalo , Modelos Animales de Enfermedad , Terapia de Reemplazo Enzimático , Lisosomas , RatonesRESUMEN
MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences ("mucin repeats") in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique, nonrepeating epitope in the ECD and another (3A5) that recognizes the repeats and binds multiple sites on each MUC16 protein. 3A5 conjugated to cytotoxic drugs exhibited superior toxicity against tumor cells in vitro and in tumor xenograft models compared with antibody-drug conjugates of 11D10. Importantly, drug conjugates of 3A5 were well tolerated in primates at levels in excess of therapeutic doses. Additionally, the presence of circulating CA125 in a rat model did not exacerbate the toxicity of 3A5 drug conjugates. We conclude that targeting the repeat MUC16 domains, thereby increasing cell-associated levels of drug-conjugated antibody, provides superior efficacy in vitro and in vivo without compromising safety.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígeno Ca-125/inmunología , Proteínas de la Membrana/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Animales , Sitios de Unión de Anticuerpos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Ratones SCID , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Association with albumin as a means to improve biodistribution and tumor deposition of a Fab was investigated using AB.Fab4D5, a bifunctional molecule derived from trastuzumab (HERCEPTIN) capable of binding albumin and tumor antigen HER2 (erbB2) simultaneously. AB.Fab4D5 was compared with trastuzumab and a trastuzumab-derived Fab (Fab4D5) for the ability to target tumors overexpressing HER2 in mouse mammary tumor virus/HER2 allograft models. Biodistribution was monitored using intravital microscopy, histology, and integrated single-photon emission computed tomography/computed tomography analysis. Fab4D5 tumor deposition was characterized by rapid but transient appearance in tumor at 2 h with little retention, followed by rapid accumulation in kidney by 6 h. Trastuzumab was slow to accumulate in tumors and slow to clear from normal tissues, although significant tumor deposition was achieved by 24 h. In contrast, AB.Fab4D5 was observed at 2 h in tumor and its presence was sustained beyond 24 h similar to trastuzumab. Intravital microscopy revealed that at peak tumor accumulation, tumor cell staining by AB.Fab4D5 was more uniform than for Fab4D5 or trastuzumab. Similar tumor deposition was achieved for both AB.Fab4D5 and trastuzumab at 48 h (35.9 +/- 1.8% and 38.2 +/- 3.1% injected dose/g); however, AB.Fab4D5 targeted tumors more rapidly and quickly cleared from blood, leading to a lower overall normal tissue exposure. Importantly, unlike Fab4D5, AB.Fab4D5 did not accumulate in kidney, suggesting that association with albumin leads to an altered route of clearance and metabolism. Rapid targeting, excellent tumor deposition and retention, coupled with high tumor to blood ratios may make AB.Fab an exceptional molecule for imaging and cancer therapy.
Asunto(s)
Albúminas/farmacocinética , Anticuerpos Monoclonales/farmacocinética , Inmunoconjugados/farmacocinética , Fragmentos de Inmunoglobulinas/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Albúminas/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunoconjugados/química , Fragmentos de Inmunoglobulinas/química , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Datos de Secuencia Molecular , Cintigrafía , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/metabolismo , TrastuzumabRESUMEN
Non-immune (naïve) antibody phage libraries have become an important source of human antibodies. The synthetic phage antibody library described here utilizes a single human framework with a template containing human consensus complementarity-determining regions (CDRs). Diversity of the libraries was introduced at select CDR positions using tailored degenerate and trinucleotide codons that mimic natural human antibodies. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 semaphorins, is expressed on endothelial cells and neurons. NRP1 is required for vascular development and is expressed widely in the developing vasculature. To investigate the possibility of function blocking antibodies to NRP1 as potential therapeutics, and study the consequence of targeting NRP1 in murine tumor models, panels of antibodies that cross-react with human and murine NRP1 were generated from a designed antibody phage library. Antibody (YW64.3) binds to the CUB domains (a1a2) of NRP1 and completely blocks Sema3A induced neuron collapse; antibody (YW107.4.87) binds to the coagulation factor V/VIII domains (b1b2) of NRP1 and blocks VEGF binding and VEGF induced cell migration. YW107.4.87 inhibits tumor growth in animal xenograft models. These antibodies have provided valuable tools to study the roles of NRP1 in vascular and tumor biology.
Asunto(s)
Anticuerpos Bloqueadores/inmunología , Neuropilina-1/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/química , Afinidad de Anticuerpos/efectos de los fármacos , Células CHO , Movimiento Celular/efectos de los fármacos , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Cricetinae , Cricetulus , Conos de Crecimiento/efectos de los fármacos , Humanos , Inmunoglobulina G/inmunología , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Neoplasias/patología , Estructura Terciaria de Proteína/efectos de los fármacos , Semaforina-3A/farmacologíaRESUMEN
Anti-HER2/CD3, a T-cell-dependent bispecific antibody (TDB) construct, induces T-cell-mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell-targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ε expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell-rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell-containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776-85. ©2018 AACR.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/farmacocinética , Complejo CD3/inmunología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Receptor ErbB-2/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Afinidad de Anticuerpos , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoterapia , Ratones , Ratones Desnudos , Ratones Transgénicos , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/patología , Distribución Tisular , Células Tumorales CultivadasRESUMEN
The apolipoprotein E E4 allele of the APOE gene is the strongest genetic factor for late-onset Alzheimer disease (LOAD). There is compelling evidence that apoE influences Alzheimer disease (AD) in large part by affecting amyloid ß (Aß) aggregation and clearance; however, the molecular mechanism underlying these findings remains largely unknown. Herein, we tested whether anti-human apoE antibodies can decrease Aß pathology in mice producing both human Aß and apoE4, and investigated the mechanism underlying these effects. We utilized APPPS1-21 mice crossed to apoE4-knockin mice expressing human apoE4 (APPPS1-21/APOE4). We discovered an anti-human apoE antibody, anti-human apoE 4 (HAE-4), that specifically recognizes human apoE4 and apoE3 and preferentially binds nonlipidated, aggregated apoE over the lipidated apoE found in circulation. HAE-4 also binds to apoE in amyloid plaques in unfixed brain sections and in living APPPS1-21/APOE4 mice. When delivered centrally or by peripheral injection, HAE-4 reduced Aß deposition in APPPS1-21/APOE4 mice. Using adeno-associated virus to express 2 different full-length anti-apoE antibodies in the brain, we found that HAE antibodies decreased amyloid accumulation, which was dependent on Fcγ receptor function. These data support the hypothesis that a primary mechanism for apoE-mediated plaque formation may be a result of apoE aggregation, as preferentially targeting apoE aggregates with therapeutic antibodies reduces Aß pathology and may represent a selective approach to treat AD.