RESUMEN
In situ clinical measurement of receptor occupancy (RO) is challenging, particularly for solid tumors, necessitating the use of mathematical models that predict tumor receptor occupancy to guide dose decisions. A potency metric, average free tissue target to initial target ratio (AFTIR), was previously described based on a mechanistic compartmental model and is informative for near-saturating dose regimens. However, the metric fails at clinically relevant subsaturating antibody doses, as compartmental models cannot capture the spatial heterogeneity of distribution faced by some antibodies in solid tumors. Here we employ a partial differential equation (PDE) Krogh cylinder model to simulate spatiotemporal receptor occupancy and derive an analytical solution, a mechanistically weighted global AFTIR, that can better predict receptor occupancy regardless of dosing regimen. In addition to the four key parameters previously identified, a fifth key parameter, the absolute receptor density (targets/cell), is incorporated into the mechanistic AFTIR metric. Receptor density can influence equilibrium intratumoral drug concentration relative to whether the dose is saturating or not, thereby influencing the tumor penetration depth of the antibody. We derive mechanistic RO predictions based on distinct patterns of antibody tumor penetration, presented as a global AFTIR metric guided by a Thiele Modulus and a local saturation potential (drug equivalent of binding potential for positron emissions tomography imaging) and validate the results using rigorous global and local sensitivity analysis. This generalized AFTIR serves as a more accurate analytical metric to aid clinical dose decisions and rational design of antibody-based therapeutics without the need for extensive PDE simulations. SIGNIFICANCE STATEMENT: Determining antibody-receptor occupancy (RO) is critical for dosing decisions in pharmaceutical development, but direct clinical measurement of RO is often challenging and invasive, particularly for solid tumors. Significant efforts have been made to develop mathematical models and simplified analytical metrics of RO, but these often require complex computer simulations. Here we present a mathematically rigorous but simplified analytical model to accurately predict RO across a range of affinities, doses, drug, and tumor properties.
Asunto(s)
Modelos Teóricos , Neoplasias , Humanos , Anticuerpos Monoclonales , Simulación por Computador , Desarrollo de Medicamentos , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológicoRESUMEN
The development of new antibody-drug conjugates (ADCs) has led to the approval of 7 ADCs by the FDA in 4 years. Given the impact of intratumoral distribution on efficacy of these therapeutics, coadministration of unconjugated antibody with ADC has been shown to improve distribution and efficacy of several ADCs in high and moderately expressed tumor target systems by increasing tissue penetration. However, the benefit of coadministration in low expression systems is less clear. TAK-164, an ADC composed of an anti-GCC antibody (5F9) conjugated to a DGN549 payload, has demonstrated heterogeneous distribution and bystander killing. Here, we evaluated the impact of 5F9 coadministration on distribution and efficacy of TAK-164 in a primary human tumor xenograft mouse model. Coadministration was found to improve the distribution of TAK-164 within the tumor, but it had no significant impact (increase or decrease) on efficacy. Experimental and computational evidence indicates that this was not a result of tumor saturation, increased binding to perivascular cells, or compensatory bystander effects. Rather, the cellular potency of DGN549 was matched with the single-cell uptake of TAK-164 making its IC50 close to its equilibrium binding affinity (KD), and as such, coadministration dilutes total DGN549 in cells below the maximum cytotoxic concentration, thereby offsetting an increased number of targeted cells with decreased ability to kill each cell. These results provide new insights on matching payload potency to ADC delivery to help identify when increasing tumor penetration is beneficial for improving ADC efficacy and demonstrate how mechanistic simulations can be leveraged to design clinically effective ADCs.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Neoplasias , Animales , Anticuerpos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Efecto Espectador , Línea Celular Tumoral , Humanos , Inmunoconjugados/farmacocinética , Ratones , Neoplasias/tratamiento farmacológicoRESUMEN
After several notable clinical failures in early generations, antibody-drug conjugates (ADC) have made significant gains with seven new FDA approvals within the last 3 years. These successes have been driven by a shift towards mechanistically informed ADC design, where the payload, linker, drug-to-antibody ratio, and conjugation are increasingly tailored to a specific target and clinical indication. However, fundamental aspects needed for design, such as payload distribution, remain incompletely understood. Payloads are often classified as "bystander" or "nonbystander" depending on their ability to diffuse out of targeted cells into adjacent cells that may be antigen-negative or more distant from tumor vessels, helping to overcome heterogeneous distribution. Seven of the 11 FDA-approved ADCs employ these bystander payloads, but the depth of penetration and cytotoxic effects as a function of physicochemical properties and mechanism of action have not been fully characterized. Here, we utilized tumor spheroids and pharmacodynamic marker staining to quantify tissue penetration of the three major classes of agents: microtubule inhibitors, DNA-damaging agents, and topoisomerase inhibitors. PAMPA data and coculture assays were performed to compare with the 3D tissue culture data. The results demonstrate a spectrum in bystander potential and tissue penetration depending on the physicochemical properties and potency of the payload. Generally, directly targeted cells show a greater response even with bystander payloads, consistent with the benefit of deeper ADC tissue penetration. These results are compared with computational simulations to help scale the data from in vitro and preclinical animal models to the clinic.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Inmunoconjugados/química , Inmunoconjugados/farmacologíaRESUMEN
With the recent approval of 3 new antibody drug conjugates (ADCs) for solid tumors, this class of drugs is gaining momentum for the targeted treatment of cancer. Despite significant investment, there are still fundamental issues that are incompletely understood. Three of the recently approved ADCs contain payloads exhibiting bystander effects, where the payload can diffuse out of a targeted cell into adjacent cells. These effects are often studied using a mosaic of antigen positive and negative cells. However, the distance these payloads can diffuse in tumor tissue while maintaining a lethal concentration is unclear. Computational studies suggest bystander effects partially compensate for ADC heterogeneity in tumors in addition to targeting antigen negative cells. However, this type of study is challenging to conduct experimentally due to the low concentrations of extremely potent payloads. In this work, we use a series of 3-dimensional cell culture and primary human tumor xenograft studies to directly track fluorescently labeled ADCs and indirectly follow the payload via an established pharmacodynamic marker (γH2A. X). Using TAK-164, an anti-GCC ADC undergoing clinical evaluation, we show that the lipophilic DNA-alkylating payload, DGN549, penetrates beyond the cell targeted layer in GCC-positive tumor spheroids and primary human tumor xenograft models. The penetration distance is similar to model predictions, where the lipophilicity results in moderate tissue penetration, thereby balancing improved tissue penetration with sufficient cellular uptake to avoid significant washout. These results aid in mechanistic understanding of the interplay between antigen heterogeneity, bystander effects, and heterogeneous delivery of ADCs in the tumor microenvironment to design clinically effective therapeutics.
Asunto(s)
Antineoplásicos Inmunológicos/farmacocinética , Efecto Espectador/efectos de los fármacos , Inmunoconjugados/farmacocinética , Animales , Biomarcadores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Monitoreo de Drogas/métodos , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Ratones , Ratones Transgénicos , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Limitations in current imaging tools have long challenged the imaging of small pancreatic islets in animal models. Here, we report the first development and in vivo validation testing of a broad-spectrum and high-absorbance near-infrared optoacoustic contrast agent, E4x12-Cy7. Our near-infrared tracer is based on the amino acid sequence of exendin-4 and targets the glucagon-like peptide-1 receptor (GLP-1R). Cell assays confirmed that E4x12-Cy7 has a high-binding affinity (dissociation constant, Kd, 4.6 ± 0.8 nM). Using the multispectral optoacoustic tomography, we imaged E4x12-Cy7 and optoacoustically visualized ß-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for ß-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.
Asunto(s)
Exenatida/química , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Rayos Infrarrojos , Técnicas Fotoacústicas/métodos , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Exenatida/farmacocinética , Femenino , Insulinoma/metabolismo , Insulinoma/patología , Ratones , Distribución TisularRESUMEN
Several antibody-drug conjugates (ADC) showing strong clinical responses in solid tumors target high expression antigens (HER2, TROP2, Nectin-4, and folate receptor alpha/FRα). Highly expressed tumor antigens often have significant low-level expression in normal tissues, resulting in the potential for target-mediated drug disposition (TMDD) and increased clearance. However, ADCs often do not cross-react with normal tissue in animal models used to test efficacy (typically mice), and the impact of ADC binding to normal tissue antigens on tumor response remains unclear. An antibody that cross-reacts with human and murine FRα was generated and tested in an animal model where the antibody/ADC bind both human tumor FRα and mouse FRα in normal tissue. Previous work has demonstrated that a "carrier" dose of unconjugated antibody can improve the tumor penetration of ADCs with high expression target-antigens. A carrier dose was employed to study the impact on cross-reactive ADC clearance, distribution, and efficacy. Co-administration of unconjugated anti-FRα antibody with the ADC-improved efficacy, even in low expression models where co-administration normally lowers efficacy. By reducing target-antigen-mediated clearance in normal tissue, the co-administered antibody increased systemic exposure, improved tumor tissue penetration, reduced target-antigen-mediated uptake in normal tissue, and increased ADC efficacy. However, payload potency and tumor antigen saturation are also critical to efficacy, as shown with reduced efficacy using too high of a carrier dose. The judicious use of higher antibody doses, either through lower DAR or carrier doses, can improve the therapeutic window by increasing efficacy while lowering target-mediated toxicity in normal tissue.
Asunto(s)
Anticuerpos/administración & dosificación , Anticuerpos/farmacología , Inmunoconjugados/metabolismo , Animales , Anticuerpos/toxicidad , Línea Celular Tumoral , Reacciones Cruzadas/inmunología , Portadores de Fármacos/química , Femenino , Inmunoconjugados/sangre , Ratones , Ratones SCID , Neoplasias/patología , Resultado del TratamientoRESUMEN
Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA-approved antibody-drug conjugates (ADC). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of prostate-specific membrane antigen-binding single-domain (Humabody) ADC constructs to demonstrate that tissue penetration of protein-drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin-binding domain to the single-domain ADCs. A construct lacking an albumin-binding domain was rapidly cleared, leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein-drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing. SIGNIFICANCE: A mechanistic study of protein-drug conjugates demonstrates that a lower potency compound is more effective in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Inmunoconjugados/farmacocinética , Modelos Biológicos , Neoplasias de la Próstata/tratamiento farmacológico , Anticuerpos de Dominio Único/farmacología , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/uso terapéutico , Línea Celular Tumoral , Simulación por Computador , Femenino , Humanos , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Masculino , Ratones , Microscopía Confocal , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/patología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/uso terapéutico , Esferoides Celulares , Relación Estructura-Actividad , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The diabetes community has long desired an imaging agent to quantify the number of insulin-secreting ß-cells, beyond just functional equivalents (insulin secretion), to help diagnose and monitor early stages of both type 1 and type 2 diabetes mellitus. Loss in the number of ß-cells can be masked by a compensatory increase in function of the remaining cells. Since ß-cells form only about 1% of the pancreas and decrease as the disease progresses, only a few imaging agents, such as exendin, have demonstrated clinical potential to detect a drop in the already scarce signal. However, clinical translation of imaging with exendin has been hampered by pancreatic uptake that is higher than expected in subjects with long-term diabetes who lack ß-cells. Exendin binds glucagonlike peptide-1 receptor (GLP-1R), previously thought to be expressed only on ß-cells, but recent studies report low levels of GLP-1R on exocrine cells, complicating ß-cell mass quantification. Methods: Here, we used a GLP-1R knockout mouse model to demonstrate that exocrine binding of exendin is exclusively via GLP-1R (â¼1,000/cell) and not any other receptor. We then used lipophilic Cy-7 exendin to selectively preblock exocrine GLP-1R in healthy and streptozotocin-induced diabetic mice. Results: Sufficient receptors remain on ß-cells for subsequent labeling with a fluorescent- or 111In-exendin. Conclusion: Selective GLP-1R blocking, which improves contrast between healthy and diabetic pancreata and provides a potential avenue for achieving the long-standing goal of imaging ß-cell mass in the clinic.
Asunto(s)
Diabetes Mellitus Tipo 1/diagnóstico por imagen , Diabetes Mellitus Tipo 1/patología , Técnicas de Inactivación de Genes , Receptor del Péptido 1 Similar al Glucagón/deficiencia , Receptor del Péptido 1 Similar al Glucagón/genética , Células Secretoras de Insulina/metabolismo , Páncreas Exocrino/diagnóstico por imagen , Animales , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Péptido 1 Similar al Glucagón/metabolismo , Ratones , Ratones Endogámicos C57BL , Páncreas Exocrino/patología , Tomografía de Emisión de PositronesRESUMEN
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that causes irreversible damage to the joints. However, effective drugs exist that can stop disease progression, leading to intense interest in early detection and treatment monitoring to improve patient outcomes. Imaging approaches have the potential for early detection, but current methods lack sensitivity and/or are time-consuming and expensive. We examined potential routes for self-administration of molecular imaging agents in the form of subcutaneous and oral delivery of an integrin binding near-infrared (NIR) fluorescent imaging agent in an animal model of RA with the long-term goal of increasing safety and patient compliance for screening. NIR imaging has relatively low cost, uses non-ionizing radiation, and provides minimally invasive spatial and molecular information. This proof-of-principle study shows significant uptake of an IRDye800CW agent in inflamed joints of a collagen antibody induced arthritis (CAIA) mouse model compared to healthy joints, irrespective of the method of administration. The imaging results were extrapolated to clinical depths in silico using a 3D COMSOL model of NIR fluorescence imaging in a human hand to examine imaging feasability. With target to background concentration ratios greater than 5.5, which are achieved in the mouse model, these probes have the potential to identify arthritic joints following oral delivery at clinically relevant depths.
Asunto(s)
Artritis Reumatoide/diagnóstico , Colorantes Fluorescentes/farmacología , Inflamación/diagnóstico , Animales , Línea Celular , Modelos Animales de Enfermedad , Fluorescencia , Humanos , Ratones , Imagen Molecular/métodos , Células RAW 264.7 , Espectroscopía Infrarroja Corta/métodosRESUMEN
Antibody-drug conjugate (ADC) development has evolved greatly over the last 3 decades, including the Food and Drug Administration (FDA) approval of several new drugs. However, translating ADCs from the design stage and preclinical promise to clinical success has been a major hurdle for the field, particularly for solid tumors. The challenge in clinical development can be attributed to the difficulty in connecting the design of these multifaceted agents with the impact on clinical efficacy, especially with the accelerated development of 'next-generation' ADCs containing a variety of innovative biophysical developments. Given their complex nature, there is an urgent need to integrate holistic ADC characterization approaches. This includes comprehensive in vivo assessment of systemic, intratumoral and cellular pharmacokinetics, pharmacodynamics, toxicodynamics, and interactions with the immune system, with the aim of optimizing the ADC therapeutic window. Pharmacokinetic/pharmacodynamic factors influencing the ADC therapeutic window include (1) selecting optimal target and ADC components for prolonged and stable plasma circulation to increase tumoral uptake with minimal non-specific systemic toxicity, (2) balancing homogeneous intratumoral distribution with efficient cellular uptake, and (3) translating improved ADC potency to better clinical efficacy. Balancing beneficial immunological effects such as Fc-mediated and payload-mediated immune cell activation against harmful immunogenic/toxic effects is also an emerging concern for ADCs. Here, we review practical considerations for tracking ADC efficacy and toxicity, as aided by high-resolution biomolecular and immunological tools, quantitative pharmacology, and mathematical models, all of which can elucidate the relative contributions of the multitude of interactions governing the ADC therapeutic window.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Inmunoconjugados/inmunología , Inmunoconjugados/farmacocinética , Animales , Diseño de Fármacos , Humanos , Inmunoconjugados/administración & dosificación , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunologíaRESUMEN
Molecular imaging is advantageous for screening diseases such as breast cancer by providing precise spatial information on disease-associated biomarkers, something neither blood tests nor anatomical imaging can achieve. However, the high cost and risks of ionizing radiation for several molecular imaging modalities have prevented a feasible and scalable approach for screening. Clinical studies have demonstrated the ability to detect breast tumors using nonspecific probes such as indocyanine green, but the lack of molecular information and required intravenous contrast agent does not provide a significant benefit over current noninvasive imaging techniques. Here we demonstrate that negatively charged sulfate groups, commonly used to improve solubility of near-infrared fluorophores, enable sufficient oral absorption and targeting of fluorescent molecular imaging agents for completely noninvasive detection of diseased tissue such as breast cancer. These functional groups improve the pharmacokinetic properties of affinity ligands to achieve targeting efficiencies compatible with clinical imaging devices using safe, nonionizing radiation (near-infrared light). Together, this enables development of a "disease screening pill" capable of oral absorption and systemic availability, target binding, background clearance, and imaging at clinically relevant depths for breast cancer screening. This approach should be adaptable to other molecular targets and diseases for use as a new class of screening agents.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Administración Oral , Animales , Línea Celular , Medios de Contraste/administración & dosificación , Modelos Animales de Enfermedad , Detección Precoz del Cáncer/métodos , Femenino , Colorantes Fluorescentes/administración & dosificación , Células HEK293 , Humanos , Verde de Indocianina/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Imagen Molecular/métodos , Espectroscopía Infrarroja Corta/métodosRESUMEN
Virus-like particles (VLPs) are nanoscale biological structures consisting of viral proteins assembled in a morphology that mimic the native virion but do not contain the viral genetic material. The possibility of chemically and genetically modifying the proteins contained within VLPs makes them an attractive system for numerous applications. As viruses are potent immune activators as well as natural delivery vehicles of genetic materials to their host cells, VLPs are especially well suited for antigen and drug delivery applications. Despite the great potential, very few VLP designs have made it through clinical trials. In this review, we will discuss the challenges of developing VLPs for antigen and drug delivery, strategies being explored to address these challenges, and the genetic and chemical approaches available for VLP engineering.
Asunto(s)
Antígenos/química , Sistemas de Liberación de Medicamentos/métodos , Ingeniería Genética/métodos , Virus/genética , Animales , Antígenos/inmunología , Humanos , Virus/químicaRESUMEN
We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.
Asunto(s)
Alginatos/química , Coenzimas/metabolismo , Enzimas Inmovilizadas/química , Biocatálisis , Coenzimas/química , Enzimas Inmovilizadas/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Deshidrogenasas del Alcohol de Azúcar/química , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Xilulosa/biosíntesisRESUMEN
Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts.