Journal of Pharmacology and Experimental Therapeutics, Apr 27, 2023
In situ clinical measurement of receptor occupancy (RO) is challenging, particularly for solid tu... more 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 sub-saturating 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 (gAFTIR), 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 PET 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.
While the recent FDA approval of six new antibody drug conjugates (ADCs) is promising, attrition ... more While the recent FDA approval of six new antibody drug conjugates (ADCs) is promising, attrition of ADCs during clinical development is still high. The inherent complexity of ADCs is a double-edged sword, providing opportunities to perfect therapeutic action while also increasing confounding factors in therapeutic failures. ADC design drives their pharmacokinetics and pharmacodynamics, requiring deeper analysis than commonly used Cmax and area under curve (AUC) metrics to scale dosing to the clinic. Common features for current FDA-approved ADCs targeting solid tumors include humanized IgG1 antibody domains, highly expressed tumor receptors, and large antibody doses. The potential consequences of these shared features for the clinical pharmacokinetics and mechanism of action are discussed, highlighting key design aspects for successful solid tumor ADCs.
Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrat... more 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 (ADCs). 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 (PSMA)-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.
This file contains information on the methods used during drug formulation, reaction, and purific... more This file contains information on the methods used during drug formulation, reaction, and purification as well as an in-depth description of in vitro assays, experimental/computational spheroid work, and tumor growth measurements. The figures and tables provide characterization of therapeutic constructs, rationale for efficacy study dosing, and Krogh Cylinder simulation parameters.
Limitations in current imaging tools have long challenged the imaging of small pancreatic islets ... more 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, E4 x12-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 E4 x12-Cy7 has a high-binding affinity (dissociation constant, Kd, 4.6 6 0.8 nM). Using the multispectral optoacoustic tomography, we imaged E4 x12-Cy7 and optoacoustically visualized b-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for b-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.
bioRxiv (Cold Spring Harbor Laboratory), Apr 30, 2020
Limitations in current imaging tools have long challenged the imaging of small pancreatic islets ... more 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 (E4x12-Cy7) 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 (IC50 = 4.6 ± 0.8 nM). Using the multispectral optoacoustic tomography (MSOT), 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.
Poor tissue penetration remains a major challenge for antibody-based therapeutics of solid tumors... more Poor tissue penetration remains a major challenge for antibody-based therapeutics of solid tumors, but proper dosing can improve the tissue penetration and thus therapeutic efficacy of these biologics. Due to dose-limiting toxicity of the small molecule payload, antibody-drug conjugates (ADCs) are administered at a much lower dose than their parent antibodies, which further reduces tissue penetration. We conducted an early-phase clinical trial (NCT02415881) and previously reported the safety of an antibody-dye conjugate (panitumumab-IRDye800CW) as primary outcome. Here, we report a retrospective exploratory analysis of the trial to evaluate whether co-administration of an unconjugated antibody could improve the intratumoral distribution of the antibody-dye conjugate in patients. By measuring the multiscale distribution of the antibody-dye conjugate, this study demonstrates improved microscopic antibody distribution without increasing uptake (toxicity) in healthy tissue when co-administered with the parent antibody, supporting further clinical investigation of the coadministration dosing strategy to improve the tumor penetration of ADCs.
Please find attached a manuscript, "Kinetics of Anti-Carcinoembryonic Antigen (CEA) Antibody Inte... more Please find attached a manuscript, "Kinetics of Anti-Carcinoembryonic Antigen (CEA) Antibody Internalization: Negligible Effects of Affinity, Bivalency, or Stability", to be considered for publication in Cancer Research. In this work, we perform careful quantitative measurements of the rate of endocytic consumption of a panel of anti-CEA scFvs and IgGs, varying in affinity, valency, and stability. CEA is a commonly targeted antigen, and yet there are widespread misconceptions regarding its cellular trafficking (e.g. that it is "noninternalizing"). The key result of this manuscript is that antibodies bound to CEA are generally internalized with a half time of 10-16 hours, regardless of valency, affinity or stability. In the accompanying manuscript by Thurber, we demonstrate that although this may seem like a slow uptake rate, it determines the depth of penetration of antibodies into LS174T spheroids. We believe that together, these two manuscripts experimentally elucidate a major factor determining the penetration of antibodies into tumor tissue.
Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrat... more 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.
Journal of Pharmacology and Experimental Therapeutics, Apr 27, 2023
In situ clinical measurement of receptor occupancy (RO) is challenging, particularly for solid tu... more 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 sub-saturating 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 (gAFTIR), 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 PET 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.
While the recent FDA approval of six new antibody drug conjugates (ADCs) is promising, attrition ... more While the recent FDA approval of six new antibody drug conjugates (ADCs) is promising, attrition of ADCs during clinical development is still high. The inherent complexity of ADCs is a double-edged sword, providing opportunities to perfect therapeutic action while also increasing confounding factors in therapeutic failures. ADC design drives their pharmacokinetics and pharmacodynamics, requiring deeper analysis than commonly used Cmax and area under curve (AUC) metrics to scale dosing to the clinic. Common features for current FDA-approved ADCs targeting solid tumors include humanized IgG1 antibody domains, highly expressed tumor receptors, and large antibody doses. The potential consequences of these shared features for the clinical pharmacokinetics and mechanism of action are discussed, highlighting key design aspects for successful solid tumor ADCs.
Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrat... more 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 (ADCs). 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 (PSMA)-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.
This file contains information on the methods used during drug formulation, reaction, and purific... more This file contains information on the methods used during drug formulation, reaction, and purification as well as an in-depth description of in vitro assays, experimental/computational spheroid work, and tumor growth measurements. The figures and tables provide characterization of therapeutic constructs, rationale for efficacy study dosing, and Krogh Cylinder simulation parameters.
Limitations in current imaging tools have long challenged the imaging of small pancreatic islets ... more 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, E4 x12-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 E4 x12-Cy7 has a high-binding affinity (dissociation constant, Kd, 4.6 6 0.8 nM). Using the multispectral optoacoustic tomography, we imaged E4 x12-Cy7 and optoacoustically visualized b-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for b-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.
bioRxiv (Cold Spring Harbor Laboratory), Apr 30, 2020
Limitations in current imaging tools have long challenged the imaging of small pancreatic islets ... more 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 (E4x12-Cy7) 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 (IC50 = 4.6 ± 0.8 nM). Using the multispectral optoacoustic tomography (MSOT), 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.
Poor tissue penetration remains a major challenge for antibody-based therapeutics of solid tumors... more Poor tissue penetration remains a major challenge for antibody-based therapeutics of solid tumors, but proper dosing can improve the tissue penetration and thus therapeutic efficacy of these biologics. Due to dose-limiting toxicity of the small molecule payload, antibody-drug conjugates (ADCs) are administered at a much lower dose than their parent antibodies, which further reduces tissue penetration. We conducted an early-phase clinical trial (NCT02415881) and previously reported the safety of an antibody-dye conjugate (panitumumab-IRDye800CW) as primary outcome. Here, we report a retrospective exploratory analysis of the trial to evaluate whether co-administration of an unconjugated antibody could improve the intratumoral distribution of the antibody-dye conjugate in patients. By measuring the multiscale distribution of the antibody-dye conjugate, this study demonstrates improved microscopic antibody distribution without increasing uptake (toxicity) in healthy tissue when co-administered with the parent antibody, supporting further clinical investigation of the coadministration dosing strategy to improve the tumor penetration of ADCs.
Please find attached a manuscript, "Kinetics of Anti-Carcinoembryonic Antigen (CEA) Antibody Inte... more Please find attached a manuscript, "Kinetics of Anti-Carcinoembryonic Antigen (CEA) Antibody Internalization: Negligible Effects of Affinity, Bivalency, or Stability", to be considered for publication in Cancer Research. In this work, we perform careful quantitative measurements of the rate of endocytic consumption of a panel of anti-CEA scFvs and IgGs, varying in affinity, valency, and stability. CEA is a commonly targeted antigen, and yet there are widespread misconceptions regarding its cellular trafficking (e.g. that it is "noninternalizing"). The key result of this manuscript is that antibodies bound to CEA are generally internalized with a half time of 10-16 hours, regardless of valency, affinity or stability. In the accompanying manuscript by Thurber, we demonstrate that although this may seem like a slow uptake rate, it determines the depth of penetration of antibodies into LS174T spheroids. We believe that together, these two manuscripts experimentally elucidate a major factor determining the penetration of antibodies into tumor tissue.
Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrat... more 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.
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