Publications || Code

# Title Authors Journal Date Figure
63 Design of high avidity and low affinity antibodies for in situ control of antibody drug conjugate targeting R. Evans, G.M. Thurber Scientific Reports 2022
Improving ADC distribution through coadministration of HALA antibody carrier doses. Administration of an ADC (single agent) produces heterogeneous perivascular distribution due to rapid binding relative to transport in the tissue in high expression tumors (e.g. at 3.6 mg/kg for Kadcyla, (A). Tumor penetration is improved when the ADC is co-administered with a saturating dose of unconjugated antibody (e.g. 8:1 ratio of antibody to ADC, (B). The two antibodies compete for binding sites increasing ADC penetration. However, in low expression systems, unconjugated antibodies can outcompete the ADC (C), lowering efficacy. Coadministration of a HALA antibody allows competition in high expression systems (B) while enabling the ADC to outcompete the HALA antibody in low expression systems (D).
62 Predictive Simulations in Preclinical Oncology to Guide the Translation of Biologics S. Dong, I. Nessler, A. Kopp, B. Rubahamya, G.M. Thurber Frontiers in Pharmacology 2022
Predictive Simulations in Development. Rather than focusing on each step in the pipeline (A), top, robust simulations of drug distribution can be employed at the earliest stages of development to forecast clinical application. During development, the predictions can be refined to improve the accuracy of the forecast or identify discrepancies (A, bottom). While predictive models for small molecule drugs typically assume tissue concentrations proportional to the plasma concentration due to fast distribution (B), the local metabolism/degradation of biologics and slow tissue penetration require alternative approaches for accurate predictions (C).
61 Cellular-Resolution Imaging of Bystander Payload Tissue Penetration from Antibody-Drug Conjugates Khera, E., S. Dong, H. Huang, L. de Bever, F.L. van Delft, G.M. Thurber Molecular Cancer Therapeutics 2021
High resolution imaging of payload penetration in spheroids. A, Schematic of pharmacodynamic marker manifestation for bystander and non-bystander ADC payloads. B, Immunofluorescence (IF) microscopy of spheroids treated with 25 nmol/L AF680-trastuzumab (green) conjugated to three different microtubule inhibitors. The pharmacodynamic signal (red) shows different extents of penetration into the spheroids. Nuclei are labeled with Hoechst (blue).
60 Simulating the Selection of Resistant Cells with Bystander Killing and Antibody Coadministration in Heterogeneous HER2 Positive Tumors Menezes, B., J.L. Linderman, G.M. Thurber Drug Metabolism and Disposition 2021
(A) The ABM environment is composed of cancer cells with different characteristics (e.g. different number of receptors or sensitivity to treatment) and blood vessels through which therapeutics are delivered. B) The model tests different regimens: 1) single agent administration (top panel) vs coadministration of antibody with ADC (bottom panel) and 2) non-bystander payloads (left column) vs bystander payloads (right column). The model can be used to examine these regimens for cell populations containing resistant cells.
59 Key metrics to expanding the pipeline of successful antibody–drug conjugates Nessler, I., B. Menezes, G.M. Thurber Trends in Pharmacological Sciences (TIPS) 2021
Scaling antibody–drug conjugates (ADCs) to the clinic.(A) There are many reasons for the failure of animal models to capture clinical results in drugdevelopment. In the case of ADCs, two specific reasons include tissue penetration and tumor saturation.In vitroassays do not capture delivery issues, and highlypotent compounds selectedin vitrooften increase toxicity, lowering tolerable dosesin vivo. The lower doses result in less tissue penetration, thus reducing efficacyin vivo(left arrow). Mice often tolerate higher doses than humans, and mouse tumor lines are sometimes less sensitive to the payload. Both of these factors can resultin higher doses being administered in preclinical animal models (better tissue penetration), which sometimes results in tumor saturation. Notably, trends identified usingsaturating doses can be the opposite of those at subsaturating doses commonly encountered in the clinic (right arrow), making it crucial to understand the saturationlevel in preclinical models and the predicted level in the clinic.
58 Reply: From mice to men: the exocrine pancreas does not matter for human GLP-1 receptor imaging G.M. Thurber Journal of Nuclear Medicine (JNM) 2021
57 Quantifying ADC bystander payload penetration with cellular resolution using pharmacodynamic mapping Khera, E., C. Cilliers, M.D. Smith, M.L. Gano, K.C. Lai, T.A. Keating, A. Kopp, I. Nessler, A.O. Abu-Yousif, G.M. Thurber Neoplasia 2021
Quantification of DGN549 bystander penetration distance in HEK293-GCC spheroids. (A) Immunohistochemistry of spheroid sections treated with TAK-164 at varying conditions showed an increasing penetration front of DGN549-mediated DSBs, indicated by the spatiotemporal increase of γ H2A.X signal in regions not targeted directly by the ADC. (B) Computational simulations showed a similar distribution pattern as the experiments spheroids. (C) High-resolution images confirm nuclear localization of the γ H2A.X signal detected beyond ADC-targeted cells and revealed formation of various staining patterns observed in literature [34] (white arrowhead = pan nuclear staining, yellow arrowhead = large foci, white arrow = small foci, yellow arrow = no foci). Scale bar = 20 μm. (D) Euclidean distance mapping of tumor spheroids showed that γ H2A.X signal (±SD) is observed well beyond the ADC-targeted (±SEM) cell layers, providing a quantitative estimate of DGN549 bystander penetration. The γ H2A.X signal appeared to be reduced in ADC-targeted cells, though it is unclear if this is a result of peripheral free payload washout, signaling-mediated suppression of γ H2A.X by 5F9 antibody, or some other factors (E) Radial plots of the computational results show similar ADC and DGN549 distribution patterns as the Euclidean map generated from experimental data.
56 Antibody Co-Administration Can Improve Systemic and Local Distribution of Antibody Drug Conjugates to Increase In Vivo Efficacy Ponte, J., L. Lanieri, E. Khera, R. Laleau, O. Ab, C. Espelin, N. Kohli, B. Matin, Y. Setiady, M.L. Miller, T.A. Keating, R. Chari, J. Pinkas, R. Gregory, G.M. Thurber Molecular Cancer Therapeutics (MCT) 2020
The schematic of half versus two skull and crossbones represents payload potency, not DAR. The high dose of potent DM4 payload in a high FRα expression (KB) tumor model can target more cells with lethal payload concentration (A, left) compared to a lower dose of highly potent DGN549 ADC (A, right). This causes complete tumor regression at clinically tolerable doses of DM4 (B, left) but only a partial response with DGN549, which is tolerated at lower total antibody doses (B, right). This is due to the lower tissue penetration from a reduced antibody dose In a low expression xenograft model (OV90), the FRα-targeting DM4 ADC saturates the tumor (C, left) but can only deliver enough DM4 payload per cell to invoke a partial response (D, left). At super-saturating antibody doses, a significant amount of the ADC washes out of the tumor without being taken up by cells. In contrast, the highly potent DGN549-ADC can penetrate deeper into the tumor tissue while maintaining a lethal dose to the targeted cells (C, right), there by efficiently kill cells with low expression at clinically tolerable doses (D, right). Animals dosed 7 days post inoculation; ADC doses were based on the payload dose (g payload per kg body weight) but are reported as mg ADC per kg body weight.
55 Co-administered Antibody Improves Penetration of Antibody-Dye Conjugate into Human Cancers with Implications for Antibody-Drug Conjugates Lu, G., N. Nishio, N. van den Berg, B. Martin, S. Fakurnejad, S. van Keulen, A. Dimitrios Colevas, G.M. Thurber, and E. Rosenthal Nature Communications 2020
a) Each tumor was gridded into small square regions and antibody penetration within each square region was quantified as the ratio of fluorescent positive area to EGFR-positive area. b) The ratio of fluorescence to EGFR over the entire tumor area was not significantly different between the LD and non-LD group (Scale bar: 5 mm). c)–e) Comparison of three metrics, including interquartile range (IQR), Pearson’s correlation coefficient between fluorescence and EGFR expression and CLCM correlation, all reflected less heterogeneity in antibody penetration across tumors in patients with loading dose. f), g) Co-administration of parent antibody could overcome binding site barrier and improve antibody–dye distribution within the tumors. f) As fluorescent antibody extravasates from blood vessels (red tube), it immediately binds EGFR in the tissue (represented by cells in the cube). g) By co-administering non-fluorescent antibody, the same amount of fluorescent antibody reaches the tissue, but these antibodies compete for binding sites locally in the tissue. This results in improved distribution of fluorescent antibody at the microscopic scale (Scale bar: 250 μm). Mann–Whitney U test (two-tailed) was used in b–e. p value = 0.025 c, 0.025 d, 0.036 e. The number of independent patient samples available for b–e: n = 12 patients in the LD group and n = 10 in the non-LD group. Graphs plotted mean with standard deviation.
54 Optoacoustic imaging of GLP-1 Receptor with a near-infrared exendin-4 analog Roberts, S., E. Khera, C. Choi, T. Navaratna, J. Grimm, G.M. Thurber and T. Reiner Journal of Nuclear Medicine (JNM) 2020
The accumulation of E4x12-Cy7 is GLP-1R mediated. (A) Optoacoustic signal at varying wavelengths (680- 900 nm, 10 nm step) taken at the kidney region (left) and the corresponding MSOT images (right). (B) Ex vivo MSOT images of organs from mice that were injected with E4x12-Cy7. From left to right are the pancreas, muscle and MIN6 (right) and the corresponding quantification (left). GLP-1R mediated accumulation. (C) Representative in vivo epifluorescent images of mice that were injected either with saline, blocking or E4x12-Cy7 (left) and quantification (right). (D) Corresponding ex vivo epifluorescent images from left to right of MIN6, muscle and pancreas that were either injected with saline, blocking or E4x12-Cy7 (left) and quantification (right).
53 Practical Guide for Quantification of In Vivo Degradation Rates for Therapeutic Proteins with Single‐Cell Resolution Using Fluorescence Ratio Imaging Nessler, I., C. Cilliers, and G.M. Thurber Pharmaceutics 2020
Dual label near-infrared fluorescence imaging technique concept. (a) Graphic depiction of dually labeled antibody binding the cell, internalizing, and degrading. The non-residualizing DDAO (red star) leaks out of the cell following protein degradation, while the residualizing IRDye (green star) is trapped. (b) DDAO and IRDye dye chemical structures, molecular weights, maximum excitation/emission, and logD (pH 7.4) calculated by MarvinSketch. (c) The plasma concentration over time of trastuzumab-IRDye, dually labeled (DDAO/IRDye or BoDIPY-FL/AF647) trastuzumab and trastuzumab (Ab, measured by ELISA).
52 Increased Tumor Penetration of Single-Domain Antibody Drug Conjugates Improves In Vivo Efficacy in Prostate Cancer Models Nessler, I., E. Khera, S. Vance, A. Kopp, Q. Qiu, T. A. Keating, A. O. Abu-Yousif, T. Sandal, J. Legg, L. Thompson, N. Goodwin, and G.M. Thurber Cancer Research 2020
In vivo tissue penetration of fluorescent antibodies. Twenty-four hours after tail vein administration of Alexa Fluor 680 antibody constructs dosed at the same level as the efficacy studies, DU145-PSMA xenografts were frozen in OCT and processed for histology. Blood vessels, shown in red, were ex vivo–labeled with Alexa Fluor 555 anti-CD31 antibody, while penetration of Alexa Fluor 680 antibody constructs is shown in green.
51 Directed Evolution Using Stabilized Bacterial Peptide Display Navaratna, T., L. Atangcho, M. Mahajan, V. Subramanian, M. Case, A. Min, D. Tresnak, and G.M. Thurber Journal of American Chemical Society 2019
Chemically stabilized peptides have attracted intense interest by academics and pharmaceutical companies due to their potential to hit currently “undruggable” targets. However, engineering an optimal sequence, stabilizing linker location, and physicochemical properties is a slow and arduous process. By pairing non-natural amino acid incorporation and cell surface click chemistry in bacteria with high-throughput sorting, we developed a method to quantitatively select high affinity ligands and applied the Stabilized Peptide Evolution by E. coli Display technique to develop disrupters of the therapeutically relevant MDM2-p53 interface. Through in situ stabilization on the bacterial surface, we demonstrate rapid isolation of stabilized peptides with improved affinity and novel structures. Several peptides evolved a second loop including one sequence (Kd = 1.8 nM) containing an i, i+4 disulfide bond. NMR structural determination indicated a bent helix in solution and bound to MDM2. The bicyclic peptide had improved protease stability, and we demonstrated that protease resistance could be measured both on the bacterial surface and in solution, enabling the method to test and/or screen for additional drug-like properties critical for biologically active compounds.
50 Severing Ties: Quantifying the Payload Release from Antibody Drug Conjugates Kopp, A. and G.M. Thurber Cell Chemical Biology 2019
An ADC (antibody shown in gray, payload in purple) undergoes receptor-mediated endocytosis after binding to target antigen (blue). Early endosomes are formed from the internalized vesicle. The endosome can be recycled back to the cell surface or proceed to a lysosome where the antibody is degraded. The payload (purple) must exit the lysosomal or endosomal compartment to reach its therapeutic target.
49 Pharmacokinetic Considerations to Maximize an ADC’s Therapeutic Index Thurber, G.M. Antibody-Drug Conjugates 2019
Image of a mouse xenograft treated with 3:1 co-administered trastuzumab:T-DM1 (white) surrounding tumor blood vessels in red (left). The simulations (middle) capture individual cell division and payload death to predict response. The results show that fractionated dosing reduces efficacy due to lower tissue penetration but can improve efficacy if higher doses are tolerated (right).
48 An Agent-Based Systems Pharmacology Model of the Antibody Drug Conjugate Kadcyla to Predict Efficacy of Different Dosing Regimens Menezes, B.*, C. Cilliers*, T. Wessler, G.M. Thurber, and J. Linderman American Association of Pharmaceutical Scientists (AAPS) 2019
Comparison between experimental and simulated drug distributions in tumor tissue. a Tumor sections from mice bearing NCI-N87 xenograft tumors dosed with 3.6 mg/kg of AlexaFluor680 labeled T-DM1 and different ratios of trastuzumab:T-DM1 were imaged after 24 hours using the same settings and then set to the same window level for different carrier dose (trastuzumab) concentrations. T-DM1 intensity decreases as it is “diluted” with trastuzumab at higher ratios and spreads out to reach more cells. b) Simulation results showing bound T-DM1. c) The same tumor sections were stained with anti-Fc labeled antibody and window-leveled the same to show total drug penetration in tumor. d) Simulation results show total antibody (T-DM1 + trastuzumab) penetration into the tumor. For all figure portions, scale bar = 200 μm
47 Antibody Coadministration as a Strategy to Overcome Binding-Site Barrier for ADCs: A Quantitative Investigation Singh, A., L. Guo, A. Verma, G. Wong, G.M. Thurber, and D. Shah American Association of Pharmaceutical Scientists (AAPS) 2019
a) A schematic diagram highlighting the hypothesis associated with the proposed coadministration strategy to overcome the binding-site barrier (BSB) for antibody-drug conjugates (ADCs). (A1): A scenario where T-DM1 (no bystander effect) is administered in HER2-low versus HER2-high tumors. More BSB will be observed in HER2-high tumors leading to restrictive distribution of ADC. (A2): A scenario where T-vc-MMAE (with bystander effect) is administered in HER2-low versus HER2-high tumors. More BSB will be observed in HER2-high tumors, however, released payload could diffuse into deeper portions of the tissues, leading to more homogeneous payload exposures. (A3): A scenario where T-DM1 is coadministered with naked Trastuzumab in HER2-low versus HER2-high tumors. Coadministration leads to deeper distribution of T-DM1 and hence more homogeneous payload exposure. (A4): A scenario where T-vc-MMAE is coadministered with naked Trastuzumab in HER2-low versus HER2-high tumors. Coadministration leads to deeper distribution of T-vc-MMAE within the tumor tissues, and the payload could also diffuse into the neighboring cells to induce more homogenous exposure. b A schematic of the proposed semi-mechanistic PK-PD model that was used to evaluate the nature of drug interaction when antibody was coadministered with ADCs during the TGI studies. The model operates with two different 2-compartment pharmacokinetic (PK) models, associated with ADC and antibody. A hybrid killing function shuttles the growing tumor cells (using biphasic saturable growth rate) to non-growing phases using a series of transit compartments, which eventually leads to tumor cell death (model equations are listed in supplementary text).
46 Blocking Glucagon Like Peptide-1 Receptors in the Exocrine Pancreas Improves Specificity for Beta Cells in a Mouse Model of Type 1 Diabetes Khera, E., L. Zhang, S. Roberts, I. Nessler, D. Sandoval, T. Reiner, and G.M. Thurber. Journal of Nuclear Medicine (JNM) 2019
Simplified schematic for β-cell imaging. (A) β-cell quantification resolution using only imaging exendin probe (label). (B) Postulated improvement in β-cell quantification resolution using preblocked Cy7-exendin followed by imaging exendin probe (low block).
45 Designing a disease screening pill: Challenges and opportunities in self-administration of optical probes Thurber, G.M. and R. Evans SPIE 2019
44 Oral and Subcutaneous Administration of a Near-Infrared Fluorescent Imaging Agent Detects Inflammation in a Mouse Model of Rheumatoid Arthritis Bhatnagar, S.*, E. Khera*, J. Liao, V. Eniola, Y. Hu, D.E. Smith, and G.M. Thurber Scientific Reports 2019
Oral versus Subcutaneous Administration of IRDye800CW Agent (a) IVIS images at 6, 24 and 48 hours of the IRDye800CW agent delivered subcutaneously (top), orally (middle), and the low affinity stereoisomer of the IRDye800CW agent delivered orally (bottom). Comparison of the (b) biodistribution and (c,d) average signals at varying times showing the specificity of the IRDye800CW agents in inflamed paws and compared to its low affinity stereoisomer. ****Denotes p < 0.0001, ***denotes p < 0.001, **denotes p < 0.01, *denotes p < 0.05 and ns stands for not significant.
43 Hitting undruggable targets: viewing stabilized peptide development through the lens of quantitative systems pharmacology Atangcho, L., T. Navaratna, and G.M. Thurber Trends in Biochemical Sciences 2019
Multiscale PBPK Model for Stabilized Peptides. This diagram is a representative illustration of a multiscale computational PBPK model for a stabilized peptide to help to predict the necessary rates and peptide properties for optimal organ distribution, tissue uptake, and target engagement. Each arrow represents a kinetic rate constant that can be measured in vitro or estimated from the literature and updated for a particular drug during the development pipeline (e.g., updating organ transport rates with preclinical biodistribution data). The cellular rate constants can be used to determine if these values are feasible for delivery within the broader organ and systemic distribution. Each organ is shown as a compartment with the relevant subcompartmental concentrations: vascular (V), interstitial (I), endothelial (E), and metabolite (M). Transport of the peptide drug between these subcompartments is shown using the heart as an example. The peptide is transported into the vascular subcompartment via the blood (Q = volumetric blood flow rate). The drug can then be transported convectively (Jiv = transport to interstitium from the vascular compartment) into the interstitial portion of the tissue and subsequently exit through the lymphatic fluid (Lorgan) or undergo metabolism (Kdeg = degradation rate constant) either directly from the vascular compartment (e.g., plasma proteases or uptake into endothelial cells and degradation) or from the interstitium (e.g., extracellular proteases or cellular uptake/degradation). It is often important to track the metabolites, particularly if these include a radiolabel or fluorophore, to properly interpret experimental data. The metabolites can re-enter the blood (Kloss) and eventually be eliminated via the kidney or liver. Distribution of the drug in the tissue of interest (e.g., the tumor), is modeled using a Krogh cylinder to analyze potential tissue-level gradients in drug concentration. The Krogh cylinder model is defined using a blood vessel radius (Rcap), cylinder radius (RKrogh), blood vessel permeability (P), and the diffusion coefficient in tissue (D). The tissue of interest is further broken down to the cellular level. The peptide drug enters the cell either by diffusion through the membrane (Kperm) or endocytosis (Kint), followed by endosomal escape (e.g., Kperm). The cytosolic peptide can then bind the target of interest (Kon, Koff). The peptide will also be degraded, either in the cytosol or in endosomes (Kdeg).
42 Pharmacokinetic and immunological considerations for expanding the therapeutic window of next-generation Antibody-Drug Conjugates Khera, E., and G.M. Thurber Biodrugs 2018
Schematic highlighting the complexity of antibody–drug conjugate (ADC) design, including different options available for the various ADC components, namely the backbone (gray), payload (green), and linker/conjugation chemistry (black)
41 Tumor drug penetration measurements could be the neglected piece of the personalized cancer treatment puzzle Bartelink, I., E. Jones, S. Shahidi-Latham, E. P. Rong, Y. Zheng, P. Vicini, L. vant Veer, D. Wolf, A. Iagaru, D. Kroetz, B. Prideaux, C. Cilliers, G. Thurber, Z. Wimana, and G. Gebhart Clinical Pharmacology & Therapeutics 2018
At clinically relevant doses, the binding of ado‐trastuzumab emtansine (T‐DM1) to human epidermal growth factor receptor 2 (HER2) expressing tumor cells is limited to the cells near functional blood vessels, and much higher doses are needed to provide a more homogeneous penetration, as shown at the microscopic level in an HER2 expressing xenograft tumor model (NCI‐N87 xenograft). (a) An immunofluorescence image of a tumor 24 hours following administration of 3.6 mg/kg of Alexa Fluor 680 tagged T‐DM1 ‐ a dose comparable to the dose used in patients ‐ to nude mice bearing NCI‐N87 flank tumors (green). Immunofluorescence staining with CD31‐AF555 (red) shows tumor vasculature, and intravenous administration and visualization of Hoechst 33342 shows functional vessels (blue) using multiplexed imaging. (b) HER2 expression (ex vivo staining with trastuzumab) in the same tumor section (white) and enlarged (c), indicating the uptake in the tumor was only sufficient to target a few cell layers. Images d, e, f show the same visualizations 24 hours following administration of 3.6 mg/kg of Alexa Fluor 680 tagged T‐DM1 and 10.8 mg/kg unlabeled trastuzumab (14.4 mg/kg total in a 1:3 ratio), indicating a more homogenous tumor penetration of T‐DM1. This dose reached many cells but did not occupy all accessible receptors in the tumor. Much higher doses up to 32 mg/kg of a combination of T‐DM1 and trastuzumab, in a 1:8 ratio (the latter to avoid antibody‐drug conjugate toxicity and improve penetration) were required in this animal model (with high HER2 expression, ~1 million receptors/cell) to reach all cells (data not shown). Red = CD31 + staining; green = 3.6 mg/kg T‐DM1‐AlexaFluor 680 (a–c) or 3.6 mg/kg T‐DM1‐AlexaFluor 680 + 10.8 mg/kg untagged trastuzumab (d–f); white = HER2 (trastuzumab labeling of histology slide); blue = functional vessels (intravenous Hoechst 33342).
40 "Standing by" for bystander effects: dual isotope imaging of Antibody-Drug Conjugates and payload distribution Cilliers, C., and G.M. Thurber Journal of Nuclear Medicine (JNM) 2018
39 Quantitative pharmacology in antibody-drug conjugate development: armed antibodies or targeted small molecules? Nessler, I., E. Khera, and G.M. Thurber Oncoscience 2018
ADC Mechanisms of Action. Integrating the contributions is necessary to identify the key attributes needed for clinical success.
38 Oral administration and detection of a near-infrared molecular imaging agent in an orthotopic mouse model for breast cancer screening Bhatnagar, S., K. Dhingra Verma, Y. Hu, E. Khera, A. Priluck, D. Smith, and G.M. Thurber Molecular Pharmaceutics 2018
Ideal properties and structure of imaging agents. (A) Schematic of an orally available systemic imaging agent technique, where the imaging agent is absorbed through the gastrointestinal tract, targets a disease site from the systemic circulation, and is detected noninvasively using near-infrared fluorescence. (B) Venn diagram showing the three main design criteria for developing orally available imaging agents. (C) Structures of the reported agents with varying physicochemical properties.
37 Improved tumor penetration and single-cell targeting of antibody-drug conjugates increases anticancer efficacy and host survival Cilliers, C., B. Menezes, I. Nessler, J. Linderman, and G.M. Thurber Cancer Research 2018
Improving T-DM1 tumor distribution through coadministration of trastuzumab. A, Administration of T-DM1 at 3.6 mg/kg (single agent) results in a heterogeneous, perivascular distribution due to rapid binding relative to transport in the tissue. B and C, The tumor penetration of a constant dose of T-DM1 is improved when coadministered with a subsaturating (B) or saturating (C) dose of trastuzumab. Trastuzumab competes for binding sites, increasing T-DM1 penetration. The middle column shows distribution of AF680-labeled T-DM1 (green) at 3.6 mg/kg, with unlabeled trastuzumab at 0:1, 3:1, and 8:1 trastuzumab:T-DM1 ratios (0, 10.8, and 28.8 mg/kg, respectively). Immunofluorescence staining with CD31-AF488 (red) shows the tumor vasculature. The right column shows immunofluorescence staining with antihuman IgG Fc-AF555 (gray). The window leveling between images is different as the intensity of the T-DM1 decreases with an increasing ratio, while the anti-Fc staining labels both trastuzumab and T-DM1, thereby maintaining a constant intensity while the penetration increases (see Supplementary Material). Scale bar, 200 μm.
36 Computational transport analysis of antibody-drug conjugate bystander effects and payload tumoral distribution: implications for therapy Khera*, E., C. Cilliers*, S. Bhatnagar, and G.M Thurber Molecular Systems Design & Engineering 2018
The computational results indicate that: 1) the heterogeneous tumoral distribution of ADCs impacts efficacy, and increasing the antibody dose improves penetration and efficacy. 2) The increased penetration of payloads with bystander effects can partially compensate for poor antibody penetration, but larger antibody doses still result in further improvement. This occurs because of the higher efficiency of direct cell killing than bystander killing. 3) Bystander effects are important for killing antigen negative cells, and an optimum in physicochemical properties exists. Payloads with a balance in cellular uptake versus tissue diffusion enter cells fast enough to avoid tumor washout but slow enough to reach distant cells. Therefore, optimizing the antibody dose, payload dose, and payload physicochemical properties results in ideal delivery to the site of action and maximum efficacy.
35 Ocular toxicity profile of ST-162 and ST-168 as novel bifunctional MEK/PI3K inhibitors Smith, A., M. Pawar, M.E. Van Dort, S. Galbán, A.R. Welton, G.M. Thurber, B.D. Ross, and C.G. Besirli Ocular Pharmacology and Therapeutics 2018
In vivo toxicity profiles of PD0325901 and bifunctional inhibitors. (A) Both 0.5 and 1 mg doses of PD0325901 led to adverse events following intravitreal injection compared to control injection with DMSO (vehicle). (B) No adverse events in the retina or retinal vasculature were observed with increasing doses of ST-162 1 week after injection (C) Pronounced toxicity difference between eyes treated with PD0325901 and ST-168 is demonstrated by retinal tear and detachment in PD0325901-treated eye only after 3 days.
34 Structure-guided design and initial studies of a bifunctional MEK/PI3K inhibitor (ST-168) Van Dort, M.E., S. Galbán, C.A. Nino, H. Hong, A.A. Apfelbaum, G.D. Luker, G.M. Thurber, L. Atangcho, C.G. Besirli, and B.D. Ross ACS Medicinal Chemistry Letters 2018
The structure-based design of a new single entity, MEK/PI3K bifunctional inhibitor (7, ST-168), which displays improved MEK1 and PI3K isoform inhibition, is described. ST-168 demonstrated a 2.2-fold improvement in MEK1 inhibition and a 2.8-, 2.7-, 23-, and 2.5-fold improved inhibition toward the PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ isoforms, respectively, as compared to a previous lead compound (4; ST-162) in in vitro enzymatic inhibition assays. ST-168 demonstrated superior tumoricidal efficacy over ST-162 in an A375 melanoma spheroid tumor model. ST-168 was comparatively more effective than ST-162 in promoting tumor control when administrated orally in a tumor therapy study conducted in an A375 melanoma mouse model confirming its bioavailability and efficacy toward combined in vivo MEK1/PI3K inhibition.
33 Tracking antibody distribution with near infrared fluorescent dyes: impact of dye structure and degree of labeling on plasma clearance Cilliers, C., I. Nessler, N. Christodolu, and G.M. Thurber Molecular Pharmaceutics 2017
Monoclonal antibodies labeled with near-infrared (NIR) fluorophores have potential use in disease detection, intraoperative imaging, and pharmacokinetic characterization of therapeutic antibodies in both the preclinical and clinical setting. Recent work has shown conjugation of NIR fluorophores to antibodies can potentially alter antibody disposition at a sufficiently high degree of labeling (DoL); however, other reports show minimal impact after labeling with NIR fluorophores. In this work, we label two clinically approved antibodies, Herceptin (trastuzumab) and Avastin (bevacizumab), with NIR dyes IRDye 800CW (800CW) or Alexa Fluor 680 (AF680), at 1.2 and 0.3 dyes/antibody and examine the impact of fluorophore conjugation on antibody plasma clearance and tissue distribution. At 0.3 DoL, AF680 conjugates exhibited similar clearance to unlabeled antibody over 17 days while 800CW conjugates diverged after 4 days, suggesting AF680 is a more suitable choice for long-term pharmacokinetic studies. At the 1.2 DoL, 800CW conjugates cleared faster than unlabeled antibodies after several hours, in agreement with other published reports. The tissue biodistribution for bevacizumab–800CW and −AF680 conjugates agreed well with literature reported biodistributions using radiolabels. However, the greater tissue autofluorescence at 680 nm resulted in limited detection above background at low (∼2 mg/kg) doses and 0.3 DoL for AF680, indicating that 800CW is more appropriate for short-term biodistribution measurements and intraoperative imaging. Overall, our work shows a DoL of 0.3 or less for non-site-specifically labeled antibodies (with a Poisson distribution) is ideal for limiting the impact of NIR fluorophores on antibody pharmacokinetics.
32 Imaging in Diabetes Zhang, L., and G.M. Thurber Imaging and Metabolism 2018
Structure of the pancreas. (a) Schematic of the pancreas with head, body, and tail portions labeled. The pancreatic duct runs along the organ and empties into the duodenum (not shown). (b) Histology slide of a mouse islet with the blood vessels (CD31) stained in brown (hematoxylin counterstain). Adapted from Keliher et al., 2012. Notice the extensive vessels on the islet surface and within. A human (c), monkey (d), and mouse (e) islet of Langerhans stained for beta cells (insulin, red), alpha cells (glucagon, green), and delta cells (somatostatin, blue). The proportion of beta cells in humans is lower (~60%) than mice (~80%). Adapted from Cabrera et al., Proc. Natl Acad. Sci. USA 103 (7), 2334-2339; © (2006) National Academy of Sciences USA
31 A helix-stabilizing linker improves subcutaneous bioavailability of a helical peptide independent of inker lipophilicity Zhang, L., T. Navaratna, and G.M. Thurber Bioconjugate Chemistry 2016
Stabilized peptides address several limitations to peptide-based imaging agents and therapeutics such as poor stability and low affinity due to conformational flexibility. There is also active research in developing these compounds for intracellular drug targeting, and significant efforts have been invested to determine the effects of helix stabilization on intracellular delivery. However, much less is known about the impact on other pharmacokinetic parameters such as plasma clearance and bioavailability. We investigated the effect of different fluorescent helix-stabilizing linkers with varying lipophilicity on subcutaneous (sc) bioavailability using the glucagon-like peptide-1 (GLP-1) receptor ligand exendin as a model system. The stabilized peptides showed significantly higher protease resistance and increased bioavailability independent of linker hydrophilicity, and all subcutaneously delivered conjugates were able to successfully target the islets of Langerhans with high specificity. The lipophilic peptide variants had slower absorption and plasma clearance than their respective hydrophilic conjugates, and the absolute bioavailability was also lower likely due to the longer residence times in the skin. Their ease and efficiency make double-click helix stabilization chemistries a useful tool for increasing the bioavailability of peptide therapeutics, many of which suffer from rapid in vivo protease degradation. Helix stabilization using linkers of varying lipophilicity can further control sc absorption and clearance rates to customize plasma pharmacokinetics.
30 Multiscale modeling of antibody-drug conjugates: connecting tissue and cellular distribution to whole animal pharmacokinetics and potential implications for efficacy Cilliers, C., H. Guo, J. Liao, N. Christodolu, and G.M. Thurber. American Association of Pharmaceutical Scientists (AAPS) 2016
Multiscale PBPK-Krogh cylinder model diagram. a PBPK model tracks systemic distribution of both antibody and ADC. Solid black lines correspond to antibody/ADC flow and green dotted lines correspond to metabolite flow. b Representative organ compartment model. All organs except the tumor and carcass are divided into vascular, interstitial, and metabolite compartments. The endothelial compartment is added in the carcass to account for FcRn recycling. c The tumor compartment is modeled by a 1-D Krogh cylinder tissue model with permeability (P) across the endothelium (extravasation) and diffusion (D) through the surrounding tissue. d Cellular-scale model showing binding, internalization, and degradation rates of both antibody and ADC
29 Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design Zhang, L.*, S. Bhatnagar*, E. Deschenes, and G.M. Thurber Scientific Reports 2016
Diagram of the interplay between imaging probe properties and target tissue properties in determining contrast. Exendin is shown with PyMol surface charge as the sample imaging agent (PDB 1JRJ).
28 Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging Zhang, L. and G.M. Thurber Molecular Imaging and Biology 2016
a) Plasma clearance is rapid for all the hydrophilic IRDye 800CW conjugates, while the lipophilic agent clears slowly. b) Biodistribution shows higher non-specific uptake of multimers in the kidney and Cy7 uptake in the liver. c) Clearance rates were not significantly different between the monomer, dimer, and trimer.
27 Tumor Effect-Site Pharmacokinetics: Mechanisms and impact on efficacy G.M. Thurber ADME and Translational PK/PD of Therapeutic Proteins: Applications in Drug Discovery and Development 2015
Steps in systemic targeting—all agents entering a tumor must (1) flow through the local blood vessels, (2) extravasate into the tissue, (3) diffuse through the interstitium, and (4) bind to their target. As these steps are occurring, the molecules are cleared (a) from the systemic circulation and (b) locally within the tissue.
26 Advances in measuring single-cell pharmacokinetics and pharmacology in vivo Vinegoni, C., J.M. Dubach, G.M. Thurber, M.A. Miller, R. Mazitschek, and R. Weissleder Drug Discovery Today 2015
Measuring key pharmacokinetic and pharmacodynamic parameters in vivo at the single cell level is likely to enhance drug discovery and development. In this review, we summarize recent advances in this field and highlight current and future capabilities.
25 Residualization rates of near infrared dyes for the rational design of molecular imaging agents Cilliers, C., J. Liao, L. Atangcho, and G.M. Thurber Molecular Imaging and Biology 2015
Confocal microscopy of cells immediately after surface labeling or after 24- and 48-h incubation. Punctate spots after incubation show the substantial internalization, while the non-residualizing dyes DDAO and Atto 740 have little signal at later times.
24 A dual-purpose linker for alpha helix stabilization and imaging agent conjugation to glucagon-like Peptide-1 receptor ligands Zhang, L., T. Navaratna, J. Liao, and G.M. Thurber Bioconjugate Chemistry 2015
Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel α-helix-stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enable this technique to potentially be used as a general method for labeling α helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents.
23 Population dynamics of islet-infiltrating cells in autoimmune diabetes Magnuson, A.M, G.M. Thurber, R.H Kohler, R. Weissleder, D. Mathis, and C. Benoist Proc Natl Acad Sci USA 2015
Monitoring cell trafficking to the pancreas. (A) Sample flow cytometry data for CD45+ cells from nonphotoconverted CLNs and 0-h, 1-min, and 24-h time points. (B) The proportion of CD45+ cells comprised of recent immigrants was measured by flow cytometry data generated at 0, 1, 3, or 7 d after photoconversion of the CLNs in 12- to 14-wk-old mice. (C) Islets in explanted pancreata from 10- to 12-wk-old mice were imaged by confocal microscopy 36 h after photoconversion of the CLNs. Kaede-red+ recent immigrants (red), β-cells (blue), and islet periphery (dashed white line). Representative z-stack projection shown here. Six pancreata per group were imaged.
22 A systems pharmacology approach towards the design of inhaled formulations of rifampicin and isoniazid for treatment of tuberculosis Cilfone, N., E. Pienaar, G.M. Thurber, D. Kirschner, and J. Linderman CPT Pharmacometrics and Systems Pharmacology 2015
Overall model structure that captures relevant dynamics across multiple compartments. (a) The pharmacokinetic (PK) model includes two transit compartments (ABS‐1 and ABS‐2) which approximate gut absorption and transit time, a plasma compartment (PLASMA), a peripheral compartment (PERIPH), a noninfected lung compartment (LUNG), and an intracellular macrophage compartment (MΦ) that is at pseudo‐steady‐state. Oral doses enter into the first transit compartment. Inhaled doses are partitioned between the noninfected lung (1‐fD) and lesion (fD) models based on representative sizes. The dose (1‐fD) into the noninfected lung compartment is further partitioned between extracellular noninfected lung and intracellular macrophage compartments. We assume no trafficking of macrophages in or out of noninfected lungs. (b) Our granuloma model, a hybrid multiscale agent‐based model, includes spatial and temporal dynamics of antibiotics and captures diffusion, extracellular degradation, cellular uptake and intracellular degradation. Antibiotics exit the plasma compartment and enter the granuloma model at vascular sources designated in the simulation grid based on vascular permeability coefficients and concentration gradients between the plasma compartment and the granuloma mode. The inhaled formulation is modeled by agent representations of each carrier. (c) Model of the behavior and release of antibiotics by inhaled carriers. Carriers move by random walk, are phagocytosed by macrophages based on size, zeta potential, and density of targeting ligand (Supplementary Figure S1b‐d), degrade in both the extra‐ and intracellular space, and release antibiotics in both the intra‐ and extracellular space. (d) The pharmacodynamics model uses Emax functions (using C50 values and Hill‐constants, H) to describe the antibacterial activity of antibiotics against multiple bacterial subpopulations (intracellular, extracellular, and nonreplicating) based on the local antibiotic concentration (C(x,y,t)). Art adapted from Servier Medical Art (‐image‐bank) provided under the Creative Commons Unported License 3.0.
21 Multichannel imaging to quantify four classes of pharmacokinetic distribution in tumors Bhatnagar, S., E. Deschenes, J. Liao, C. Cilliers, and G.M. Thurber Pharmaceutical Sciences 2014
Simulations based on a Krogh cylinder model with axial and radial gradients (a). Four different agents were simulated with their predicted class of behavior show in parentheses. The results for BODIPY‐FL in tumors (b) and liver (c) demonstrate axial blood flow limitations. The monoclonal antibody cetuximab has slow extravasation with rapid perivascular binding (d). The high plasma protein binding of Hoechst 33342 diminishes axial gradients, whereas slow diffusion into the tissue and rapid cellular uptake results in perivascular distribution (e). Integrisense 680 was simulated at a saturating dose, which results in no transport limitations and uptake equivalent to the receptor concentration (f).
20 Effect of small molecule modification on single cell pharmacokinetics of PARP inhibitors Thurber, G.M., T. Reiner, K.S. Yang, R.H. Kohler, and R. Weissleder Molecular Cancer Therapeutics (MCT) 2014
Molecular properties and cellular imaging. A, the measured inhibitory concentrations of the three PARP inhibitors with the structure shown in B. C, in vitro images of olaparib-BODIPY FL and olaparib-BODIPY 650 show similar patterns of uptake. Before washing, the majority of the probe is located in the perinuclear endoplasmic reticulum, and, after washing, the nuclear signal (with higher PARP concentrations in the nucleolus) is more prominent. The kinetics of distribution are significantly different between the two probes, with the olaparib-BODIPY 650 rates around 10 times slower. The ratio of uptake in the ER to the nucleus before washing is also much higher with the olaparib-BODIPY 650 probe. The contrast is identical for olaparib-BODIPY FL before and after the wash, but the contrast was increased after 2 hours of washing to show the nuclear-specific staining, which is much lower than the perinuclear signal before washing. Scale bar, 20 μm.
19 Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo Thurber, G.M., K.S. Yang, T. Reiner, R.H. Kohler, P. Sorger, T. Mitchison, and R. Weissleder Nature Communications 2013
Precursor compounds are conjugated with cell permeable, small fluorophores to generate therapeutically active fluorescent companion imaging drugs. Using a window chamber model, in vivo microscopy enabled the detection of drugs (green) with subcellular resolution and frame rates of several seconds. Scale bar, 50 μm in an MDA-MB-231 breast cancer cell line expressing a fluorescent H2B protein in the nucleus (red).
18 A mechanistic compartmental model for antibody uptake in tumors Thurber, G.M. and K.D. Wittrup Theoretical Biology 2012
Diagram of the compartmental model and Krogh cylinder simulations. (A) Arrows indicate transport between compartments. The dotted line surrounds the two-compartment model for normal tissue. (B) Diagram of the Krogh cylinder model with the equations and boundary conditions within the tissue.
17 Noise suppressed, multifocus image fusion for enhanced intraoperative navigation Feruglio P.F., C. Vinegoni, L. Fexon, G.M. Thurber, A. Sbarbati, R. Weissleder Biophotonics 2012
Fluorescent all-in-focus imaging for intravital sur-gery. During intraoperative surgery, the use of all-in-focusimaging in fluorescent mode, would enable the detectionof extended tumors and small lesions within tissue mar-gins (d, f). Panel (c, e) demonstrates the impossibility ofidentifying small lesions or metastases when the imagesare not in focus. If small tumoral masses lie at differentdepths, scanning along the axis of the microscope lens isnecessary for accurate detection. Panels (a–b) show the lo-cation of the tumors within the mouse in white light andfluorescent mode respectively.
16 Efficient 18F-Labeling of Synthetic Exendin-4 Analogues for Imaging Beta Cells Keliher, E.J., T. Reiner, G.M. Thurber, R. Upadhyay, R. Weissleder Chemistry Open 2012
In vivo imaging of insulinoma. A) PET‐CT and PET only scans of 18F‐E4Tz12 7 in a C57BL/6 mouse bearing 916‐1 tumor xenografts. B) Average uptake of 18F‐E4Tz12 7 in different tumors and in muscle tissue [injected dose per gram of tissue (% ID g−1)]; tumor xenograft (916‐1), intestines (I), kidney (K), bladder (B). C) Western blot of 916‐1, NIT1, and WTRT2 cell lines against GLP‐1R and GAPDH.
15 Reactive Polymer Enables Efficient In Vivo Bioorthogonal Chemistry Devaraj NK*, G.M. Thurber*, E.J. Keliher, B. Marinelli, R. Weissleder Proc Natl Acad Sci USA 2012
In Vivo Bioorthogonal Reactions. (A) Tetrazine cycloaddition with trans-cyclooctene forming a dihydropyrazine. (B) Schematic of PMT used in this study. The scaffold consists of dextran that has been aminated to allow attachment of tetrazine reactive groups as well as imaging agents such as near-infrared fluorophores and radioisotopes. (C) In vivo multistep delivery of imaging agent. A slow clearing targeting agent is administered first (green) and is given 24 h for localization and background clearance. Next, a lower molecular weight secondary agent (red) is delivered that rapidly reacts and is cleared from the background tissue much faster than the primary agent. (D) Kinetic parameters of consideration for in vivo clicking. The secondary tetrazine agent reacts with transcyclooctene antibodies at a given rate (kreaction). This rate is in competition with other rates including the clearance of the secondary agent from the body (kclearance) and internalization of the antibody (kendocytocis).
14 Practical Theoretic Guidance for the Design of Tumor Targeting Agents Wittrup K.D., G.M. Thurber, M.M. Schmidt, J.J. Rhoden Methods in Enzymology 2012
13 A Systems Approach for Tumor Pharmacokinetics Thurber, G.M.*, R. Weissleder* PLoS ONE 2011
(A) Space filling models of oxygen, FDG, doxorubicin, and an IgG for size comparison. (B) Diagram of a Krogh cylinder labeled with the four fundamental steps in tumor localization.
12 Accurate Measurement of Beta Cell Mass Using a Second-Generation Fluorescent Exendin-4 Analog Reiner T., G.M. Thurber, J. Gaglia, et al. Proc Natl Acad Sci USA 2011
GFP and probe colocalization in E4×12-VT750–injected MIP-GFP mice. (A and B) Fluorescence, histology of harvested pancreas (green, MIP-GFP; red, E4×12-VT750). The arrow points to H&E staining of a single islet (C) and antiinsulin staining of the same islet (D) using adjacent sections. (E) Correlation of islet sizes estimated via the MIP-GFP reporter (488 nm) vs. the GLP-1R probe E4×12-VT750 (750 nm). (F) Correlation of islet sizes observed with the MIP-GFP reporter (488 nm) vs. signal intensity observed at 750 nm in the same area.
11 Kinetics of Antibody Penetration into Tumors Thurber G.M Targeted Radionuclide Therapy 2011
10 Quantitating Antibody Uptake In Vivo: Conditional Dependence on Antigen Expression Levels Thurber, G.M. and R. Weissleder Molecular Imaging and Biology 2011
Effect of antigen expression on uptake. When the Clearance modulus and Thiele modulus are less than 1, the tumor concentration is a function of the antigen expression level. At subsaturating concentrations, the uptake is a function of transport to the tumor (a). Targeting of antibodies is similar between moderately expressed EGFR and highly expressed EpCAM at subsaturating concentrations (b). Image of a tumor targeted by anti-EpCAM and anti-EGFR antibodies showing qualitatively similar uptake (c). Scale bar = 5 mm.
9 Detection Limits of Intraoperative Near Infrared Imaging for Tumor Resection Thurber, G.M., J.L. Figueiredo, R. Weissleder Surgical Oncology 2010
Imaging requirements vary depending on the surgical objective. B: The ability to image the surgical status of a resection varies with the fluorescent imaging set‐up, each having strengths and limitations.
8 Multicolor Fluorescent Intravital Live Microscopy (FILM) for Surgical Tumor Resection in a Mouse Xenograft Model Thurber, G.M., J.L. Figueiredo, and R. Weissleder PLoS One 2009
A) Tumor schematic. B) Mechanisms of probe localization. Numbers indicate wavelength of fluorescent channel. C) Imaging agent properties. MW = molecular weight, Ex = excitation maximum, Em = emission maximum. α and β are plasma clearance rate constants, with A being the fraction of the α phase. D) Simulated time course of antibody and protease sensor localization. Tumor to background ratios were estimated based on a characteristic normal tissue compartment. Different scales are used on the TBR axis.
7 Hybrid in vivo FMT-CT imaging of protease activity in atherosclerosis with customized nanosensors Nahrendorf, M., P. Waterman, G.M. Thurber, K. Groves, M. Rajopadhye, P. Panizzi, B Marinelli, E. Aikawa, M.J. Pittet, F.K. Swirski, and R. Weissleder Arteriosclerosis, Thrombosis, and Vascular Biology 2009
The protease sensor with the highest sensitivity, PS-40, was used in a therapy trial to investigate its potential as a noninvasive imaging biomarker in drug development. A and B, FMT-CT data sets in respective treatment groups. C, Fluorescence activity in the aortic root measured by FMT, 24 hours after injection of PS-40. The protease sensor detected effects of atorvastatin treatment. *P<0.05. D and E, Ex vivo fluorescence reflectance imaging of excised aortas corroborated in vivo FMT data. *P<0.05. F, Quantitative PCR analysis showed lower translation of cathepsin B in apoE−/− treated with atorvastatin, confirming the imaging data. *P<0.05. G, Plasma cholesterol levels in respective trial groups.
6 18F Labeled Nanoparticles for in vivo PET-CT Imaging Devaraj, N.K., E.J. Keliher, G.M. Thurber, M. Nahrendorf, and R. Weissleder Bioconjugate Chemistry 2009
Preparation of 18F-CLIO. (A) Derivatization of primary amines on CLIO-VT680 with the NHS ester of 1-azido-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oic acid followed by chemoselective “click” of 18F-PEG3 radiotracer. (B) Schematic of 18F-CLIO.
5 Kinetics of Anti-Carcinoembryonic Antigen Antibody Internalization: Effects of Affinity, Bivalency, and Stability Schmidt, M.M., G.M. Thurber, and K.D. Wittrup Cancer Immunology and Immunotherapy 2008
Imaging anti-CEA antibody uptake. a Anti-CEA antibodies are trafficked into intracellular pools at 37°C. Trypsinized LS174T cells were surface labeled with Alexa-488 labeled IgG M85151a on ice and incubated for 24 h at 4°C or 37°C. Cells were then labeled with goat-anti-mouse-PE and imaged on a deconvolution microscope. When incubated at 37°C a significant fraction of the 488 labeled anti-CEA antibodies are endocytosed into an intracellular pool where they are not labeled by the secondary antibody. Scale bar, 20 μm. b Internalized anti-CEA antibodies partially colocalize with markers of endocytosis. LS174T cells were incubated overnight at 37°C with fluorescently labeled anti-CEA scFvs. Cells were then washed and incubated for 1 h with fluorescently labeled markers of endocytic and lysosomal pathways. Dual label images were taken on a deconvolution microscope. The anti-CEA scFv shows partial but incomplete colocalization with all endocytic pathway markers. Each image is a clump of 5–10 adherent LS174T cells. Scale bar, 10 μm
4 Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance Thurber, G.M., M.M. Schmidt, and K.D. Wittrup Advanced Drug Delivery Reviews 2008
A diagram showing the balance between antibody penetration and systemic or local clearance. A) A sequence of four time points illustrates the penetration of antibody into the tumor while free antibody remains in the blood. Once the plasma concentration has cleared, no more free antibody exists to diffuse deeper into the tumor. A clearance modulus greater than unity indicates systemic clearance occurs before all cells out to the target distance R are targeted. B) Catabolism of antibody counteracts penetration into tumor tissue. In this illustration, the steady-state concentration of plasma antibody is only sufficient to target one cell layer away from the capillary. The green shaded area indicates the region saturated with antibody, while several individual antibodies are shown to explicitly depict catabolism. The Thiele modulus value greater than one indicates that the antibody is catabolized before reaching all of the cells out to the target distance R.
3 Quantitative Spatiotemporal Analysis of Antibody Fragment Diffusion and Endocytic Consumption in Tumor Spheroids Thurber, G.M. and K.D. Wittrup Cancer Research 2008
Live sm3E diffusion with binding in spheroids. Top, radius of the moving scFv front as a function of time (•). Black line, theoretical prediction; gray shaded area, ±SD from the theoretical prediction due to error in the experimentally measured variables. Bottom, differential interference contrast (DIC) image and three fluorescence images at various time points. The high background is due to the 100 nmol/L fluorescent scFv present in the bulk solution. Scale bar, 200 μm.
2 Factors Determining Antibody Distribution in Tumors Thurber, G.M., M.M. Schmidt, and K.D. Wittrup Trends in Pharmacological Sciences 2008
Processes of antibody distribution in tumor tissue. (1) Extravasation of antibody across the tumor blood vessel walls is a function of the vessel permeability and occurs slowly. (2) Once in the tumor tissue, the antibodies diffuse freely in the extracellular matrix (a), with their motion retarded by formation of immobile complexes owing to binding (b) and dissociation (c), which are functions of affinity. (3) Antibody administered in the plasma, which supplies free antibody to the tumor, is cleared through several mechanisms, depending on its size, charge and Fc receptor binding. (4) While bound to surface antigens, local clearance of antibodies occurs owing to internalization and degradation at a rate determined by the target antigen.
1 Theoretic Criteria for Antibody Penetration into Solid Tumors and Micrometastases Thurber, G.M., S.C. Zajic, and K.D. Wittrup Journal of Nuclear Medicine (JNM) 2007
Full numeric simulations were performed with biexponential clearance and catabolism in tumor tissue. Antibody concentration was varied to change clearance modulus, and endocytosis rate was varied to independently change Thiele modulus. met = metastasis.