Comparative Medicine Copyright 2016 by the American Association for Laboratory Animal Science
Vol 66, No 2 April 2016 Pages 90–99
Original Research
Biodistribution Analyses of a Near-Infrared, Fluorescently Labeled, Bispecific Monoclonal Antibody Using Optical Imaging Norman C Peterson,1,* George G Wilson,1 Qihui Huang,2 Nazzareno Dimasi,3 and Kris F Sachsenmeier2 In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies. Abbreviations: EGFR, epidermal growth factor receptor; EIBS, EGFR–IGF1R bispecific monoclonal antibody; FMT, fluorescence molecular tomography; IGF1R, insulin-like growth factor 1 receptor; NIRF, near-infrared fluorophore; PET, positron emission tomography
Over the past 25 y, bispecific antibodies have been added to the arsenal of humanized antibodies that have been developed to treat cancer, infectious diseases, and inflammatory diseases. Bispecific biotherapeutics simultaneously recognize 2 different epitopes on the same or different cells or bacteria. This multispecificity offers several advantages over their monospecific counterparts in that cell or tissue specificity can be increased and thus toxicity reduced, 2 (or more) receptor pathways can be targeted simultaneously, and immune-mediated responses at clinically affected sites may be activated (cancer) or deactivated (inflammatory disease).13,18 As the size and structure of monoclonal antibodies are altered from their natural state to create biotherapeutics, such as bispecific antibodies,8 the pharmacokinetic properties of the products may differ from that of their parental (natural) antibodies. Aside from the potential to increase immunogenicity or aggregate, very little is known about how these structural differences affect pharmacokinetics and biodistribution. Receptormediated uptake and cellular processing both are likely to play a role,20 but identifying the tissues or cell types involved in recognizing these altered proteins is a necessary first step toward developing engineering strategies that can circumvent these impediments. Received: 04 Aug 2015. Revision requested: 04 Sep 2015. Accepted: 23 Sep 2015. Departments of 1Translational Sciences, 2Cancer Biology, and 3Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, Maryland * Corresponding author. Email: [email protected]
At the organ or tissue level, the use of radiolabeling and imaging in clinical and preclinical studies has greatly facilitated our understanding of the pharmacokinetics and tissue distribution of biotherapeutics.7,21 This advance is in part due to the ability to track and monitor the distribution of labeled compounds in the same animal over time. The increased sensitivity of positron emission tomography (PET) and the ability of radioisotope signals to travel through tissues without being significantly attenuated or diffracted has made PET the leading modality for preclinical and clinical biodistribution analyses. Disadvantages of PET are that its application is more expensive and requires skill in chemical conjugation, imaging technology, and radiation safety. Given that these resources are often limited or unavailable at several institutions (ours included), near-infrared optical imaging with improved tissue penetrance and quantitative imaging algorithms are demonstrating value when compared with ‘high-end’ imaging modalities.19,33 The accessibility of a variety commercially available fluorescently labeled targeting proteins and peptides, activatable probes, and fluorescent protein labeling kits has made this technology more readily available. In addition, because multiple fluorophores of nonoverlapping emission spectrums can be administered simultaneously, colocalization studies and direct comparisons of 2 or more labeled compounds can be performed in the same animal. Here we show how fluorescence-based optical imaging was used to identify potential tissue depots or sinks and characterize the biodistribution profile of a bispecific antibody under preclinical development.
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Materials and Methods
Antibodies. The recombinant antibodies used in this study were cloned, expressed and purified at MedImmune as described previously.8 The antibody EIBS is a humanized bispecific antibody engineered to concurrently bind to insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR). The antiIGF1R antibody is cross-reactive to both mouse and human, whereas the antiEGFR portion is human-specific. This bispecific antibody has a structure similar to the bispecific antibody prototype (Bs2Ab), in that an antiIGF1R single-chain variable fragment is engineered on the amino terminal of the heavy chains of the antiEGFR antibody.8 Humanized monoclonal antibody R347, developed at Medimmune, is an IgG1 that binds to HIV and was used as a control in all studies. EIBS and control antibodies were purified to homogeneity, approximately 99% monomeric, and formulated in PBS pH 7.2. The antibodies were labeled with Alexa Fluor 680 or 750 according to the manufacturer’s instructions (SAIVI Rapid Antibody Labeling Kit, Life Technologies, Grand Island, NY) Cells. NCI-H358 human-derived nonsmall-cell lung carcinoma cells were obtained from ATCC (Manassas, VA) and were cultured in RPMI1640 media (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen) in humidified incubators at 37 °C and 5% CO2. Cells (5 × 106) were injected in the subcutaneous tissue of the right flank of the mice and allowed to grow until the tumor was approximately 1000 mm3 before imaging. Flow cytometry. NCI-H358 cells (ATCC, Manassas, VA) were grown in flasks containing RPMI1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Life Technologies) in a humidified incubator at 37 °C with 5% CO2. Cells were released from culture, washed, and resuspended in 2% BSA (Roche Diagnostics, Indianapolis, IN) in PBS (Life Technologies). Single-cell suspensions were distributed in 96-well plates (2 × 105 cells/well). Cells were incubated with fluorescently labeled EIBS (or control antibody) for 1 h on ice and washed in buffer prior to analysis. Cells were analyzed by flow cytometry (LSR II, Becton Dickinson, San Jose, CA), and data were analyzed by using FlowJo software (TreeStar, Ashland, OR). Serum immunoglobulin assay. Serum immunoglobulin titers were determined by sandwich ELISA using mouse antihuman IgG (Fc-specific, Jackson Immunoresearch, West Grove, PA). After incubation with serum and repeated washing, either goat antihuman IgG λ–horseradish peroxidase (R347 detection; Southern Biotech, Birmingham, AL) or goat antihuman IgG κ-horseradish peroxidase (EIBS detection; Southern Biotech) was added to microtiter plates containing the bound antibodies. After repeated washing, SureBlue TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD) was added to each well, and absorbance at 450 nm was determined. Animals. Female athymic (nu/nu) mice approximately 5 wk of age were obtained from Harlan (Indianapolis, IN) and were housed in individually ventilated cages, on hardwood bedding, and fed a commercially available diet (HarlanTeklad 2918 Diet, 18% Global Protein Diet, Harlan, Indianapolis, IN). According to results from vendor and quarterly institutional health surveillance programs, the mice were free of mouse adenovirus, cilia-associated respiratory bacillus, ectromelia virus, rotavirus, lymphocytic choriomeningitis virus, mouse hepatitis virus, reovirus, pneumonia virus of mice, Mycoplasma pulmonis, minute virus of mice, mouse parvovirus, cytomegalovirus, polyoma virus,
Sendai virus, norovirus and Helicobacter spp. Environmental conditions were: temperature, 21 to 22 °C; relative humidity, 40% to 60%; and 12:12-h light:dark cycle. All procedures were run in accordance with the Guide for the Care and Use of Laboratory Animals16 and institutional standards in our AAALAC-accredited facility and were approved by MedImmune’s IACUC. Imaging. Mice received a single injection into the tail vein of antibody labeled with Alexa Fluor 680 or 750 (3 mg/kg; SAIVI Rapid Antibody Labeling Kit, Life Technologies, Grand Island, NY). To accommodate logistics with fewer animals and minimize interanimal variability, a mixture of equal amounts of control and test antibody (1 to 3 mg/kg) with different labels were injected into the each mouse.27 Mice were anesthetized with 2% isoflurane and scanned in a Fluorescent Molecular Tomography 2500 system (PerkinElmer, Waltham, Massachusetts) on either 680 nm or 750 nm channels at 3 mm density and medium laser intensity. Images were reconstructed using TrueQuant (version 3.1) software (PerkinElmer), and regions of interest were defined visually. Surface fluorescence from anesthetized mice and tissues (ex vivo) was analyzed by using an IVIS Spectrum (Perkin Elmer) set at medium binning, F-stop 1, and auto exposure. In vivo surface fluorescence of manually defined regions of interest was quantitated as average radiance efficiency ([p/s/cm2/sr] / [µW/cm2]) by using Living Image software (Perkin Elmer). Fluorescent signals were standardized across mice and presented as a proportion of the total surface fluorescence (in vivo) or the fluorescence from the heart (ex vivo). Data were analyzed with GraphPad 6.03 software (Prism, La Jolla, CA). ANOVA; Bonferroni posthoc analyses were preformed to determine statistical significance (defined as a P value less than 0.05).
Results
Localization of antiIGF1R–EGFR bispecific antibody in tumors. To increase specificity and simultaneously target 2 tumor-associated proteins that are commonly and highly expressed in several tumor types (pancreatic, breast, colorectal, and lung), the bispecific IGF1R–EGFR1 antibody EIBS was developed from antibodies that recognize each receptor independently. The antibodies were labeled with commercially available near-infrared fluorophores (NIRF), and the degree of labeling (ratio of antibody to label) remained within the range of 1.8 to 3.5 for each batch tested. Flow cytometry demonstrated that NIRF-labeled EIBS bound to nonsmall-cell lung cancer cells (H358 cells) in vitro (Figure 1) and that there was no detectable difference in affinity between EIBS labeled with either NIRF. In addition, preliminary data indicated that the antiEGFR arm does not cross react with the mouse protein, whereas the antiIGF1R arm does. To more specifically evaluate the tumor-targeting ability of EIBS in vivo and assess nonspecific tissue binding, imaging biodistribution studies were conducted by using optical imaging modalities. Mice with subcutaneously implanted H358 cells were intravenously injected with a mixture of EIBS and a control antibody that were labeled with different NIRFs; the spectra of the 2 NIRFs used did not overlap (Alexa Fluor 750 and 680, respectively). Comparison of clearance of total body fluorescence in these mice as estimated by abdominal fluorescent molecular tomography (FMT) scans revealed that 50% of the EIBS had cleared from the mice in the first 24 h and that EIBS-associated fluorescence continued to decline, whereas total body fluorescence remained unchanged for the con-
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Figure 1. Flow cytometric analysis of H358 cells exposed to EIBS and control (Ctl) antibody labeled with Alexa Fluor 680 or 750 at specified concentrations. The assay was performed in duplicate, and the mean fluorescent intensity ratio (MFIR) was plotted. The MFIR was calculated by dividing the mean fluorescence of EIBS-680 at each concentration by the average mean fluorescence of Ctl-680, and likewise for the 750-labeled antibodies.
trol antibody (Figure 2 A). Analysis of the 2- and 24-h antibody concentrations in the blood confirmed that plasma clearance of EIBS was more rapid than that of the control antibody (80% compared with 67% cleared, respectively (P < 0,05); Figure 2 B). Despite the rapid clearance of EIBS from the blood and decreased total body fluorescence, the amount of EIBS remained unchanged in the tumor (Figure 2 C). This dynamic resulted in significantly (P < 0.05) higher tumor:blood antibody ratios in EIBS-injected mice when compared with controls (Figure 2 D). Although the tumor:blood ratios suggested that EIBS was retained in the tumor, the average percentage of injected dose for tumors with EIBS did not exceed that of controls (Figure 2 C). In our experience, nonspecific retention of control antibodies in tumor tissue is typically greater than that of most other tissues and ranges from 10% to 20% of the injected dose per gram of tissue. To determine whether tumor exposure to the antibodies was limited by their retention in tissues, ex vivo surface fluorescence of the major organs from these mice was measured (Figure 3) by FMT. Biodistribution profiles showed that when compared with controls, significantly (P < 0.05) and markedly more EIBS was retained in the liver shortly after injection (Figure 3 A), with a trend suggestive of continued retention through at least 24 h(P = 0.39) (Figure 3 B). The control antibody showed nonspecific retention in the skin (P < 0,05) (Figure 3 B and C). To confirm these results, further studies were designed such that each antibody was compared with an alternative NIRF (Alexa Fluor 680 or 750), and each mouse was injected with a single labeled antibody (that is, EIBS labeled with Alexa Fluor 680 [EIBS-680], EIBS-750, Control-680, or Control-750). Early FMT scans (1 and 5 h after injection) in 2 trials failed to yield consistent reconstructed images for all mice in each group when mice that received doses of 3 and 4 mg/kg were analyzed. This issue likely was due to either insufficient or saturated signal and was partially resolved by 24 h, when all ventrodorsal scans (except of mice given Control-680) yielded 3D constructions of fluorescence data. By 48 h, 3D reconstructions were generated from all scans (Figure 4). Comparison of FMT scans revealed greater fluorescence from the livers of EIBS-750–treated mice than from those of Control-750 injected mice (Figure 4 A and B; Figure 5 A through
D); this difference reaching significance (P < 0.05) by 24 h after injection (Figure 4 B, lateral scan). However, the liver-associated signal did not differ when EIBS-680– and Control-680–injected mice were compared. The fluorescence from the tumors was similar for both labeled EIBS antibodies but was lower than their controls (Figure 4 C), presumably reflecting exposure levels in the blood. Whereas in vivo biodistribution analysis with FMT was somewhat limited at early time points, early surface epifluorescence images yielded sufficient fluorescence from the organs to provide meaningful data. Regions of interest on the lateral surface approximating the liver, kidney, tumor, and thorax could be defined by regional contrast in surface fluorescence until 5 h after injection (Figures 5 E and 6). In vivo surface fluorescence further supported our FMT results in that significantly more EIBS-750 than Control-750 was retained in the liver (Figure 6 A). This difference was not observed when EIBS-680 and Control-680 injected mice were compared. In addition, surface images showed that, for both labels, EIBS-injected mice had significantly more fluorescence in the region over the kidneys (Figure 6 B) when compared with their counterpart controls. As previously observed, tumor fluorescence did not differ across the groups (Figure 6 C). In addition, surface fluorescence emitted from the thorax, liver, stomach, intestine, and bladder regions was evident in early ventrodorsal images (Figures 5 F and 7). Ventrodorsal surface fluorescence from the area of the liver was concordant with the lateral results (Figures 6 A and 7 A). In the stomach (Figures 7 B and 8) and intestine (Figure 7 D) regions, fluorescence averages from EIBS-injected mice were greater than that of their counterpart controls for both labels, but the differences were not significant. Fluorescence presumably emitting from urine in the bladder was readily apparent in several of the mice, and despite being highly variable, was more pronounced in EIBS-injected mice for both labels (Figure 5 F and 7 C). Ex vivo surface fluorescence of the livers of mice injected with EIBS-750 was markedly higher than those of mice injected with EIBS-680 or either control at 24 and 48 h after injection (Figure 8). Results from the ex vivo surface fluorescence of livers taken at 24 h correlated well with those obtained in vivo by FMT (r = 0.89; P < 0.0001). The kidneys of mice injected with either EIBS labeled antibody had significantly more signal than did their counterpart controls when assessed in vivo and ex vivo (Figures 5 and 8). In addition, the isolated stomachs of EIBS-680–injected mice were significantly (P < 0.0001) more fluorescent than were those from the other groups. Blood was collected from each mouse prior to tissue collection. Because, in an attempt to optimize FMT imaging in these sequential studies, the concentration of antibody used in the tumorbearing mice (3 mg/kg) differed from that of nontumor-bearing animals (4 mg/kg), direct comparison between these 2 groups of studies is difficult. However, at each time point, regardless of presence of tumor or the type of label, the concentration of EIBS was significantly lower (P < 0.05) than that of the controls (Figure 9). The clearance of each antibody was unaffected by the type of label used, because there was no difference in serum concentration when EIBS-680 was compared with EIBS-750 or when Control-680 was compared with Control-750.
Discussion
Our initial biodistribution studies used FMT to image mice injected with a mixture of EIBS and control antibodies labeled
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Figure 2. FMT localization of antibody in the tumor and body. Fluorescence emitted from the body scans of mice injected with EIBS-750 and Ctl-680 at indicated times (A) with corresponding plasma antibody concentrations (B) and fluorescence from tumors(C). The retention of antibody in tumor tissue was assessed by calculating tumor fluorescence relative to plasma clearance for each mouse(D). Data shown (mean ± 1 SD) represent 3 mice per group per time point; brackets indicate significant differences (P < 0.05) between values. Naïve mice showed no fluorescence in fluorescence molecular tomography.
with NIRF with nonoverlapping spectra. Although this strategy can decrease variability caused by tumor or model heterogeneity27 and reduce animal use, it has limitations in that comparisons may be affected by using dissimilar NIRF labels on each antibody and each antibody could potentially alter the other’s physiologic interactions (PK, PD, and Fc receptor binding, etc) To rule out these possibilities, we then injected individual antibodies with each of 2 labels and performed additional analyses by using an IVIS Spectrum imaging system. In our initial FMT analysis, we noted that the large difference in total fluorescence coming from the bodies of the mice during the first 2 h was due to the extravasation of EIBS-750 into the tissues and presumably to quenching of the Control-680, which remained primarily in the blood vessels30 (Figure 2 A). The higher plasma antibody titers from the Control-680–injected mice support this assumption (Figure 2B), and ex vivo tissue surface fluorescence demonstrated that the liver was largely responsible for the increased abdominal fluorescence in the EIBS-750–injected mice.
Further studies using the IVIS Spectrum system in mice that received individually labeled antibodies were consistent with our previous findings, in that EIBS-750 was rapidly cleared from the blood and highly retained in the liver. Although the pharmacokinetics were similar between EIBS-680 and EIB-750, the fluorescence from the livers of EIBS-680–injected mice was not significantly higher than that of either of the control groups. These results suggest that EIBS-750 (or its fluorescent metabolites) was retained in the liver longer than was EIBS-680. Our results are consistent with those of a previous study,5 which showed similar retention in the liver of an antibody that was labeled with the NIRF IRDye800, when compared with the unlabeled antibody. It is unclear why there are differences in the tissue retention among fluorophores, but they may be the result of differences in hydrophobicity, charge density, or charge distribution.4 In our studies, we did not find that the NIRF label was the driving force for antibody disposition in the liver, given that the 2 labeled control antibodies distributed similarly.
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Figure 3. Ex vivo tissue surface fluorescence. Major organs were harvested from mice euthanized at (A) 2 h, (B) 24 h, and (C) 48 h after injection of EIBS-750 and control (Ctl)-680 in each of the mice, and surface fluorescence was measured in the FMT on each channel. Data shown (mean ± 1 SD) represent tissue fluorescence relative to that of heart muscle. Bar, significant difference (P < 0.05) between values.
Because plasma levels of EIBS-750 and EIBS-680 were similar, either EIBS-680 was cleared by a different mechanism at a similar rate as EIBS-750 or EIBS-680 fluorescent metabolites were processed from the liver tissue faster and were thus not as readily
Figure 4. In vivo FMT 3D biodistribution analysis. Fluorescence from (A and B) the liver region and (C) subcutaneously implanted tumors of mice injected with EIBS and control antibodies labeled with Alexa Fluor 680 and 750 were compared. Mice were scanned in the (A and D) ventrodorsal and (B and C) lateral recumbency. Bracket represents significant difference (P < 0.05) between values. (D) An example of FMT scans of the liver region of mice.
detected as were EIBS-750 byproducts. The greater fluorescence of EIBS-680 in the stomach supports this latter assumption. Metabolites or fluorophores from EIBS-680 that are cleared by the liver are likely excreted via the biliary system into the gastrointestinal tract.26 Retrograde travel and Fc receptors expressed on the gastric epithelial cells2 could potentially result in the retention of fluorophore-conjugated antibodies and Fc metabolites in the
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Figure 5. Examples of in vivo images. FMT whole-body scans showing fluorescence from the liver of mice which were injected with the indicated NIRF labeled antibodies 24 h prior to imaging (A-D). IVIS spectrum surface fluorescence images of mice (E) in lateral recumbency at 5 h after injection and (F) in ventrodorsal recumbence taken 24 h after injection of EIBS-750.
stomach. This situation does not occur with EIBS-750, because the majority of the fluorophore was entrapped at the liver. Although autofluorescence from the stomach contents can be problematic15 and should be considered, it accounted for less than 10% of the fluorescence detected in all tissues. In addition, the fluorescence from the stomachs of Control-680–injected mice was relatively low. The liver is one of the largest organs and receives and filters approximately 25% of the cardiac output.31 As this volume moves through the liver making numerous cell surface contacts, slight differences in retention, binding, and metabolism are likely to
profoundly affect the pharmacokinetics and pharmacodynamics of therapeutically administered antibodies.24 Retention of radiolabeled antibodies in the liver is often evident in immunoPET studies, and this feature has been largely attributed to the radiolabeling process that was used, but hepatic recognition and processing of immunoglobulin moieties likely are involved also.17,25 Similar to our results, another study14 found significant accumulation of a “bifunctional homodimeric diabody” in the liver 15 min after intravenous injection, which also subsided by 24 h. A predominant factor in influencing antibody half-life in the blood is the role of FcRn receptors.6 These receptors bind
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Figure 6. In vivo lateral surface fluorescence (IVIS spectrum). Regions of interest over and approximating the (A) liver, (B) kidneys, and (C) tumor were defined in anesthetized mice injected with the specified labeled antibodies. After 5 h, signals from the liver and kidney region were similar to background (thoracic region) and not graphed. Fluorescence (mean ± 1 SD) of tissues from 4 mice are plotted; bracket indicates a significant difference (P < 0.05) between values.
immunoglobulins and protect them from cellular catabolism. The binding affinity for FcRn is largely dependent on immunoglobulin isotype and structure. Because FcRn receptors are expressed in hepatocytes,20,29 their affinities for different types of immunoglobulin-like molecules will likely influence their retention time as they pass through the liver. The antiEGFR arm of EIBS was not cross-reactive with mouse EGFR and was an unlikely contributor to the antibody’s retention in the liver. In RT-PCR organ expression analyses, the IGF1R was not detected in the adult liver but was observed in the kidney.9 In our studies, the kidney had elevated EIBS regardless of label (Figure 6 B). However, interpretation of the significance of fluorescence from the kidneys can be challenging, because it is difficult to differentiate released fluorophore and fluorophorebound breakdown products filtered through the kidneys from signals due to intact antibody that is bound to renal tissue. The high signal intensities from both the kidneys and bladder regions of these mice (Figures 6 and 7) suggest that the fluorescence in these tissues may be the result of urinary clearance of antibody metabolites or of released label. Further analyses at the cellular level are needed to determine whether EIBS is binding to IGF1R that may be expressed in the kidneys. In this study, tumors (Figures 3 and 8) and skin (Figure 3) tend to be sites of nonspecific control antibody accumulation. The tendency for labeled antibodies to get ‘trapped’ in subcutaneous xenografts has been reported in early literature,1 and nonspecific antibody accumulation may be largely influenced by tumor ‘leakiness.’32 Pharmacokinetic modeling comparisons in FcRn knockout and wildtype mice suggest that FcRn receptors play a role in immunoglobulin retention and catabolism in the skin.11 Other authors showed that FcRn receptors are actively expressed in the capillaries of the skin.3 Remarkably, 30% endogenous plasma IgG in humans is located in dermal interstitial fluid transported from capillary beds.28 Consistent with these earlier findings, our studies demonstrate that a considerable fraction of the labeled antibodies administered to mice travel to the skin and are retained there for at least 24 h (Figure 3). Differences in FcRn processing or recycling may have affected the retention of the labeled antibodies in tissues. In support of this theory, FcRn-mediated transport pathways have been reported to shunt immunoglobulins toward lysosomal degradation instead of recycling.23 Studies with dendritic cells showed that FcRn-bound multimeric immune complexes were directed to lysosomes, whereas monomeric immune complexes were recycled.23 Conceivably, structural differences between our control antibodies and bispecific antibodies may have led to alternative FcRn-mediated processing pathways and therefore affected tissue retention/biodistribution. Work by another group10 suggests that the FcRIIb on liver sinusoidal endothelial cells plays a major role in clearing small immune complexes. After EIBS enters the blood stream or hepatic sinusoids, the environmental effects on its structure are unknown. Theoretically, the bispecific antibody could form aggregates or complex with serum proteins either before entering or within the hepatic sinusoids and therefore be bound by FcRIIb. Further studies at the cellular level will be useful in elucidating how Fc receptors in the liver interact with immunotherapeutics to result in their hepatic retention and poor pharmacokinetics. Other factors that could affect the biodistribution of the antibodies studied here include their molecular weight and
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Figure 7. In vivo ventrodorsal surface fluorescence (IVIS spectrum) in nontumor-bearing mice. Areas over the (A) liver, (B) stomach, (C) bladder, and (D) intestines of anesthetized, nontumor-bearing mice injected with the specified labeled antibodies were identified as having differential fluorescence, and regions of interest were defined for comparison. Fluorescence (mean ± 1 SD) from regions of interest of 4 mice are plotted; bracket indicates a significant difference (P < 0.05) between values.
carbohydrate content. The molecular weight of our control antibody is estimated to be 150 kD, whereas the bispecific antibodies have a molecular weight of 210 kD. The liver, kidneys, and spleen have either discontinuous arteriole supply or pores that might differentially influence tissue or receptor exposure to our antibodies.24 Hepatic cells also have asialoglycoprotein receptors that can remove immunoglobulins containing galactosyl moieties.22 Mannose receptors expressed on Kupffer cells, endothelium, macrophages, and dendritic cells in the liver function to clear immunoglobulins laden with mannose residues.24 Further analysis of our antibodies and their interactions with these tissues is needed to elucidate how and whether differences in carbohydrate content affect their biodistribution. A secondary objective of the current studies was to evaluate optical imaging applications for antibody biodistribution analysis in preclinical studies. FMT uses laser-stimulated excitation of fluorophores and the detection and collection of emittance over multiple points for algorithm-mediated reconstruction of quantifiable 3D images. Although increased tissue penetrance and
quantification of labeled compound within a 3D space maybe advantageous with this transillumination modality, we found that some scans at early time points did not reconstruct to produce images. This difficulty was most apparent with the control antibody and the ventrodorsal positioning. Perhaps under these circumstances there was insufficient contrast to generate a reconstruction. In addition, the skin (Figure 3) is a common site of antibody retention. High levels of fluorescence at the animal’s surface could affect transillumination image readouts.19 However, when 3D images could be reconstructed, there was good correlation of fluorescence data from the liver in vivo and ex vivo. Ex vivo analyses of tissues are performed for comparison with in vivo results and, in some cases, may provide increased sensitivity, because the signal is not attenuated by other tissues in the light path. In addition, tissue borders can readily be defined by using the superimposed normal light reflectance image, whereas regions of interest are only approximated in FMT scans,30 thus increasing variability. Although we were able to detect strong fluorescent signals from the liver by using epifluorescence, deeper or weaker
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Fluor 750). In light of these findings and in consideration of the limitations we have described, we find that optical imaging provides a convenient relatively inexpensive modality for assessing the biodistribution of biotherapeutics in mouse models.
Acknowledgments
The authors thank Ryan Fleming, Binyam Bezabeh, and Chansghou Gao (ADPE MedImmune) for their assistance in developing the bispecific proteins described here. Rakesh Dixit provided valuable editorial suggestions.
References
Figure 8. Ex vivo organ surface fluorescence from mice injected 48 h previously with labeled antibodies. Fluorescence (mean ± 1 SD) from tissues of 4 mice are plotted; bracket indicates a significant difference (P < 0.05) between values.
Figure 9. Plasma antibody concentrations from the mice imaged. Two studies are represented: one in tumor-bearing mice (xenograft +) that received 3 mg/kg of labeled antibody, and the other in mice without tumors (xenograft –) that received 4 mg/kg of labeled antibody. Data are shown as the mean ± 1 SD of 4 plasma samples.
near-infrared signals are unlikely to be detected at the animal’s surface. Likewise, the average fluorescent intensity values of large, highly heterogeneous tumors that predominately bind labeled antibody at their periphery12 may differ when IVIS Spectrum images (2D, surface) are compared with FMT scans (3D, volumetric). Although a direct comparison with radioisotope labeling and PET imaging would have been helpful, this modality was not readily available, and running an externally funded project was costprohibitive. However, using optical imaging, we found that the liver was a major organ of retention for our bispecific antibody and the likely cause of its poor pharmacokinetics. The study also suggested that the kidney may be a site of target specific or nonspecific retention. Furthermore, we have shown that the choice of fluorescent label can affect tissue signal retention. Although additional time and resources may be necessary, performing biodistribution analyses with 2 different labels is very informative. In addition, we found that tissue depots were more readily identified by using fluorophores with prolonged tissue retention (for example, Alexa
1. Bale WF, Contreras MA, Grady ED. 1980. Factors influencing localization of labeled antibodies in tumors. Cancer Res 40:2965–2972. 2. Ben Suleiman Y, Yoshida M, Nishiumi S, Tanaka H, Mimura T, Nobutani K, Yamamoto K, Takenaka M, Aoganghua A, Miki I, Ota H, Takahashi S, Matsui H, Nakamura M, Blumberg RS, Azuma T. 2012. Neonatal Fc receptor for IgG (FcRn) expressed in the gastric epithelium regulates bacterial infection in mice. Mucosal Immunol 5:87–98. 3. Borvak J, Richardson J, Medesan C, Antohe F, Radu C, Simionescu M, Ghetie V, Ward ES. 1998. Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice. Int Immunol 10:1289–1298. 4. Choi HS, Nasr K, Alyabyev S, Feith D, Lee JH, Kim SH, Ashitate Y, Hyun H, Patonay G, Strekowski L, Henary M, Frangioni JV. 2011. Synthesis and in vivo fate of zwitterionic near-infrared fluorophores. Angew Chem Int Ed Engl 50:6258–6263. 5. Conner KP, Rock BM, Kwon GK, Balthasar JP, Abuqayyas L, Wienkers LC, Rock DA. 2014. Evaluation of near-infrared fluorescent labeling of monoclonal antibodies as a tool for tissue distribution. Drug Metab Dispos 42:1906–1913. 6. Dall’Acqua WF, Woods RM, Ward ES, Palaszynski SR, Patel NK, Brewah YA, Wu H, Kiener PA, Langermann S. 2002. Increasing the affinity of a human IgG1 for the neonatal Fc receptor: Biological consequences. J Immunol 169:5171–5180. 7. Dijkers EC, Oude Munnink TH, Kosterink JG, Brouwers AH, Jager PL, de Jong JR, van Dongen GA, Schroder CP, Lub-de Hooge MN, de Vries EG. 2010. Biodistribution of 89Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin Pharmacol Ther 87:586–592. 8. Dimasi N, Gao C, Fleming R, Woods RM, Yao XT, Shirinian L, Kiener PA, Wu H. 2009. The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators. J Mol Biol 393:672–692. 9. Freeman TC, Dixon AK, Campbell EA, Tait TM, Richardson PJ, Rice KM, Maslen GL, Metcalfe AD, Streuli CH, Bentley DR. [Internet] 1998. Expression mapping of mouse genes. [Cited 19 February 2016]. Available at: http://www.informatics.jax.org/reference/J:46439. 10. Ganesan LP, Kim J, Wu Y, Mohanty S, Phillips GS, Birmingham DJ, Robinson JM, Anderson CL. 2012. FcγRIIb on liver sinusoidal endothelium clears small immune complexes. J Immunol 189:4981–4988. 11. Garg A, Balthasar JP. 2007. Physiologically based pharmacokinetic (PBPK) model to predict IgG tissue kinetics in wild-type and FcRnknockout mice. J Pharmacokinet Pharmacodyn 34:687–709. 12. Girgis MD, Olafsen T, Kenanova V, McCabe KE, Wu AM, Tomlinson JS. 2011. Targeting CEA in pancreas cancer xenografts with a mutated scFv–Fc antibody fragment. EJNMMI Res 1:24. 13. Grandjenette C, Dicato M, Diederich M. 2015. Bispecific antibodies: an innovative arsenal to hunt, grab, and destroy cancer cells. Curr Pharm Biotechnol 16:670–683. 14. Hemmerle T, Wulhfard S, Neri D. 2012. A critical evaluation of the tumor- targeting properties of bispecific antibodies based on quantitative biodistribution data. Protein Eng Des Sel. 25:851–854. 15. Inoue Y, Izawa K, Kiryu S, Tojo A, Ohtomo K. 2008. Diet and abdominal autofluorescence detected by in vivo fluorescence imaging of living mice. Mol Imaging 7:21–27.
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16. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 17. Johansson AG, Lovdal T, Magnusson KE, Berg T, Skogh T. 1996. Liver cell uptake and degradation of soluble immunoglobulin G immune complexes in vivo and in vitro in rats. Hepatology 24:169–175. 18. Kontermann RE, Brinkmann U. 2015. Bispecific antibodies. Drug Discov Today.20:838–847. 19. Leblond F, Davis SC, Valdes PA, Pogue BW. 2010. Preclinical wholebody fluorescence imaging: review of instruments, methods, and applications. J Photochem Photobiol B 98:77–94. 20. Lobo ED, Hansen RJ, Balthasar JP. 2004. Antibody pharmacokinetics and pharmacodynamics. J Pharm Sci 93:2645–2668. 21. McCabe KE, Wu AM. 2010. Positive progress in immunoPET—not just a coincidence. Cancer Biother Radiopharm 25:253–261. 22. Meier W, Gill A, Rogge M, Dabora R, Majeau GR, Oleson FB, Jones WE, Frazier D, Miatkowski K, Hochman PS. 1995. Immunomodulation by LFA3TIP, an LFA3–IgG1 fusion protein: cell-line–dependent glycosylation effects on pharmacokinetics and pharmacodynamic markers. Ther Immunol 2:159–171. 23. Qiao SW, Kobayashi K, Johansen FE, Sollid LM, Andersen JT, Milford E, Roopenian DC, Lencer WI, Blumberg RS. 2008. Dependence of antibody-mediated presentation of antigen on FcRn. Proc Natl Acad Sci USA 105:9337–9342. 24. Rojko J, Price-Schiavi S. 2008. Physiologic IgG biodistribution, transport, and clearance: implications of monoclonal antibody products, p. 241. In: Cavagnaro JA, editor. Preclinical safety evaluation of biopharmaceuticals: a science-based approach to facilitating clinical trials. Hoboken (NY): John Wiley and Sons.
25. Sands H, Jones PL. 1987. Methods for the study of the metabolism of radiolabeled monoclonal antibodies by liver and tumor. J Nucl Med 28:390–398. 26. Shen BQ, Bumbaca D, Yue Q, Saad O, Tibbitts J, Khojasteh SC, Girish S. 2015. Nonclinical disposition and metabolism of DM1, a component of trastuzumab emtansine (T-DM1), in Sprague Dawley rats. Drug Metab Lett 9:119–131. 27. Sun Y, Shukla G, Pero SC, Currier E, Sholler G, Krag D. 2012. Single-tumor imaging with multiple antibodies targeting different antigens. Biotechniques 52:1–3. 28. Svedman C, Yu BB, Ryan TJ, Svensson H. 2002. Plasma proteins in a standardised skin mini-erosion (I): permeability changes as a function of time. BMC Dermatol 2:3. 29. Telleman P, Junghans RP. 2000. The role of the brambell receptor (FcRB) in liver: protection of endocytosed immunoglobulin G (IgG) from catabolism in hepatocytes rather than transport of IgG to bile. Immunology 100:245–251. 30. Vasquez KO, Casavant C, Peterson JD. 2011. Quantitative wholebody biodistribution of fluorescent-labeled agents by noninvasive tomographic imaging. PLoS One 6:e20594. 31. Vollmar B, Menger MD. 2009. The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair. Physiol Rev 89:1269–1339. 32. Wolfgang WA, Kiessling F. 2011. Imaging in oncology research, p 549. In: Kiessling F,Pichler PJ, editors. Small animal imaging: basics and practical guide. Heidelberg (Germany): Springer– Verlag. 33. Zelmer A, Ward TH. 2013. Noninvasive fluorescence imaging of small animals. J Microsc 252:8–15.
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Original Research
Biodistribution Analyses of a Near-Infrared, Fluorescently Labeled, Bispecific Monoclonal Antibody Using Optical Imaging Norman C Peterson,1,* George G Wilson,1 Qihui Huang,2 Nazzareno Dimasi,3 and Kris F Sachsenmeier2 In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies. Abbreviations: EGFR, epidermal growth factor receptor; EIBS, EGFR–IGF1R bispecific monoclonal antibody; FMT, fluorescence molecular tomography; IGF1R, insulin-like growth factor 1 receptor; NIRF, near-infrared fluorophore; PET, positron emission tomography
Over the past 25 y, bispecific antibodies have been added to the arsenal of humanized antibodies that have been developed to treat cancer, infectious diseases, and inflammatory diseases. Bispecific biotherapeutics simultaneously recognize 2 different epitopes on the same or different cells or bacteria. This multispecificity offers several advantages over their monospecific counterparts in that cell or tissue specificity can be increased and thus toxicity reduced, 2 (or more) receptor pathways can be targeted simultaneously, and immune-mediated responses at clinically affected sites may be activated (cancer) or deactivated (inflammatory disease).13,18 As the size and structure of monoclonal antibodies are altered from their natural state to create biotherapeutics, such as bispecific antibodies,8 the pharmacokinetic properties of the products may differ from that of their parental (natural) antibodies. Aside from the potential to increase immunogenicity or aggregate, very little is known about how these structural differences affect pharmacokinetics and biodistribution. Receptormediated uptake and cellular processing both are likely to play a role,20 but identifying the tissues or cell types involved in recognizing these altered proteins is a necessary first step toward developing engineering strategies that can circumvent these impediments. Received: 04 Aug 2015. Revision requested: 04 Sep 2015. Accepted: 23 Sep 2015. Departments of 1Translational Sciences, 2Cancer Biology, and 3Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, Maryland * Corresponding author. Email: [email protected]
At the organ or tissue level, the use of radiolabeling and imaging in clinical and preclinical studies has greatly facilitated our understanding of the pharmacokinetics and tissue distribution of biotherapeutics.7,21 This advance is in part due to the ability to track and monitor the distribution of labeled compounds in the same animal over time. The increased sensitivity of positron emission tomography (PET) and the ability of radioisotope signals to travel through tissues without being significantly attenuated or diffracted has made PET the leading modality for preclinical and clinical biodistribution analyses. Disadvantages of PET are that its application is more expensive and requires skill in chemical conjugation, imaging technology, and radiation safety. Given that these resources are often limited or unavailable at several institutions (ours included), near-infrared optical imaging with improved tissue penetrance and quantitative imaging algorithms are demonstrating value when compared with ‘high-end’ imaging modalities.19,33 The accessibility of a variety commercially available fluorescently labeled targeting proteins and peptides, activatable probes, and fluorescent protein labeling kits has made this technology more readily available. In addition, because multiple fluorophores of nonoverlapping emission spectrums can be administered simultaneously, colocalization studies and direct comparisons of 2 or more labeled compounds can be performed in the same animal. Here we show how fluorescence-based optical imaging was used to identify potential tissue depots or sinks and characterize the biodistribution profile of a bispecific antibody under preclinical development.
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Materials and Methods
Antibodies. The recombinant antibodies used in this study were cloned, expressed and purified at MedImmune as described previously.8 The antibody EIBS is a humanized bispecific antibody engineered to concurrently bind to insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR). The antiIGF1R antibody is cross-reactive to both mouse and human, whereas the antiEGFR portion is human-specific. This bispecific antibody has a structure similar to the bispecific antibody prototype (Bs2Ab), in that an antiIGF1R single-chain variable fragment is engineered on the amino terminal of the heavy chains of the antiEGFR antibody.8 Humanized monoclonal antibody R347, developed at Medimmune, is an IgG1 that binds to HIV and was used as a control in all studies. EIBS and control antibodies were purified to homogeneity, approximately 99% monomeric, and formulated in PBS pH 7.2. The antibodies were labeled with Alexa Fluor 680 or 750 according to the manufacturer’s instructions (SAIVI Rapid Antibody Labeling Kit, Life Technologies, Grand Island, NY) Cells. NCI-H358 human-derived nonsmall-cell lung carcinoma cells were obtained from ATCC (Manassas, VA) and were cultured in RPMI1640 media (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen) in humidified incubators at 37 °C and 5% CO2. Cells (5 × 106) were injected in the subcutaneous tissue of the right flank of the mice and allowed to grow until the tumor was approximately 1000 mm3 before imaging. Flow cytometry. NCI-H358 cells (ATCC, Manassas, VA) were grown in flasks containing RPMI1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Life Technologies) in a humidified incubator at 37 °C with 5% CO2. Cells were released from culture, washed, and resuspended in 2% BSA (Roche Diagnostics, Indianapolis, IN) in PBS (Life Technologies). Single-cell suspensions were distributed in 96-well plates (2 × 105 cells/well). Cells were incubated with fluorescently labeled EIBS (or control antibody) for 1 h on ice and washed in buffer prior to analysis. Cells were analyzed by flow cytometry (LSR II, Becton Dickinson, San Jose, CA), and data were analyzed by using FlowJo software (TreeStar, Ashland, OR). Serum immunoglobulin assay. Serum immunoglobulin titers were determined by sandwich ELISA using mouse antihuman IgG (Fc-specific, Jackson Immunoresearch, West Grove, PA). After incubation with serum and repeated washing, either goat antihuman IgG λ–horseradish peroxidase (R347 detection; Southern Biotech, Birmingham, AL) or goat antihuman IgG κ-horseradish peroxidase (EIBS detection; Southern Biotech) was added to microtiter plates containing the bound antibodies. After repeated washing, SureBlue TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD) was added to each well, and absorbance at 450 nm was determined. Animals. Female athymic (nu/nu) mice approximately 5 wk of age were obtained from Harlan (Indianapolis, IN) and were housed in individually ventilated cages, on hardwood bedding, and fed a commercially available diet (HarlanTeklad 2918 Diet, 18% Global Protein Diet, Harlan, Indianapolis, IN). According to results from vendor and quarterly institutional health surveillance programs, the mice were free of mouse adenovirus, cilia-associated respiratory bacillus, ectromelia virus, rotavirus, lymphocytic choriomeningitis virus, mouse hepatitis virus, reovirus, pneumonia virus of mice, Mycoplasma pulmonis, minute virus of mice, mouse parvovirus, cytomegalovirus, polyoma virus,
Sendai virus, norovirus and Helicobacter spp. Environmental conditions were: temperature, 21 to 22 °C; relative humidity, 40% to 60%; and 12:12-h light:dark cycle. All procedures were run in accordance with the Guide for the Care and Use of Laboratory Animals16 and institutional standards in our AAALAC-accredited facility and were approved by MedImmune’s IACUC. Imaging. Mice received a single injection into the tail vein of antibody labeled with Alexa Fluor 680 or 750 (3 mg/kg; SAIVI Rapid Antibody Labeling Kit, Life Technologies, Grand Island, NY). To accommodate logistics with fewer animals and minimize interanimal variability, a mixture of equal amounts of control and test antibody (1 to 3 mg/kg) with different labels were injected into the each mouse.27 Mice were anesthetized with 2% isoflurane and scanned in a Fluorescent Molecular Tomography 2500 system (PerkinElmer, Waltham, Massachusetts) on either 680 nm or 750 nm channels at 3 mm density and medium laser intensity. Images were reconstructed using TrueQuant (version 3.1) software (PerkinElmer), and regions of interest were defined visually. Surface fluorescence from anesthetized mice and tissues (ex vivo) was analyzed by using an IVIS Spectrum (Perkin Elmer) set at medium binning, F-stop 1, and auto exposure. In vivo surface fluorescence of manually defined regions of interest was quantitated as average radiance efficiency ([p/s/cm2/sr] / [µW/cm2]) by using Living Image software (Perkin Elmer). Fluorescent signals were standardized across mice and presented as a proportion of the total surface fluorescence (in vivo) or the fluorescence from the heart (ex vivo). Data were analyzed with GraphPad 6.03 software (Prism, La Jolla, CA). ANOVA; Bonferroni posthoc analyses were preformed to determine statistical significance (defined as a P value less than 0.05).
Results
Localization of antiIGF1R–EGFR bispecific antibody in tumors. To increase specificity and simultaneously target 2 tumor-associated proteins that are commonly and highly expressed in several tumor types (pancreatic, breast, colorectal, and lung), the bispecific IGF1R–EGFR1 antibody EIBS was developed from antibodies that recognize each receptor independently. The antibodies were labeled with commercially available near-infrared fluorophores (NIRF), and the degree of labeling (ratio of antibody to label) remained within the range of 1.8 to 3.5 for each batch tested. Flow cytometry demonstrated that NIRF-labeled EIBS bound to nonsmall-cell lung cancer cells (H358 cells) in vitro (Figure 1) and that there was no detectable difference in affinity between EIBS labeled with either NIRF. In addition, preliminary data indicated that the antiEGFR arm does not cross react with the mouse protein, whereas the antiIGF1R arm does. To more specifically evaluate the tumor-targeting ability of EIBS in vivo and assess nonspecific tissue binding, imaging biodistribution studies were conducted by using optical imaging modalities. Mice with subcutaneously implanted H358 cells were intravenously injected with a mixture of EIBS and a control antibody that were labeled with different NIRFs; the spectra of the 2 NIRFs used did not overlap (Alexa Fluor 750 and 680, respectively). Comparison of clearance of total body fluorescence in these mice as estimated by abdominal fluorescent molecular tomography (FMT) scans revealed that 50% of the EIBS had cleared from the mice in the first 24 h and that EIBS-associated fluorescence continued to decline, whereas total body fluorescence remained unchanged for the con-
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Figure 1. Flow cytometric analysis of H358 cells exposed to EIBS and control (Ctl) antibody labeled with Alexa Fluor 680 or 750 at specified concentrations. The assay was performed in duplicate, and the mean fluorescent intensity ratio (MFIR) was plotted. The MFIR was calculated by dividing the mean fluorescence of EIBS-680 at each concentration by the average mean fluorescence of Ctl-680, and likewise for the 750-labeled antibodies.
trol antibody (Figure 2 A). Analysis of the 2- and 24-h antibody concentrations in the blood confirmed that plasma clearance of EIBS was more rapid than that of the control antibody (80% compared with 67% cleared, respectively (P < 0,05); Figure 2 B). Despite the rapid clearance of EIBS from the blood and decreased total body fluorescence, the amount of EIBS remained unchanged in the tumor (Figure 2 C). This dynamic resulted in significantly (P < 0.05) higher tumor:blood antibody ratios in EIBS-injected mice when compared with controls (Figure 2 D). Although the tumor:blood ratios suggested that EIBS was retained in the tumor, the average percentage of injected dose for tumors with EIBS did not exceed that of controls (Figure 2 C). In our experience, nonspecific retention of control antibodies in tumor tissue is typically greater than that of most other tissues and ranges from 10% to 20% of the injected dose per gram of tissue. To determine whether tumor exposure to the antibodies was limited by their retention in tissues, ex vivo surface fluorescence of the major organs from these mice was measured (Figure 3) by FMT. Biodistribution profiles showed that when compared with controls, significantly (P < 0.05) and markedly more EIBS was retained in the liver shortly after injection (Figure 3 A), with a trend suggestive of continued retention through at least 24 h(P = 0.39) (Figure 3 B). The control antibody showed nonspecific retention in the skin (P < 0,05) (Figure 3 B and C). To confirm these results, further studies were designed such that each antibody was compared with an alternative NIRF (Alexa Fluor 680 or 750), and each mouse was injected with a single labeled antibody (that is, EIBS labeled with Alexa Fluor 680 [EIBS-680], EIBS-750, Control-680, or Control-750). Early FMT scans (1 and 5 h after injection) in 2 trials failed to yield consistent reconstructed images for all mice in each group when mice that received doses of 3 and 4 mg/kg were analyzed. This issue likely was due to either insufficient or saturated signal and was partially resolved by 24 h, when all ventrodorsal scans (except of mice given Control-680) yielded 3D constructions of fluorescence data. By 48 h, 3D reconstructions were generated from all scans (Figure 4). Comparison of FMT scans revealed greater fluorescence from the livers of EIBS-750–treated mice than from those of Control-750 injected mice (Figure 4 A and B; Figure 5 A through
D); this difference reaching significance (P < 0.05) by 24 h after injection (Figure 4 B, lateral scan). However, the liver-associated signal did not differ when EIBS-680– and Control-680–injected mice were compared. The fluorescence from the tumors was similar for both labeled EIBS antibodies but was lower than their controls (Figure 4 C), presumably reflecting exposure levels in the blood. Whereas in vivo biodistribution analysis with FMT was somewhat limited at early time points, early surface epifluorescence images yielded sufficient fluorescence from the organs to provide meaningful data. Regions of interest on the lateral surface approximating the liver, kidney, tumor, and thorax could be defined by regional contrast in surface fluorescence until 5 h after injection (Figures 5 E and 6). In vivo surface fluorescence further supported our FMT results in that significantly more EIBS-750 than Control-750 was retained in the liver (Figure 6 A). This difference was not observed when EIBS-680 and Control-680 injected mice were compared. In addition, surface images showed that, for both labels, EIBS-injected mice had significantly more fluorescence in the region over the kidneys (Figure 6 B) when compared with their counterpart controls. As previously observed, tumor fluorescence did not differ across the groups (Figure 6 C). In addition, surface fluorescence emitted from the thorax, liver, stomach, intestine, and bladder regions was evident in early ventrodorsal images (Figures 5 F and 7). Ventrodorsal surface fluorescence from the area of the liver was concordant with the lateral results (Figures 6 A and 7 A). In the stomach (Figures 7 B and 8) and intestine (Figure 7 D) regions, fluorescence averages from EIBS-injected mice were greater than that of their counterpart controls for both labels, but the differences were not significant. Fluorescence presumably emitting from urine in the bladder was readily apparent in several of the mice, and despite being highly variable, was more pronounced in EIBS-injected mice for both labels (Figure 5 F and 7 C). Ex vivo surface fluorescence of the livers of mice injected with EIBS-750 was markedly higher than those of mice injected with EIBS-680 or either control at 24 and 48 h after injection (Figure 8). Results from the ex vivo surface fluorescence of livers taken at 24 h correlated well with those obtained in vivo by FMT (r = 0.89; P < 0.0001). The kidneys of mice injected with either EIBS labeled antibody had significantly more signal than did their counterpart controls when assessed in vivo and ex vivo (Figures 5 and 8). In addition, the isolated stomachs of EIBS-680–injected mice were significantly (P < 0.0001) more fluorescent than were those from the other groups. Blood was collected from each mouse prior to tissue collection. Because, in an attempt to optimize FMT imaging in these sequential studies, the concentration of antibody used in the tumorbearing mice (3 mg/kg) differed from that of nontumor-bearing animals (4 mg/kg), direct comparison between these 2 groups of studies is difficult. However, at each time point, regardless of presence of tumor or the type of label, the concentration of EIBS was significantly lower (P < 0.05) than that of the controls (Figure 9). The clearance of each antibody was unaffected by the type of label used, because there was no difference in serum concentration when EIBS-680 was compared with EIBS-750 or when Control-680 was compared with Control-750.
Discussion
Our initial biodistribution studies used FMT to image mice injected with a mixture of EIBS and control antibodies labeled
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Figure 2. FMT localization of antibody in the tumor and body. Fluorescence emitted from the body scans of mice injected with EIBS-750 and Ctl-680 at indicated times (A) with corresponding plasma antibody concentrations (B) and fluorescence from tumors(C). The retention of antibody in tumor tissue was assessed by calculating tumor fluorescence relative to plasma clearance for each mouse(D). Data shown (mean ± 1 SD) represent 3 mice per group per time point; brackets indicate significant differences (P < 0.05) between values. Naïve mice showed no fluorescence in fluorescence molecular tomography.
with NIRF with nonoverlapping spectra. Although this strategy can decrease variability caused by tumor or model heterogeneity27 and reduce animal use, it has limitations in that comparisons may be affected by using dissimilar NIRF labels on each antibody and each antibody could potentially alter the other’s physiologic interactions (PK, PD, and Fc receptor binding, etc) To rule out these possibilities, we then injected individual antibodies with each of 2 labels and performed additional analyses by using an IVIS Spectrum imaging system. In our initial FMT analysis, we noted that the large difference in total fluorescence coming from the bodies of the mice during the first 2 h was due to the extravasation of EIBS-750 into the tissues and presumably to quenching of the Control-680, which remained primarily in the blood vessels30 (Figure 2 A). The higher plasma antibody titers from the Control-680–injected mice support this assumption (Figure 2B), and ex vivo tissue surface fluorescence demonstrated that the liver was largely responsible for the increased abdominal fluorescence in the EIBS-750–injected mice.
Further studies using the IVIS Spectrum system in mice that received individually labeled antibodies were consistent with our previous findings, in that EIBS-750 was rapidly cleared from the blood and highly retained in the liver. Although the pharmacokinetics were similar between EIBS-680 and EIB-750, the fluorescence from the livers of EIBS-680–injected mice was not significantly higher than that of either of the control groups. These results suggest that EIBS-750 (or its fluorescent metabolites) was retained in the liver longer than was EIBS-680. Our results are consistent with those of a previous study,5 which showed similar retention in the liver of an antibody that was labeled with the NIRF IRDye800, when compared with the unlabeled antibody. It is unclear why there are differences in the tissue retention among fluorophores, but they may be the result of differences in hydrophobicity, charge density, or charge distribution.4 In our studies, we did not find that the NIRF label was the driving force for antibody disposition in the liver, given that the 2 labeled control antibodies distributed similarly.
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Figure 3. Ex vivo tissue surface fluorescence. Major organs were harvested from mice euthanized at (A) 2 h, (B) 24 h, and (C) 48 h after injection of EIBS-750 and control (Ctl)-680 in each of the mice, and surface fluorescence was measured in the FMT on each channel. Data shown (mean ± 1 SD) represent tissue fluorescence relative to that of heart muscle. Bar, significant difference (P < 0.05) between values.
Because plasma levels of EIBS-750 and EIBS-680 were similar, either EIBS-680 was cleared by a different mechanism at a similar rate as EIBS-750 or EIBS-680 fluorescent metabolites were processed from the liver tissue faster and were thus not as readily
Figure 4. In vivo FMT 3D biodistribution analysis. Fluorescence from (A and B) the liver region and (C) subcutaneously implanted tumors of mice injected with EIBS and control antibodies labeled with Alexa Fluor 680 and 750 were compared. Mice were scanned in the (A and D) ventrodorsal and (B and C) lateral recumbency. Bracket represents significant difference (P < 0.05) between values. (D) An example of FMT scans of the liver region of mice.
detected as were EIBS-750 byproducts. The greater fluorescence of EIBS-680 in the stomach supports this latter assumption. Metabolites or fluorophores from EIBS-680 that are cleared by the liver are likely excreted via the biliary system into the gastrointestinal tract.26 Retrograde travel and Fc receptors expressed on the gastric epithelial cells2 could potentially result in the retention of fluorophore-conjugated antibodies and Fc metabolites in the
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Figure 5. Examples of in vivo images. FMT whole-body scans showing fluorescence from the liver of mice which were injected with the indicated NIRF labeled antibodies 24 h prior to imaging (A-D). IVIS spectrum surface fluorescence images of mice (E) in lateral recumbency at 5 h after injection and (F) in ventrodorsal recumbence taken 24 h after injection of EIBS-750.
stomach. This situation does not occur with EIBS-750, because the majority of the fluorophore was entrapped at the liver. Although autofluorescence from the stomach contents can be problematic15 and should be considered, it accounted for less than 10% of the fluorescence detected in all tissues. In addition, the fluorescence from the stomachs of Control-680–injected mice was relatively low. The liver is one of the largest organs and receives and filters approximately 25% of the cardiac output.31 As this volume moves through the liver making numerous cell surface contacts, slight differences in retention, binding, and metabolism are likely to
profoundly affect the pharmacokinetics and pharmacodynamics of therapeutically administered antibodies.24 Retention of radiolabeled antibodies in the liver is often evident in immunoPET studies, and this feature has been largely attributed to the radiolabeling process that was used, but hepatic recognition and processing of immunoglobulin moieties likely are involved also.17,25 Similar to our results, another study14 found significant accumulation of a “bifunctional homodimeric diabody” in the liver 15 min after intravenous injection, which also subsided by 24 h. A predominant factor in influencing antibody half-life in the blood is the role of FcRn receptors.6 These receptors bind
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Figure 6. In vivo lateral surface fluorescence (IVIS spectrum). Regions of interest over and approximating the (A) liver, (B) kidneys, and (C) tumor were defined in anesthetized mice injected with the specified labeled antibodies. After 5 h, signals from the liver and kidney region were similar to background (thoracic region) and not graphed. Fluorescence (mean ± 1 SD) of tissues from 4 mice are plotted; bracket indicates a significant difference (P < 0.05) between values.
immunoglobulins and protect them from cellular catabolism. The binding affinity for FcRn is largely dependent on immunoglobulin isotype and structure. Because FcRn receptors are expressed in hepatocytes,20,29 their affinities for different types of immunoglobulin-like molecules will likely influence their retention time as they pass through the liver. The antiEGFR arm of EIBS was not cross-reactive with mouse EGFR and was an unlikely contributor to the antibody’s retention in the liver. In RT-PCR organ expression analyses, the IGF1R was not detected in the adult liver but was observed in the kidney.9 In our studies, the kidney had elevated EIBS regardless of label (Figure 6 B). However, interpretation of the significance of fluorescence from the kidneys can be challenging, because it is difficult to differentiate released fluorophore and fluorophorebound breakdown products filtered through the kidneys from signals due to intact antibody that is bound to renal tissue. The high signal intensities from both the kidneys and bladder regions of these mice (Figures 6 and 7) suggest that the fluorescence in these tissues may be the result of urinary clearance of antibody metabolites or of released label. Further analyses at the cellular level are needed to determine whether EIBS is binding to IGF1R that may be expressed in the kidneys. In this study, tumors (Figures 3 and 8) and skin (Figure 3) tend to be sites of nonspecific control antibody accumulation. The tendency for labeled antibodies to get ‘trapped’ in subcutaneous xenografts has been reported in early literature,1 and nonspecific antibody accumulation may be largely influenced by tumor ‘leakiness.’32 Pharmacokinetic modeling comparisons in FcRn knockout and wildtype mice suggest that FcRn receptors play a role in immunoglobulin retention and catabolism in the skin.11 Other authors showed that FcRn receptors are actively expressed in the capillaries of the skin.3 Remarkably, 30% endogenous plasma IgG in humans is located in dermal interstitial fluid transported from capillary beds.28 Consistent with these earlier findings, our studies demonstrate that a considerable fraction of the labeled antibodies administered to mice travel to the skin and are retained there for at least 24 h (Figure 3). Differences in FcRn processing or recycling may have affected the retention of the labeled antibodies in tissues. In support of this theory, FcRn-mediated transport pathways have been reported to shunt immunoglobulins toward lysosomal degradation instead of recycling.23 Studies with dendritic cells showed that FcRn-bound multimeric immune complexes were directed to lysosomes, whereas monomeric immune complexes were recycled.23 Conceivably, structural differences between our control antibodies and bispecific antibodies may have led to alternative FcRn-mediated processing pathways and therefore affected tissue retention/biodistribution. Work by another group10 suggests that the FcRIIb on liver sinusoidal endothelial cells plays a major role in clearing small immune complexes. After EIBS enters the blood stream or hepatic sinusoids, the environmental effects on its structure are unknown. Theoretically, the bispecific antibody could form aggregates or complex with serum proteins either before entering or within the hepatic sinusoids and therefore be bound by FcRIIb. Further studies at the cellular level will be useful in elucidating how Fc receptors in the liver interact with immunotherapeutics to result in their hepatic retention and poor pharmacokinetics. Other factors that could affect the biodistribution of the antibodies studied here include their molecular weight and
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Figure 7. In vivo ventrodorsal surface fluorescence (IVIS spectrum) in nontumor-bearing mice. Areas over the (A) liver, (B) stomach, (C) bladder, and (D) intestines of anesthetized, nontumor-bearing mice injected with the specified labeled antibodies were identified as having differential fluorescence, and regions of interest were defined for comparison. Fluorescence (mean ± 1 SD) from regions of interest of 4 mice are plotted; bracket indicates a significant difference (P < 0.05) between values.
carbohydrate content. The molecular weight of our control antibody is estimated to be 150 kD, whereas the bispecific antibodies have a molecular weight of 210 kD. The liver, kidneys, and spleen have either discontinuous arteriole supply or pores that might differentially influence tissue or receptor exposure to our antibodies.24 Hepatic cells also have asialoglycoprotein receptors that can remove immunoglobulins containing galactosyl moieties.22 Mannose receptors expressed on Kupffer cells, endothelium, macrophages, and dendritic cells in the liver function to clear immunoglobulins laden with mannose residues.24 Further analysis of our antibodies and their interactions with these tissues is needed to elucidate how and whether differences in carbohydrate content affect their biodistribution. A secondary objective of the current studies was to evaluate optical imaging applications for antibody biodistribution analysis in preclinical studies. FMT uses laser-stimulated excitation of fluorophores and the detection and collection of emittance over multiple points for algorithm-mediated reconstruction of quantifiable 3D images. Although increased tissue penetrance and
quantification of labeled compound within a 3D space maybe advantageous with this transillumination modality, we found that some scans at early time points did not reconstruct to produce images. This difficulty was most apparent with the control antibody and the ventrodorsal positioning. Perhaps under these circumstances there was insufficient contrast to generate a reconstruction. In addition, the skin (Figure 3) is a common site of antibody retention. High levels of fluorescence at the animal’s surface could affect transillumination image readouts.19 However, when 3D images could be reconstructed, there was good correlation of fluorescence data from the liver in vivo and ex vivo. Ex vivo analyses of tissues are performed for comparison with in vivo results and, in some cases, may provide increased sensitivity, because the signal is not attenuated by other tissues in the light path. In addition, tissue borders can readily be defined by using the superimposed normal light reflectance image, whereas regions of interest are only approximated in FMT scans,30 thus increasing variability. Although we were able to detect strong fluorescent signals from the liver by using epifluorescence, deeper or weaker
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Fluor 750). In light of these findings and in consideration of the limitations we have described, we find that optical imaging provides a convenient relatively inexpensive modality for assessing the biodistribution of biotherapeutics in mouse models.
Acknowledgments
The authors thank Ryan Fleming, Binyam Bezabeh, and Chansghou Gao (ADPE MedImmune) for their assistance in developing the bispecific proteins described here. Rakesh Dixit provided valuable editorial suggestions.
References
Figure 8. Ex vivo organ surface fluorescence from mice injected 48 h previously with labeled antibodies. Fluorescence (mean ± 1 SD) from tissues of 4 mice are plotted; bracket indicates a significant difference (P < 0.05) between values.
Figure 9. Plasma antibody concentrations from the mice imaged. Two studies are represented: one in tumor-bearing mice (xenograft +) that received 3 mg/kg of labeled antibody, and the other in mice without tumors (xenograft –) that received 4 mg/kg of labeled antibody. Data are shown as the mean ± 1 SD of 4 plasma samples.
near-infrared signals are unlikely to be detected at the animal’s surface. Likewise, the average fluorescent intensity values of large, highly heterogeneous tumors that predominately bind labeled antibody at their periphery12 may differ when IVIS Spectrum images (2D, surface) are compared with FMT scans (3D, volumetric). Although a direct comparison with radioisotope labeling and PET imaging would have been helpful, this modality was not readily available, and running an externally funded project was costprohibitive. However, using optical imaging, we found that the liver was a major organ of retention for our bispecific antibody and the likely cause of its poor pharmacokinetics. The study also suggested that the kidney may be a site of target specific or nonspecific retention. Furthermore, we have shown that the choice of fluorescent label can affect tissue signal retention. Although additional time and resources may be necessary, performing biodistribution analyses with 2 different labels is very informative. In addition, we found that tissue depots were more readily identified by using fluorophores with prolonged tissue retention (for example, Alexa
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