Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17-34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.
Age-related Macular Degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly in developed countries. Neovascular/exudative (wet) AMD is the aggressive form of AMD and can involve choroidal neovascularization and vascular leakage. Anti-vascular endothelial growth factor (anti-VEGF) medications have significantly improved treatment of wet-AMD. However, only approximately 40% of patients obtain full benefit from anti-VEGF therapy and the medications are given by intravitreal injection. Axitinib, a small molecule multi-receptor tyrosine kinase inhibitor used for the treatment of advanced renal cell carcinoma, is taken orally and inhibits VEGF activity by blocking VEGF receptors. Axitinib also has the advantage of blocking platelet derived growth factor (PDGF) receptors which play a role in neovascularization. Using in vitro human retinal microvascular endothelial cells (HRMVECs), human brain vascular pericytes (HBVRs), 3D co-culture vessel sprout assay, and in vivo laser induced rat choroidal neovascularization (CNV) models, the effect of axitinib on neovascularization was evaluated. Axitinib inhibited neovascularization better than anti-VEGF and/or anti-hPDGF-B mAb in the in vitro models demonstrating that combined inhibition of both VEGF and PDGF pathways may be synergistic in treating wet-AMD. Additionally, axitinib showed good efficacy at a low dose (0.875 mg/day) in laser-induced CNV model in rats. In conclusion our data shows that axitinib, an inhibitor of VEGF and PDGF-B pathways may be useful in ameliorating wet-AMD therapy.
P-cadherin-LP-DART is a bispecific antibody targeting P-cadherin expressed on the tumor cells and CD3 on the T-cells. Previously we demonstrated the development and efficacy of P-cadherin-LP-DART in in vitro and in vivo models. Here, we evaluated the three pillars: exposure, targeting specificity and pharmacodynamic modulation for P-cadherin-LP-DART using fluorescence molecular tomography (FMT). Bispecific antibodies and T-cells were conjugated with a near-infrared fluorophores: VivoTag ® 680XL (VT680) and CellVue ® NIR815 (CV815), respectively. In vitro binding and cytotoxic T-lymphocyte assay demonstrated that P-cadherin-LP-DART significantly retained its properties after VT680 conjugation. In vivo FMT imaging was performed to determine the bispecific biodistribution and T-cell trafficking in HCT-116 xenograft model. Peak tumor exposure (2.71%ID) was observed at 96 hr postinjection with measurable quantity even at 240 hr (1.46%ID) (Pillar 1). P-cadherin-LP-DART accumulation in tumor was 20-25 fold higher compared to Control-LP-DART demonstrating the targeting specificity (Pillar 2). Imaging after engraftment of CV815 labeled T-cells showed P-cadherin-LP-DART mediated T-cell trafficking in tumors (Pillar 3). This study harnessed the multichannel capability of FMT and demonstrated the targeting of drug and trafficking of T cells to tumors, simultaneously. Our results show the impact of molecular imaging in demonstrating three pillars of pharmacology, longitudinally and non-invasively.
<div>Abstract<p>Understanding a drug's whole-body biodistribution and tumor targeting can provide important information regarding efficacy, safety, and dosing parameters. Current methods to evaluate biodistribution include <i>in vivo</i> imaging technologies like positron electron tomography and single-photon emission computed tomography or <i>ex vivo</i> quantitation of drug concentrations in tissues using autoradiography and standard biochemical assays. These methods use radioactive compounds or are cumbersome and do not give whole-body information. Here, for the first time, we show the utility of fluorescence molecular tomography (FMT) imaging to determine the biodistribution and targeting of an antibody–drug conjugate (ADC). An anti–5T4-antibody (5T4-Ab) and 5T4-ADC were conjugated with a near-infrared (NIR) fluorophore VivoTag 680XL (VT680). Both conjugated compounds were stable as determined by SEC-HPLC and plasma stability studies. Flow cytometry and fluorescence microscopy studies showed that VT680-conjugated 5T4-ADC specifically bound 5T4-expressing cells <i>in vitro</i> and also exhibited a similar cytotoxicity profile as the unconjugated 5T4-ADC. <i>In vivo</i> biodistribution and tumor targeting in an H1975 subcutaneous xenograft model demonstrated no significant differences between accumulation of VT680-conjugated 5T4-Ab or 5T4-ADC in either normal tissues or tumor. In addition, quantitation of heart signal from FMT imaging showed good correlation with the plasma pharmacokinetic profile suggesting that it (heart FMT imaging) may be a surrogate for plasma drug clearance. These results demonstrate that conjugation of VT680 to 5T4-Ab or 5T4-ADC does not change the behavior of native biologic, and FMT imaging can be a useful tool to understand biodistribution and tumor-targeting kinetics of antibodies, ADCs, and other biologics. <i>Mol Cancer Ther; 15(10); 2530–40. ©2016 AACR</i>.</p></div>
<p>Supplemental Figure 1: Standard curve of VT680 showing the 3-D quantitation and linearity by FMT imaging; Supplemental Figure 2: Evaluation of VT680 conjugated 5T4-Ab and 5T4-ADC; Supplemental Figure 3: Determination of antibody binding capacity (ABC) and receptor saturation in H1975 and MDA-MB-468 cells by flow cytometry; Supplemental Figure 4: Ex vivo FMT imaging showing the accumulation of 5T4-Ab-VT680, 5T4-ADC-VT680, Isotype control-Ab-VT680 and VT680 alone 48 h post-injection in tumor (A) and liver (B).</p>
Introduction: Currently bio-distribution of biologic drugs is evaluated by PET imaging, autoradioraphy using radio-labeled molecules or ex vivo methods. Advances in optical probes and non-invasive imaging technologies have given us an opportunity to conduct such studies without the use of radio-labeled materials or by traditional pharmacokinetic (PK) studies. 5T4 (also known as TPBG or oncofetal antigen) is a transmembrane glycoprotein expressed highly on tumor-initiating cells. Anti-5T4-mcMMAF used in these studies is an anti-5T4-antibody drug conjugate (ADC) that reacts to human, cyno and marmoset orthologs of 5T4. Previously, we showed the efficacy of anti-5T4-ADC in pre-clinical models (Sapra et al.,). In this study we show the utility of Fluorescence Molecular Tomography (FMT) imaging in bio-distribution studies with this ADC using a H1975 non-small cell lung cancer (NSCLC) xenograft model. Methods: For the H1975 xenograft model, five million cells in 50% matrigel were injected into the subcutaneous flanks of the female nu/nu mice, and a biodistribution study was initiated when the tumors reached ∼500 mm3. The anti-5T4-ADC and a control ADC (non-binding) were conjugated with near-IR fluorophore VivoTag680XL. The labeling efficiency and quality was determined by Nanodrop-8000 spectrophotometer and binding assays. FMT imaging was performed longitudinally at 5min, 6hr, 24hr, 48hr, 96hr and 240hrs post injection of labeled ADCs. Ex vivo imaging of organs was performed at intermittent time points after perfusing with PBS. Data was analyzed using TrueQuant software. Plasma and tissues were collected at various time points and analyzed by GyrolabTM workstation and LCMS methods. Results: VivoTag680XL conjugation was efficient and achieved degree of labeling between 2-3. Three-dimensional quantitative analysis of FMT data showed significant specific targeting of anti-5T4-ADC to the tumors (ex vivo and in vivo comparison), relative to the control non-binding ADC. The peak accumulation in tumor was observed at 48hrs post injection and the concentration decreased in later time points. Liver was the major organ for the non-specific accumulation of these antibodies/ADCs, followed by kidneys and lung. Both 5T4-ADC and Control-ADC showed similar accumulation at 48hrs and 96hrs in liver, whereas it decreased significantly at 240hrs. The FMT imaging data was comparative and correlated with the traditional plasma PK profile data. Conclusion: These results show that anti-5T4-ADC targets the tumor better than non-binding ADC. This study also shows the utility of FMT in bio-distribution studies of biologics. Since the fluorophore can be conjugated to any protein/peptide, this novel approach can become a platform technology in conducting biodistribution studies of all biologic drugs. Citation Format: Anand Giddabasappa, Rand Norberg, Mauricio Leal, David Paterson, Kush Lalwani, Ted Levkoff, Stella Rapa, Puja Sapra, Michael Ritche, Joann Wentland, Brian Rago, Jeetendra Eswaraka. Whole-body bio-distribution of anti-5T4-mcMMAF (anti-5T4-ADC) using fluorescence molecular tomography (FMT) imaging in a non-small cell lung cancer mice model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4293. doi:10.1158/1538-7445.AM2014-4293
Introduction: Previously we have shown the utility of Fluorescent Molecular Tomography (FMT) imaging in evaluating bio-distribution of biologics. P-cadherin LP-DART is a bi-specific Dual Affinity Re-Targeting (DART®) molecule targeting CD3 expressed on T-cells and P-cadherin expressed on tumors. In this study we evaluated the bio-distribution and tumor targeting of P-cadherin LP-DART using FMT imaging in a colorectal xenograft model. Methods: NSG or athymic nude mice with subcutaneous HCT-116 xenografts were used. Studies that included engraftment of T-cells received either PBMNCs or T-cells isolated from healthy human volunteers. Bio-distribution studies were initiated when the tumors reached 300-500 mm3. P-cadherin LP-DART or a negative control-DART (non-targeted domain x CD3 binding domain) was conjugated with a near-infrared fluorophore VivoTag680XL (VT680), and the labeling efficiency was determined by spectrophotometer. T-cells used in trafficking studies were labeled with CellVue815. Cell surface P-cadherin expression and P-cadherin LP-DART binding was determined by flow cytometry. T-cell activity was measured with cytotoxic T-lymphocyte (CTL) assays. FMT imaging was performed longitudinally post injection of labeled bi-specifics. Data was analyzed using TrueQuant software. Plasma and tissues were collected for PK analysis by ELISA or histology. Results: VT680 conjugation to P-cadherin LP-DART did not significantly affect the binding to P-cadherin, whereas CD3 binding was decreased. In vivo FMT imaging revealed high levels of P-cadherin LP-DART accumulation in the tumors. The in vivo kinetics revealed that the peak accumulation in tumors was 96hrs post-injection. At 240hrs post-injection, there was still measurable P-cadherin LP-DART detected in tumors. Ex vivo imaging showed 20-25 fold increase in accumulation of P-cadherin LP-DART compared to negative control DART. Comparison of P-cadherin LP-DART accumulation between PBMNC engrafted and non-engrafted model showed no significant difference in quantity or kinetics. There was no significant difference in the kinetics of elimination in the whole-body, heart or liver between P-cadherin LP-DART or negative control. Ex vivo comparison of accumulation in various organs showed no difference between P-cadherin LP-DART or negative control. Cell trafficking studies with CellVue labeled T-cells showed the co-localization of T-cells and P-cadherin LP-DART in tumors. Conclusion: FMT imaging showed that P-cadherin LP-DART specifically targeted HCT-116 tumors. Cell trafficking studies showed that engrafted T-cells accumulated in tumors. This study shows the utility of FMT in bio-distribution studies of biologics and in vivo cell trafficking. Citation Format: Anand Giddabasappa, Vijay Gupta, Timothy S. Fisher, John David, Norberg Rand, Allison Rohner, Justin Cohen, Tracey Clark, Nahor Haddish-Berhane, Adam Root, Chad May. Bio-distribution and tumor targeting of a P-cadherin x CD3 bi-specific redirected T-cell molecule using fluorescence molecular tomography imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2015-5111
<div>Abstract<p>Understanding a drug's whole-body biodistribution and tumor targeting can provide important information regarding efficacy, safety, and dosing parameters. Current methods to evaluate biodistribution include <i>in vivo</i> imaging technologies like positron electron tomography and single-photon emission computed tomography or <i>ex vivo</i> quantitation of drug concentrations in tissues using autoradiography and standard biochemical assays. These methods use radioactive compounds or are cumbersome and do not give whole-body information. Here, for the first time, we show the utility of fluorescence molecular tomography (FMT) imaging to determine the biodistribution and targeting of an antibody–drug conjugate (ADC). An anti–5T4-antibody (5T4-Ab) and 5T4-ADC were conjugated with a near-infrared (NIR) fluorophore VivoTag 680XL (VT680). Both conjugated compounds were stable as determined by SEC-HPLC and plasma stability studies. Flow cytometry and fluorescence microscopy studies showed that VT680-conjugated 5T4-ADC specifically bound 5T4-expressing cells <i>in vitro</i> and also exhibited a similar cytotoxicity profile as the unconjugated 5T4-ADC. <i>In vivo</i> biodistribution and tumor targeting in an H1975 subcutaneous xenograft model demonstrated no significant differences between accumulation of VT680-conjugated 5T4-Ab or 5T4-ADC in either normal tissues or tumor. In addition, quantitation of heart signal from FMT imaging showed good correlation with the plasma pharmacokinetic profile suggesting that it (heart FMT imaging) may be a surrogate for plasma drug clearance. These results demonstrate that conjugation of VT680 to 5T4-Ab or 5T4-ADC does not change the behavior of native biologic, and FMT imaging can be a useful tool to understand biodistribution and tumor-targeting kinetics of antibodies, ADCs, and other biologics. <i>Mol Cancer Ther; 15(10); 2530–40. ©2016 AACR</i>.</p></div>
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