scholarly journals A paired-agent fluorescent molecular imaging strategy for quantifying antibody drug target engagement in in vivo window chamber xenograft models

2020 ◽  
Author(s):  
Elif Kayaalp-Nalbant ◽  
Cody Rounds ◽  
Negar Sadeghipour ◽  
Boyu Meng ◽  
Margaret R. Folaron ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Drug Target ◽  
Target Engagement ◽  
Imaging Strategy ◽  
Xenograft Models ◽  
Window Chamber ◽  
Antibody Drug

scholarly journals Multi-Modal Multi-Spectral Intravital Microscopic Imaging of Signaling Dynamics in Real-Time during Tumor–Immune Interactions

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 499
Author(s):  
Tracy W. Liu ◽  
Seth T. Gammon ◽  
David Piwnica-Worms
Keyword(s):  
Molecular Imaging ◽  
Single Cell ◽  
Real Time ◽  
Bioluminescence Imaging ◽  
Emission Spectra ◽  
Ccd Camera ◽  
Microscopic Imaging ◽  
Signaling Dynamics ◽  
Window Chamber

Intravital microscopic imaging (IVM) allows for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo. Current IVM strategies primarily use fluorescence imaging; however, with the advances in bioluminescence imaging and the development of new bioluminescent reporters with expanded emission spectra, the applications for bioluminescence are extending to single cell imaging. Herein, we describe a molecular imaging window chamber platform that uniquely combines both bioluminescent and fluorescent genetically encoded reporters, as well as exogenous reporters, providing a powerful multi-plex strategy to study molecular and cellular processes in real-time in intact living systems at single cell resolution all in one system. We demonstrate that our molecular imaging window chamber platform is capable of imaging signaling dynamics in real-time at cellular resolution during tumor progression. Importantly, we expand the utility of IVM by modifying an off-the-shelf commercial system with the addition of bioluminescence imaging achieved by the addition of a CCD camera and demonstrate high quality imaging within the reaches of any biology laboratory.

scholarly journals Proof of concept of a multimodal intravital molecular imaging system for tumour transpathology investigation

2021 ◽  
Author(s):  
Zhen Liu ◽  
Tao Cheng ◽  
Stephan Düwel ◽  
Ziying Jian ◽  
Geoffrey J. Topping ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Imaging System ◽  
Ex Vivo ◽  
Fiducial Markers ◽  
Proof Of Concept ◽  
Registration Accuracy ◽  
Microscopic Imaging ◽  
Window Chamber

Abstract Background Transpathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation. Methods The proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin–eosin (HE) stained for comparison with multimodal in vivo imaging. Results The final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology. Conclusions The established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

Use of an antibody-drug conjugate targeting tissue factor to induce complete tumor regression in xenograft models with heterogeneous target expression.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3066-3066 ◽  
Author(s):  
Esther CW Breij ◽  
David Satijn ◽  
Sandra Verploegen ◽  
Bart de Goeij ◽  
Danita Schuurhuis ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tumor Cells ◽  
Tumor Regression ◽  
Antibody Drug Conjugate ◽  
Xenograft Models ◽  
Anti Tumor Activity ◽  
Complete Tumor ◽  
Antibody Drug

3066 Background: Tissue factor (TF) is the main initiator of coagulation, that starts when circulating factor VII(a) (FVII(a)) binds membrane bound TF. In addition, the TF:FVIIa complex can initiate a pro-angiogenic signaling pathway by activation of PAR-2. TF is aberrantly expressed in many solid tumors, and expression has been associated with poor prognosis. TF-011-vcMMAE, an antibody-drug conjugate (ADC) under development for the treatment of solid tumors, is composed of a human TF specific antibody (TF-011), a proteaseEcleavable valine-citrulline (vc) linker and the microtubule disrupting agent monomethyl auristatin E (MMAE). Methods: TF-011 and TF-011-vcMMAE were functionally characterized using in vitro assays. In vivo anti-tumor activity of TF-011-vcMMAE was assessed in human biopsy derived xenograft models, which genetically and histologically resemble human tumors. TF expression in xenografts was assessed using immunohistochemistry. Results: TF-011 inhibited TF:FVIIa induced intracellular signaling and efficiently killed tumor cells by antibody dependent cell-mediated cytoxicity in vitro, but showed only minor inhibition of TF procoagulant activity. TF-011 was rapidly internalized and targeted to the lysosomes, a prerequisite for intracellular MMAE release and subsequent tumor cell killing by the ADC. Indeed, TF-011-vcMMAE efficiently and specifically killed TF-positive tumors in vitro and in vivo. Importantly, TF-011-vcMMAE showed excellent anti-tumor activity in human biopsyEderived xenograft models derived from bladder, lung, pancreas, prostate, ovarian and cervical cancer (n=7). TF expression in these models was heterogeneous, ranging from 25-100% of tumor cells. Complete tumor regression was observed in all models, including cervical and ovarian cancer xenografts that showed only 25-50% TF positive tumor cells. Conclusions: TF-011-vcMMAE is a promising new ADC with potent anti-tumor activity in xenograft models that represent the heterogeneity of human tumors, including heterogeneous TF expression. The functional characteristics of TF-011-vcMMAE allow efficient tumor targeting, with minimal impact on coagulation.

scholarly journals An engineered chimeric toxin that cleaves activated mutant and wild-type RAS inhibits tumor growth

2020 ◽  
Vol 117 (29) ◽  
pp. 16938-16948 ◽  
Author(s):  
Vania Vidimar ◽  
Greg L. Beilhartz ◽  
Minyoung Park ◽  
Marco Biancucci ◽  
Matthew B. Kieffer ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Protein Delivery ◽  
Tumor Burden ◽  
Target Engagement ◽  
Wild Type ◽  
Downstream Signaling ◽  
Oncogenic Ras ◽  
Broad Array ◽  
Xenograft Models

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1–specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutantRAS. Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.

scholarly journals 783 SGN-PDL1V, a novel, investigational PD-L1-directed antibody-drug conjugate for the treatment of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A818-A818
Author(s):  
Byron Kwan ◽  
Megan Ramirez ◽  
Steven Jin ◽  
Changpu Yu ◽  
Serena Wo ◽  
...  
Keyword(s):  
Immune Checkpoint ◽  
Safety Profile ◽  
Cell Lymphoma ◽  
Brentuximab Vedotin ◽  
Programmed Death ◽  
Xenograft Models ◽  
Human Primate ◽  
Antibody Drug

BackgroundPD-1/PD-L1 immune checkpoint inhibitors have transformed oncology, but a significant unmet need persists for patients with relapsed/refractory tumors following PD-1/PD-L1 treatment. PD-L1 is expressed in patients across a broad spectrum of tumor types and displays limited normal tissue expression, highlighting the potential of PD-L1 as a target for antibody-drug conjugates (ADCs) in addition to its role as an immune checkpoint. SGN-PDL1V is a PD-L1-directed ADC currently under preclinical investigation, which is comprised of an anti-PD-L1 antibody conjugated to the vedotin drug-linker. The vedotin drug-linker, consists of the microtubule disrupting agent, monomethyl auristatin E (MMAE), and a protease-cleavable peptide linker, which has been clinically validated in multiple ADC programs including brentuximab vedotin, enfortumab vedotin and polatuzumab vedotin.1–3 The proposed SGN-PDL1V primary mechanism of action is direct cytotoxicity against PD-L1-expressing malignant cells through delivery of the MMAE payload. Additionally, MMAE induces immunogenic cell death, leading to subsequent immune activation in the tumor microenvironment.4 Here, we characterize the preclinical activity and tolerability of SGN-PDL1V.MethodsSGN-PDL1V cytotoxicity was evaluated using PD-L1 expressing tumor cell lines in vitro and xenograft tumor models in vivo. Inhibition of the PD-1/PD-L1 immune checkpoint was assessed in a luminescent reporter system in vitro and a syngeneic tumor model in vivo. The tolerability and safety profile of SGN-PDL1V was determined in a non-human primate study.ResultsIn vitro, SGN-PDL1V demonstrated internalization and potent cytotoxic activity against PD-L1 expressing tumor cells. In vivo, SGN-PDL1V achieved tumor regressions in multiple tumor xenograft models at doses as low as 1 mg/kg when dosed weekly for a total of three doses. This activity was observed in immunocompromised mice, which lack responses to PD-1/PD-L1 inhibition. Notably, activity was observed even in xenograft models with low, heterogeneous PD-L1 expression, supporting the possibility to treat patients across a wide range of PD-L1 expression levels. Additionally, SGN-PDL1V exhibited potential to inhibit the PD-1/PD-L1 checkpoint in vitro and in vivo. The tolerability and safety profile of SGN-PDL1V were assessed in a non-human primate study and found to be comparable to other FDA-approved vedotin ADCs.ConclusionsSGN-PDL1V is a promising PD-L1 directed ADC with a unique cytotoxic mechanism of action among other PD-L1-targeted therapeutics. SGN-PDL1V demonstrated robust activity in multiple preclinical models and comparable tolerability and safety profile to other vedotin ADCs in non-human primates. Collectively, these data support further evaluation of SGN-PDL1V in a planned, first-in-human Phase 1 study.AcknowledgementsWe would like to thank Kerry Klussman for assay support and Jamie Mitchell for conjugation support.Trial RegistrationN/AReferencesSenter PD, Sievers EL. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat Biotechnol 2012;30(7):631–7. Epub 2012/07/12. doi: 10.1038/nbt.2289. PubMed PMID: 22781692.Rosenberg JE, O'Donnell PH, Balar AV, McGregor BA, Heath EI, Yu EY, et al. Pivotal trial of enfortumab vedotin in urothelial carcinoma after platinum and anti-programmed death 1/Programmed death ligand 1 therapy. J Clin Oncol 2019;37(29):2592–600. Epub 2019/07/30. doi: 10.1200/JCO.19.01140. PubMed PMID: 31356140; PubMed Central PMCID: PMC6784850.Tilly H, Morschhauser F, Bartlett NL, Mehta A, Salles G, Haioun C, et al. Polatuzumab vedotin in combination with immunochemotherapy in patients with previously untreated diffuse large B-cell lymphoma: an open-label, non-randomised, phase 1b-2 study. Lancet Oncol 2019;20(7):998–1010. Epub 2019/05/19. doi: 10.1016/S1470-2045(19)30091-9. PubMed PMID: 31101489.Klussman K, Tenn E, Higgins S, Mazahreh R, Snead K, Hamilton J, Grogan B, Sigurjonsson J, Cao A, Gardai S, Liu B. 618 Vedotin ADCs induce ER stress and elicit hallmarks of ICD across multiple cancer indications. J Immunother Cancer 2020;8(Suppl 3):A372. DOI:10.1136/jitc-2020-SITC2020.0618.Ethics ApprovalAll animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen or the external testing facility that conducted the studies.

scholarly journals Quantitating drug-target engagement in single cells in vitro and in vivo

2016 ◽  
Vol 13 (2) ◽  
pp. 168-173 ◽  
Author(s):  
J Matthew Dubach ◽  
Eunha Kim ◽  
Katherine Yang ◽  
Michael Cuccarese ◽  
Randy J Giedt ◽  
...  
Keyword(s):  
Drug Target ◽  
Single Cells ◽  
Target Engagement

Abstract 804: Noninvasive Integrated SPECT/CT Molecular Imaging of Activated Factor XIII Activity in Thrombosis

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Farouc A Jaffer ◽  
Jose L Figueiredo ◽  
Gregory Wojtkiewicz ◽  
Hanwen Zhang ◽  
Purvish Patel ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Human Plasma ◽  
Half Life ◽  
Factor Xiii ◽  
Control Agent ◽  
Isotropic Resolution ◽  
Novel Approach ◽  
Imaging Strategy

Background : Activated factor XIII (FXIIIa) is a blood transglutaminase that mediates fibrinolytic resistance and is a hallmark of acute thrombi. Noninvasive molecular imaging of FXIIIa may offer a novel approach to identify acute thrombi and to gauge fibrinolytic resistance in vivo. Here we developed and validated a FXIIIa thrombosis imaging strategy using noninvasive integrated SPECT/CT. Methods: A FXIIIa-targeted peptide agent (F13) was synthesized using NQEQVSPLTLLK chelated to DOTA and then labeled with 111 InCl 3 . A control agent (C13, 111 In-NAEQVSPLTLLK) was analogously synthesized. In vitro validation of the F13 agent was performed in human plasma clots. Next, the in vivo blood-half life of F13 was determined in mice (n=4). In vivo thrombosis studies (n=15 mice) were then performed using 10% ferric chloride jugular venous thrombi aged 1 hour or 16 hours. Mice were intravenously injected with 200 μCi of F13 or C13. After 4 hours, mice underwent integrated CT angiography (72 μm isotropic resolution) and SPECT imaging (32 minute acquisition). In situ thrombi were then resected for radioactivity and weight measurements. Results : Human plasma clots incubated with F13 showed 280–740% greater counts per minute (CPM) than controls (p<0.01). F13 binding was dose-dependent and >90% inhibited by pretreatment with iodoacetamide, an alkylating agent. The blood half-life of F13 was calculated to be 16 minutes. In one hour thrombi, in vivo SPECT/CT imaging revealed strong focal F13 SPECT signal in the co-registered ipsilateral venous thrombi but not the contralateral normal jugular vein. One hour thrombi in the F13 group had 15-fold greater radioactivity than the C13 group (4.6±3.6% vs. 0.3±0.2% injected dose per gram tissue, IDGT, p<0.01). Compared to 1 hour thrombi, 16 hour old thrombi had 4-fold less F13 radioactivity (1.1%±0.1% IDGT, p<0.05). Conclusions : Blood transglutaminase FXIIIa can be noninvasively detected using a FXIIIa-sensitive and specific imaging agent for integrated SPECT/CT. The current in vivo results further validate that activated factor XIII is a hallmark of acute thrombi and declines in activity over time. This clinically translatable imaging strategy could permit visualization of FXIIIa in patients with thrombotic syndromes.

Analytical considerations for quantitative LC–MS strategies for measuring antibody drug–target engagement in tissues

Bioanalysis ◽  
2017 ◽  
Vol 9 (23) ◽  
pp. 1845-1847 ◽  
Author(s):  
Petia Shipkova ◽  
Eugene Ciccimaro ◽  
Timothy V Olah
Keyword(s):  
Drug Target ◽  
Target Engagement ◽  
Antibody Drug
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