In Vivo Imaging With Two-Photon Microscope For Assessing Tumor Selective Binding Of Anti-CD137 Switch Antibody

STA551, a novel anti-CD137 switch antibody, binds to CD137 in an extracellular ATP (exATP) concentration dependent manner. Although STA551 was assumed to show higher target binding in tumor than normal tissues, quantitative detection of the target binding of switch antibody in vivo is technically challenging. In this study, we investigated the target binding of STA551 in vivo using intravital imaging with two-photon microscopy. Tumor-bearing human CD137 knock-in mice were intravenously administered 1 mg/kg of fluorescent-labeled antibodies at day 0 and 3. Flow cytometry analysis of antibody-binding cells and intravital imaging using two-photon microscopy was conducted at day4. Higher CD137 expression in tumor than spleen was detected by flow cytometry analysis, and T cells and NK cells were major CD137 expressing cells. In the intravital imaging experiment, conventional and switch anti-CD137 antibody showed binding in tumor. However, in spleen, the fluorescence of switch antibody was much weaker than conventional anti-CD137 antibody and comparable with isotype control. In conclusion, we could assess switch antibody biodistribution in vivo through intravital imaging with two-photon microscopy. These results suggested that the tumor selective binding of STA551 leads to a wide therapeutic window and potent antitumor efficacy without systemic immune activation.

Two Stage Intravital Imaging Mouse Model to Assess Venous Thromboembolism in Sickle Cell Disease

Sickle cell disease (SCD) patients have an increased risk of venous thromboembolism (VTE). Population-based studies demonstrated that VTE has a cumulative incidence rate of approximately 25% in adult SCD patients and is associated with higher risk of mortality. VTE in SCD is most commonly manifested as deep vein thrombosis (DVT) with associated pulmonary embolism (PE). Although autopsy studies have regularly discovered pulmonary thromboembolic lesions in SCD patients, the pathophysiology of VTE in SCD remains largely unknown mostly due to the lack of relevant animal VTE model. Understanding the mechanisms that promote VTE in SCD is imperative to identify its prevention and treatment measures. To elucidate the cellular, molecular and biophysical mechanisms of VTE in SCD we developed an innovative two stage intravital imaging analysis experimental model in SCD mice. DVT in SCD mice was induced by surgical ligation of femoral vein. Venous thrombus formation was visualized using intravital multi-photon-excitation (MPE) microscopy. Venous thrombus took the form of a large mass of elliptical shape which extended in the long-axis direction of the femoral vein. It was composed of fibrin, erythrocytes and sparse platelets. Over time, thrombus was infiltrated by migrating neutrophils. To trigger acute PE, femoral vein ligation was removed and the venous thrombus was observed to spontaneously detach and travel to the SCD mouse lung. This method allowed real time visualization of acute PE in vivo using MPE microscopy of intact lung in live breathing mice. Acute PE involved embolization of the pulmonary arterioles and the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries. The embolization of arteriolar circulation led to loss of blood flow in the arterioles and the down-stream capillaries. Herein we introduce an intravital microscopy approach to probe VTE in SCD live mouse. Our model has potential application in investigating the molecular determinants of VTE associated with SCD as well as evaluating efficacy of new antithrombotic drugs. Disclosures Sundd: Bayer: Research Funding; CSL Behring Inc: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Direct Intravital Imaging of the Bone Marrow and Splenic Hematopoietic Niches in Individual Mice to Define the Early Engraftment Kinetics Following HSC-Transplant

Hematopoietic stem cells (HSCs) give rise to and maintain the entire hematopoietic system for the life of an organism. This remarkable feat has established HSC transplant as an indispensable tool for treating a variety of hematological disorders. Yet the initial steps of homing, engraftment, and clonal expansion, which lead to eventual long-term hematopoietic recovery after HSC transplant, remain incompletely characterized. Given the determinative effect that early HSC activity has on transplant outcomes, a more complete understanding of initial engraftment dynamics is imperative for bettering HSC therapies. Preliminary studies aimed at functional characterization of classic HSC and hematopoietic stem and progenitor cell (HSPC) populations-namely the CD150 +CD48 -Sca-1 +c-Kit +Lin - (SLAM SKL) and Sca-1 +c-Kit +Lin - (SKL) populations, respectively-revealed that these populations exhibit disparate early engraftment dynamics. Using previously developed intravital imaging techniques, we were able to partially characterize early HSC and HSPC engraftment dynamics in mice competitively transplanted with GFP + SLAM SKL and DsRed + SKL cells. The SKL population was found to primarily engraft in the bone marrow, completely recapitulating engraftment behavior of transplanted whole bone marrow. In contrast, the more purified SLAM SKL population engrafted poorly in the marrow space and instead preferentially engrafted in the spleen, where it produced the majority of donor-derived blood at early stages (7 days) after competitive transplant into lethally irradiated mice. However, by 14 days post-transplant, SLAM SKL-derived cells migrated from the spleen to repopulate the majority of bone marrow space. These results reflect the dynamic nature of hematopoietic recovery in a myeloablative model and highlight the need for in vivo imaging techniques to fully understand hematopoietic reconstitution by the SLAM SKL population. In order to further dissect the interactive processes of bone marrow hematopoiesis and splenic extramedullary hematopoiesis, we have developed a novel, multi-organ intravital imaging technique that allows for simultaneous analysis of defined hematopoietic compartments in a single animal. Our multimodal imaging approach combines direct visualization of fluorescently labeled hematopoietic cells in the spleen via our recently developed spleen window, with concomitant observation of hematopoietic cells in tibia marrow environment. Our spleen window is a specially engineered biocompatible ring with an affixed coverslip to allow for direct, non-invasive microscopic visualization of labeled hematopoietic cells in the spleen. The spleen window can be installed with the tibia window in an individual mouse. Multimodal mice can be visualized repeatedly over a minimum of 7 days post-HSC transplant to follow individual cell behaviors within the living recipient. Preliminary results from competitive repopulation assays utilizing our multimodal imaging approach suggest that the SLAM SKL population is an active one that confers rapid hematopoietic recovery in lethally irradiated recipients primarily from extramedullary hematopoiesis in the spleen (CFU-S). The results of ongoing work characterizing the active use of the splenic and marrow niches will be presented. Disclosures Cogle: Celgene: Membership on an entity's Board of Directors or advisory committees; Aptevo therapeutics: Research Funding.