Frederick O. Cope

Discovery of anticancer agents of diverse natural origin.

A collaborative multidisciplinary research project is described in which new natural product anticancer drug leads are obtained from a diverse group of organisms, constituted by tropical plants, aquatic cyanobacteria, and filamentous fungi. Information is provided on how these organisms are collected and processed. The types of bioassays are indicated in which crude extracts of these acquisitions are tested. Progress made in the isolation of lead bioactive secondary metabolites from three tropical plants is discussed.

A Phase 2 Study of 99mTc-Tilmanocept in the Detection of Sentinel Lymph Nodes in Melanoma and Breast Cancer

BackgroundSeveral 99mTc-labeled agents that are not approved by the U.S. Food and Drug Administration are used for lymphatic mapping. A new low-molecular-weight mannose receptor–based, reticuloendothelial cell-directed, 99mTc-labeled lymphatic imaging agent, 99mTc-tilmanocept, was used for lymphatic mapping of sentinel lymph nodes (SLNs) from patients with primary breast cancer or melanoma malignancies. This novel molecular species provides the basis for potentially enhanced SLN mapping reliability.MethodsIn a prospectively planned, open-label phase 2 clinical study, 99mTc-tilmanocept was injected into breast cancer and cutaneous melanoma patients before intraoperative lymphatic mapping. Injection technique, preoperative lymphoscintigraphy (LS), and intraoperative lymphatic mapping with a handheld gamma detection probe were performed by investigators per standard practice.ResultsSeventy-eight patients underwent 99mTc-tilmanocept injection and were evaluated (47 melanoma, 31 breast cancer). For those whom LS was performed (55 patients, 70.5%), a 99mTc-tilmanocept hot spot was identified in 94.5% of LS patients before surgery. Intraoperatively, 99mTc-tilmanocept identified at least one regional SLN in 75 (96.2%) of 78 patients: 46 (97.9%) of 47 in melanoma and 29 (93.5%) of 31 in breast cancer cases. Tissue specificity of 99mTc-tilmanocept for lymph nodes was 100%, displaying 95.1% mapping sensitivity by localizing in 173 of 182 nodes removed during surgery. The overall proportion of 99mTc-tilmanocept-identified nodes that contained metastatic disease was 13.7%. Five procedure-related serious adverse events occurred, none related to 99mTc-tilmanocept.ConclusionsOur results demonstrate the safety and efficacy of 99mTc-tilmanocept for use in intraoperative lymphatic mapping. The high intraoperative localization and lymph node specificity of 99mTc-tilmanocept and the identification of metastatic disease within the nodes suggest SLNs are effectively identified by this novel mannose receptor–targeted molecule.

γ-Tilmanocept, a New Radiopharmaceutical Tracer for Cancer Sentinel Lymph Nodes, Binds to the Mannose Receptor (CD206)

γ-Tilmanocept (99mTc-labeled-tilmanocept or [99mTc]-tilmanocept) is the first mannose-containing, receptor-directed, radiolabeled tracer for the highly sensitive imaging of sentinel lymph nodes in solid tumor staging. To elucidate the mannose-binding receptor that retains tilmanocept in this microenvironment, human macrophages were used that have high expression of the C-type lectin mannose receptor (MR; CD206). Cy3-labeled tilmanocept exhibited high specificity binding to macrophages that was nearly abolished in competitive inhibition experiments. Furthermore, Cy3-tilmanocept binding was markedly reduced on macrophages deficient in the MR by small interfering RNA treatment and was increased on MR-transfected HEK 293 cells. Finally, confocal microscopy revealed colocalization of Cy3-tilmanocept with the macrophage membrane MR and binding of labeled tilmanocept to MR+ cells (macrophages and/or dendritic cells) in human sentinel lymph node tissues. Together these data provide strong evidence that CD206 is a major binding receptor for γ-tilmanocept. Identification of CD206 as the γ-tilmanocept–binding receptor enables opportunities for designing receptor-targeted advanced imaging agents and therapeutics for cancer and other diseases.

Mannose receptor high, M2 dermal macrophages mediate nonhealing Leishmania major infection in a Th1 immune environment

The origin and functional specialization of dermal macrophages in cutaneous infections have been little studied. In this paper, we show that a strain of Leishmania major (L. major Seidman [LmSd]) that produces nonhealing cutaneous lesions in conventionally resistant C57BL/6 mice was more efficiently taken up by M2-polarized bone marrow (BM)–derived macrophages (BMDMs) in vitro and by mannose receptor (MR)hi dermal macrophages in vivo compared with a healing strain (L. major Friedlin V1). Both in steady and in T helper type 1 (Th1) cell–driven inflammatory states, the MRhi dermal macrophages showed M2 characteristics. The dermal macrophages were radio resistant and not replaced by monocytes or adult BM-derived cells during infection, but were locally maintained by IL-4 and IL-10. Notably, the favored infection of M2 BMDMs by LmSd in vitro was MR dependent, and genetic deletion of MR or selective depletion of MRhi dermal macrophages by anti–CSF-1 receptor antibody reversed the nonhealing phenotype. We conclude that embryonic-derived, MRhi dermal macrophages are permissive for parasite growth even in a strong Th1-immune environment, and the preferential infection of these cells plays a crucial role in the severity of cutaneous disease.

Corrigendum to the inextricable axis of targeted diagnostic imaging and therapy: An immunological natural history approach [Nucl Med Biol 43 (2016) 215–225]

Corrigendum to the inextricable axis of targeted diagnostic imaging and therapy: An immunological natural history approach [Nucl Med Biol 43 (2016) 215–225] Frederick O. Cope⁎, Bonnie Abbruzzese, James Sanders, Wendy Metz, Kristyn Sturms, David Ralph, Michael Blue, Jane Zhang, Paige Bracci, Wiam Bshara, Spencer Behr, Toby Maurer, Kenneth Williams, Joshua Walker, Allison Beverly, Brooke Blay, Anirudh Damughatla, Mark Larsen, Courtney Mountain, Erin Neylon, Kaeli Parcel, Kapil Raghuraman, Kevin Ricks, Lucas Rose, Akhilesh Sivakumar, Nicholas Streck, Bryan Wang, Christopher Wasco, Amifred Williams, Larry S. Schlesinger , Abul Azad, Murugesan V.S. Rajaram, Wael Jarjour, Nicholas Young, Thomas Rosol , Michael McGrath

Abstract 5026: Tumor associated targeting with Manocept: HIV associated Kaposi's sarcoma as a model system

Introduction Inflammation appears to play a critical role in tumor development of HIV-associated Kaposi9s sarcoma (KS). Specifically, emerging data show that KS tumor cells that co-express various macrophage (MO) antigens become resistant to current anti-viral therapies used to treat KS and AIDS. MOs also are a known source of KS tumor cell growth factors and substantial evidence suggests that tumor associated macrophages (TAMs) may represent a reservoir for HIV and its evolved retroviral variants. The MO pool driving these two pathological pathways share a common element rooted in the MOs, the CD206 human macrophage mannose receptor. Manocept, a molecular targeting agent, binds and enters MOs via pinocytosis of holo-CD206, providing a cell portal for the evaluation of Manocept as a MO and KS targeting agent. Methods Manocept was prepared as a synthetic molecule with a dextran backbone, and 12-20 mannose moieties. The reporter molecule (Cy3) was included as a fluorescent tracer for locating Manocept on and in MOs and KS tumor cells. Binding and localization Cy3-Manocept was assessed through intracellular visualization and flow cytometric quantitation of Cy3-Manocept binding and uptake using in vitro generation of monocyte-derived CD206+ MOs. The fresh HIV+ KS tumor tissue culture followed by immunofluorescence staining and confocal imaging was performed to confirm Cy3-Manocept uptake in KS tumor cells and TAMs. Results In vitro culture showed that Cy3-Manocept binds avidly to CD206 expressing MOs. Time course studies of increasing Cy3-Manocept concentrations confirmed continuous and ongoing uptake of Manocept into CD206+ MOs at 37°C. Fresh HIV+ KS tissue culture studies showed both Manocept uptake and CD206 staining of TAMs as well as KS tumor spindle cells (HHV8+ cells). Cy3-Manocept co-localized with CD206 in nearly all KS-associated cells expressing HHV8 and/or CD68 (Tissue MO marker), confirming that CD206 acts both as a target and Manocept concentrating receptor for TAMs and KS tumor cells. Conclusions Our results support the potential use of CD206 macrophage mannose receptor-targeted Manocept for imaging KS tumor cells, TAMs, and more importantly, for delivery of therapeutic/diagnostic agents capable of targeting all KS-associated cells including a potential MO reservoir for HIV. Citation Format: Rongzhen Zhang, Frederick O. Cope, Paige M. Bracci, Michael S. McGrath. Tumor associated targeting with Manocept: HIV associated Kaposi9s sarcoma as a model system. [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 5026. doi:10.1158/1538-7445.AM2015-5026

Abstract 4932: Kaposi's sarcoma represents a dynamic pathogenic process involving ongoing macrophage replenishment with both tumor cells as well as macrophages expressing CD206, a target for the recently approved imaging agent, Tilmanocept

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction In patients with Kaposis sarcoma (KS), plasma elevation of macrophage (MO)-produced growth factors such as EGF and bFGF suggests a role for tumor associated MOs (TAM) in KS pathogenesis. Goals of the current study were twofold: 1) To define MO subsets using M1 (MAC387), M2 (CD163, CD206) and pan macrophage CD68 antibodies in tissue microarray (TMA) analysis of all forms of KS including plaque, oral and visceral tumors. 2) To test whether CD206, the macrophage mannose receptor recognized by the recently approved imaging agent Tilmanocept (Navidea), is present on TAMs and KS tumor cells. If TAMs and KS tumor cells are CD206+, Tilmanocept could allow KS specific imaging for purposes of tumor staging, a procedure heretofore unapproachable. Methods A KS TMA with 66 KS cases and controls was obtained from the AIDS and Cancer Specimen Resource (ACSR). MO antigens were identified by IHC studies and results were standardized to the proportion of KSHV LANA+ cells (KS tumor specific marker). ACSR KS specimens from Kenya and South Africa (where KS is a common cause of death) were tested in a follow-up study. Results Most TAMs in KS tissues were identified with the M2 specific anti-CD163 antibody whereas the M1 anti-MAC387 antibody identified a smaller subset of cells. The CD68 antibody also identified a large number of TAMs in more than 90% of tumors. KS tumor spindle cells in general expressed macrophage antigens; however the most prevalent antigen for both KS tumor cells (LANA+) and TAMs was CD206 molecule. Expression of MO antigens and CD206 in relation to level of LANA within tumor tissues was similar across all tissue forms of KS (plaque, oral, visceral). In general, KS tissues from Africa contained higher relative levels of TAMs than tissues from US cases. Conclusions KS pathogenesis appears to involve an ongoing replenishment of TAMs from recent blood derived monocytes that express either M1 (MAC-387) or M2 (CD163) antigens. Both MO antigens identify cells that are turned over within days, suggesting that KS while appearing relatively indolent has a very dynamic MO migration component, apparently required for KS pathogenesis. This study also confirmed a preliminary study (Uccini et al. AJP 1997) that found both TAMs and KS tumor cells to express the CD206 macrophage mannose receptor. Tilmanocept, a technetium labeled ligand for CD206 recently approved by the FDA for human imaging use, may allow effective imaging of KS involved nodes and other visceral sites of disease. Considering the current inability of clinicians to determine the degree of KS spread beyond the presence of obvious skin lesions, the potential for using Tilmanocept to define tumor burden may allow earlier tumor specific treatment beyond the current use of anti-retroviral therapy alone which is proving ineffective in growing numbers of KS patients worldwide. Citation Format: Michael S. McGrath, Paige M. Bracci, Frederick O. Cope, Rongzhen Zhang. Kaposis sarcoma represents a dynamic pathogenic process involving ongoing macrophage replenishment with both tumor cells as well as macrophages expressing CD206, a target for the recently approved imaging agent, Tilmanocept. [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 4932. doi:10.1158/1538-7445.AM2014-4932

Abstract 2700: The use of a novel receptor targeted (CD206) Tc99m-labeled intraoperative node mapping agent for preoperative SPECT/CT in imaging head/neck cancer, breast cancer and melanoma Phase 3 trials: Localization and concordance in lymph nodes

The intraoperative lymphaticmapping (ILM) concept, based on evidence that colored agents injected into tissues leads to tracing of the agents into lymphatic structures, has carried over from observations dating back to the 1920s. The modern concept of using the pathological status of the lymph nodes to decide whether regional lymphatic clearance should be done was clearly articulated by Cabanas in 1977. Refinement of the concept as it would be reduced to a practical methodology of lymph node identification both in Europe and the United States began in earnest in the 1990s. Development of the application of sentinel node mapping has been built upon the concept of an anatomic/biologic nexus between the solid tumor bed and the immediate versus the distal lymphatic structures. It is the “biological proximity” that defines the probability of found disease in the adnexa, between the tumor bed and the lymphatic structures. It is inferred that the probability of disseminated disease in any lymphatic anatomy is highest when anatomic or biologic nexuses actually exist, as opposed to the lack of any anatomic nexuses where disease dissemination probabilities in lymphatic structures are lowest. To date, agents that have been employed in this procedure bear only simple designs that utilize simple physiologic pulse/draining of the lymphatics and provide no specificity for targeting the lymphatic tissue of interest. We present here patient results of three Phase III studies in head/neck squamous cell carcinoma, breast cancer, and melanoma where a novel 99mTc-labeled receptor targeted agent (99mTc-tilmanocept) not only out performed standard of care agents, but lent itself to remarkable concordance of intraoperative nodal localization with preoperative SPECT/CT imaging. The conclusion of these studies is that 99mTc-tilmanocept provides higher localization rates, degree of localization, lower failed detection rates, and reliable concordance between imaging and intraoperative findings. This work was supported by Neoprobe Corporation, Dublin, OH 43017 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2700. doi:1538-7445.AM2012-2700

Abstract LB-216: Receptor targeted (CD206) Tc99m tilmanocept localization in sentinel nodes in breast cancer and melanoma is concordant with preoperative imaging and a reduction in missed disease

Purpose: Tilmanocept is wholly synthetic molecule composed of a dextran-10 backbone with both multiple mannose side moieties (for receptor biding) and DTPA (for chelation of divalent cations). Tilmanocept is designed as a high affinity ligand for the human mannose binding receptor (CD206) of reticuloendothelial cells (RECs), and more specifically, those RECs that reside in tumor-draining lymph nodes. Tilmanocept is designed to provide high sensitivity and specificity for intraoperative lymphatic mapping in melanoma, breast cancer, head/neck squamous cell carcinoma, and other solid tumors. This study was designed to evaluate the correlation of preoperative imaging using Tc99m tilmanocept and intraoperative tumor bed-adnexed node findings, along with metatstatic disease finding for Tc99m tilmaocept with its “truth” comparator, Lymphazurin. Results/Conclusions: The results of combined prospective phase 3 clinical trials in breast cancer and melanoma indicate that there is a near 99% correlation between the preoperative imaging (lymphoscintigraphy) based on the use of the CD206 targeted Tc99m tilmanocept and the intraoperative/surgical findings of tumor-adnexed lymph nodes. Additionally, these findings provide exceptional guidance for nodal extraction in contrast to the “truth” mapping agent Lymphazurin, where Tc99m provides a significantly lower failed detection rate leading to a ∼10 percent improved staging rate for breast cancer and mealnoma patients (less missed disease and appropriate post-surgical treatment program). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-216. doi:1538-7445.AM2012-LB-216

Abstract 5231: Tilmaocept: A novel biotargeted agent for enhanced intraoperative discrimination of sentinel lymph nodes in breast cancer, melanoma, and head & neck SCC

Sentinel lymph node (SLN) mapping and identification is a primary component of surgical therapies for breast cancers, melanomas, pediatric soft tissue sarcomas, and certain other cancers. The current standard of care for identifying SLNs to inject into a tumor (or near a tumor) a blue dye (often called “vital blue dye”), of which several are available, in conjunction with a radiolabeled ( 99m Tc) tracer, available as either a technetium sulfur colloid or as a technetium labeled albumin formulation. The exact formulations used vary, especially between those used in the United States and Europe. All such compounds are effectively agents of passive transport in the lymphatic ducts and are “inert” with regard to any biomarker recognition specificity. Allegedly, such agents move passively from the tumor/tumor basin into lymph nodes with a presumptive tumor-node anatomic nexus and the nodes are elucidated as blue and/or radioactive, the former based on excision line-of-sight, the latter based on intraoperative evaluation with a gamma-detecting probe. But because such components do not bear any specificity, that is, bind or accumulate lymph nodes bearing targets, they either remain static at the injection site and/or quickly drain away, reducing their real-time clinical effectiveness. A novel 99m Tc-labeled, prospectively designed, low M r molecule ( 99m Tc DTPA Mannosyl Dextran; Tilmanocept) targeted as a ligand for the mannose binding receptor (MBR) of lymphatic reticuloendothelial cells and intended for real-time intraoperative gamma mapping of tumor-lymphatic nexuses has been synthesized and clinically tested in Phase 3 clinical studies in breast cancer and melanoma. The results of the development process and clinical testing indicate: 1) The prospective molecular designs resulted in safety and clinical performance characteristics that provided times from agent injection to patient evaluation that were significantly reduced compared to currently applied 99m Tc-labeled agents; and 2) The specificity, sensitivity, and performance with regard to anatomic localization, false negative rates, and receptor retention exceed those of other colorimetric and 99m Tc-labeled agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5231. doi:10.1158/1538-7445.AM2011-5231