FAP Radioligand Linker Optimization Improves Tumor Dose and Tumor-to-Healthy Organ Ratios in 4T1 Syngeneic Model.

Fibroblast activation protein (FAP) has attracted considerable attention as a possible target for the radiotherapy of solid tumors. Unfortunately, initial efforts to treat solid tumors with FAP-targeted radionuclides have yielded only modest clinical responses, suggesting that further improvements in the molecular design of FAP-targeted radiopharmaceutical therapies (RPT) are warranted. In this study, we report several advances on the previously described FAP6 radioligand that increase tumor retention and accelerate healthy tissue clearance.

Design of a Fibroblast Activation Protein-Targeted Radiopharmaceutical Therapy with High Tumor-to-Healthy-Tissue Ratios.

Because of upregulated expression on cancer-associated fibroblasts, fibroblast activation protein (FAP) has emerged as an attractive biomarker for the imaging and therapy of solid tumors. Although many FAP ligands have already been developed for radiopharmaceutical therapies (RPTs), most suffer from inadequate tumor uptake, insufficient tumor residence times, or off-target accumulation in healthy tissues, suggesting a need for further improvements. A new FAP-targeted RPT with a novel ligand (FAP8-PEG-IP-DOTA) was designed by combining the desirable features of several previous ligand-targeted RPTs.

In Vivo Labeling and Detection of Circulating Tumor Cells in Mice Using OTL38.

Purpose: We recently developed an optical instrument to non-invasively detect fluorescently labeled circulating tumor cells (CTCs) in mice called 'Diffuse in vivo Flow Cytometry' (DiFC). OTL38 is a folate receptor (FR) targeted near-infrared (NIR) contrast agent that is FDA approved for use in fluorescence guided surgery of ovarian and lung cancer. In this work, we investigated the use OTL38 for in vivo labeling and detection of FR + CTCs with DiFC.

Tumor-specific activation of folate receptor beta enables reprogramming of immune cells in the tumor microenvironment.

Folate receptors can perform folate transport, cell adhesion, and/or transcription factor functions. The beta isoform of the folate receptor (FRβ) has attracted considerable attention as a biomarker for immunosuppressive macrophages and myeloid-derived suppressor cells, however, its role in immunosuppression remains uncharacterized. We demonstrate here that FRβ cannot bind folate on healthy tissue macrophages, but does bind folate after macrophage incubation in anti-inflammatory cytokines or cancer cell-conditioned media.

Fibroblast Activation Protein-Targeted Radioligand Therapy for Treatment of Solid Tumors.

Fibroblast activation protein (FAP) has received increasing attention as an oncologic target because of its prominent expression in solid tumors but virtual absence from healthy tissues. Most radioligand therapies (RLTs) targeting FAP, however, suffer from inadequate tumor retention or clearance from healthy tissues. Herein we report a FAP-targeted RLT comprising an FAP6 ligand conjugated to DOTA and an albumin binder (4--iodophenylbutyric acid, or IP) for enhanced pharmacokinetics.

Nanoparticles Targeted to Fibroblast Activation Protein Outperform PSMA for MRI Delineation of Primary Prostate Tumors.

Accurate delineation of gross tumor volumes remains a barrier to radiotherapy dose escalation and boost dosing in the treatment of solid tumors, such as prostate cancer. Magnetic resonance imaging (MRI) of tumor targets has the power to enable focal dose boosting, particularly when combined with technological advances such as MRI-linear accelerator. Fibroblast activation protein (FAP) is overexpressed in stromal components of >90% of epithelial carcinomas.

Efficient capture of circulating tumor cells with low molecular weight folate receptor-specific ligands.

Retrieval of circulating tumor cells (CTC) has proven valuable for assessing a patient's cancer burden, evaluating response to therapy, and analyzing which drug might treat a cancer best. Although most isolation methods retrieve CTCs based on size, shape, or capture by tumor-specific antibodies, we explore here the use of small molecule tumor-specific ligands linked to magnetic beads for CTC capture. We have designed folic acid-biotin conjugates with different linkers for the capture of folate receptor (FR) + tumor cells spiked into whole blood, and application of the same technology to isolate FR + CTCs from the peripheral blood of both tumor-bearing mice and non-small cell lung patients.

Design and characterization of fibroblast activation protein targeted pan-cancer imaging agent for fluorescence-guided surgery of solid tumors.

Tumor-targeted fluorescent dyes have been shown to significantly improve a surgeon's ability to locate and resect occult malignant lesions, thereby enhancing a patient's chances of long term survival. Although several tumor-targeted fluorescent dyes have been developed for imaging specific subsets of human cancers, no tumor-targeted dye has been designed that can image all cancer types. Based on observations that fibroblast activation protein (FAP) is upregulated on cancer-associated fibroblasts (CAFs) that infiltrate essentially all solid tumors, we have undertaken to develop a FAP-targeted fluorescent dye that can image CAFs without accumulating in healthy cells or fibroblasts.

Folate Receptor Beta for Macrophage Imaging in Rheumatoid Arthritis.

Non-invasive imaging modalities constitute an increasingly important tool in diagnostic and therapy response monitoring of patients with autoimmune diseases, including rheumatoid arthritis (RA). In particular, macrophage imaging with positron emission tomography (PET) using novel radiotracers based on differential expression of plasma membrane proteins and functioning of cellular processes may be suited for this. Over the past decade, selective expression of folate receptor β (FRβ), a glycosylphosphatidylinositol-anchored plasma membrane protein, on myeloid cells has emerged as an attractive target for macrophage imaging by exploiting the high binding affinity of folate-based PET tracers.

Design, Synthesis, and Targeted Delivery of an Immune Stimulant that Selectively Reactivates Exhausted CAR T Cells.

Although chimeric antigen receptor (CAR) T cells have demonstrated significant promise in suppressing hematopoietic cancers, their applications in treating solid tumors have been limited by onset of CAR T cell exhaustion that accompanies continuous CAR T cell exposure to tumor antigen. To address this limitation, we have exploited the abilities of recently designed universal CARs to bind fluorescein and internalize a fluorescein-TLR7 agonist conjugate by CAR-mediated endocytosis. We demonstrate here that anti-fluorescein CAR-mediated uptake of a fluorescein-TLR7-3 conjugate can reactivate exhausted CAR T cells, leading to dramatic reduction in T cell exhaustion markers (PD-1 Tim-3 ) and shrinkage of otherwise resistant tumors without inducing systemic activation of the immune system.

Design of a Near Infrared Fluorescent Ureter Imaging Agent for Prevention of Ureter Damage during Abdominal Surgeries.

The inadvertent severing of a ureter during surgery occurs in as many as 4.5% of colorectal surgeries. To help prevent this issue, several near-infrared (NIR) dyes have been developed to assist surgeons with identifying ureter location.

Efficacy and tolerability of folate-aminopterin therapy in a rat focal model of multiple sclerosis.

Background: Activated macrophages in the experimental model of multiple sclerosis (MS) express folate receptor-β (FR-β), representing a promising target for the treatment of MS. Here, we both evaluated the efficacy of a novel folate-aminopterin construct (EC2319) in a rat focal model of multiple sclerosis (MS) and investigated the utility of Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated folate (Ga-FOL) for assessing inflammatory lesions. In addition, we investigated whether FR-β is expressed in the brain of patients with MS.

A universal dual mechanism immunotherapy for the treatment of influenza virus infections.

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells.

Sensitive manipulation of CAR T cell activity using a chimeric endocytosing receptor.

Background: Most adoptive cell therapies (ACTs) suffer from an inability to control the therapeutic cell's behavior following its transplantation into a patient. Thus, efforts to inhibit, activate, differentiate or terminate an ACT after patient reinfusion can be futile, because the required drug adversely affects other cells in the patient.

Methods: We describe here a two domain fusion receptor composed of a ligand-binding domain linked to a recycling domain that allows constitutive internalization and trafficking of the fusion receptor back to the cell surface.

Targeted inhibition of PI3 kinase/mTOR specifically in fibrotic lung fibroblasts suppresses pulmonary fibrosis in experimental models.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an average life expectancy of 3 to 5 years. IPF is characterized by progressive stiffening of the lung parenchyma due to excessive deposition of collagen, leading to gradual failure of gas exchange. Although two therapeutic agents have been approved from the FDA for IPF, they only slow disease progression with little impact on outcome.

Radiosynthesis and preclinical evaluation of [Ga]Ga-NOTA-folate for PET imaging of folate receptor β-positive macrophages.

Folate receptor β (FR-β), a marker expressed on macrophages, is a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [Ga]Ga-NOTA-folate (Ga-FOL). After determining the affinity of Ga-FOL using cells expressing FR-β, we studied atherosclerotic mice with Ga-FOL and F-FDG PET/CT.

Reprogramming of profibrotic macrophages for treatment of bleomycin-induced pulmonary fibrosis.

Fibrotic diseases cause organ failure that lead to ~45% of all deaths in the United States. Activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize collagen and extracellular matrix proteins. Although suppression of macrophage-derived cytokine production can halt progression of fibrosis, therapeutic agents that prevent release of these cytokines (e.

Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for Diffuse In Vivo Flow Cytometry.

Purpose: We recently developed a new instrument called "diffuse in vivo flow cytometry" (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC.

Evaluation of a Neurokinin-1 Receptor-Targeted Technetium-99m Conjugate for Neuroendocrine Cancer Imaging.

Purpose: Neuroendocrine tumors (NETs) have reasonably high 5-year survival rates when diagnosed at an early stage but are significantly more lethal when discovered only after metastasis. Although several imaging modalities such as computed tomography (CT), positron emission tomography, and magnetic resonance imaging can detect neuroendocrine tumors, their high false positive rates suggest that more specific diagnostic tests are required. Targeted imaging agents such as Octreoscan® have met some of this need for improved specificity, but their inability to image poorly differentiated NETs suggests that improved NET imaging agents are still needed.

Regulation of CAR T cell-mediated cytokine release syndrome-like toxicity using low molecular weight adapters.

Although chimeric antigen receptor (CAR) T cell therapies have demonstrated considerable success in treating hematologic malignancies, they have simultaneously been plagued by a cytokine release syndrome (CRS) that can harm or even kill the cancer patient. We describe a CAR T cell strategy in which CAR T cell activation and cancer cell killing can be sensitively regulated by adjusting the dose of a low molecular weight adapter that must bridge between the CAR T cell and cancer cell to initiate tumor eradication. By controlling the concentration and dosing schedule of adapter administration, we document two methods that can rapidly terminate (<3 h) a pre-existing CRS-like toxicity and two unrelated methods that can pre-emptively prevent a CRS-like toxicity that would have otherwise occurred.

Use of a Single CAR T Cell and Several Bispecific Adapters Facilitates Eradication of Multiple Antigenically Different Solid Tumors.

Most solid tumors are comprised of multiple clones that express orthogonal antigens, suggesting that novel strategies must be developed in order to adapt chimeric antigen receptor (CAR) T-cell therapies to treat heterogeneous solid tumors. Here, we utilized a cocktail of low-molecular-weight bispecific adapters, each comprised of fluorescein linked to a different tumor-specific ligand, to bridge between an antifluorescein CAR on the engineered T cell and a unique antigen on the cancer cell. This formation of an immunologic synapse between the CAR T cell and cancer cell enabled use of a single antifluorescein CAR T cell to eradicate a diversity of antigenically different solid tumors implanted concurrently in NSG mice.

Bone-Fracture-Targeted Dasatinib-Oligoaspartic Acid Conjugate Potently Accelerates Fracture Repair.

Approximately 6.3 million bone fractures occur annually in the United States, resulting in considerable morbidity, deterioration in quality of life, loss of productivity and wages, and sometimes death (e.g.

Targeted Tubulysin B Hydrazide Conjugate for the Treatment of Luteinizing Hormone-Releasing Hormone Receptor-Positive Cancers.

The targeted delivery of chemotherapeutic agents to receptors that are overexpressed on cancer cells has become an attractive strategy to concentrate drugs in cancer cells while avoiding uptake by healthy cells. Luteinizing hormone-releasing hormone receptor (LHRH-R) has attracted considerable interest for this application, since LHRH-R is upregulated in ∼86% of prostate cancers, ∼80% of endometrial cancers, ∼80% of ovarian cancers, and ∼50% of breast cancers, but virtually absent from normal tissues. Although LHRH and related peptides have been used to deliver cytotoxic drugs to LHRH-R overexpressing cancer cells, they have suffered from off-target delivery of the therapeutic agents to the liver and kidneys.