Therapeutic efficacy and safety of a human fusion construct targeting the TWEAK receptor Fn14 and containing a modified granzyme B.

Background: Antibody-drug conjugates are an exceptional and useful therapeutic tool for multiple diseases, particularly for cancer treatment. We previously showed that the fusion of the serine protease granzyme B (GrB), the effector molecule or T and B cells, to a binding domain allows the controlled and effective delivery of the cytotoxic payload into the target cell. The production of these constructs induced the formation of high molecular aggregates with a potential impact on the efficacy and safety of the protein.

Development of a human immuno-oncology therapeutic agent targeting HER2: targeted delivery of granzyme B.

Background: Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade.

Methods: We designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy.

Light-enhanced VEGF/rGel: A tumor targeted modality with vascular and immune-mediated efficacy.

Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells.

Targeting of Tumor Neovasculature with GrB/VEGF, a Novel Cytotoxic Fusion Protein.

Angiogenesis is a critical process in numerous diseases, and intervention in neovascularization has therapeutic value in several disease settings, including ocular diseases, arthritis, and in tumor progression and metastatic spread. Various vascular targeting agents have been developed, including those that inhibit growth factor receptor tyrosine kinases, blocking antibodies that interfere with receptor signal transduction, and strategies that trap growth factor ligands. Limited anti-tumor efficacy studies have suggested that the targeted delivery of the human pro-apoptotic molecule Granzyme B to tumor cells has significant potential for cancer treatment.

Development of human serine protease-based therapeutics targeting Fn14 and identification of Fn14 as a new target overexpressed in TNBC.

The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)-containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines.

Photochemical internalization augments tumor vascular cytotoxicity and specificity of VEGF(121)/rGel fusion toxin.

Vascular targeting for cancer is increasingly recognized as a therapeutic strategy although the lack of objective responses and the development of resistance are major limitations for clinically-available drugs. Endothelial targeted toxins exert increased toxicity compared to antiangiogenic drugs and may therefore overcome these limitations. The specificity and toxicity of targeted toxins may be increased by utilization of a drug delivery system which provides selective release of the targeted toxins in the target cells.

The functionalized human serine protease granzyme B/VEGF₁₂₁ targets tumor vasculature and ablates tumor growth.

The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF₁₂₁ binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF₁₂₁ fusion protein and characterized its properties in vitro and in vivo.

Construction and characterization of novel, completely human serine protease therapeutics targeting Her2/neu.

Immunotoxins containing bacterial or plant toxins have shown promise in cancer-targeted therapy, but their long-term clinical use may be hampered by vascular leak syndrome and immunogenicity of the toxin. We incorporated human granzyme B (GrB) as an effector and generated completely human chimeric fusion proteins containing the humanized anti-Her2/neu single-chain antibody 4D5 (designated GrB/4D5). Introduction of a pH-sensitive fusogenic peptide (designated GrB/4D5/26) resulted in comparatively greater specific cytotoxicity although both constructs showed similar affinity to Her2/neu-positive tumor cells.

The TWEAK receptor Fn14 is a therapeutic target in melanoma: immunotoxins targeting Fn14 receptor for malignant melanoma treatment.

Fibroblast growth factor-inducible protein 14 (Fn14), the cell surface receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is overexpressed in various human solid tumor types and can be a negative prognostic indicator. We detected Fn14 expression in ∼60% of the melanoma cell lines we tested, including both B-Raf WT and B-Raf(V600E) lines. Tumor tissue microarray analysis indicated that Fn14 expression was low in normal skin, but elevated in 173/190 (92%) of primary melanoma specimens and in 86/150 (58%) of melanoma metastases tested.

Pharmacodynamics, tissue distribution, toxicity studies and antitumor efficacy of the vascular targeting fusion toxin VEGF121/rGel.

As a part of an ongoing assessment of its mechanism of action, we evaluated the in vivo pharmacokinetics, tissue distribution, toxicity and antitumor efficacy of VEGF(121)/rGel, a novel fusion protein. Pharmacokinetic studies showed that VEGF(121)/rGel cleared from the circulation in a biphasic manner with calculated half-lives of 0.3 and 6h for the alpha and beta phases, respectively.

Noninvasive monitoring of orthotopic glioblastoma therapy response using RGD-conjugated iron oxide nanoparticles.

Noninvasive imaging techniques have been considered important strategies in the clinic to monitor tumor early response to therapy. In the present study, we applied RGD peptides conjugated to iron oxide nanoparticles (IONP-RGD) as contrast agents in magnetic resonance imaging (MRI) to noninvasively monitor the response of a vascular disrupting agent VEGF(121)/rGel in an orthotopic glioblastoma model. RGD peptides were firstly coupled to IONPs coated with a crosslinked PEGylated amphiphilic triblock copolymer.

Cell-targeting fusion constructs containing recombinant gelonin.

Therapeutic agents capable of targeting tumor cells present as established tumors and micrometastases have already demonstrated their potential in clinical trials. Immunotoxins targeting hematological malignancies and solid tumors have additionally demonstrated excellent clinical activity. This review focuses on our design and characterization studies of constructs composed of recombinant gelonin toxin fused to either growth factors or single-chain antibodies targeting solid tumor cells, tumor vasculature or hematological malignancies.

Cytotoxicity of VEGF(121)/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2.

Background: The fusion protein VEGF(121)/rGel composed of the growth factor VEGF(121) and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF(121)/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.

Noncovalent functionalization of carbon nanovectors with an antibody enables targeted drug delivery.

Current chemotherapeutics are characterized by efficient tumor cell-killing and severe side effects mostly derived from off-target toxicity. Hence targeted delivery of these drugs to tumor cells is actively sought. We previously demonstrated that poly(ethylene glycol)-functionalized carbon nanovectors are able to sequester paclitaxel, a widely used hydrophobic cancer drug, by simple physisorption and thereby deliver the drug for killing of cancer cells.

Sensitive angiogenesis imaging of orthotopic bladder tumors in mice using a selective magnetic resonance imaging contrast agent containing VEGF121/rGel.

Objectives: To investigate the efficiency of magnetic resonance imaging (MRI) contrast agents employing vascular endothelial growth factor (VEGF121)/rGel conjugated MnFe2O4 nanocrystals for imaging of neovasculature using a bladder tumor model.

Materials And Methods: VEGF121/rGel was conjugated to MnFe2O4 nanoparticles (MNPs). The targeting efficiency and detection capability of the VEGF121/rGel-MNPs were investigated in both KDR-deficient (253JB-V) and KDR-overexpressing (PAE/KDR) cells using MRI.

Inhibition of prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature.

Purpose: A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF(121)/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting nontumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa.

Multiplexed PET probes for imaging breast cancer early response to VEGF₁₂₁/rGel treatment.

In this study, we applied multiplexed positron emission tomography (PET) probes to monitor glucose metabolism, cellular proliferation, tumor hypoxia and angiogenesis during VEGF₁₂₁/rGel therapy of breast cancer. Two doses of 12 mg/kg VEGF₁₂₁/rGel, administered intraperitoneally, resulted in initial delay of tumor growth, but the growth resumed 4 days after tumor treatment was stopped. The average tumor growth rate expressed as V/V(0), were 1.

Characterization and mechanistic studies of a novel melanoma-targeting construct containing IκBa for specific inhibition of nuclear factor-κB activity.

The transcription factor nuclear factor-κB (NF-κB) is a central mediator of growth and homeostasis for both normal and neoplastic cells. IκBα is the natural intracellular inhibitor of NF-κB and can effectively complex with and thereby inhibit the biologic activity and translocation of NF-κB to the nucleus. We designed a fusion protein designated IκBα/scFvMEL composing of human IκBα and the single-chain antibody scFvMEL, targets melanoma gp240 antigen.

Imaging of VEGF receptor in a rat myocardial infarction model using PET.

Unlabelled: Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factor's receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject.

Multimodality molecular imaging of glioblastoma growth inhibition with vasculature-targeting fusion toxin VEGF121/rGel.

Unlabelled: Vascular endothelial growth factor A (VEGF-A) and its receptors, Flt-1/FLT-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2), are key regulators of tumor angiogenesis and tumor growth. The purpose of this study was to determine the antiangiogenic and antitumor efficacies of a vasculature-targeting fusion toxin (VEGF(121)/rGel) composed of the VEGF-A isoform VEGF(121) linked with a G(4)S tether to recombinant plant toxin gelonin (rGel) in an orthotopic glioblastoma mouse model by use of noninvasive in vivo bioluminescence imaging (BLI), MRI, and PET.

Methods: Tumor-bearing mice were randomized into 2 groups and balanced according to BLI and MRI signals.

Overexpression of biologically active VEGF121 fusion proteins in Escherichia coli.

Vascular endothelial growth factor-A (VEGF) exists as five different isoforms, which exert their growth stimulatory effects through interaction with the FLK and KDR receptors. The VEGF(121) isoform has been employed as a highly selective carrier of therapeutic agents to target tumor endothelial cells resulting in inhibition of tumor growth and metastasis. VEGF(121) and VEGF(121)/rGel fusion toxin containing hexa-histidine tags were expressed in Escherichia coli AD494 (DE3) pLysS.

PET of vascular endothelial growth factor receptor expression.

Unlabelled: For solid tumors and metastatic lesions, tumor vascularity is a critical factor in assessing response to therapy. Here we report the first example, to our knowledge, of (64)Cu-labeled vascular endothelial growth factor 121 (VEGF(121)) for PET of VEGF receptor (VEGFR) expression in vivo.

Methods: VEGF(121) was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and then labeled with (64)Cu for small-animal PET of mice bearing different sized U87MG human glioblastoma xenografts.

Inhibition of prostate tumor growth and bone remodeling by the vascular targeting agent VEGF121/rGel.

The pathophysiology of tumor growth following skeletal metastases and the poor response of this type of lesion to therapeutic intervention remains incompletely understood. Vascular endothelial growth factor (VEGF)-A and its receptors play a role in both osteoclastogenesis and tumor growth. Systemic (i.

The vascular-targeting fusion toxin VEGF121/rGel inhibits the growth of orthotopic human bladder carcinoma tumors.

Vascular endothelial growth factor (VEGF) and its receptors (FLT-1 and KDR) are overexpressed by human bladder cancer cells and tumor endothelial cells, respectively. Strategies that target VEGF receptors hold promise as antiangiogenic therapeutic approaches to bladder cancer. A fusion protein of VEGF121 and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity.

Vascular targeting of ocular neovascularization with a vascular endothelial growth factor121/gelonin chimeric protein.

Tumors provide an extremely abnormal microenvironment that stimulates neovascularization from surrounding vessels and causes altered gene expression within vascular cells. Up-regulation of vascular endothelial growth factor (VEGF) receptors has allowed selective destruction of tumor vessels by administration of a chimeric protein consisting of VEGF121 coupled to the toxin gelonin (VEGF/rGel). We sought to determine whether there is sufficient up-regulation of VEGF receptors in endothelial cells participating in ocular neovascularization to permit a similar strategy.