Highly Specific and Effective Targeting of EGFRvIII-Positive Tumors with TandAb Antibodies.

To harness the cytotoxic capacity of immune cells for the treatment of solid tumors, we developed tetravalent, bispecific tandem diabody (TandAb) antibodies that recognize EGFRvIII, the deletion variant III of the epidermal growth factor receptor (EGFR), and CD3 on T-cells, thereby directing immune cells to eliminate EGFRvIII-positive tumor cells. Using phage display, we identified scFv antibodies selectively binding to EGFRvIII. These highly EGFRvIII-specific, fully human scFv were substantially improved by affinity maturation, achieving s in the picomolar range, and were used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties.

Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia.

Purpose: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a molecular weight exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs.

Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity.

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis.

LRP/LR Antibody Mediated Rescuing of Amyloid-β-Induced Cytotoxicity is Dependent on PrPc in Alzheimer's Disease.

The neuronal perturbations in Alzheimer's disease are attributed to the formation of extracellular amyloid-β (Aβ) neuritic plaques, composed predominantly of the neurotoxic Aβ42 isoform. Although the plaques have demonstrated a role in synaptic dysfunction, neuronal cytotoxicity has been attributed to soluble Aβ42 oligomers. The 37kDa/67kDa laminin receptor has been implicated in Aβ42 shedding and Aβ42-induced neuronal cytotoxicity, as well as internalization of this neurotoxic peptide.

A phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma.

AFM13 is a bispecific, tetravalent chimeric antibody construct (TandAb) designed for the treatment of CD30-expressing malignancies. AFM13 recruits natural killer (NK) cells via binding to CD16A as immune effector cells. In this phase 1 dose-escalation study, 28 patients with heavily pretreated relapsed or refractory Hodgkin lymphoma received AFM13 at doses of 0.

A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19(+) tumor cells.

To harness the potent tumor-killing capacity of T cells for the treatment of CD19(+) malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies.

The 37kDa/67kDa laminin receptor acts as a receptor for Aβ42 internalization.

Neuronal loss is a major neuropathological hallmark of Alzheimer's disease (AD). The associations between soluble Aβ oligomers and cellular components cause this neurotoxicity. The 37 kDa/67 kDa laminin receptor (LRP/LR) has recently been implicated in Aβ pathogenesis.

Anti-LRP/LR specific antibody IgG1-iS18 impedes adhesion and invasion of liver cancer cells.

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated.

A novel tetravalent bispecific TandAb (CD30/CD16A) efficiently recruits NK cells for the lysis of CD30+ tumor cells.

To improve recruitment and activation of natural killer (NK) cells to lyse tumor cells, we isolated a human anti-CD16A antibody with similar affinity for the CD16A 158F/V allotypes, but no binding to the CD16B isoform. Using CD16A-targeting Fv domains, we constructed a tetravalent bispecific CD30/CD16A tandem diabody (TandAb®) consisting solely of Fv domains. This TandAb has two binding sites for CD16A and two for CD30, the antigen identifying Hodgkin lymphoma cells.

Anti-LRP/LR specific antibodies and shRNAs impede amyloid beta shedding in Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia. The amyloid beta (Aβ) peptide is the predominant candidate aetiological agent and is generated through the sequential proteolytic cleavage of the Amyloid Precursor Protein (APP) by beta (β) and gamma (γ) secretases. Since the cellular prion protein (PrP(c)) has been shown to regulate Aβ shedding, we investigated whether the cellular receptor for PrP(c), namely the 37 kDa/67 kDa Laminin Receptor (LRP/LR) played a role in Aβ shedding.

Anti-LRP/LR specific antibody IgG1-iS18 and knock-down of LRP/LR by shRNAs rescue cells from Aβ42 induced cytotoxicity.

Alzheimer's disease (AD) is characterized by neurofibrillary tangles, senile plaques and neuronal loss. Amyloid beta (Aβ) is proposed to elicit neuronal loss through cell surface receptors. As Aβ shares common binding partners with the 37 kDa/67 kDa laminin receptor (LRP/LR), we investigated whether these proteins interact and the pathological significance of this association.

Adhesion and Invasion of Breast and Oesophageal Cancer Cells Are Impeded by Anti-LRP/LR-Specific Antibody IgG1-iS18.

Adhesion and invasion have been identified as the two key components of metastasis. The 37 kDa/67 kDa laminin receptor (LRP/LR) is thought to enhance these two processes thus endorsing the progression of cancer. Here we report on LRP/LR and the metastatic potential of MDA-MB 231 breast and WHCO1 oesophageal cancer cells.

In vitro inhibition of angiogenesis by antibodies directed against the 37kDa/67kDa laminin receptor.

The 37kDa/67kDa laminin receptor (LRP/LR) is a central receptor mediating interactions between tumour cells and the basement membrane and is thereby a key player in adhesion and invasion, essential processes in metastatic cancer. To affect continued tumour growth, tumours induce angiogenesis for the constant delivery of nutrients and oxygen. This study aims to determine the blocking effect of the anti-LRP/LR specific antibody, W3 on the angiogenic potential of HUVE (human umbilical vein endothelial) cells.

Anti-LRP/LR-specific antibody IgG1-iS18 significantly reduces adhesion and invasion of metastatic lung, cervix, colon and prostate cancer cells.

The 37-kDa/67-kDa laminin receptor [laminin receptor precursor/high-affinity laminin receptor (LRP/LR)] is thought to play a major role in invasion and adhesion, key components of metastatic cancer. Lung cancer, cervical cancer, colon cancer and prostate cancer are among the top 10 cancer types worldwide. Here, we report that LRP/LR levels on the surface of lung cancer cells, cervical cancer cells, colon cancer cells and prostate cancer cells are significantly increased compared to non-tumorigenic fibroblasts.

68Ga-labelled recombinant antibody variants for immuno-PET imaging of solid tumours.

Purpose: Recombinant antibodies isolated from human antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of recombinant antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope (68)Ga.

Methods: In order to combine the promising tumour targeting properties of antibodies with (68)Ga, four antibody variants with the same specificity and origin only differing in molecular weight were constructed for comparison.

Delivery of single-chain antibodies (scFvs) directed against the 37/67 kDa laminin receptor into mice via recombinant adeno-associated viral vectors for prion disease gene therapy.

The 37/67 kDa laminin receptor (LRP/LR) acts as a receptor for prions providing a promising target for the treatment of prion diseases. Recently, we selected anti-LRP/LR single-chain antibodies (scFvs) and proved a reduction of the peripheral PrP(Sc) propagation by passive immunotransfer into scrapie-infected mice. Here, we report the development of an in vivo gene delivery system based on adeno-associated virus (AAV) vectors expressing scFvs-S18 and -N3 directed against LRP/LR.

Tetrafluorophenolate of HBED-CC: a versatile conjugation agent for 68Ga-labeled small recombinant antibodies.

Purpose: The success of (68)Ga-labeled peptides for positron emission tomography of neuroendocrine tumors is mainly depending on the complex chemistry of this radioisotope. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the chelator of choice has however limitations if its application is expanded to heat-sensitive proteins. Recombinant antibodies like single chain Fv or diabodies belong to this class of proteins.

Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor.

The 37-kDa/67-kDa laminin receptor precursor/laminin receptor (LRP/LR) acting as a receptor for prions and viruses is overexpressed in various cancer cell lines, and their metastatic potential correlates with LRP/LR levels. We analyzed the tumorigenic fibrosarcoma cell line HT1080 regarding 37-kDa/67-kDa LRP/LR levels and its invasive potential. Compared to the less invasive embryonic fibroblast cell line NIH3T3, the tumorigenic HT1080 cells display approximately 1.

Single chain Fv antibodies directed against the 37 kDa/67 kDa laminin receptor as therapeutic tools in prion diseases.

Transmissible spongiform encephalopathies are a group of neurological disorders associated with the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(c). The 37 kDa/67 kDa laminin receptor (LRP/LR) has been identified as a prion receptor and several lines of evidence strongly suggest that this protein plays a role during prion pathogenesis. Here we report the selection of recombinant single chain antibodies (scFvs) directed against LRP from naïve and synthetic phage scFv libraries for therapeutic application.

Specific inhibition of interleukin-13 activity by a recombinant human single-chain immunoglobulin domain directed against the IL-13 receptor alpha1 chain.

Interleukin-13 (IL-13) is a T-cell-derived pleiotropic cytokine of particular medical importance because of its critical role in the development of allergic asthma. The effects of IL-13 on its target cells are mediated through a dimeric transmembrane receptor (IL-13R), which shares the IL-4Ralpha subunit with the IL-4R system, but contains as a specific component the IL-13Ralpha1 chain. We have generated a set of single-chain Fv fragments with specific binding capacity to the extracellular domain of the human IL-13Ralpha1 receptor.

Single-chain antibodies for the conformation-specific blockade of activated platelet integrin alphaIIbbeta3 designed by subtractive selection from naive human phage libraries.

Binding of fibrinogen to platelet integrin alphaIIbbeta3 mediates platelet aggregation, and thus inhibition of alphaIIbbeta3 represents a powerful therapeutic strategy in cardiovascular medicine. However, the currently used inhibitors of alphaIIbbeta3 demonstrate several adverse effects like thrombocytopenia and bleeding, which are associated with their property to bind to non-activated alphaIIbbeta3. To circumvent these problems, we designed blocking single-chain antibody-fragments (scFv) that bind to alphaIIbbeta3 exclusively in its activated conformation.

Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma.

To target NK cells against non-Hodgkin's lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcgammaRIII (CD16). Bacterially produced CD19 x CD16 BsDb specifically interacted with both CD19(+) and CD16(+) cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs.

Identification of the gene coding for the largest subunit of RNA polymerase I (A) of Drosophila melanogaster.

The gene coding for the largest subunit (RPA1) of RNA polymerase I (A) of Drosophila melanogaster (DmRPA1) was cloned and sequenced. The gene is interrupted by seven small introns and the cDNA reveals an open reading frame of 4932 nucleotides. The deduced polypeptide consists of 1644 amino acids with a calculated molecular weight of 185 kDa.