T and NK cells of B cell NHL patients exert cytotoxicity against lymphoma cells following binding of bispecific tetravalent antibody CD19 × CD3 or CD19 × CD16.

Bispecific tetravalent antibodies (TandAb) directed against the B cell surface marker CD19 and activating receptors on T or NK cells (CD19 × CD3 or CD19 × CD16) have shown promising effects in vitro and in preclinical studies. Here, we examine the cytotoxic efficacy of T and NK cells from patients with B cell Non-Hodgkin's Lymphoma (NHL) against B-lymphoma cells following the binding of the matching TandAb. The addition of CD19 × CD16 TandAb led to a threefold increase in NK cell activation in the presence of B-lymphoma cells. Similarly, T cells displayed a sevenfold increase in cytotoxic activity after the addition of CD19 × CD3 TandAb. Comparison of T and NK cell effector function of patients and healthy controls showed comparable levels of cytotoxic activity in response to lymphoma cells and no reduction in functional activity due to age, disease stage or the type and amount of previous therapy. Thus, T and NK cells of patients with B cell NHL are fully capable of being activated by therapeutic crosslinking antibodies. These results provide a rationale for the use of TandAbs for patients with B cell NHL, particularly in cases where remission with minimal residual disease could be achieved by cytotoxic chemotherapy.

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. Therefore, the binding domains of a single-chain fragment variable (scFv) isolated from a human naïve antibody library were modified genetically to construct the respective full-length IgG, the tria- and diabody variants. These molecules were conjugated with the bifunctional chelating agent N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) to enable (68)Ga labelling at ambient temperature and compared in biodistribution and immuno-PET imaging experiments. RESULTS: The antibody variants with identical specificity proved to have the correct molecular weight, high binding affinity and specificity to their antigen, EpCAM. Radiometal complexation was efficiently performed at room temperature leading to (68)Ga-labelled antibodies with unchanged binding properties compared to the original antibody variants. The best targeting properties were obtained with the scFv and especially with the diabody. The triabody showed higher absolute tumour uptake but only moderate clearance from circulation. CONCLUSION: The antibody variants differed considerably in normal organ uptake, clearance from circulation and tumour accumulation. The data demonstrate the feasibility of imaging solid tumours with the (68)Ga-labelled diabody format. This type of recombinant protein might be a promising carrier even for the short-lived radiometal (68)Ga to support e.g. the management of immunotherapy which may provide important information regarding receptor expression of solid tumours.

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. Western blotting and FACS analysis confirmed a specific LRP/LR recognition pattern of the two selected scFvs S18 and N3. Both scFvs specifically interfered with the PrP/LRP interaction in vitro. High yield production of the scFvs of approx. 1mg/l of culture medium was achieved in E. coli. Passive immunotransfer of the scFv S18 antibody reduced PrP(Sc) levels by approx. 40% in the spleen of scrapie infected C57BL/6 mice 90 days post scFv injection, suggesting that scFv S18 interferes with peripheral PrP(Sc) propagation, without a significant prolongation of incubation and survival times.

LRP/LR Antibody Mediated Rescuing of Amyloid-ß-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-ß (Aß) neuritic plaques, composed predominantly of the neurotoxic Aß42 isoform. Although the plaques have demonstrated a role in synaptic dysfunction, neuronal cytotoxicity has been attributed to soluble Aß42 oligomers. The 37kDa/67kDa laminin receptor has been implicated in Aß42 shedding and Aß42-induced neuronal cytotoxicity, as well as internalization of this neurotoxic peptide. As the cellular prion protein binds to both LRP/LR and Aß42, the mechanism underlying this cytotoxicity may be indirectly due to the PrPc-Aß42 interaction with LRP/LR. The effects of this interaction were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assays. PrPc overexpression significantly enhanced Aß42 cytotoxicity in vitro, while PrP-/-  cells were more resistant to the cytotoxic effects of Aß42 and exhibited significantly less cell death than PrPc expressing N2a cells. Although anti-LRP/LR specific antibody IgG1-iS18 significantly enhanced cell viability in both pSFV1-huPrP1-253 transfected and non-transfected cells treated with exogenous Aß42, it failed to have any cell rescuing effect in PrP-/-  HpL3-4 cells. These results suggest that LRP/LR plays a significant role in Aß42-PrPc mediated cytotoxicity and that anti-LRP/LR specific antibodies may serve as potential therapeutic tools for Alzheimer's disease.

Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies.

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.

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. Two naive phage libraries were created: a natural phage library, based on human lymphocyte cDNA, and a synthetic library, with randomized VHCDR3. We performed serial rounds of subtractive panning with depletion on non-activated and selection on activated alphaIIbbeta3, which were provided on resting and ADP-stimulated platelets and CHO cells, expressing wild-type or mutated and thereby activated alphaIIbbeta3. In contrast to isolated, immobilized targets, as generally used for phage display, this unique cell-based approach for panning allowed the preservation of functional integrin conformation. Thereby, we obtained several scFv-clones that demonstrated exclusive binding to activated platelets and complete inhibition of fibrinogen binding and platelet aggregation. Interestingly, all activation-specific clones contained an RXD pattern in the HCDR3. Binding studies on transiently expressed point mutants and mouse-human domain-switch mutants of alphaIIbbeta3 indicate a binding site similar to fibrinogen. In conclusion, we generated human activation-specific scFvs against alphaIIbbeta3, which bind selectively to activated alphaIIbbeta3 and thereby potently inhibit fibrinogen binding to alphaIIbbeta3 and platelet aggregation.

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. An essential component of telomerase, hTERT is overexpressed in 85-90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.

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. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19(+) cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19(+) malignancies with an advantageous safety risk profile and anticipated dosing regimen.

Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody.

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.

Immunosuppressive properties of anti-CD3 single-chain Fv and diabody.

The mouse anti-human CD3 monoclonal antibody OKT3 is a potent immunosuppressive agent used in clinical transplantation. However, OKT3 therapy is associated with unpleasant and often serious side effects which appear to result from cytokine release, complement activation and a human anti-mouse antibody (HAMA) response. To decrease these adverse side effects, we constructed antibody fragments comprising OKT3 variable domains without any constant domains. Single-chain Fv (scFv) monomers, dimers and trimers were generated by changing the linker length between the V(H) and V(L) domains. The linkers used were the natural extensions of the V(H) into the C(H)1 domain. The dimeric molecules (diabodies) demonstrated the best CD3-binding activity. The diabody with the six amino acid linker was produced in bacteria with a tenfold higher yield than other scFvs and possessed CD3-binding affinity approaching that of the parental mAb. In contrast to OKT3 mAb, the anti-CD3 diabody and scFv monomer did not cause any T-cell activation and cytokine release in vitro, while demonstrating CD3 modulation. In mixed lymphocyte cultures, both diabody and scFv, but not the monoclonal antibody OKT3, were able to suppress T-cell activation and secretion of IL-2 and IFN-gamma in a dose-dependent manner. The anti-CD3 diabody may provide a potent immunosuppressive drug with low toxicity and immunogenicity.

Recombinant chimeric OKT3/IgM antibodies for immune suppression: evaluation in a human CD3 transgenic mouse model.

ScOKT3-gammaDeltaIgM VAEVD is a recombinant chimeric anti-CD3 antibody variant consisting of the light and heavy variable binding domains of the OKT3 monoclonal antibody and the CH3 and CH4 domains of a human IgM mutation linked by a human IgG3 hinge region. Due to the IgM Fc domains, scOKT3-gammaDeltaIgM VAEVD antibodies are able to form polymeric structures. Independent of their polymerization state, they possess in vitro CD3 modulating and immunosuppressive properties while inducing only minimal T cell activation compared to their monoclonal counterpart. To evaluate the in vivo efficacy of the antibodies, an adjuvant-induced chronic inflammation was established in human CD3 transgenic mice. Administration of four doses of 15 microg of isolated scOKT3-gammaDeltaIgM VAEVD monomers and pentamers significantly reduced diameters of inflamed ankle joints in a manner comparable to the monoclonal antibody OKT3. Additionally, the antibody treatment lead to a significant reduction of the cytokine levels (IL-2, TNF-alpha and INF-gamma) in the mice's sera. These results suggest that scOKT3-gammaDeltaIgM VAEVD antibodies may provide a useful alternative to the OKT3 mAb for clinical immunosuppressive treatment for auto-aggressive diseases or for organ-transplantation.

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. The binding and cytotoxicity of the TandAb were compared with antibodies with identical anti-CD30 domains: (1) a native IgG, (2) an IgG optimized for binding to Fc receptors, and (3) a bivalent bispecific CD30/CD16A diabody. Due to its CD16A-bivalency and reduced koff, the TandAb was retained longer on the surface of NK cells than the IgGs or the diabody. This contributed to the higher potency and efficacy of the TandAb relative to those of the other anti-CD30 antibodies. TandAb cytotoxicity was independent of the CD16A allotype, whereas the anti-CD30 IgGs were substantially less cytotoxic when NK cells with low affinity CD16A allotype were employed. TandAb activation of NK cells was strictly dependent on the presence of CD30(+) target cells. Therefore, the CD30/CD16A TandAb may represent a promising therapeutic for the treatment of Hodgkin's lymphoma; further, anti-CD16A TandAbs may function as potent immunotherapeutics that specifically recruit NK cells to destroy cancer cells.

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. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson's correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction.

The 37kDa/67kDa laminin receptor acts as a receptor for Aß42 internalization.

Neuronal loss is a major neuropathological hallmark of Alzheimer's disease (AD). The associations between soluble Aß oligomers and cellular components cause this neurotoxicity. The 37 kDa/67 kDa laminin receptor (LRP/LR) has recently been implicated in Aß pathogenesis. In this study the mechanism underlying the pathological role of LRP/LR was elucidated. Försters Resonance Energy Transfer (FRET) revealed that LRP/LR and Aß form a biologically relevant interaction. The ability of LRP/LR to form stable associations with endogenously shed Aß was confirmed by pull down assays and Aß-ELISAs. Antibody blockade of this association significantly lowered Aß42 induced apoptosis. Furthermore, antibody blockade and shRNA mediated downregulation of LRP/LR significantly hampered Aß42 internalization. These results suggest that LRP/LR is a receptor for Aß42 internalization, mediating its endocytosis and contributing to the cytotoxicity of the neuropeptide by facilitating intra-cellular Aß42 accumulation. These findings recommend anti-LRP/LR specific antibodies and shRNAs as potential therapeutic tools for AD treatment.

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. Western blot analysis revealed a significant increase in total laminin receptor precursor (LRP) levels of breast and oesophageal cancer cells in comparison to non-invasive MCF-7 breast cancer cells, whereas LRP/LR cell surface levels in both cell lines were not significantly different to those of MCF-7 cells as analysed by flow cytometry. Incubation of breast and oesophageal cancer cells with the anti-LRP/LR specific antibody, IgG1-iS18, resulted in significant reduction in the adhesive potential of WHCO1 and MDA-MB 231 cells by 92% and 16%, respectively. Moreover, invasion was significantly impeded by 98% and 25% for WHCO1 and MDA-MB 231 cells, respectively. Pearson's correlation coefficients proved a positive correlation between total LRP/LR levels and invasive potential as well as between the adhesive and invasive potential of breast and oesophageal cancer cells. Our findings suggest that through interference of the LRP/LR-laminin-1 interaction, anti-LRP/LR specific antibody IgG1-iS18 may act as a possible alternative therapeutic tool for metastatic breast and oesophageal cancer treatment.

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. Flow cytometric analysis revealed that 97% of HUVE cells display cell surface LRP/LR. An angiogenesis assay was conducted employing HUVE cells seeded on the basement membrane reconstituent Matrigel™ supplemented with the pro-angiogenic factor vascular endothelial growth factor (VEGF). Post 18h incubation at 37°C tubular structures, namely tube lengths were assessed. Treatment of established tubular structures with 100 µg/ml anti-LRP/LR specific antibody completely blocked angiogenesis. Our findings suggest a central role of the 37kDa/67kDa LRP/LR in tube formation and recommends anti-LRP/LR specific antibodies as potential therapeutic tools for treatment of tumour angiogenesis.

Anti-LRP/LR specific antibody IgG1-iS18 and knock-down of LRP/LR by shRNAs rescue cells from Aß42 induced cytotoxicity.

Alzheimer's disease (AD) is characterized by neurofibrillary tangles, senile plaques and neuronal loss. Amyloid beta (Aß) is proposed to elicit neuronal loss through cell surface receptors. As Aß 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. An LRP/LR-Αß42 interaction was assessed by immunofluorescence microscopy and pull down assays. The cell biological effects were investigated by 3-(4,5-Dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide and Bromodeoxyuridine assays. LRP/LR and Αß42 co-localised on the cell surface and formed immobilized complexes suggesting an interaction. Antibody blockade by IgG1-iS18 and shRNA mediated down regulation of LRP/LR significantly enhanced cell viability and proliferation in cells co-treated with Αß42 when compared to cells incubated with Αß42 only. Results suggest that LRP/LR is implicated in Αß42 mediated cytotoxicity and that anti-LRP/LR specific antibodies and shRNAs may serve as potential therapeutic tools for AD.

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 (Aß) peptide is the predominant candidate aetiological agent and is generated through the sequential proteolytic cleavage of the Amyloid Precursor Protein (APP) by beta (ß) and gamma (γ) secretases. Since the cellular prion protein (PrP(c)) has been shown to regulate Aß shedding, we investigated whether the cellular receptor for PrP(c), namely the 37 kDa/67 kDa Laminin Receptor (LRP/LR) played a role in Aß shedding. Here we show that LRP/LR co-localises with the AD relevant proteins APP, ß- and γ-secretase, respectively. Antibody blockage and shRNA knock-down of LRP/LR reduces Aß shedding, due to impediment of ß-secretase activity, rather than alteration of APP, ß- and γ-secretase levels. These findings indicate that LRP/LR contributes to Aß shedding and recommend anti-LRP/LR specific antibodies and shRNAs as novel therapeutic tools for AD treatment.

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. Adhesion of lung cancer cells, cervical cancer cells, colon cancer cells and prostate cancer cells to laminin-1 is significantly reduced, employing the anti-LRP/LR-specific antibody IgG1-iS18. Invasion of these cell lines into the Matrigel™ matrix was significantly impeded with IgG1-iS18. The Pearson's correlation coefficient proves a correlation between LRP/LR cell-surface levels and invasion potential, as well as adhesion and invasion, respectively. Our findings suggest that IgG1-iS18 antibody might act as alternative therapeutic tool for treatment of various metastatic cancer types.