Biomedical subjects

Hinrich P Hansen

Publications and source records attributed to Hinrich P Hansen.

10 recordsLinked to original sources

PubMed2007

ADAM10 inhibition of human CD30 shedding increases specificity of targeted immunotherapy in vitro.

CD30 is a transmembrane protein selectively overexpressed on many human lymphoma cells and therefore an interesting target for antibody-based immunotherapy. However, binding of therapeutic antibodies stimulates a juxtamembrane cleavage of CD30 leading to a loss of target antigen and an enhanced release of the soluble ectodomain of CD30 (sCD30). Here, we show that sCD30 binds to CD30 ligand (CD153)-expressing non-target cells. Because antibodies bind to sCD30, this results in unwanted antibody binding to these cells via sCD30 bridging. To overcome shedding-dependent damage of normal cells in CD30-specific immunotherapy, we analyzed the mechanism involved in the release. Shedding of CD30 can be enhanced by protein kinase C (PKC) activation, implicating the disintegrin metalloproteinase ADAM17 but not free cytoplasmic calcium. However, antibody-induced CD30 shedding is calcium dependent and PKC independent. This shedding involved the related metalloproteinase ADAM10 as shown by the use of the preferential ADAM10 inhibitor GI254023X and by an ADAM10-deficient cell line generated from embryonically lethal ADAM10(-/-) mouse. In coculture experiments, the antibody-induced transfer of sCD30 from the human Hodgkin's lymphoma cell line L540 to the CD30-negative but CD153-expressing human mast cell line HMC-1 was inhibited by GI254023X. These findings suggest that selective metalloproteinase inhibitors blocking antibody-induced shedding of target antigens could be of therapeutic value to increase the specificity and reduce side effects of immunotherapy with monoclonal antibodies.

ADAM Proteins↗

PubMed2005

A novel bispecific protein (ULBP2-BB4) targeting the NKG2D receptor on natural killer (NK) cells and CD138 activates NK cells and has potent antitumor activity against human multiple myeloma in vitro and in vivo.

The inability of the immune system to recognize and kill malignant plasma cells in patients with multiple myeloma (MM) has been attributed in part to the ineffective activation of natural killer (NK) cells. In order to activate and target NK cells to the malignant cells in MM we designed a novel recombinant bispecific protein (ULBP2-BB4). While ULBP2 binds the activating NK receptor NKG2D, the BB4 moiety binds to CD138, which is overexpressed on a variety of malignancies, including MM. ULBP2-BB4 strongly activated primary NK cells as demonstrated by a significant increase in interferon-gamma (IFN-gamma) secretion. In vitro, ULBP2-BB4 enhanced the NK-mediated lysis of 2 CD138+ human MM cell lines, U-266 and RPMI-8226, and of primary malignant plasma cells in the allogenic and autologous setting. Moreover, in a nude mouse model with subcutaneously growing RPMI-8226 cells, the cotherapy with ULBP-BB4 and human peripheral blood lymphocytes abrogated the tumor growth. These data suggest potential clinical use of this novel construct in patients with MM. The use of recombinant NK receptor ligands that target NK cells to tumor cells might offer new approaches for other malignancies provided a tumor antigen-specific antibody is available.

Animals↗

PubMed2004

Metalloproteinase inhibition augments antitumor efficacy of the anti-CD30 immunotoxin Ki-3(scFv)-ETA' against human lymphomas in vivo.

There is increasing evidence that the shedding of extracellular antigen domains impedes selective immunotherapy. One example is CD30, which is overexpressed on the surface of malignant lymphoma cells and has been identified as a promising target for antibody-based immunotherapy. However, CD30 is cleaved from the surface of target cells and the resulting soluble ectodomain (sCD30) is protecting the cells from antibody binding. Shedding can be inhibited by hydroxamate inhibitors of metalloproteinases such as BB-3644. We thus evaluated the influence of BB-3644 on the efficacy of the anti-CD30 single-chain immunotoxin Ki-3(scFv)-ETA'. In vitro, the addition of BB-3644 augmented the antitumor effect of Ki-3(scFv)-ETA' against Hodgkin-derived L540Cy cells by a factor of 2.75. Severe combined immunodeficiency (SCID) mice challenged with CD30-positive L540Cy cells were treated with the immunotoxin. One single nontoxic dose of BB-3644 increased the mean survival time of animals treated concomitantly with Ki-3(scFv)-ETA' to 93 days as compared with 35 days in the control (p = 0.0017). When BB-3644 was continuously delivered using subcutaneously implanted pumps, this effect was even more pronounced with no observed tumor growth in the animals within 200 days. Thus, concomitant application of metalloproteinase inhibitors might become clinically relevant in antibody-based immunotherapy against targets known to be shed from tumor cells.

Aminopyridines↗

PubMed2004

Depletion of cellular cholesterol and lipid rafts increases shedding of CD30.

CD30, a lymphoid activation marker, is shed into the cell environment after endoproteolytic cleavage of its ectodomain. Soluble (s)CD30 is able to suppress the Th1-type immune response. Because high serum levels of sCD30 and cholesterol-lowering drugs seem to be beneficial in some Th1-type autoimmune diseases, we focused on a link between CD30 shedding and the amount of cellular cholesterol. Cholesterol depletion of human Hodgkin lymphoma- and non-Hodgkin lymphoma-derived cell lines by methyl-beta-cyclodextrin led to a down-regulation of membrane-bound CD30 and increased release of sCD30. Additionally, the cholesterol-interfering drugs lovastatin, cholesterol oxidase, and filipin increased CD30 shedding. Both the down-regulation of membrane-anchored CD30 and the release of sCD30 were dependent on metalloproteinases. Using specific inhibitors, we detected TNF-alpha converting enzyme (TACE) as the leading enzyme responsible for cholesterol-dependent CD30 shedding. A Triton X-100-based method for lipid raft isolation revealed that CD30 was partially present in lipid rafts, whereas TACE was localized in the nonraft fractions. Disintegration of lipid rafts by cholesterol depletion might therefore lead to dynamic interactions of CD30 with TACE, resulting in enhanced shedding of CD30. Our results suggest a possible role of cholesterol-dependent shedding of CD30 in the pathogenesis of immune diseases.

ADAM Proteins↗

PubMed2004

The ectodomain shedding of CD30 is specifically regulated by peptide motifs in its cysteine-rich domains 2 and 5.

Tumor necrosis factor (TNF)-alpha converting enzyme (TACE) is responsible for the ectodomain release of various membrane proteins by proteolytic cleavage in close proximity to the cell membrane. Despite the wide spectrum of possible substrates, selective cleavage can be achieved by substrate cross-linking. To explore the underlying mechanism, we studied the TACE-mediated shedding of CD30. Whereas the constitutive release of the soluble ectodomain of CD30 (sCD30) from the lymphoma cell line Karpas 299 was enhanced by most anti-CD30 antibodies, it was inhibited by antibodies Ber-H2 and Ki-4. On the basis of the recognized epitopes, shedding seemed to depend on the availability of the cysteine-rich domains (CRD) 2 and 5 of the CD30 ectodomain. CRD2 and 5 have almost identical amino acid sequences and are localized distant from the TACE-targeted cleavage site. Soluble CD30, the product of this enzyme reaction, did not inhibit, but on the contrary, it stimulated CD30 shedding in a CRD2/5-dependent manner. This process could also be induced by CRD2/5-derived peptides but not by a CRD1-derived control peptide. This example of a product-activation was CD30 selective since other TACE substrates such as TNFR1 or TNF-alpha were not affected. These data suggest that CD30 shedding is stimulated by an elevated local availability of CRD2 or 5, possibly by forming a docking station for the releasing enzyme through substrate aggregation.

ADAM Proteins↗

PubMed2004

Selective killing of B-cell hybridomas targeting proteinase 3, Wegener's autoantigen.

Wegener's granulomatosis (WG) is a rare disease characterized by granulomatous lesions, small vessel vasculitis and the presence of anti-neutrophil cytoplasmic autoantibodies (C-ANCAs) in the sera of affected patients. Their main target antigen is proteinase 3 (PR3), a neutrophil and monocyte-derived neutral serine protease. Since the standard treatment of this severe autoimmune disease, with cyclophosphamide and corticosteroids, is associated with potential side-effects, the development of a more specific immunotherapeutic agent is warranted. The key role of ANCA in the pathogenesis of vasculitis and the effectiveness of anti-CD20 antibodies in patients with refractory WG points towards the importance of B cells in WG. We thus evaluated a new approach to selectively eliminate PR3-specific autoreactive B cells by targeting the B-cell receptor. For this purpose we used a bifunctional recombinant fusion protein consisting of the antigen PR3 and a toxin. The cytotoxic component of this novel fusion protein was the ribonuclease angiogenin, a human toxin with low immunogenicity. The toxin was stabilized by exchanging the catalytically relevant histidine in position 44 with glutamine to eliminate the autoproteolytic activity. PR3H44Q was fused either to the N terminus or to the C terminus of angiogenin. The recombinant proteins were expressed in 293T cells. Binding assays demonstrated the appropriate size and recognition by anti-PR3 antibodies. Using TUNEL technology, we demonstrated that these autoantigen toxins kill proteinase 3-specific B-cell hybridomas selectively by inducing apoptosis. The data indicate that autoantigen-toxins are promising tools in the treatment or co-treatment of autoimmune diseases in which the antigen is known.

Antibodies, Monoclonal↗

PubMed2002

Inhibition of metalloproteinases enhances the internalization of anti-CD30 antibody Ki-3 and the cytotoxic activity of Ki-3 immunotoxin.

CD30 is selectively expressed on the tumor cells of a variety of malignant disorders of the immune system and can therefore be used as a target for an anti-CD30 antibody-based immunotherapy. However, CD30 is cleaved at the cell surface by tumor necrosis factor-alpha converting enzyme (TACE). This metalloproteinase releases the soluble ectodomain of CD30 (sCD30), which is able to neutralize immunotherapeutic agents before these reach their target cells. Such constitutive CD30 cleavage is enhanced after binding of most anti-CD30 antibodies, leading to a downregulation of CD30 and an increase of sCD30 in the cell environment. Here, we demonstrate that CD30 shedding from the cell line Karpas 299 could effectively be blocked by the hydroxamic acid-based metalloproteinase inhibitors BB-3644 (IC50 = 180 nM), BB-2116 (IC50 = 230 nM), BB-94 (batimastat, IC50 = 230 nM) and BB-2516 (marimastat, IC50 = 1 microM). This inhibition reduced the concentration of sCD30 in the cell environment to the background level, prolonged the persistence of the anti-CD30 antibody Ki-3 on Karpas 299 cells and favored its internalization. Moreover, a nontoxic concentration of the inhibitor BB-3644 significantly increased the cytotoxic activity of the anti-CD30 ricin A-chain immunotoxin Ki-3.dgA towards the CD30(+) Hodgkin-derived cell line L540. Hence, the metalloproteinase inhibitor BB-3644 may be a promising compound to improve the immunotherapy of CD30(+) malignancies.

Aminopyridines↗

PubMed2002

Vitronectin- and fibronectin-containing immune complexes in primary systemic vasculitis.

In primary systemic vasculitis anti endothelial cell autoantibodies (AECA) have been described frequently. They represent a heterogeneous group of autoantibodies whose target antigens are mostly unknown. We tried to find AECA-antigens by a co-operative binding assay with a panel of monoclonal antibodies (mAb) directed to human umbilical vein endothelial cells (HUVEC) and extracellular matrix proteins. The mAb were used to bind antigens from lysate of endothelial cells, and binding of human antibodies to these antigens was measured. mAb directed to Vitronectin (VN) and Fibronectin (FN) resulted in enhanced binding of antibodies in sera from patients with Churg Strauss Syndrome (CSS) and Wegener's Granulomatosis (WG) compared to normal sera. Neither free autoantibodies against VN or FN could be detected nor did the addition of endothelial cell lysate influence the binding activity from the patients' sera. This suggests that preformed VN and FN-containing immune complexes (IC) are present in the patient sera. The amount of IC was decreased by incubation with HUVEC, demonstrating that these IC can bind to endothelial cells. However, their involvement in the pathogenesis of the disease is not clearly defined. Our data suggest that there are preformed IC present in sera of patients with CSS and WG that contain VN and FN and bind to endothelial cells.

Antibodies, Monoclonal↗

PubMed2002

Soluble CD30 plasma concentrations correlate with disease activity in patients with atopic dermatitis.

The levels of soluble CD30 in 79 patients with atopic dermatitis were compared with those found in 54 patients with psoriasis and 36 control individuals (no psoriasis, no atopic dermatitis). In relation to the control group, patients with atopic dermatitis were found to exhibit an increased concentration of sCD30 of at least 1.5-fold (p < 0.001). In addition, sCD30 concentrations were shown to correlate with the severity of the disease as measured by the score index for atopic dermatitis and different stages of disease activity, such as acute, subacute, or chronic forms, and localized or generalized distribution of atopic dermatitis. The application of topical glucocorticoid therapy for a period of 2 weeks resulted in a decrease in the level of sCD30 by 46% in 8 patients, especially in the acute, generalized form of atopic dermatitis. Psoriasis patients showed no significant differences in sCD30 levels in relation to the control group. This study demonstrates a correlation between sCD30 concentration and the activity of the disease and therefore suggests sCD30 as a prognostic marker, being superior to predictions from measurements of IgE or eosinophil cationic protein.

Administration, Topical↗

PubMedDate not supplied

Human CD30: structural implications from epitope mapping and modeling studies.

The human CD30 molecule is expressed transiently at very low levels on intrafollicular and perifollicular T and B cell blasts in lymphoid tissues, but is specifically upregulated on certain tumor cells, e.g. Hodgkin and Reed-Sternberg (H-RS) cells. With its specific expression pattern and easy accessibility on the surface of H-RS cells CD30 is a valuable diagnostic marker and holds considerable promise as a target for in vivo immunotherapy. Knowledge of epitopes on the CD30 molecule is expected to facilitate the design of novel non-immunogenic anti-CD30 reagents. Therefore, we have mapped the epitopes of several monoclonal antibodies (mAb) applying a peptide array of overlapping CD30-derived peptides. For the mAb Ber-H2, two linear epitopes with identical sequence were found, while the mAb Ki-2 and the single chain Fv fragment R4-4 each recognized a single linear antigenic determinant, respectively. On the other hand, the mAb Ki-1 bound to a discontinuous epitope composed of two regions, one located near the N-terminus and the other near the membrane-spanning region of CD30. Using molecular modeling, it was possible to visualize the location of the epitopes on exposed loop regions of the molecule within the N-terminal domain. Finally, the results obtained with the mAb Ki-1 imply that the ends of the N- and C-terminal parts of the extracellular portion of CD30 are in close vicinity of each other, suggesting a flower-like structure for the membrane-bound homotrimeric CD30 molecule.

Alanine↗