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1.
Pharm Res ; 35(6): 118, 2018 Apr 17.
Article in English | MEDLINE | ID: mdl-29666962

ABSTRACT

PURPOSE: Triple-negative breast cancer (TNBC) is related with a poor prognosis as patients do hardly benefit from approved therapies. CD138 (Syndecan-1) is upregulated on human breast cancers. Indatuximab ravtansine (BT062) is an antibody-drug-conjugate that specifically targets CD138-expressing cells and has previously shown clinical activity in multiple myeloma. Here we show indatuximab ravtansine as a potential mono- and combination therapy for TNBC. METHODS: The effects of indatuximab ravtansine were assessed in vitro in SK-BR-3 and T47D breast cancer cell lines. The in vivo effects of indatuximab ravtansine alone and in combination with docetaxel or paclitaxel were assessed in MAXF401, MAXF1384 and MAXF1322 xenograft TNBC models. RESULTS: CD138+ SK-BR-3 and T47D cells were highly sensitive to indatuximab ravtansine. The high CD138-expressing MAXF401 xenograft model demonstrated strong inhibition of tumor growth with 4 mg/kg indatuximab ravtansine. High doses of indatuximab ravtansine (8 mg/kg), docetaxel and the combination of both led to complete remission. In the low CD138-expressing MAXF1384 xenograft model, only combination of indatuximab ravtansine and docetaxel demonstrated a significant efficacy. In the MAXF1322 xenograft model, indatuximab ravtansine alone and in combination with paclitaxel elicited complete remission. CONCLUSIONS: These data demonstrate potential use of indatuximab ravtansine in combination with docetaxel or paclitaxel for CD138-positive TNBC.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Immunoconjugates/pharmacology , Syndecan-1/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast/pathology , Cell Line, Tumor , Docetaxel/pharmacology , Docetaxel/therapeutic use , Female , Humans , Immunoconjugates/therapeutic use , Maytansine/analogs & derivatives , Maytansine/pharmacology , Maytansine/therapeutic use , Mice , Mice, Nude , Paclitaxel/therapeutic use , Syndecan-1/metabolism , Treatment Outcome , Triple Negative Breast Neoplasms/pathology , Xenograft Model Antitumor Assays
2.
J Hematol Oncol ; 10(1): 13, 2017 01 11.
Article in English | MEDLINE | ID: mdl-28077160

ABSTRACT

Indatuximab ravtansine is a monoclonal antibody-linked cytotoxic agent that specifically targets CD138-expressing cells. Monotherapy has been shown to significantly inhibit multiple myeloma tumour growth in vivo and improve host survival. Here, we show that in most cell lines tested, indatuximab ravtansine acts additively or even synergistically with clinically approved therapies for treatment of multiple myeloma. In addition, in vivo mouse xenograft models confirmed the activity of indatuximab ravtansine in combination with lenalidamide and lenalidomide/dexamethasone. Indatuximab ravtansine may therefore be a suitable combination partner for multiple myeloma, and a clinical study is ongoing.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Immunoconjugates/therapeutic use , Multiple Myeloma/drug therapy , Animals , Dexamethasone/therapeutic use , Drug Evaluation, Preclinical , Humans , Lenalidomide , Maytansine/analogs & derivatives , Maytansine/therapeutic use , Mice , Syndecan-1/antagonists & inhibitors , Syndecan-1/immunology , Thalidomide/analogs & derivatives , Thalidomide/therapeutic use
3.
J Cell Mol Med ; 20(7): 1287-94, 2016 07.
Article in English | MEDLINE | ID: mdl-27008316

ABSTRACT

Many B-cell acute and chronic leukaemias tend to be resistant to killing by natural killer (NK) cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance. Here, we extend our previous observations on the resistance of malignant lymphoblasts to NK-92 cells, a continuously growing NK cell line, showing that anti-CD19-CAR (αCD19-CAR) engineered NK-92 cells can regain significant cytotoxicity against CD19 positive leukaemic cell lines and primary leukaemia cells that are resistant to cytolytic activity of parental NK-92 cells. The 'first generation' CAR was generated from a scFv (CD19) antibody fragment, coupled to a flexible hinge region, the CD3ζ chain and a Myc-tag and cloned into a retrovirus backbone. No difference in cytotoxic activity of NK-92 and transduced αCD19-CAR NK-92 cells towards CD19 negative targets was found. However, αCD19-CAR NK-92 cells specifically and efficiently lysed CD19 expressing B-precursor leukaemia cell lines as well as lymphoblasts from leukaemia patients. Since NK-92 cells can be easily expanded to clinical grade numbers under current Good Manufactoring Practice (cGMP) conditions and its safety has been documented in several phase I clinical studies, treatment with CAR modified NK-92 should be considered a treatment option for patients with lymphoid malignancies.


Subject(s)
B-Lymphocytes/immunology , Genetic Engineering , Killer Cells, Natural/immunology , Lymphoma, B-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Cell Line, Tumor , Cell Lineage , Cytotoxicity, Immunologic , Humans , Lymphoma, B-Cell/pathology , Retroviridae/metabolism , Single-Chain Antibodies/metabolism , Tissue Donors , Transfection
4.
Immunol Cell Biol ; 93(4): 396-405, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25512343

ABSTRACT

CD4(+)CD25(+) regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.


Subject(s)
Antibodies, Monoclonal/pharmacology , CD4 Antigens/metabolism , Epitopes, B-Lymphocyte/metabolism , Immunosuppressive Agents/pharmacology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Amino Acid Sequence , Antibodies, Monoclonal, Humanized , Cells, Cultured , Crystallography, X-Ray , Humans , Interleukin-2 Receptor alpha Subunit/metabolism , Lymphocyte Activation , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Conformation , Signal Transduction , Transforming Growth Factor beta/metabolism
5.
J Cell Mol Med ; 16(3): 569-81, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21595822

ABSTRACT

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.


Subject(s)
Antibody-Dependent Cell Cytotoxicity/immunology , Jejunal Neoplasms/therapy , Killer Cells, Natural/metabolism , Neuroblastoma/therapy , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , CD3 Complex/genetics , CD3 Complex/immunology , Cell Line, Tumor , Child , Gangliosides/genetics , Gangliosides/immunology , Gene Expression , Genetic Engineering , Genetic Vectors , Humans , Immunotherapy, Adoptive , Jejunal Neoplasms/immunology , Jejunal Neoplasms/secondary , Jejunum/immunology , Jejunum/pathology , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Neuroblastoma/immunology , Neuroblastoma/secondary , Receptors, Antigen/genetics , Receptors, Antigen/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Retroviridae , Transduction, Genetic
6.
Clin Cancer Res ; 15(12): 4028-37, 2009 Jun 15.
Article in English | MEDLINE | ID: mdl-19509164

ABSTRACT

PURPOSE: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. EXPERIMENTAL DESIGN: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). RESULTS: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G(2)-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. CONCLUSION: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Phytogenic/therapeutic use , Immunoconjugates/therapeutic use , Maytansine/therapeutic use , Multiple Myeloma/drug therapy , Syndecan-1/immunology , Animals , Bone Marrow Cells/metabolism , Cell Line, Tumor , Humans , Insulin-Like Growth Factor I/metabolism , Interleukin-6/metabolism , Mice , Mice, SCID , Multiple Myeloma/immunology , Multiple Myeloma/pathology , Stromal Cells/metabolism , Xenograft Model Antitumor Assays
7.
Nucl Med Biol ; 35(5): 579-88, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18589302

ABSTRACT

INTRODUCTION: The objective of this study was to label the human natural killer (NK) cell line NK-92 with [(18)F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. METHODS: NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [(18)F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [(18)F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. RESULTS: The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [(18)F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x10(6) [(18)F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x10(6) NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. CONCLUSION: The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [(18)F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography.


Subject(s)
Fluorodeoxyglucose F18 , Killer Cells, Natural/diagnostic imaging , Neoplasms, Experimental/diagnostic imaging , Neoplasms, Experimental/genetics , Radiopharmaceuticals , Receptor, ErbB-2/genetics , 3T3 Cells , Animals , Autoradiography , CD57 Antigens/metabolism , Cell Line, Tumor , Female , Humans , Hypoglycemic Agents/pharmacology , Immunohistochemistry , Insulin/pharmacology , Mice , Mice, Inbred C57BL , Mice, Nude , Monocytes/diagnostic imaging , Neoplasm Transplantation , Protein Engineering , Radionuclide Imaging
8.
Cancer Immunol Immunother ; 57(3): 411-23, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17717662

ABSTRACT

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.


Subject(s)
Antigens, CD20/immunology , Cytotoxicity, Immunologic/immunology , Killer Cells, Natural/immunology , Leukemia/immunology , Lymphoma/immunology , Receptors, Antigen, B-Cell/biosynthesis , Animals , Cell Proliferation , Cytotoxicity, Immunologic/genetics , Humans , Immunoglobulin Variable Region/immunology , Kinetics , Leukemia/pathology , Lymphoma/pathology , Mice , Mice, Inbred NOD , Mice, SCID , NIH 3T3 Cells , Receptors, Antigen, B-Cell/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Tumor Cells, Cultured
9.
Eur Radiol ; 15(1): 4-13, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15616814

ABSTRACT

The purpose of this study is to optimize labeling of the human natural killer (NK) cell line NK-92 with iron-oxide-based contrast agents and to monitor the in vivo distribution of genetically engineered NK-92 cells, which are directed against HER2/neu receptors, to HER2/neu positive mammary tumors with magnetic resonance (MR) imaging. Parental NK-92 cells and genetically modified HER2/neu specific NK-92-scFv(FRP5)-zeta cells, expressing a chimeric antigen receptor specific to the tumor-associated ErbB2 (HER2/neu) antigen, were labeled with ferumoxides and ferucarbotran using simple incubation, lipofection and electroporation techniques. Labeling efficiency was evaluated by MR imaging, Prussian blue stains and spectrometry. Subsequently, ferucarbotran-labeled NK-92-scFv(FRP5)-zeta (n=3) or parental NK-92 cells were intravenously injected into the tail vein of six mice with HER2/neu-positive NIH 3T3 mammary tumors, implanted in the mammary fat pad. The accumulation of the cells in the tumors was monitored by MR imaging before and 12 and 24 h after cell injection (p.i.). MR data were correlated with histopathology. Both the parental NK-92 and the genetically modified NK-92-scFv(FRP5)-zeta cells could be labeled with ferucarbotran and ferumoxides by lipofection and electroporation, but not by simple incubation. The intracellular cytoplasmatic iron-oxide uptake was significantly higher after labeling with ferucarbotran than ferumoxides (P<0.05). After intravenous injection of 5 x 10(6) NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, MR showed a progressive signal decline in HER2/neu-positive mammary tumors at 12 and 24 h (p.i.). Conversely, injection of 5 x 10(6) parental NK-92 control cells, not directed against HER2/neu receptors, did not cause significant signal intensity changes of the tumors. Histopathology confirmed an accumulation of the former, but not the latter cells in tumor tissue. The human natural killer cell line NK-92 can be efficiently labeled with clinically applicable iron-oxide contrast agents, and the accumulation of these labeled cells in murine tumors can be monitored in vivo with MR imaging. This MR cell tracking technique may be applied to monitor NK-cell based immunotherapies in patients in order to assess the presence and extent of NK-cell tumor accumulations and, thus, to determine therapy response early and non-invasively.


Subject(s)
Killer Cells, Natural/immunology , Magnetic Resonance Imaging , Mammary Neoplasms, Animal/therapy , Receptor, ErbB-2 , Analysis of Variance , Animals , CD56 Antigen/analysis , Cell Movement , Cells, Cultured , Contrast Media , Dextrans , Female , Ferrosoferric Oxide , Genetic Engineering , Iron , Magnetite Nanoparticles , Mammary Neoplasms, Animal/immunology , Mammary Neoplasms, Animal/pathology , Mice , Mice, Inbred BALB C , Oxides
10.
Cancer Immunol Immunother ; 53(3): 217-26, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14704833

ABSTRACT

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells' potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.


Subject(s)
Immunoglobulin Fragments/therapeutic use , Immunotoxins/therapeutic use , Killer Cells, Natural/immunology , T-Lymphocytes, Cytotoxic/immunology , ErbB Receptors/immunology , Genes, erbB-2/immunology , Humans , Recombinant Proteins/therapeutic use
11.
Blood ; 100(4): 1265-73, 2002 Aug 15.
Article in English | MEDLINE | ID: mdl-12149207

ABSTRACT

The continuously growing natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origins without affecting normal human cells. Based on this selectivity, the potential of NK-92 cells for adoptive therapy is currently being investigated in phase I clinical studies. To further enhance the antitumoral activity of NK-92 cells and expand the range of tumor entities suitable for NK-92-based therapies, here by transduction with a retroviral vector we have generated genetically modified NK-92 cells expressing a chimeric antigen receptor specific for the tumor-associated ErbB2 (HER2/neu) antigen, which is overexpressed by many tumors of epithelial origin. The chimeric antigen receptor consists of the ErbB2-specific scFv(FRP5) antibody fragment, a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3 zeta chain. Transduced NK-92-scFv(FRP5)-zeta cells express high levels of the fusion protein on the cell surface as determined by fluorescence-activated cell-scanning (FACS) analysis. In europium release assays, no difference in cytotoxic activity of NK-92 and NK-92-scFv(FRP5)-zeta cells toward ErbB2-negative targets was found. However, even at low effector-to-target ratios, NK-92-scFv(FRP5)-zeta cells specifically and efficiently lysed established and primary ErbB2-expressing tumor cells that were completely resistant to cytolytic activity of parental NK-92 cells. These results demonstrate that efficient retargeting of NK-92 cytotoxicity can be achieved and might allow the generation of potent cell-based therapeutics for the treatment of ErbB2-expressing malignancies.


Subject(s)
Killer Cells, Natural/immunology , Neoplasms/immunology , Receptor, ErbB-2/immunology , Apoptosis , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Breast Neoplasms/therapy , CD3 Complex/genetics , CD8 Antigens/genetics , Cell Division , Cell Line , Cytotoxicity, Immunologic , Flow Cytometry , Humans , Immunoglobulin Variable Region/genetics , Immunotherapy, Adoptive , In Situ Nick-End Labeling , Receptor, ErbB-2/analysis , Receptor, ErbB-2/genetics , Receptors, Antigen/genetics , Recombinant Fusion Proteins , Tumor Cells, Cultured
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