Genetically engineered attenuated measles virus specifically infects and kills primary multiple myeloma cells

The applicability of cytoreductive treatment of malignant diseases using recombinant viruses strongly depends on specific recognition of surface receptors to target exclusively neoplastic cells. A recently generated monoclonal antibody (mAb), Wue-1, specifically detects CD138+ multiple myeloma (MM) cells. In this study, a haemagglutinin (H) protein that was receptor-blinded (i.e. did not bind to CD46 and CD150) was genetically re-engineered by fusing it to a single-chain antibody fragment (scFv) derived from the Wue-1 mAb open reading frame (scFv-Wue), resulting in the recombinant retargeted measles virus (MV)-Wue. MV-Wue efficiently targeted and fully replicated in primary MM cells, reaching titres similar to those seen with non-retargeted viruses. In agreement with its altered receptor specificity, infection of target cells was no longer dependent on CD150 or CD46, but was restricted to cells that had been labelled with Wue-1 mAb. Importantly, infection with MV-Wue rapidly induced apoptosis in CD138+ malignant plasma cell targets. MV-Wue is the first fully retargeted MV using the restricted interaction between Wue-1 mAb and primary MM cells specifically to infect, replicate in and deplete malignant plasma cells.

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Fully Retargeted Oncolytic Measles Virus for Multiple Myeloma Therapy.

Blood ◽

10.1182/blood.v108.11.5474.5474 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5474-5474

Author(s):

Horst D. Hummel ◽

Gaby Kuntz ◽

Takafumi Nakamura ◽

Axel Greiner ◽

Stephen J. Russell ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Cell Lines ◽

Plasma Cell ◽

Measles Virus ◽

Plasma Cells ◽

Treatment Modalities ◽

Single Chain ◽

Gfp Expression ◽

Frequent Relapses

Abstract Multiple Myeloma (MM) is a disseminated plasma cell malignancy with approximately 14,600 new cases diagnosed in the USA annually. Despite recent progress in current therapeutical options the median survival is 3 to 5 years and cure is extremely rare. Therefore the evaluation of new treatment modalities for MM is highly warranted. An attractive approach to treat Myeloma with a minimum of undesired side effects is the use of a tumour antigen specific for MM cells. Wue-1, a monoclonal antibody binds very selectively normal and malignant plasma cells (50 of 51 MM samples, 14 of 15 immunocytoma and 13 of 13 MALT type lymphomas with plasma cell differentiation were Wue-1 positive, normal tissue including hematopoietic cells were negative) and offers the possibility to define MM cells as targets. The tool for selective killing of MM cells recognized by Wue-1 monoclonal antibody is in this study the measles virus vaccine strain Edmonston B in an ablated variant (MV-Wue) which no longer binds the usual measles receptors CD46 and CD150 (SLAM) expressed on almost every human cell type displaying a single-chain antibody (scFv) derived from the monoclonal Wue-1-antibody which has been tethered to the C-terminus of the H protein to restrict and retarget its interaction to malignant plasma cells especially MM cells. In addition, MV-Wue encodes EGFP facilitating the read out of infected cells. To determine if the fully retargeted MV-Wue would be able to infect MM cell lines and primary MM cells selectively an array of infection assays were performed using the MM cell lines U266 as well as primary CD138 positive MM cells expressing the Wue-1 antigen as expected targets and CD138 negative cells and normal B cells as controls negative for Wue-1. In these experiments selective infections of the MM cell line and primary MM cells were observed whereas the control cells were not infected with MV-Wue. In all cell types GFP expression indicating replicative infection correlated with the expression of the Wue-1 antigen determined by FACS. Infection experiments performed in the presence of monoclonal Wue-1 antibody showed a decreased GFP expression of about 78% in CD138 positive MM cells demonstrating specificity of the infection by MV-Wue. These results indicate that the engineered virus can be a safe and potential curative oncolytic agent to face the main problem in Multiple Myeloma which is responsible for frequent relapses, the minimal residual disease (MRD).

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The CD317-Targeted Immunotoxin HM1.24-ETA′ Efficiently Eliminates Malignant Plasma Cells in Vitro and in Vivo

Blood ◽

10.1182/blood.v120.21.1838.1838 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1838-1838

Author(s):

Matthias Staudinger ◽

Pia Glorius ◽

Christian Kellner ◽

Andreas Guenther ◽

Roland Repp ◽

...

Keyword(s):

Multiple Myeloma ◽

Plasma Cells ◽

Annexin V ◽

Treatment Modalities ◽

Parp Cleavage ◽

Target Cells ◽

Single Chain ◽

Promising Target

Abstract Abstract 1838 Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma still needs improvement. Recently antibody-based targeted therapies with a toxic payload have documented impressing activity. HM1.24 (CD317), a surface molecule overexpressed on malignant plasma cells, is efficiently internalized and may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the generation and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described. HM1.24-ETA′ was generated by genetic fusion of a CD317-specific single-chain Fv antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and the protein was purified to homogeneity by affinity chromatography. HM1.24-ETA′ efficiently inhibited growth of myeloma cell lines (INA-6, RPMI8226, U266) analyzed in MTT assays. Half maximal growth inhibition was observed at low nanomolar concentrations. Target cell killing occurred via induction of apoptosis as indicated by annexin V / propidium iodide staining and analysis of PARP cleavage. The delivery of HM1.24-ETA′ to target cells is antigen-specific, because excess of unconjugated parental antibody completely blocked the cytotoxic effect. The proliferation of IL-6 dependent INA-6 was efficiently reduced by HM1.24-ETA′ even in co-culture experiments with bone marrow stromal cells that otherwise strongly support tumor cell growth. Importantly, HM1.24-ETA′ strongly triggered apoptosis (up to 80% annexin V-positive cells) of freshly isolated tumor cells from 5 of 5 myeloma patients. In a xenograft SCID mouse model, establishment of INA-6 plasma cell tumors was efficiently abrogated by treatment with HM1.24-ETA′ immunotoxin (p < 0.04). Thus, HM1.24-ETA′ immunotoxin in vitro and in the preclinical xenograft model in vivo demonstrates that the CD317 antigen may represent a promising target structure for immunotherapy of multiple myeloma using immunoconjugates with toxic payloads. Disclosures: No relevant conflicts of interest to declare.

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Llama Single-Chain Antibody That Blocks Lipopolysaccharide Binding and Signaling: Prospects for Therapeutic Applications

Clinical and Vaccine Immunology ◽

10.1128/cvi.00107-06 ◽

2006 ◽

Vol 13 (10) ◽

pp. 1079-1086 ◽

Cited By ~ 25

Author(s):

Mohamed El Khattabi ◽

Hendrik Adams ◽

Erik Heezius ◽

Pim Hermans ◽

Frank Detmers ◽

...

Keyword(s):

Inner Core ◽

Antibody Fragment ◽

Target Cells ◽

Gram Negative Bacteria ◽

Single Chain ◽

Therapeutic Approaches ◽

Single Chain Antibody ◽

Legitimate Target ◽

Single Chain Antibody Fragment ◽

Lps Signaling

ABSTRACT Sepsis is a considerable health problem and a burden on the health care system. Endotoxin, or lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria, is responsible for more than 50% of the sepsis cases and is, therefore, a legitimate target for therapeutic approaches against sepsis. In this study, we selected and characterized a llama single-chain antibody fragment (VHH) directed to Neisseria meningitidis LPS. The VHH, designated VHH 5G, showed affinity to purified LPS as well as to LPS on the surfaces of the bacteria. Epitope mapping using a panel of N. meningitidis mutants revealed that VHH 5G recognizes an epitope in the inner core of LPS, and as expected, the VHH proved to have broad specificity for LPS from different bacteria. Furthermore, this VHH blocked binding of LPS to target cells of the immune system, resulting in the inhibition of LPS signaling in whole blood. Moreover, it was found to remove LPS efficiently from aqueous solutions, including serum. The selected anti-LPS VHH is a leading candidate for therapies against LPS-mediated sepsis.

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MeV-Stealth: A CD46-specific oncolytic measles virus resistant to neutralization by measles-immune human serum

PLoS Pathogens ◽

10.1371/journal.ppat.1009283 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009283 ◽

Cited By ~ 1

Author(s):

Miguel Ángel Muñoz-Alía ◽

Rebecca A. Nace ◽

Alexander Tischer ◽

Lianwen Zhang ◽

Eugene S. Bah ◽

...

Keyword(s):

Measles Virus ◽

Malignant Tumors ◽

Antibody Fragment ◽

Immune Serum ◽

Single Chain ◽

Single Chain Antibody ◽

Tumor Bearing ◽

20 Nm ◽

Single Chain Antibody Fragment

The frequent overexpression of CD46 in malignant tumors has provided a basis to use vaccine-lineage measles virus (MeV) as an oncolytic virotherapy platform. However, widespread measles seropositivity limits the systemic deployment of oncolytic MeV for the treatment of metastatic neoplasia. Here, we report the development of MeV-Stealth, a modified vaccine MeV strain that exhibits oncolytic properties and escapes antimeasles antibodies in vivo. We engineered this virus using homologous envelope glycoproteins from the closely-related but serologically non-cross reactive canine distemper virus (CDV). By fusing a high-affinity CD46 specific single-chain antibody fragment (scFv) to the CDV-Hemagglutinin (H), ablating its tropism for human nectin-4 and modifying the CDV-Fusion (F) signal peptide we achieved efficient retargeting to CD46. A receptor binding affinity of ~20 nM was required to trigger CD46-dependent intercellular fusion at levels comparable to the original MeV H/F complex and to achieve similar antitumor efficacy in myeloma and ovarian tumor-bearing mice models. In mice passively immunized with measles-immune serum, treatment of ovarian tumors with MeV-Stealth significantly increased overall survival compared with treatment with vaccine-lineage MeV. Our results show that MeV-Stealth effectively targets and lyses CD46-expressing cancer cells in mouse models of ovarian cancer and myeloma, and evades inhibition by human measles-immune serum. MeV-Stealth could therefore represent a strong alternative to current oncolytic MeV strains for treatment of measles-immune cancer patients.

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Simultaneous Silencing of Two Independent Signaling Pathways Essential for Angiogenesis Using Bispecific, Tetravalent Intra-Diabodies.

Blood ◽

10.1182/blood.v104.11.5284.5284 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5284-5284

Author(s):

Nina Jendreyko ◽

Mikhail Popkov ◽

Christoph Rader ◽

Carlos F. Barbas ◽

Gerhard Gaedicke

Keyword(s):

Signaling Pathways ◽

Antibody Fragment ◽

Single Chain ◽

Single Chain Antibody ◽

Alternative Pathways ◽

Surface Receptors ◽

Surface Depletion ◽

Precise Tool

Abstract Vascular endothelial growth factor and its receptors (VEGF-R) have been implicated in promoting tumor growth and metastasis. Emerging evidence suggest that angiogenesis also play an important role in the growth of leukemias and lymphomas. Antiangiogenic therapies that interfere with the VEGF/VEGF-R pathways represent novel approaches to effective treatment for certain leukemia, such as myeloid leukemias. But not all types of tumors respond to interruption of the VEGF/VEGF-R pathway. This suggest that alternative pathways for vascular growth exist and can drive tumor angiogenesis. Intrabodies have been used to specifically target intracellular proteins and manipulate biological processes,as well as for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. In order to generate a precise tool for the simultaneous silencing ot two independent signaling pathways essential for angiogenesis, we developed an endoplasmatic-reticulum (ER) targeted bispecific, tetravalent intrabody format, targeting VEGF-R2 and Tie-2. Comparison of the ER-targeted intradiabody with the corresponding conventional ER-targeted single-chain antibody fragment (scFv) intrabodies showed that the intradiabody is significantly more efficient with respect to efficiency and duration of surface depletion of VEGF-R2 and Tie-2. In vitro and in vivo data demonstrate the development of a very effective antiangiogenic intrabody format for the simultaneous functional knockout of two cell surface receptors using an adenovirus-mediated gene delivery system. Our findings suggest that simultaneous interference with the VEGF and the Tie-2 receptor pathway results in at least additive antiangiogenic effects. Our study provides a novel antiangiogenic therapeutic tool for the silencing of two independent signaling pathways involved in angiogenesis.

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