Oncolytic measles viruses displaying a single-chain antibody against CD38, a myeloma cell marker

Live attenuated measles virus (MV-Edm) has potent oncolytic activity against myeloma xenografts in mice. Therapy of multiple myeloma, a disseminated plasma cell malignancy, would require systemic administration of the virus. Thus, the virus should ideally be targeted to infect only myeloma cells to minimize collateral damage to normal tissues: viral binding to its natural receptors must be ablated and a new specificity domain that targets entry into myeloma cells be added. This study covers 2 critical steps toward generating such a retargeted virus: (1) a new specificity domain against the plasma cell marker CD38 was constructed in the form of a single-chain antibody (scFv) and (2) display of that scFv on the measles viral envelope glycoprotein successfully redirected virus entry through CD38 expressed on target cells devoid of the natural MV receptors. The anti-CD38 scFv was tethered to the C-terminus of the hemagglutinin (H) glycoprotein of MV-Edm through a Factor Xa protease cleavable linker. Immunoblot analysis demonstrated that the scFv was efficiently incorporated into recombinant viral particles. Replication of MV-αCD38 was not hindered by the scFv, reaching titers comparable to MV-Edm. Chinese hamster ovary (CHO) cells were resistant to infection by MV-Edm and MV-αCD38. In contrast, CHO cells expressing CD38 became susceptible to infection by MV-αCD38 but not MV-Edm. Removal of the displayed scFv rendered MV-αCD38 noninfectious on CHO-CD38 cells. Tumorigenicity of CHO-CD38 cells in immunocompromised mice was significantly attenuated by MV-αCD38, resulting in enhanced survival of these mice compared with the control group.

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Detection and characterization of syndecan-1-associated heparan sulfate 6-O-sulfated motifs overexpressed in multiple myeloma cells using single chain antibody variable fragments

Human Antibodies ◽

10.3233/hab-2012-0259 ◽

2012 ◽

Vol 21 (1-2) ◽

pp. 29-40 ◽

Cited By ~ 7

Author(s):

Marc Delcommenne ◽

Hans-G. Klingemann

Keyword(s):

Multiple Myeloma ◽

Heparan Sulfate ◽

Single Chain ◽

Single Chain Antibody ◽

Myeloma Cells

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

Journal of General Virology ◽

10.1099/vir.0.007302-0 ◽

2009 ◽

Vol 90 (3) ◽

pp. 693-701 ◽

Cited By ~ 13

Author(s):

Horst-Dieter Hummel ◽

Gabriele Kuntz ◽

Stephen J. Russell ◽

Takafumi Nakamura ◽

Axel Greiner ◽

...

Keyword(s):

Multiple Myeloma ◽

Measles Virus ◽

Plasma Cells ◽

Antibody Fragment ◽

Genetically Engineered ◽

Target Cells ◽

Single Chain ◽

Single Chain Antibody ◽

Reading Frame ◽

Surface Receptors

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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Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell–activating polypeptide

Blood ◽

10.1182/blood.v99.7.2342 ◽

2002 ◽

Vol 99 (7) ◽

pp. 2342-2350 ◽

Cited By ~ 63

Author(s):

Marielle Maurice ◽

Els Verhoeyen ◽

Patrick Salmon ◽

Didier Trono ◽

Stephen J. Russell ◽

...

Keyword(s):

T Cell ◽

Gene Transfer ◽

Gene Delivery ◽

Genetic Material ◽

Lentiviral Vectors ◽

Target Cells ◽

Single Chain ◽

Single Chain Antibody ◽

Blood Lymphocytes ◽

Hiv 1

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1–derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G0 to G1 phases of the cell cycle. Compared to unmodified HIV-1–based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

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Targeting of human immunodeficiency virus-infected cells by CD8+ T lymphocytes armed with universal T-cell receptors

Blood ◽

10.1182/blood.v84.9.2878.bloodjournal8492878 ◽

1994 ◽

Vol 84 (9) ◽

pp. 2878-2889 ◽

Cited By ~ 2

Author(s):

MR Roberts ◽

L Qin ◽

D Zhang ◽

DH Smith ◽

AC Tran ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cell ◽

T Cell Receptors ◽

Target Cells ◽

Single Chain ◽

Single Chain Antibody ◽

Retroviral Transduction ◽

Cell Receptors ◽

Immunodeficiency Virus ◽

Infected Cells

We have developed an immunotherapeutic approach with potential application in the treatment of viral and malignant disease. We show that primary CD8+ T cells isolated from peripheral blood can be genetically modified by retroviral transduction to express high levels of universal (major histocompatibility complex-unrestricted) chimeric T- cell receptors specific for human immunodeficiency virus (HIV) antigens. Two classes of HIV-specific URs in which the antigen-binding domain is comprised of either CD4 or a single-chain antibody are capable of activating a number of T-cell effector functions in response to target cells, including cytolysis, in a highly sensitive and specific manner. Importantly, we have addressed a number of issues which, although particularly relevant to the clinical application of this approach in the treatment of HIV infection, may also impact on the potential of UR immunotherapy for other disease targets. The UR immunotherapeutic system is particularly suited for evaluation in the clinical setting.

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PSIX-14 Active and passive immunity in calves vaccinated with a subunit targeted vaccine against BVDV

Journal of Animal Science ◽

10.1093/jas/skab235.486 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 265-266

Author(s):

Mauro Venturini ◽

Demian Bellido ◽

Josefina Baztarrica ◽

Lucia Rocha ◽

Viviana Parreño ◽

...

Keyword(s):

Subunit Vaccine ◽

Economic Losses ◽

Passive Immunity ◽

Control Group ◽

Structural Protein ◽

Single Chain ◽

Single Chain Antibody ◽

Diarrhea Virus ◽

Inactivated Vaccines ◽

Antibody Levels

Abstract Bovine viral diarrhea virus (BVDV) infects ruminants, with a worldwide distribution, the virus causes a broad spectrum of clinical diseases and economic losses. Vaccination against BVDV is an important component of prevention and control programs. Currently, only modified live vaccines (MLV) and inactivated vaccines are used. Both have historical disadvantages; MLV in terms of safety and inactivated vaccines in terms of immunogenicity. We have previously reported the development and efficacy trials of the first targeted subunit vaccine against BVDV. The core of the vaccine is a fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the MHC-II present on antigen-presenting cells. The goal of this work was to evaluate passive immunity through colostrum and active immunity of calves immunized with the novel vaccine. 24 A. angus heifers were divided into two groups, 12 immunized with the vaccine and 12 received placebo, in the last trimester of gestation. Serum samples from calves were taken at day 30 of age and analyzed by competitive ELISA. In the vaccinated group, 92% of the d30 old calves maintained medium (25%) or high (67%) antibody levels against BVDV, while 50% of the animals in the control group presented low antibody level (Pearson’s chi-squared p:0,07) (Table 1). At 5 months of age, calves of both groups were immunized with the targeted vaccine. Thirty days later, 96% of the calves had medium or high antibody levels against BVDV, which was independent of their respective heifer vaccine status. These results confirm the new targeted subunit vaccine against BVDV is safe and efficacious to be used in pregnant cattle and can passively immunize newborn calves. They also show that these maternal antibodies do not interfere with the active immunization of calves.

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Targeting the Tumor Microenvironment with Fluorescence-Activatable Bispecific Endoglin/Fibroblast Activation Protein Targeting Liposomes

Pharmaceutics ◽

10.3390/pharmaceutics12040370 ◽

2020 ◽

Vol 12 (4) ◽

pp. 370 ◽

Cited By ~ 2

Author(s):

Felista L. Tansi ◽

Ronny Rüger ◽

Ansgar M. Kollmeier ◽

Markus Rabenhold ◽

Frank Steiniger ◽

...

Keyword(s):

Tumor Microenvironment ◽

Targeted Delivery ◽

Near Infrared ◽

Fibroblast Activation Protein ◽

Target Cells ◽

Single Chain ◽

High Concentration ◽

Single Chain Antibody ◽

Stromal Fibroblasts ◽

Fibroblast Activation

Liposomes are biocompatible nanocarriers with promising features for targeted delivery of contrast agents and drugs into the tumor microenvironment, for imaging and therapy purposes. Liposome-based simultaneous targeting of tumor associated fibroblast and the vasculature is promising, but the heterogeneity of tumors entails a thorough validation of suitable markers for targeted delivery. Thus, we elucidated the potential of bispecific liposomes targeting the fibroblast activation protein (FAP) on tumor stromal fibroblasts, together with endoglin which is overexpressed on tumor neovascular cells and some neoplastic cells. Fluorescence-quenched liposomes were prepared by hydrating a lipid film with a high concentration of the self-quenching near-infrared fluorescent dye, DY-676-COOH, to enable fluorescence detection exclusively upon liposomal degradation and subsequent activation. A non-quenched green fluorescent phospholipid was embedded in the liposomal surface to fluorescence-track intact liposomes. FAP- and murine endoglin-specific single chain antibody fragments were coupled to the liposomal surface, and the liposomal potentials validated in tumor cells and mice models. The bispecific liposomes revealed strong fluorescence quenching, activatability, and selectivity for target cells and delivered the encapsulated dye selectively into tumor vessels and tumor associated fibroblasts in xenografted mice models and enabled their fluorescence imaging. Furthermore, detection of swollen lymph nodes during intra-operative simulations was possible. Thus, the bispecific liposomes have potentials for targeted delivery into the tumor microenvironment and for image-guided surgery.

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Amplified anticoagulant activity of tissue factor-targeted thrombomodulin

Thrombosis and Haemostasis ◽

10.1160/th06-04-0219 ◽

2006 ◽

Vol 96 (09) ◽

pp. 317-324 ◽

Cited By ~ 9

Author(s):

Chenliang Wu ◽

Jon Vincelette ◽

Baby Martin-McNulty ◽

Serene Alexander ◽

Brent Larsen ◽

...

Keyword(s):

Tissue Factor ◽

Neutralizing Antibodies ◽

Factor Viia ◽

Anticoagulant Activity ◽

Coagulation Cascade ◽

Control Group ◽

Therapeutic Effects ◽

Single Chain ◽

Single Chain Antibody

SummaryTissue factor (TF) exposure isa potent pro-thrombotic trigger that initiates activation of the coagulation cascade, while thrombomodulin (TM) is a potent anticoagulant protein that limits the extent of activation. Both TF neutralizing antibodies and soluble TM (sTM) are effective anticoagulants. We have developed a novel anticoagulant fusion protein, Ab(TF)-TM, by fusing a TF-neutralizing single-chain antibody, Ab(TF), to an active fragment of TM. Ab(TF)-TM is a novel anticoagulant targeting to sites of TF exposure with a dual mechanism of action. The Ab(TF) portion of the molecule inhibits TF/factor VIIa mediated activation of FIX and FX, and the TM portion of the molecule acts as a cofactor for activation of protein C. In-vitro coagulation assays show that Ab(TF)-TM more potently inhibits TF-initiated coagulation (prothrombin time) than can its individual components, Ab(TF) (20-fold) and sTM (80-fold) alone, or in combination (10-fold). In contrast, the potency of Ab(TF)-TM in the activated partial thromboplastin and thrombin clotting time assays was similar to sTM alone. Ina rat model of disseminated intravascular coagulation (DIC), intravenous injection of a human TF-containing thromboplastin reagent (0.5 ml/kg) resulted in an immediate death in ∼60% of the animals and a clinical score of ∼2.5. Pre-injection of Ab(TF)-TM or Ab(TF) and sTM, given alone or in combination, showed dose-dependent efficacy. At a dose of 0.7 nmol/kg, Ab(TF)-TM completely prevented death and reduced clinical scores by 79%, while neither Ab(TF) nor sTM, given alone or in combination, showed significant therapeutic effects. Calculated effective doses that reduced mortality by 50% relative to that in the control group (ED50, nmol/kg) were 0.21 for Ab(TF)-TM, 3.2 for an equimolar mixture of Ab(TF) and sTM, 4.3 for sTM and 20 for Ab(TF). Thus, Ab(TF)-TM presented 10– to 100-fold enhancement of the anticoagulant potency, relative to the ED50 in Ab(TF) and sTM given either alone or in combination, in a rat DIC model.

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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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