scholarly journals Highly Effective Recombinant Format of a Humanized IgG-like Bispecific Antibody for Cancer Immunotherapy with Retargeting of Lymphocytes to Tumor Cells

2007 ◽  
Vol 282 (38) ◽  
pp. 27659-27665 ◽  
Author(s):  
Ryutaro Asano ◽  
Yasuhiro Watanabe ◽  
Hiroko Kawaguchi ◽  
Hidesuke Fukazawa ◽  
Takeshi Nakanishi ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Growth Inhibition ◽  
Mammalian Cells ◽  
Bispecific Antibody ◽  
Cell Phenotype ◽  
Inhibition Assay ◽  
Single Chain ◽  
Lymphokine Activated Killer Cells ◽  
Effector Cells ◽  
Single Chain Fv

We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgG1 (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1–100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced antitumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense antitumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed.

Efficient Lysis of Myeloma Cells by CD16 Positive Effector Cells with Recombinant Single Chain Bispecific Antibodies Targeting HM1.24.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2752-2752
Author(s):  
Matthias Peipp ◽  
Claudia Ehlert ◽  
Matthias Staudinger ◽  
Joerg Bruenke ◽  
Georg Fey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Bispecific Antibody ◽  
Lytic Activity ◽  
High Dose ◽  
Antigen Specificity ◽  
Single Chain ◽  
Myeloma Cells ◽  
Effector Cells ◽  
Single Chain Fv

Abstract Different therapeutic options are available for the treatment of multiple myeloma patients, but conventional chemotherapy often is not able to completely eradicate the tumor. After high dose chemotherapy, complete remission with only minimal residual disease could be achieved in many patients, but additional targeted strategies may help to eradicate residual cells and improve prognosis. Therapy with monoclonal antibodies is well established in CD20 positive B-cell lymphomas, but in contrast not many suitable target antigens are defined / expressed by multiple myeloma cells. HM1.24 a surface molecule expressed on terminally differentiated B-lineage cells represents a promising candidate antigen that is overexpressed on multiple myeloma cells. Here the development of a recombinant bispecific single chain Fv HM1.24 × CD16 antibody (tandem format) with novel features is presented. The HM1.24×CD16 bispecific antibody was expressed in 293T cells and purified to homogeneity by two-step affinity chromatography. Binding to HM1.24 and CD16 was demonstrated by immunofluorescence staining and flow cytometry with antigen positive and negative cells. The lytic activity of the bispecific HM1.24×CD16 scFv was evaluated in an antibody-dependent cellular cytotoxicity (ADCC) assay with different myeloma cell lines (RPMI 8226, INA-6, U266, JK6L) and primary patient derived cells as targets. Mononuclear cells (MNC), isolated from healthy donors served as effector cells. The bispecific HM1.24×CD16 scFv mediated efficient lysis of all tested cell lines at concentrations as low as 1 nM. In direct comparison to an HM1.24-IgG1 control molecule, the recombinant bispecific antibody demonstrated superior lytic activity at saturating concentrations and showed significant enhanced killing capacity. In conclusion, the recombinant bispecific HM1.24×CD16 retained its antigen specificity and demonstrated efficient lytic activity against patient-derived tumor cell lines and primary material. These results indicate that the bispecific antibody may be promising as a new therapeutic strategy in multiple myeloma.

scholarly journals Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Zackary Tajin Park
Keyword(s):  
Phage Display ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Phage Display Library ◽  
Single Chain ◽  
Phage Display Technology ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

Expressing Intracellular Single-Chain Fv Fragments in Mammalian Cells

2001 ◽  
pp. 755-774
Author(s):  
Silvia Biocca ◽  
Alessio Cardinale ◽  
Antonino Cattaneo
Keyword(s):  
Mammalian Cells ◽  
Single Chain ◽  
Single Chain Fv

Anti-hepatoma human single-chain Fv antibody and adriamycin conjugates with potent antitumor activity

2014 ◽  
Vol 18 (1) ◽  
pp. 20-26 ◽  
Author(s):  
Lin Chen ◽  
Yan-Hong Liu ◽  
Yue-Hui Li ◽  
Yan Jiang ◽  
Ping-Li Xie ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Single Chain ◽  
Single Chain Fv ◽  
Fv Antibody ◽  
Single Chain Fv Antibody

An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv

2004 ◽  
Vol 324 (4) ◽  
pp. 1165-1172 ◽  
Author(s):  
Päivi Pihkala ◽  
Masahiro Kawahara ◽  
Hiroshi Ueda ◽  
Teruyuki Nagamune
Keyword(s):  
Mammalian Cells ◽  
Selection System ◽  
Single Chain ◽  
Single Chain Fv

scholarly journals A CD19/CD3 bispecific antibody for effective immunotherapy of chronic lymphocytic leukemia in the ibrutinib era

Blood ◽  
2018 ◽  
Vol 132 (5) ◽  
pp. 521-532 ◽  
Author(s):  
Hannah R. Robinson ◽  
Junpeng Qi ◽  
Erika M. Cook ◽  
Cydney Nichols ◽  
Eman L. Dadashian ◽  
...  
Keyword(s):  
T Cells ◽  
Bispecific Antibody ◽  
Lymphocytic Leukemia ◽  
Single Chain ◽  
Prior Therapy ◽  
Resistant Disease ◽  
Single Chain Fv ◽  
Key Points

Key Points A CD19/CD3 single-chain Fv-Fc bsAb mediated potent killing of CLL cells by autologous T cells in vitro and in vivo. bsAb-mediated cytotoxicity was enhanced by prior therapy with ibrutinib and extended to ibrutinib-resistant disease.

scholarly journals An antibody-tumor necrosis factor fusion protein that synergizes with oxaliplatin for treatment of colorectal cancer

2019 ◽  
Author(s):  
Davor Bajic ◽  
Kerry A. Chester ◽  
Dario Neri
Keyword(s):  
Colorectal Cancer ◽  
Tumor Necrosis Factor ◽  
Fusion Protein ◽  
Mammalian Cells ◽  
Tumor Necrosis ◽  
Human Antibody ◽  
Single Chain ◽  
Single Chain Fv ◽  
Immunocompetent Mice ◽  
Necrosis Factor

ABSTRACTWe have cloned and characterized a novel fusion protein (Sm3E-TNF), consisting of the monoclonal antibody Sm3E in single-chain Fv fragment format, fused to murine tumor necrosis factor. The protein, which was expressed in mammalian cells and purified as a non-covalent stable homotrimer, bound to the cognate carcinoembryonic antigen (CEA) and retained tumor necrosis factor activity. A quantitative biodistribution experiment, performed in immunocompetent mice with CT26 colon carcinomas transfected with human CEA, revealed that Sm3E-TNF was able to preferentially accumulate in the tumors with excellent selectivity (tumor:blood ratio = 56:1, twenty-four hours after intravenous administration). The fusion protein mediated a rapid hemorrhagic necrosis of a large portion of the tumor mass, but a rim survived and eventually regrew. Surprisingly, the combination of Sm3E-TNF with 5-fluorouracil led to a reduction of therapeutic activity, while a combination with oxaliplatin led to a prolonged stabilization, with complete tumor eradication in 40% of treated mice. These therapy results were confirmed in a second immunocompetent mouse model of colorectal cancer (CEA-transfected C51 tumors) and provide a rationale for the possible clinical use of oxaliplatin in combination with fully-human antibody-TNF fusions.

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