Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody. 2011

Ryutaro Asano, and Keiko Ikoma, and Ippei Shimomura, and Shintaro Taki, and Takeshi Nakanishi, and Mitsuo Umetsu, and Izumi Kumagai
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan.

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.

UI MeSH Term Description Entries
D003603 Cytotoxins Substances that are toxic to cells; they may be involved in immunity or may be contained in venoms. These are distinguished from CYTOSTATIC AGENTS in degree of effect. Some of them are used as CYTOTOXIC ANTIBIOTICS. The mechanism of action of many of these are as ALKYLATING AGENTS or MITOSIS MODULATORS. Cytolysins,Cytotoxic Agent,Cytotoxic Agents,Cytotoxin,Agent, Cytotoxic
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000970 Antineoplastic Agents Substances that inhibit or prevent the proliferation of NEOPLASMS. Anticancer Agent,Antineoplastic,Antineoplastic Agent,Antineoplastic Drug,Antitumor Agent,Antitumor Drug,Cancer Chemotherapy Agent,Cancer Chemotherapy Drug,Anticancer Agents,Antineoplastic Drugs,Antineoplastics,Antitumor Agents,Antitumor Drugs,Cancer Chemotherapy Agents,Cancer Chemotherapy Drugs,Chemotherapeutic Anticancer Agents,Chemotherapeutic Anticancer Drug,Agent, Anticancer,Agent, Antineoplastic,Agent, Antitumor,Agent, Cancer Chemotherapy,Agents, Anticancer,Agents, Antineoplastic,Agents, Antitumor,Agents, Cancer Chemotherapy,Agents, Chemotherapeutic Anticancer,Chemotherapy Agent, Cancer,Chemotherapy Agents, Cancer,Chemotherapy Drug, Cancer,Chemotherapy Drugs, Cancer,Drug, Antineoplastic,Drug, Antitumor,Drug, Cancer Chemotherapy,Drug, Chemotherapeutic Anticancer,Drugs, Antineoplastic,Drugs, Antitumor,Drugs, Cancer Chemotherapy
D013601 T-Lymphocytes Lymphocytes responsible for cell-mediated immunity. Two types have been identified - cytotoxic (T-LYMPHOCYTES, CYTOTOXIC) and helper T-lymphocytes (T-LYMPHOCYTES, HELPER-INDUCER). They are formed when lymphocytes circulate through the THYMUS GLAND and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T Cell,T Lymphocyte,T-Cells,Thymus-Dependent Lymphocytes,Cell, T,Cells, T,Lymphocyte, T,Lymphocyte, Thymus-Dependent,Lymphocytes, T,Lymphocytes, Thymus-Dependent,T Cells,T Lymphocytes,T-Cell,T-Lymphocyte,Thymus Dependent Lymphocytes,Thymus-Dependent Lymphocyte
D017252 CD3 Complex Complex of at least five membrane-bound polypeptides in mature T-lymphocytes that are non-covalently associated with one another and with the T-cell receptor (RECEPTORS, ANTIGEN, T-CELL). The CD3 complex includes the gamma, delta, epsilon, zeta, and eta chains (subunits). When antigen binds to the T-cell receptor, the CD3 complex transduces the activating signals to the cytoplasm of the T-cell. The CD3 gamma and delta chains (subunits) are separate from and not related to the gamma/delta chains of the T-cell receptor (RECEPTORS, ANTIGEN, T-CELL, GAMMA-DELTA). Antigens, CD3,CD3 Antigens,T3 Antigens,CD3 Antigen,T3 Antigen,T3 Complex,Antigen, CD3,Antigen, T3,Antigens, T3
D057127 Single-Chain Antibodies A form of antibodies consisting only of the variable regions of the heavy and light chains (FV FRAGMENTS), connected by a small linker peptide. They are less immunogenic than complete immunoglobulin and thus have potential therapeutic use. Fv Antibody Fragments, Single-Chain,ScFv Antibodies,Single-Chain Fv,Single-Chain Fv Antibody,Single-Chain Fv Antibody Fragments,Single-Chain Variable Fragment,Single-Chain Variable Fragment Antibodies,Single-Chain Variable Fragment Antibody,Single-Chain Variable Fragments,Antibodies, ScFv,Antibodies, Single-Chain,Antibody, Single-Chain Fv,Fragment, Single-Chain Variable,Fragments, Single-Chain Variable,Fv Antibody Fragments, Single Chain,Fv Antibody, Single-Chain,Fv, Single-Chain,Single Chain Antibodies,Single Chain Fv,Single Chain Fv Antibody,Single Chain Fv Antibody Fragments,Single Chain Variable Fragment,Single Chain Variable Fragment Antibodies,Single Chain Variable Fragment Antibody,Single Chain Variable Fragments,Variable Fragment, Single-Chain,Variable Fragments, Single-Chain
D018033 Antibodies, Bispecific Antibodies, often monoclonal, in which the two antigen-binding sites are specific for separate ANTIGENIC DETERMINANTS. They are artificial antibodies produced by chemical crosslinking, fusion of HYBRIDOMA cells, or by molecular genetic techniques. They function as the main mediators of targeted cellular cytotoxicity and have been shown to be efficient in the targeting of drugs, toxins, radiolabeled haptens, and effector cells to diseased tissue, primarily tumors. Bifunctional Antibodies,Bispecific Antibodies,Bispecific Monoclonal Antibodies,Antibodies, Bifunctional,Antibodies, Bispecific Monoclonal,Monoclonal Antibodies, Bispecific
D066246 ErbB Receptors A family of structurally related cell-surface receptors that signal through an intrinsic PROTEIN-TYROSINE KINASE. The receptors are activated upon binding of specific ligands which include EPIDERMAL GROWTH FACTORS, and NEUREGULINS. EGF Receptor,Epidermal Growth Factor Receptor,Epidermal Growth Factor Receptor Family Protein,Epidermal Growth Factor Receptor Protein-Tyrosine Kinase,ErbB Receptor,HER Family Receptor,Receptor, EGF,Receptor, Epidermal Growth Factor,Receptor, TGF-alpha,Receptor, Transforming-Growth Factor alpha,Receptor, Urogastrone,Receptors, Epidermal Growth Factor-Urogastrone,TGF-alpha Receptor,Transforming Growth Factor alpha Receptor,Urogastrone Receptor,c-erbB-1 Protein,erbB-1 Proto-Oncogene Protein,EGF Receptors,Epidermal Growth Factor Receptor Family Proteins,Epidermal Growth Factor Receptor Kinase,HER Family Receptors,Proto-oncogene c-ErbB-1 Protein,Receptor Tyrosine-protein Kinase erbB-1,Receptor, ErbB-1,Receptors, Epidermal Growth Factor,Epidermal Growth Factor Receptor Protein Tyrosine Kinase,ErbB-1 Receptor,Family Receptor, HER,Family Receptors, HER,Proto oncogene c ErbB 1 Protein,Proto-Oncogene Protein, erbB-1,Receptor Tyrosine protein Kinase erbB 1,Receptor, ErbB,Receptor, ErbB 1,Receptor, HER Family,Receptor, TGF alpha,Receptor, Transforming Growth Factor alpha,Receptors, EGF,Receptors, Epidermal Growth Factor Urogastrone,Receptors, ErbB,Receptors, HER Family,c erbB 1 Protein,c-ErbB-1 Protein, Proto-oncogene,erbB 1 Proto Oncogene Protein

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