New type recombinant antibody single chain variable fragment (scFv) is formed by the joined VH and VL domains of immunoglobulin with the used of a polypeptide linker that is at least 12 residues in length. scFv is the smallest functional unit of antibody and has shown a fine prospect for the radioimmunoscintigraphy of cancer because of its special characteristics including increased tumour penetration and fast clearance rates compared with parent Ig. A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (Diabody). Reducing the linker length below three residues can force scFv association into trimers (Triabody) or tetramers (Tetrabody) depending on linker length, composition and V-domain orientation. This review describes linker length and V-domain orientation of scFv, expression and stability of scFv multimers, size of scFv multimers and its effect on in vivo pharmacokinetics, flexibility and avidity of scFv multimers, in vitro application of multimeric murine scFv, multispecific scFv multimers and cancer targeting.
Antibodies, Bispecific ; Immunoglobulin Fragments ; Immunoglobulin Heavy Chains ; chemistry ; Immunoglobulin Light Chains ; chemistry ; Immunoglobulin Variable Region ; Immunotherapy ; Neoplasms ; immunology ; therapy ; Protein Engineering ; Recombinant Fusion Proteins

OBJECTIVETo induce anti-B-cell acute lymphoblastic leukemia (B-ALL) cytotoxic T lymphocyte response against immunoglobulin heavy chain frame-derived nonapeptide.

METHODSThe peptide, QLVQSGAEV, containing IgHV1 frame region 3rd-11th amino acids (IgHV1(3-11)), was synthesized. IgHV1(3-11)-T2 binding tests were performed. HLA-A * 0201-positive normal peripheral blood mononuclear cells (PBMNC) were stimulated by IgHV1(3-11)-loaded antigen presenting cells three times at weekly intervals. HLA-A * 0201/IgHV1(3-11) tetramers were used to detect the proliferation of IgHV1(3-11)-specific CD8(+) T cells in the culture. Seven IgHV gene families of B-ALL patients were respectively amplified by PCR and the PCR products were sequenced to select IgHV1 and IgHV3 family monoallelic functional rearrangements. Among them, HLA-A * 0201 positive individuals were subsequently identified. Cytotoxicity of IgHV1(3-11)-specific CD8(+) T cells against HLA-A * 0201-positive IgHV1/IgHV3 family B-ALL cells was measured by MTT assay.

RESULTSThe synthesized IgHV1(3-11) up-regulated HLA-A * 0201 expression on T2 cell surface by 1.63-folds. The percentage of IgHV1(3-11)-specific CD8(+) T cells in HLA-A * 0201-positive normal PBMNC was increased from 1.64% after second stimulation to 82.57% after third stimulation. At effector: target ratio of 20:1, the killing rate of IgHV1(3-11)-specific CD8(+) T cells against IgHV1 family B-ALL cells was 18.24%, being 1.8-folds as that against IgHV3 family B-ALL cells (P = 0.01).

CONCLUSIONCytolytic T lymphocytes generated in vitro against immunoglobulin heavy chain frame-derived nonapeptides can kill B-ALL cells expressing these peptides.

Cells, Cultured ; Humans ; Immunoglobulin Heavy Chains ; immunology ; pharmacology ; Leukemia, B-Cell ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology

OBJECTIVETo develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology

OBJECTIVETo construct a human phage display antibody library, which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic. This can provide a platform for human antibody preparation and diagnosis, prophylaxis and therapy of RSV infection in children.

METHODSPeripheral blood lymphocytes were isolated from 52 children with RSV infection. cDNA was synthesized from the total RNA of lymphocytes. The light and heavy chain Fd (VH-CH1) fragments of immunoglobulin gene were amplified by RT-PCR. The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue. The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs. The plasmids extracted from amplified E.coli were digested with restriction endonucleases Sac I, Xba I, Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes. RSV virions were utilized as antigens to screen Fab antibodies.

RESULTSBy recombination of light and heavy chain genes, an immune Fab phage display antibody library against RSV containing 2.08x10(7) different clones was constructed, in which 70% clones had light chains and heavy chain Fd genes. The capacity of Fab phage antibody gene library was 1.46x10(7) and the titre of the original Fab antibody library was about 1.06x10(12) pfu/mL. The antibody library gained an enrichment in different degrees after the preliminary panning.

CONCLUSIONSUtilizing the technology of phage display, an immune Fab phage display antibody library against RSV was successfully constructed in this study, which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis, therapy and prophylaxis of RSV infection in children.

Antibodies, Viral ; genetics ; Bacteriophages ; genetics ; Child ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Peptide Library ; Respiratory Syncytial Viruses ; immunology

OBJECTIVETo identify immune epitopes existed in the framework region (FR) of the IgHV protein of B-cell malignance, and explore the use of these FR-derived peptides to induce the family specific immune response in vitro, in order to explore the possibility of a new IgHV gene family-specific immunotherapy for B-cell malignance.

METHODSBioinformatics was used for predicting T cell epitopes in IgHV protein. Peptides of interest were synthesized in vitro. T2 cell binding assay was performed to determine the binding ability of the peptides to HLA-A*0201 molecules. Peptide/HLA tetramer staining was used to detect the number of peptide-specific cytotoxic T lymphocytes (CTLs). Cytotoxicity assay was used to determine killing activity.

RESULTSTwelve peptides that were common to seven IgHV gene subfamilies were identified, and 10 (83%) of them were located in the FR of IgHV protein. The synthesized peptides up-regulated HLA*0201 molecules fluorescence intensity on cell surfaces of T2. By using an antigen-specific T-cell expansion system in vitro, the peripheral blood mononuclear cells (PBMNC) from a healthy HLA-A0201 donor were stimulated weekly by autologous PBMNC loaded with the peptide as antigen presenting cells (APC), and the peptide-specific CTLs were demonstrated to be generated successfully in the healthy donors. The frequency of CD8 and peptide/HLA tetramer double positive cells in the gated lymphocyte population was 0.38% before stimulation and increased to 49.38 % after four times stimulation. Cytotoxicity assay indicated that these CTLs were capable of killing the HLA-A*0201, IgHV1 (+) lymphoma cells. Furthermore, the generated CTL could not kill the target cell loaded with the IgHV3 peptide, indicating that the cytotoxicity is family-specific.

CONCLUSIONPeptides derived from the IgHV protein FR can successfully induce the generation of peptide-specific CTLs in vitro. These CTLs are capable of killing the lymphoma cell belonged to the same subfamily in a peptide-specific and MHC-restricted way. These findings could potentially form the basis of broadening application of immunoglobulin-directed immunotherapy in B-cell malignancies.

Cells, Cultured ; Epitopes, B-Lymphocyte ; immunology ; Humans ; Immunoglobulin Heavy Chains ; immunology ; Immunoglobulin Variable Region ; immunology ; Lymphoma, B-Cell ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

BACKGROUND: Korea is an endemic area of scrub typhus and it is a common seasonal febrile illness. Although, various humoral immune responses to scrub typhus have been documented, no association between gammopathy and scrub typhus has ever been reported. We analyzed the incidences and types of monoclonal and biclonal gammopathies in scrub typhus for better coping with those gammopathies in scrub typhus. METHODS: Anti-Orientia tsutsugamushi antibody-positive sera identified by indirect immunofluorescence assay were acquired from 40 patients with confirmed scrub typhus. Monoclonal and biclonal gammopathies were screened by protein electrophoresis and were confirmed using immunofixation electrophoresis (IFE). Laboratory findings on admission of the patients with monoclonal or biclonal gammopathy were investigated retrospectively to characterize the gammopathies. RESULTS: Monoclonal or biclonal gammopathies were detected in 30% (12/40) of patients with scrub typhus (IgG-lambda, 40%; IgG-kappa, 30%; IgM-kappa, 10%; IgM-lambda, 10%; IgA-kappa, 5%; IgA-lambda, 5%). Concentrations of clonal immunoglobulin were less than 3 g/dL in all gammopathies, and hypercalcemia was not detected in any of the patients. CONCLUSIONS: Our results suggest possible association between gammopathies and scrub typhus. Further studies in larger series will be needed for exact incidence and clinical course of gammopathies in scrub typhus.
Adult ; Aged ; Humans ; Immunoglobulin A/blood ; Immunoglobulin G/blood ; Immunoglobulin Heavy Chains/blood ; Immunoglobulin Light Chains/blood ; Immunoglobulin M/blood ; Incidence ; Middle Aged ; Orientia tsutsugamushi/immunology ; Paraproteinemias/complications/*diagnosis/epidemiology/immunology ; Retrospective Studies ; Scrub Typhus/complications/*diagnosis/immunology

OBJECTIVETo construct a personalized full-length fully human antibody mammalian display library for children with systemic lupus erythematosus (SLE).

METHODSThe total RNA was isolated from the PBMCs of SLE children. The heavy chain variable region and kappa light chain (VH and LCκ) of the antibody genes were amplified by RT-PCR and inserted into the pDGB-HC-TM vector separately to construct the heavy chain and light chain libraries. The library DNAs were transfected into 293T cells and the expression of full-length fully human antibody on the surface of 293T cells was analyzed by flow cytometry.

RESULTSUsing 0.8 µg total RNA as the template, the VH and LCκ were amplified and the full-length fully human antibody mammalian display library was constructed. The VH and LCκ gene libraries had a size of 9.4×10(4) and 8.4×10(4), respectively. Sequence analysis of 10 clones randomly selected from the VH and LCκ gene libraries each showed that 8 heavy chain clones and 7 light chain clones contained correct open reading frames, and flow cytometry demonstrated that all the 15 clones express full-length antibodies on 293T cell surfaces. 293T cells co-transfected with the VH and LCκ gene libraries expressed the full-length antibodies on the cell surface.

CONCLUSIONThe personalized full-length fully human antibody library for SLE children constructed allows display of the full-length antibodies on mammalian cell surfaces, thus providing a valuable platform for analyzing the autoantibodies, their etiological role, and their clinical implications in SLE.

Amino Acid Sequence ; Child ; Gene Library ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Lupus Erythematosus, Systemic ; genetics ; immunology ; Membrane Proteins ; genetics

OBJECTIVETo construct full-length human bladder cancer-specific antibody libraries for efficient display of full-length antibodies on the surface of mammalian cells.

METHODSThe total RNA was isolated from peripheral blood mononuclear cells from patients with bladder cancer. The repertoires of IgG1 heavy chain variable region (VH) and Kappa light chain were amplified by RT-PCR using specific primers. The antibody genes were inserted into the vector pDGB-HC-TM to construct the bladder-cancer-specific antibody libraries of heavy chains and light chains. Ten clones from each library were randomly picked for gene sequencing and transient transfection into FCHO cells to analyze antibody display on mammalian cell surface by flow cytometry after staining with corresponding fluorescent labeled antibodies.

RESULTSThe libraries of bladder-cancer-specific antibody heavy chain (IgG1) and light chain (LCk) were successfully constructed. Seven out of the 10 clones randomly selected from the heavy chain library and 9 out of the 10 clones from the light chain library showed correct open reading frame, coding for 7 unique VH and 9 unique LCk. The combinatory library size reached 3.32×10(11).

CONCLUSIONWe have successfully constructed a full-length human bladder-cancer-specific antibody library with a combinatory diversity of 3.32×10(11) based on mammalian display technology, which can be used for screening monoclonal antibodies against bladder-cancer-associated antigens.

Amino Acid Sequence ; Animals ; Antibodies ; genetics ; Cell Surface Display Techniques ; Gene Library ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Peptide Library ; Urinary Bladder Neoplasms ; genetics ; immunology

Functional heavy-chain antibodies (HCAbs) lacking light chains occur naturally in camels. The variable domain of heavy chain of heavy-chain antibody is referred to VHH. The VHH gene family is homologous to human VH subgroup III. The single-domain VHH antibodies are constructed by cloning the variable domains of HCAbs. Compared to human VHs, VHH germ-line sequences contain some hallmark substitutions in framework region 2, including V37F(Y), G44 E, L45 R, W47G. The substitutions at positions 44, 45, 47 are often used to camelise the human VHs. Being a small binders, VHH antibodies are well expressed, extremely stable and very soluble. Camelised human VHs are proved to exhibit the same qualities as those of VHH antibodies. The single-domain VHH antibodies will be useful in the drug development and basic research.
Animals ; Binding Sites, Antibody ; Camelus ; immunology ; metabolism ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.
Animals ; Antibodies, Monoclonal/*biosynthesis/immunology ; Chromatography, Affinity/veterinary ; Electrophoresis, Gel, Two-Dimensional/veterinary ; Immunoblotting/veterinary ; Immunoglobulin Heavy Chains/immunology ; Immunoglobulin Light Chains/immunology ; Immunoglobulins/blood/*immunology ; Perciformes/blood/*immunology