The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.
Codon/genetics* ; Protein Sorting Signals/genetics* ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Interferon-alpha/metabolism* ; Immunoglobulin Heavy Chains/immunology* ; Cell Line ; Immunoglobulin Variable Region/immunology*

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.
Antibodies ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin Variable Region ; Intercellular Adhesion Molecule-1 ; immunology ; Peptide Library ; Single-Chain Antibodies

OBJECTIVETo clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs).

METHODSThe VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells.

RESULTSThe VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired.

CONCLUSIONHuman anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

Antibodies, Monoclonal ; genetics ; Cloning, Molecular ; Genetic Vectors ; Humans ; Hybridomas ; Immunoglobulin Variable Region ; genetics ; Interleukin-1 Receptor Accessory Protein ; immunology ; Plasmids ; Polymerase Chain Reaction ; Receptors, Antigen ; genetics ; Single-Chain Antibodies

To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system. The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood. The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF' with the Hte gene. Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times. The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations. The library was calculated to have a diversity of 1.7 x 10(11) and validated by the selection of antibodies against six different protein antigens. This library provides the basis for further selection of antibody-based drugs. It is the first time to report that XL2-blue MRF' can be used to improve the diversity of the library in the recombination system.
Adult ; Escherichia coli ; genetics ; immunology ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Infant, Newborn ; Integrases ; metabolism ; Lymphocytes ; immunology ; Peptide Library ; Recombination, Genetic ; genetics ; Single-Chain Antibodies ; genetics ; metabolism ; Transformation, Genetic

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.
Amino Acid Sequence ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; Antibody Affinity ; Asthma ; blood ; Base Sequence ; DNA, Complementary ; metabolism ; Escherichia coli ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lymphocytes ; chemistry ; Peptide Library ; RNA, Messenger ; isolation & purification ; Recombination, Genetic ; genetics ; Ribosomes ; chemistry ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; isolation & purification ; Transformation, Genetic

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Animals ; Antibody Affinity ; Cells, Cultured ; Cytidine Deaminase ; genetics ; metabolism ; HEK293 Cells ; Humans ; Immunoglobulin Variable Region ; genetics ; immunology ; Mice ; Mutation ; Single-Chain Antibodies ; chemistry ; genetics ; immunology ; Somatic Hypermutation, Immunoglobulin ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo investigate the overrepresentation of specific gene segments of immunoglobulin heavy chain variable region (IgVH) among unmutated and mutated chronic lymphocytic leukemia (CLL) patients and its prognostic implication.

METHODSMultiplex PCR was used to identify the expression of IgVH segment and its mutation status in CLL.

RESULTSAnalyses were successfully performed in 80 of 85 samples. Marked skewed IgVH families were disclosed. The most commonly used VH was VH3 (40.0%), followed by VH4 (30.0%), VHI (13.8%), VH2 (10.0%) and VH5, VH7 (2.5%). Fifty-six patients (70.0%) had mutated VH, 24 (30.0%) unmutated VH. Nine cases (11.3%) were with 100% germline sequence. Fifteen cases (15/24, 62.5%) in VH4, 29 (29/32, 90.7%) in VH3, and 4 (4/11, 36.3%) in VH1 had mutated VH. The most frequently used IgVH gene was VH4-39 (13.8%), and VH4-34 (8.8%). J4 (36/66, 54.5%) and D3 (25/66, 37.8%) were the most frequently used in J and D genes. The progression-free survival (PFS) was 82 and 17 months (P = 0.000), and the overall survival (OS) was 90 and 41 months (P = 0.009), respectively, for mutated and unmutated cases. Recurrent CDR3 sequences were found in our patients and 2 patients with VH1-69 had CDR3 sequences highly similar to those reported in literature.

CONCLUSIONThere is difference in IgVH gene segment usage and mutational status in different area CLL patients. Recurrent CDR3 sequences were found in specific IgVH gene segments, which highlights the importance of immunoglobulin mediated stimulation in the development of CLL.

Adult ; Aged ; Aged, 80 and over ; DNA Mutational Analysis ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; immunology ; pathology ; Male ; Middle Aged ; Mutation

OBJECTIVETo observe the function of gamma delta T lymphocytes and the polymorphism of T cell receptor V delta chain in the lungs of asthmatic patients and explore the role of gamma delta T cells in airway inflammation.

METHODSBronchoalveolar lavage fluid BALF was obtained from 7 asthmatic patients and 7 healthy control individuals. The percentage of gamma delta T cell in BALF was measured by flow cytometry. The gamma delta T cell in BALF was purified by magnetic labeled beads. Proliferous activity was examined by MTT assay. Cytokines secreted by gamma delta T cells in medium was assessed by enzyme-linked immunosorbent assay. Polymorphism of T cell receptor V delta chain was detected by RT-PCR and gene scan analysis.

RESULTSThe proportion of gamma delta T cell in the BALF of asthmatic patients [(6.39+/-0.71)%] was significantly higher than that in control subjects [(2.62+/-0.37)%] (P<0.01). The proportion of macrophage in the BALF of asthmatic patients [(81+/-4)] was significantly lower than that in control subjects [(86+/-2)] (P<0.05). The proliferation rate of asthmatic patients [(284.2+/-43.6)%] was significantly higher than that of control subjects [(217.5+/-59.5)%] (P<0.05). Interleukin-4 secreted by gamma delta T cells of asthmatic patients [(18.9+/-3.1) pg/ml)] significantly increased when compared with the control subjects [(14.1+/-3.0) pg/ml] (P<0.05). The polymorphism of T cell receptor V delta chain was not significantly different between these two groups.

CONCLUSIONSThe increase of gamma delta T cells in the lung of asthmatic patients further exacerbates Th1/Th2 disturbance and airway inflammation. Antigen recognition by gamma delta T cells is non-specific.

Adult ; Asthma ; genetics ; immunology ; Bronchoalveolar Lavage Fluid ; cytology ; Case-Control Studies ; Cell Proliferation ; Cytokines ; metabolism ; Female ; Genes, T-Cell Receptor delta ; genetics ; Genes, T-Cell Receptor gamma ; genetics ; Humans ; Immunoglobulin Variable Region ; genetics ; Lung ; immunology ; Male ; Middle Aged ; Polymorphism, Genetic ; T-Lymphocyte Subsets ; immunology ; metabolism ; Th1-Th2 Balance

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.
Erythrocytes ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

OBJECTIVETo construct a human phage antibody library and screen the single chain variable fragment (ScFv) antibudies to peroxiredoxin I (Prx I) of lung adenocarcinoma.

METHODSThe total RNA was isolated from the lymph nodes of lung cancer patients to amplify V(H) and V(L) genes by RT-PCR. V(H) and V(L) were linked with a DNA linker by SOE-PCR to construct the single chain variable fragment gene. The ScFvs were coloned into the phage vector pCANTAB5E. The insert ratio of the ScFv antibody library was identified by PCR, and the products were digested by SfiI/NotI and analyzed with 1% agarose gel electrophoresis. Three rounds of panning against lung adenocarcinoma cell line A549 and Prx I were performed, and the positive clones were identified for soluble expression. The soluble antibodies were identified by SDS-PAGE and Western blotting, and ELISA and immunocytochemistry were used to characterize the activity of the antibodies.

RESULTSA recombination phage antibody library was constructed. The insert ratio of ScFv gene was 77% (23/30), and enzyme digestion identified the target product. The sixth phage harvest resulted in a yield 180 folds of that of the first one. Positive reactions to A549 cells were detected in 6 of 10 random clones, with a positivity rate of 60%. The soluble human ScFvs against Prx I of lung adenocarcinoma were expressed in E. coli HB2151 and confirmed by SDS-PAGE and Western blotting. ELISA and immunocytochemistry demonstrated a relative specific affinity of the soluble antibodies to A549 cells.

CONCLUSIONScFv antibodies against lung adenocarcinoma have been acquired by phage display antibody library technique, and the soluble antibodies have a relative avidity specific to human lung adenocarcinoma A549 cells overexpressing PrxI.

Adenocarcinoma ; immunology ; pathology ; Antibodies, Neoplasm ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; Cell Line, Tumor ; Humans ; Immunoglobulin Variable Region ; immunology ; Lung Neoplasms ; immunology ; pathology ; Peptide Library ; Peroxiredoxins ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; immunology