A single-chain antibody fragment specific for the Plasmodium berghei ookinete protein Pbs21 confers transmission blockade in the mosquito midgut

Mouse monoclonal antibody 13.1 (mAb 13.1) directed against Pbs21, a 21-kDa sexual-stage surface protein of Plasmodium berghei, is known to inhibit oocyst development from gametocytes and ookinetes in the mosquito midgut. To examine the properties and potential uses of a single-chain antibody fragment (scFv) for blocking transmission of malaria parasites to mosquitoes, we have cloned and sequenced the genes encoding variable regions of the immunoglobulin heavy and light chains (V(H) and V(L)) of mAb 13.1. The V(H) and V(L) genes were assembled as an scFv gene, and expressed in a baculovirus expression system. Following purification of 13.1 scFv, Western blotting and inhibition ELISA assays confirmed that 13.1 scFv retained the binding specificity of the parent mAb 13.1 for Pbs21. Furthermore, 13.1 scFv bound to the surface of P. berghei ookinetes, and blocked oocyst development in the mosquito midgut by at least 93%, as assessed by oocyst counts in mosquitoes. We suggest that the 13.1 scFv gene could be useful not only in studying the mechanism of transmission blockade, but also in generating, by mosquito germline transformation, a model system to evaluate the production of mosquitoes refractory to malaria.     

Characterization of a new murine monoclonal antibody against human DP antigens

Murine Hybridoma cell line BraFB6 was prepared producing monoclonal antibody reactive with an antigen identified as DP. This identification is based on the results of immunoprecipitation from radiolabeled cell lysates, competitive binding-inhibition assay with a reference anti-DP antibody B7/21, sequential immunoprecipitation and specific binding to the purified DP glycoprotein. In addition to this anti-DP monoclonal antibody, two anti-DR and two anti-DR +DP monoclonals were obtained.     

Characterization of a monoclonal antibody that recognizes a determinant unique to human haemoglobin beta-chain

Methodologies are described for the production and characterization of monoclonal antibodies to human haemoglobin. Three monoclonal antibodies are described, two of which recognize distinct determinants on the alpha-chain subunit. A third monoclonal antibody, Hb-2d, recognizes a determinant expressed on human beta-chain. The Hb-2d determinant is shared by human and baboon haemoglobins, but is not expressed by haemoglobins from beef, goose, pig, rabbit, sheep, dog, rat or mouse. Monoclonal antibody Hb-2d will bind to haemoglobin A2 but not to foetal haemoglobin suggesting that delta-but not gamma-chain also expresses the Hb-2d determinant. The results of testing a limited panel of human haemoglobin variants is presented.     

Characterization of antigens on mosquito midgut stages of Plasmodium gallinaceum. III. Changes in zygote surface proteins during transformation to mature ookinete

Proteins expressed on the surface of zygotes of Plasmodium gallinaceum during their development from fertilization to mature ookinetes have been examined by lactoperoxidase catalysed surface radioiodination and immunoprecipitation with stage-specific immune rabbit sera and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Surface-labelled proteins of apparent Mr equal to or greater than 55 000 on the female gametes and newly fertilized zygotes were shed during transformation and were recovered quantitatively and apparently intact from the culture supernatant. Zygote surface proteins of Mr 50 000, 19 000 and 17 000 remained bound to the surface throughout the transformation. Three proteins were expressed de novo on the surface of the mature ookinete of which Mr 26 000 and 28 000 represented major surface components and Mr 52 000 a relatively minor component.     

A unique human B lymphocyte antigen defined by a monoclonal antibody

We produced a hybridoma designated 4G7 from a mouse immunized with chronic lymphocytic leukemia cells. The 4G7 hybridoma secretes an IgG1 antibody that is specific for normal and malignant B lymphocytes. Using dual color immunofluorescence staining, this antibody reacted with all immunoglobulin-positive cells but no T cells in normal peripheral blood. There was no detectable 4G7 antigen on monocytes, platelets, red cells, granulocytes, or phytohemagglutinin-activated T cells. When PBL were depleted of 4G7 positive cells and stimulated with pokeweed mitogen, secreted immunoglobulin levels fell to less than 10% of control values on Day 5 and less than 1% of control on Day 7. This antibody was reactive with 155 of 176 B lineage neoplasms on which it was screened. Thirty-five cases of myeloid or T-lymphoid malignancy were negative. Our studies show that the 4G7 antigen modulates in the presence of excess antibody. Free 4G7 antigen was not found circulating in human serum. The cell surface antigen identified by 4G7 was sensitive to pronase proteolysis but resistant to trypsin and chymotrypsin digestion. A comparison of 4G7 with other known B-cell antibodies indicates that the 4G7 antigen has not been previously identified. This antibody is of use for the identification of normal B lymphocytes, the study of B-cell differentiation, and the characterization of lymphoid malignancies.     

Assembly of targeting complexes driven by a single-chain antibody

Rapid development in design and production of recombinant antibodies and antibody fragments specific for cell surface markers opens new opportunities for targeted delivery of therapeutic or imaging agents. However, the progress in this field is slowed by inactivation of many antibodies by chemical conjugation of payloads and by lack of internalization of complexes formed on the cell surface. Here, we describe conversion of a non-internalizing single chain Fv (scFv) antibody P4G7 specific for vascular endothelial growth factor receptor 2 (VEGFR-2) into a targeting protein (Hu-P4G7) for assembly of a novel type of targeting complexes. Hu-P4G7 contains an N-terminal "docking" Hu-tag, a 15-aa fragment of human RNase I, capable of high affinity binding of S-protein fragment of human RNase I or bovine RNase A. Purified Hu-P4G7 and complexes of Hu-P4G7 with S-protein bind both soluble and full-length cellular VEGFR-2. To assemble targeted DNA delivery complexes, S-protein modified with a DNA condensing agent was "docked" to Hu-P4G7, and then loaded with luciferase plasmid DNA. As expected for a non-internalizing targeting protein, Hu-P4G7-based complexes did not deliver DNA in VEGFR-2 expressing cells. However, in the presence of vascular endothelial growth factor (VEGF), these complexes selectively delivered DNA into the cells overexpressing VEGFR-2 suggesting that even a non-internalizing scFv antibody can be used for targeted intracellular drug delivery.     

In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library

Background: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv. Objectives: To isolate and characterize human single chain Fvs which bind to c-erbB-2, an oncogene product overexpressed by 30–50% of breast carcinomas and other adenocarcinomas. Study design: A non-immune human single-chain Fv phage antibody library was selected on human c-erbB extracellular domain and sFv characterized with respect to affinity, binding kinetics, and in vivo pharmacokinetics in tumor-bearing scid mice. Results: A human single-chain Fv (C6.5) was isolated which binds specifically to c-erbB-2. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. C6.5 binds to immobilized c-erbB-2 extracellular domain with a K_d of 1.6 × 10⁻⁸ M and to c-erbB-2 on SK-OV-3 cells with a K_d of 2.0 × 10⁻⁸ M, an affinity that is similar to sFv produced against the same antigen from hybridomas. Biodistribution studies demonstrate 1.47% injected dose/g tumor 24 h after injection of ¹²⁵I-C6.5 into scid mice bearing SK-OV-3 tumors. Tumor:normal organ ratios range from 8.9:1 for kidney to 283:1 for muscle. Conclusions: These results are the first in vivo biodistribution studies using an sFv isolated from a nonimmune human repertoire and confirm the specificity of sFv produced in this manner. The use of phage display to produce C6.5 mutants with higher affinity and slower k_off would permit rigorous evaluation of the role of antibody affinity and binding kinetics in tumor targeting, and could result in the production of a therapeutically useful targeting protein for radioimmunotherapy and other applications.     

Cell surface antigens of human trophoblast: definition of an apparently unique system with a monoclonal antibody.

An epitope with apparent specificity for the surface of human syncytiotrophoblast was defined by a murine monoclonal antibody, FDO46B (IgG1, kappa). The epitope was predominantly expressed during the first trimester of pregnancy. Binding was detected on frozen tissue sections and on cultured trophoblast by the immunoperoxidase technique. It was also detected on the surface of a small percentage (less than 10%) of cultured choriocarcinoma cells (JEG-3). A panel of human tissues was negative, as were normal and malignant human lymphocytes. The antigen bearing the FDO46B epitope was still expressed by trophoblast after culture in the presence of tunicamycin, indicating that it is possibly protein in nature. This antigen may have potential utility as a target for a contraceptive vaccine.     

人源性抗HBsAg单链抗体在大肠杆菌中的表达及活性测定

目的 :制备有活性的人源性抗HBsAg单链抗体。方法 :应用噬菌体表面呈现技术获得人抗乙型肝炎表面抗原(HBsAg)抗体的Fab片段 ,并将VH 和VL 基因以 (Gly4Ser) 3linker连接成单链 ,插入表达载体pQE40 ,筛选大肠杆菌高表达菌株。结果 :工程菌表达优化试验表明 ,在OD6 0 0 为 0 6时开始诱导 ,持续 6h ,目的蛋白表达量最高可达菌体总蛋白的 31%。包涵体变性后上样镍离子螯合层析柱一步纯化 ,收集目的峰 ,透析复性 ,得纯度为 97%的重组单链抗体 ,其亲和常数为 0 2 3× 10 8mol L。结论 :抗HBsAg单链抗体在大肠杆菌内获得高效表达 ,经变性和复性 ,得到有活性的单链抗体 ,氨基酸组成正确。为进行临床前研究打下基础。     

Enhanced phagocytosis of Candida species mediated by opsonization with a recombinant human antibody single-chain variable fragment

Specific antibody opsonization significantly enhances the level of phagocytosis of Candida in the absence of complement. Furthermore, we have described a system using a recombinant human antibody single-chain variable fragment that allows a comparative study of phagocytosis of multiple Candida species opsonized via a common antigen.     

Targeted identification of a unique glycan epitope of Schistosoma mansoni egg antigens using a diagnostic antibody

The eggs of Schistosoma mansoni express a plethora of glycoconjugate antigens. A specific subset of these antigens can be detected in the serum or urine of infected individuals by a diagnostic sandwich ELISA using the anti-carbohydrate monoclonal antibody (mAb) 114-4D12-A [Nourel Din MS, Nibbeling R, Rotmans JP, Polderman AM, Krijger FW, Deelder AM. Quantitative determination of circulating soluble egg antigen in urine and serum of Schistosoma mansoni-infected individuals using a combined two-site enzyme-linked immunosorbent assay. Am J Trop Med Hyg 1994;50:585-94]. We used affinity chromatography to isolate the 114-4D12-binding glycoprotein subset from soluble egg antigens (SEA) of S. mansoni. SEA and the isolated SEA-subset (SEA-4D12) were subjected to reductive beta-elimination and hydrazinolysis to release intact glycans and glycan fragments, respectively, from the protein backbones. The released glycans were characterised by matrix-assisted laser-desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS), liquid-chromatography (LC)-MS and gas chromatography (GC)-MS linkage analysis. Glycans released by reductive beta-elimination from SEA-4D12 were larger and more heavily fucosylated than glycans released from SEA. Most SEA-4D12 glycans contained a branched O-glycan core structure carrying up to 4 N-acetylhexosamines per chain which were substituted with maximum 12 fucose residues. Hydrazinolysis of SEA-4D12 resulted in the release of fucosylated antenna fragments. After 2-aminobenzamide (2AB)-labelling these fragments were subjected to 114-4D12-affinity purification. Normal phase (NP)-LC analysis of the flow-through and retained fractions indicated that the Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1- element forms the epitope of mAb 114-4D12. Most O-glycans from SEA-4D12 contain this structural element. Epitope-bearing N-glycans have not been found. In terms of abundance in total SEA, only a minority of all glycans possesses the epitope. This multifucosylated motif has so far only been found in schistosomes, providing a structural basis for the high specificity of the diagnostic antibody.     

Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library

Traditionally, the selection of phage-display libraries is performed on purified antigens (Ags), immobilized to a solid substrate. However, this approach may not be applicable for some Ags, such as membrane proteins, which for structural integrity strongly rely on their native environment. Here we describe an approach for the selection of phage-libraries against membrane proteins. The envelope glycoproteins (Env) of the Human Immunodeficiency Virus type-1 (HIV-1) were used as a model for a type-1 integral membrane protein. HIV-1IHI Env, expressed on the surface of Rabbit Kidney cells (RK13) with a recombinant vaccinia virus (rVV), was solubilized using the non-ionic detergent n-Octyl beta-D-glucopyranoside (OG). Membrane associated Env was reconstituted into vesicles by the simultaneous removal of detergent and free monomeric Env subunits by gel-filtration. The resulting antigen preparation, termed OG-P1IHI, was captured on microtiter plates coated with Galanthus nivalis agglutinin (GNA) and used for rounds of selection (panning) of a well-characterized phage-display library derived from an HIV-1 seropositive donor. Simultaneously, an identical experiment was performed with OG-P1IHI vesicles disrupted by Nonidet P-40 (NP-P1IHI). Both membrane-associated and soluble Ags were selected for vaccinia-specific clones (OG-P1IHI: 59/75 and NP-P1IHI: 1/75) and HIV-1-specific clones (OG-P1IHI: 11/75 and NP-P1IHI: 65/75) using our approach. Hence, the novel panning strategy described here may be applicable for selection of phage-libraries against membrane proteins.     

Production and characterization of human monoclonal antibody Fab fragments to vaccinia virus from a phage-display combinatorial library.

A combinatorial, phage-display library of human Fab antibody fragments was generated from IgG heavy chain (HC) and light chain (LC) genes cloned from the lymphocytes of a vaccinia virus (VACV)-immune donor. To ascertain the complexity of the library, nucleotide sequences of the variable regions of the HC and LC genes were determined. Fourteen distinct HC and 18 distinct LC (7 kappa and 11 lambda) that formed a combinatorial library of 22 Fabs were identified. Immune-precipitation of radiolabeled VACV revealed that at least six different VACV proteins were recognized by the antibodies. Plaque-reduction neutralization demonstrated that six of the Fabs neutralized VACV in the presence of anti-human antibody. ELISA studies indicated that 15 of the Fabs were cross-reactive with monkeypox virus.     

大容量噬菌体抗体库的构建及鉴定

目的　构建大容量噬菌体单链抗体库 ,从中筛选人源单链抗体 (ScFv)。方法　从正常成人外周血和新生儿脐血分离淋巴细胞 ,用RT PCR扩增轻链可变区基因 (VL)和重链可变区基因(VH) ,通过重叠PCR法将VH 和VL 拼接形成ScFv基因 ,并克隆入噬菌体表达载体PDF ,得到ScFv初级噬菌体抗体库。以高MOI超感染cre 菌株BS136 5 ,通过loxp cre定位重组系统 ,介导轻重链的组合配对 ,得到大容量抗体库 ,用多种抗原对抗体库进行生物淘筛 ,鉴定抗体库的性能。结果　获得了 6×10 10 的大容量单链噬菌体抗体库。分别用卵清蛋白、胃蛋白酶、铁蛋白、人角蛋白、人TNF α、地高辛等6种抗原进行筛选 ,均得到多样性的特异性噬菌体抗体。结论　经loxp cre定位重组系统在单细胞内重组成功地构建了大容量单链噬菌体抗体库 ,初步尝试对 6种抗原进行筛选均获成功 ,提示该抗体库可用于制备具有应用前景的人源抗体     

Humanization of a highly stable single-chain antibody by structure-based antigen-binding site grafting

The murine single-chain variable fragment F8 (scFv(F8)) is endowed with high intrinsic thermodynamic stability and can be functionally expressed in the reducing environment of both prokaryotic and eukaryotic cytoplasm. The stability and intracellular functionality of this molecule can be ascribed mostly to its framework regions and are essentially independent of the specific sequence and structure of the supported antigen-binding site. Therefore, the scFv(F8) represents a suitable scaffold to construct stable scFv chimeric molecules against different antigens by in vitro evolution or antigen-binding site grafting. Thanks to the favourable pharmacokinetic properties associated to a high thermodynamic stability of antibody fragments, such scFv(F8) variants may be exploited for a wide range of biomedical applications, from in vivo diagnosis to therapy, as well as to interfere with the function of intracellular proteins and pathogens, and for functional genomics studies. However, the potential immunogenicity of the murine framework regions represents a limitation for their exploitation in therapeutic applications. To overcome this limitation, we humanized a derivative of the scFv(F8), the anti-lysozyme scFv(11E), which is endowed with even higher thermodynamic stability than the parent antibody. The humanization was carried out by substituting the framework residues differing from closely related V(H) and V(L) domains of human origin with their human counterparts. Site-directed mutagenesis generated the fully humanized product and four intermediate scFvs, which were analyzed for protein expression and antigen binding. We found that the substitution Tyr 90-->Phe in the V(H) domain dramatically reduced the bacterial expression of all mutants. The back-mutation of Phe H90 to Tyr led to the final humanized variant named scFv(H5)H90Tyr. This molecule comprises humanized V(H) and V(L) framework regions and is endowed with HEL-binding affinity, stability in human serum and functionality under reducing conditions comparable to the murine cognate antibody. Consequently, the humanized scFv(H5)H90Tyr represents a suitable scaffold onto which new specificities towards antigens of therapeutic interest can be engineered for biomedical applications.     

Characterization of a single-chain intrabody directed against the human receptor tyrosine kinase Ron

A large human nonimmune phage antibody library was screened by affinity chromatography to select single-chain antibodies directed against the human receptor tyrosine kinase (RTK) Ron. As antigen, we used a GST fusion protein (GST-IRP(-)) containing the whole intracellular portion of Ron except for the carboxyl-terminal arginine-proline-rich motif. One selected phage was highly specific for Ron when tested in an enzyme-linked immunosorbent assay (ELISA). We report here the immunological characterization of this anti-Ron single-chain antibody (sc7) and show that it recognizes both denatured and native forms of the receptor. The epitope bound by sc7 maps within the first 50 amino acid residues of the juxtamembrane domain of Ron. This monoclonal fragment does not cross-react with other receptor tyrosine kinases including the closely related human proto-oncogene Met. We demonstrate that the isolated antibody fragment interacts in vivo with the intracellular domain of Ron in mammalian cells.     

Penetration of the mosquito midgut wall by the ookinetes ofPlasmodium yoelii nigeriensis

The penetration route of ookinetes ofPlasmodium yoelii nigeriensis in the midgut of a mosquito,Anopheles omorii, was investigated by electron microscopy. Within 15–18 h after an infective blood meal, ookinetes could be seen in the midgut lumen in the process of entering the midgut wall, or lodged between the basement membrane and the basal lamina. The morphology of the ookinetes and their transformation into early oocysts were found to be similar to those previously reported. Ookinetes penetrated the midgut wall by the intercellular route; however, the intracellular occurrence of the parasite was also observed. Vacuoles appeared around the penetrating ookinetes during the penetration process, but no change in the electron density of the rhoptry-microneme complex was noted.Abbreviations in the figures bl Basal lamina - bm basement membrane - c cristalloid - cj cellular junction - d ductuli - dm disrupted mitochondrion - f pedicular fold - ic intestinal content - im intercellular membrane - l labyrinth - mtoc microtubule organizing centre - mc midgut cell - mv microvilli - n nucleus - o ookinete - ow oocyst wall - p pigment bar - pm peritrophic membrane - rm rhoptry-microneme complex - v vacuole     

Monosomy 6 in a human lymphoma line induced by selection with a monoclonal antibody

The human Epstein Barr Virus-superinfected B lymphoma cell line BJAB-B95.8.6 was mutagenized by gamma irradiation, and HLA mutants were selected with the HLA-Bw6-specific monoclonal antibody SFR8-B6. One of the mutants obtained, BM19, had lost one of the chromosomes 6 present in the wild type cells. Electrophoretic analysis of phosphoglucomutase isozyme PGM3 and erythrocyte glyoxalase 1 from both cells supports this conclusion. The HLA antigens expressed on BM19 were HLA-A2, B13, Bw4, C-, DR2 (questionable), DRw52 (weak) and DQw1. This constitutes one of the haplotypes of the wild type cells, the other (lost from BM19 cells) being HLA-A1, B35, Bw6, Cw4, DR5, DRw52 (strong) and DQw3. Possibilities to employ BM19 cells for the analysis of the major histocompatibility complex and other chromosome 6-encoded genes as well as their products are discussed.     

Recognition by a murine monoclonal antibody of a unique epitope specific for the human alloantigen HLA-B8

A cytotoxic murine monoclonal antibody recognizing a specific HLA alloantigen was produced from the spleen cells of a BALB/c immunized with partially purified class I glycoproteins from an HLA-A1,B8 homozygous b-lymphoblastoid cell line. The antibody, designated P8.1, was tested against cells from 521 unrelated donors. It reacted with each of the 83 donors known to be HLA-B8 positive and with no HLA-B8 negative donors (sensitivity, 100%; specificity, 100%). Immunoprecipitation with antibody P8.1 and polyacrylamide gel electrophoresis confirmed that the antigen recognized was a class I structure. Although most murine monoclonal anti-HLA antibodies previously described have recognized “public” or supertypic specificities, the identification of a monoclonal antibody specific for a “private” HLA alloantigen indicates first that the BALB/c mouse has the appropriate immune response repertoire for recognizing certain HLA allospecificities and second that HLA-B8 can be defined by a single unique epitope.     

Generation and characterization of a single-chain anti-EphA2 antibody

Abstract

Recombinant antibody phage library technology provides multiple advantages, including that human antibodies can be generated against proteins that are highly conserved between species. We used this technology to isolate and characterize an anti-EphA2 single-chain antibody. We show that the antibody binds the antigen with 1:1 stoichiometry and has high specificity for EphA2. The crystal structure of the complex reveals that the antibody targets the same receptor surface cavity as the ephrin ligand. Specifically, a lengthy CDR-H3 loop protrudes deep into the ligand-binding cavity, with several hydrophobic residues at its tip forming an anchor-like structure buried within the hydrophobic Eph pocket, in a way similar to the ephrin receptor-binding loop in the Eph/ephrin structures. Consequently, the antibody blocks ephrin binding to EphA2. Furthermore, it induces apoptosis and reduces cell proliferation in lymphoma cells lines. Since Ephs are important mediators of tumorigenesis, such antibodies could have applications both in research and therapy.