Nucleotide sequence analysis of CDR3 elements of a panel of anti-peptide monoclonal antibodies recognizing parathyroid hormone-related protein.

Nucleotide sequences of heavy (VH) and light (VL) chain variable region complementarity determining regions have been determined from in vitro amplified mRNA isolated from a panel of monoclonal antibodies (mAb) raised to a synthetic 34mer peptide representing the N-terminal portion of human parathyroid hormone-related protein (PTHrP or parathyrin) reported to contain an immunodominant epitope. These mAb vary in affinity for the synthetic peptide and native PTHrP (Ka between 5.9 x 10(8) and 1.9 x 10(11)l/M). All 10 mAb studied were found were found to utilized restricted VH2, V kappa 2, JH4 and J kappa 1 family genes. Significant differences in the length and sequence of D elements were found; however 9/10 mAb utilize members of the DSP2 family. Significantly, two broad ranges of affinity could be determined based on the presence of Asp or Ala at residue 101 in JH.     

人源抗狂犬病毒单链抗体库的构建及体外亲和筛选

目的 :构建人源噬菌体展示单链抗体 (scFv)库 ,筛选抗狂犬病毒特异性、高亲和力的scFv。方法 :应用重组噬菌体抗体技术 ,从经狂犬病毒WISTARPM株疫苗免疫者的外周血淋巴细胞中 ,分离并构建scFv基因。将其克隆入噬粒载体pCANTAB 5E中 ,转化于大肠杆菌TG1,通过辅助噬菌体M13K0 7援救构建噬菌体单链抗体库。采用狂犬病毒Vero疫苗亲和富集法 ,淘选阳性重组噬菌体 ,经鉴定后对其进行序列分析。用竞争ELISA ,初步检测重组scFv的特异性抗原结合活性。结果 :成功地构建了库容量约为 7× 10 8抗狂犬病毒噬菌体scFv库 ,筛选到 1株新的抗狂犬病毒的scFv S12。结论 :噬菌体展示scFv库的成功构建及人源抗狂犬病毒特异性scFv的获得 ,为进一步研制抗狂犬病毒的高特异性、高亲和力的基因工程抗体奠定了基础     

Cloning and expression of the variable regions of mouse myeloma protein MOPC315 in E. coli: recovery of active FV fragments.

Recombinant DNA techniques were used to clone and express the FV portion of MOPC315, a mouse myeloma protein with a high affinity for 2,4-dinitrophenyl (DNP). The FV fragment consists of a heterodimer of heavy and light chain variable domains (VH and VL). Two separate bacterial plasmid constructs, containing either a variable region cDNA for the light chain or a variable region synthetic gene for the heavy chain demonstrated high levels of expression (150-200 mg/L) under control of the bacteriophage T7 promoter. Recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8 M urea, combined together, and refolded by subsequent chaotrope removal. Biologically active FV was affinity purified from the chain mixture by specific binding to DNP-lysine-Sepharose. Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis. The purified FV displayed a binding affinity of 4.8 +/- 0.3 x 10(-7) M which was identical to the native FV. Chimeric FVs composed of recombinant and native chain mixtures yielded similar results. Recombinant MOPC315 FV activity was also obtained using a single chain construct (sFV), in which recombinant VH and VL were linked via a (Gly4Ser)3 spacer region. Binding affinity of the sFV was shown to be the same as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 FV makes this system useful in the study of the optimization of antibody production in bacteria.     

抗Pgp基因工程抗体ScFv的构建、表达及其活性测定

目的：构建表达抗PgpScFv，进行体外活性的测定。方法：利用RT-PCR方法，克隆抗Pgp杂交瘤细胞PHMAO2的重链可变区基因(VH)，轻链可变区基因(VL)，拼接为单链抗体(ScFv)，再克隆到具有强启动子的PET28a( )载体上进行表达，并进行了表达产物体外活性的测定。结果：构建了表达质粒PET28a( )．ScFvPGP，表达产物可与表达Pgp抗原的K562／A02细胞特异性结合，并不抑制Pgp外排泵的功能。结论：构建表达的抗PgpScFv只有针对Pgp抗原的识别功能，并无抑制作用，因此应用于人体后不会干扰正常细胞的排泄分泌功能，无论是作为靶向诊断治疗的载体还是作为双功能抗体的一臂，均具有广泛的应用前景。     

具有GPX活性的单克隆抗体可变区基因的克隆和序列分析

目的利用分泌具有GPX活性的单克隆抗体(mAb)的杂交瘤细胞3G5,克隆其mAb体的可变区基因。方法提取杂交瘤细胞的总RNA ,分离mRNA ,反转录合成cDNA。经PCR扩增VH 基因和VL 基因 ,将VH 基因和VL基因与载体 pGEM T连接后 ,进行酶切鉴定和序列分析。结果构建了2个分别含有VH 和VL 基因的重组质粒。序列分析表明 ,VH 和VL 分别属于mouscheavychainsubgroupIII和mouselightchainsubgroupV亚群 ,长度为372和324bp ,编码124和108个氨基酸 ,在高变区分别有4和2个丝氨酸。结论克隆的VH 和VL 基因符合功能性重排的鼠抗体可变区基因特征 ,为将来制备具有GPX活性的单链抗体提供了可靠的基因材料。     

Active site contributions to fluorescein binding determined by in vitro heavy and light chain reassociations

In addition to prior crystallographic studies that determined antigen contact residues for high affinity murine monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 1.7 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal anti-Fl antibody 9-40 (Ka = 5.7 x 10(7) M-1) possessed identical Fl contact residues with the exception of L34 His for Arg. Mab 9-40 L34 His was germ-line encoded and 4-4-20 L34 Arg correlated with increased 4-4-20 affinity and enhanced Fl quenching. To better define L34 Arg and L96 Trp contributions to antigen binding, in vitro H and L chain reassociation experiments were performed. Following reassociations, affinity purified and homologous chain reassociated proteins 4-4-20 (H4-4-20 L4-4-20) and 9-40 (H9-40 L9-40) yielded identical idiotypes (greater than 91% related), Qmax values (91% and 44.7%), affinity constants (approximately 2.0 x 10(10) M-1 and 5.5 x 10(7) M-1) and iodide quenching values (1.2% and 2.1%), respectively. Although heterologous reassociated proteins were idiotypically related to prototypic proteins (greater than or equal to 87.1%), differences in Fl-binding characteristics were observed. Recombinant H4-4-20 L9-40 expressed Ka and Qmax values similar to 9-40 and implicated L34 Arg in increased 4-4-20 Ka and Qmax. Residue L34 Arg replacement in the 9-40 active site (H9-40 L4-4-20). however, exhibited low Qmax (44.4%) and slightly increased affinity (3.7 x 10(8) M-1). In addition, L96 Leu substitution into 4-4-20 (H4-4-20 L-04-01) and 9-40 (H9-40 L04-01) resulted in lower Qmax values (15.1% and 20.5%, respectively) and significantly reduced Fl affinity (approximately 10(3)-fold). These results demonstrated: (1) L34 Arg was responsible for increased 4-4-20 affinity, (2) L96 Trp was critical for an intermediate Fl affinity (approximately 10(7) M-1) and (3) active site Fl contact residue orientations were potentially affected by differences in H chain complementarity-determining region 3 and CH 1 residues.     

Bacterial expression of immunoglobulin VH proteins

A bacterial expression system in Escherichia coli has been developed that produces as much as 10 mg/l of culture of the VH protein associated with monoclonal antibodies specific for the 5-dimethylaminonaphthalene-2-sulfonyl (Dns) group. This system has been applied to the expression of the VH genes derived from a low-affinity, IgM-producing hybridoma and from a high-affinity, IgG-producing cell line. The plasmid vectors (contributed by Dr William F. Studier) utilize a T7 expression cassette whose activity is initiated by infection with a lambda phage derivative carrying the T7 RNA polymerase gene. The VH proteins were extracted from the bacterial pellet in 8 M urea and purified by chromatography in 8 M urea. Recombinants with the homologous light (L) chains were prepared to yield VHL molecules. These were used to measure intrinsic affinity for Dns-lysine by resonance energy transfer. The association constants were 7 x 10(6) M-1 and 7 x 10(9) M-1 for the low- and high-affinity systems, respectively. These values are not significantly different from those observed with monoclonal antibodies secreted from the corresponding cell lines. This system lends itself to the quantitative evaluation of the binding properties of the VH protein itself as well as the modulation of affinity by site-directed mutagenesis.     

Generation of a panel of related human scFv antibodies with high affinities for human CEA

Background: A human single chain Fv (scFv) specific for human carcinoembryonic antigen (CEA) has been isolated from a 2.0 × 10⁹ phage display library from unimmunised human donors. The dissociation constant of the scFv has been measured by surface plasmon resonance (SPR) and found to be 7.7 x 10⁻⁹ M, with an off-rate component of 6.2 × 10⁻³ s⁻¹. In order to investigate directly whether increased affinity leads to improved targeting of CEA-positive tumours, this scFv has been affinity matured by both targeted mutagenesis of the CDRs of heavy and light chains, and by light chain shuffling. Study design: A partial randomisation scheme, biased towards amino acids commonly found as somatic mutations of germline antibody sequences, was used for directed diversification of VH and VL CDR3s. Diversification of the entire VL region was also introduced by light chain shuffling of the parental anti-CEA scFv. Selection of the mutagenised repertoires was carried out to enrich for antibodies with a reduced k_off. Results: Sequencing the selected clones identified a number of amino acid changes in the VH CDR3, one of which gave a four-fold reduction in k_off. Stringent selection of the light chain shuffled library resulted in several clones with a two to three-fold reduction in k_off. It has been possible to combine the selected changes from both mutagenesis approaches by using the mutagenised heavy chain and a light chain derived by shuffling to give a human scFv with a dissociation constant for human CEA of 6.0 x 10⁻¹⁰ M. Conclusion: A panel of human anti-CEA scFvs has been generated with differing dissociation constants for antigen, which will allow the correlation between tumour targeting efficiency in relation to binding affinity to be assessed directly. The scFv panel will be valuable in the optimisation of human antibodies for immunotherapy.     

抗人纤维蛋白噬菌体单链抗体库的构建和鉴定

目的应用噬菌体展示技术构建抗人纤维蛋白单链抗体(scFv)文库,筛选高亲和力抗人纤维蛋白scFv并进行鉴定。方法利用人纤维蛋白免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸聚合酶链反应(PCR)将VH和VL基因拼接成scFv基因,SfiⅠ/NotⅠ双酶切克隆入pCANTAB 5E噬菌粒载体,转化E.coli TG1构建成库,采用人纤维蛋白原对抗体库进行负筛选,人纤维蛋白进行正筛选,酶联免疫吸附分析(ELISA)检测阳性克隆的抗原特异性并进行测序分析。结果构建了库容为8.7×106的抗人纤维蛋白scFv库,ELISA测定显示scFv具有较高的抗原特异性;抗人纤维蛋白scFv基因序列长732 bp,编码244个氨基酸,VH和VL基因均有明确的3个互补决定区和4个骨架区。结论成功构建了抗人纤维蛋白scFv文库,并筛选到高亲和力的抗人纤维蛋白scFv,为新型血栓显像剂的开发奠定了实验基础。     

HBV Pre S2 3B9单抗轻、重链可变区基因的分离,序列分析及单链可变区抗体基因的构建

3B9(杂交瘤细胞)是一株分泌HBV Pre S2抗原的单抗细胞,为从分子水平分析、了解3B9株进而为制备新一代基因工程抗体奠定基础,用分子生物学技术,提取3B9杂交瘤细胞总RNA,经反转录、PCR分离获得了3B9单抗的轻、重链可变区基因,并利用PCR及双脱氧核苷酸链末端终止法对3B9单抗的可变区基因的核苷酸序列进行了分析。结果表明,3B9单抗轻链隶属小鼠Ig Kappa轻链第Ⅱ亚组,JK_1基因重排;而3B9单抗重链隶属小鼠Ⅰg重链第Ⅱc亚组。在此基础上,作者用一编码疏水性多肽接头的DNA片断将3B9单抗轻、重链可变区基因连接,成功地构建了3B9单抗单链可变区抗体基因。为下一步克隆、表达有抗原结合活性的单链抗体打下了良好的物质基础。     

抗人核内不均一核糖核蛋白A2/B1抗体可变区基因克隆、串联和表达

目的克隆抗人核内不均一核糖核蛋白A2/B1(heterogeneousnuclearribonucleoproteinA2/B1,HnRNPA2/B1)单克隆抗体的重链可变区(variableregionofheavychain,VH)和轻链可变区(variableregionoflightchain,VL)基因,构建抗HnRNPA2/B1单链抗体(singlechainFv,ScFv)基因,并在大肠杆菌表达。方法采用斑点ELISA、Western印迹和免疫组化检测抗HnRNPA2/B1单克隆抗体3E8的特异性,并通过逆转录-聚合酶链反应、重叠延伸PCR来克隆、串联VH和VL基因,构建抗HnRNPA2/B1ScFv基因pET28(a )表达载体,在大肠杆菌BL21中诱导表达,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳、竞争抑制ELISA检测表达产物。结果3E8抗体能特异性地与HnRNPA2/B1合成肽以及3株人肺癌细胞内HnRNPA2/B1结合;克隆的VH基因为345bp,VL基因为309bp,符合小鼠抗体可变区特性;抗HnRNPA2/B1ScFv蛋白以包涵体形式表达,分子量约28000,并具有与抗原结合活性。结论成功构建了抗HnRNPA2/B1ScFv基因克隆,并在大肠杆菌中获得了功能性表达,为阐明HnRNPA2/B1与肺癌相关性奠定了实验基础。     

Anti-V region framework antibodies affect the ligand binding of VL dimer.

The effect of antibodies to the light chain variable region (VL) of protein MOPC-315 (alpha, lambda 2), on the binding of hapten by VL315 dimer or Fv315 (VL + VH) was studied by equilibrium dialysis. Anti-VL did not change the binding properties of Fv but affected the binding properties of VL dimer. At pH 5, the binding properties of VL in the presence or absence of anti-VL were the same, whereas at pH 8, anti-VL reduced the number of ligands bound to VL from two to one. It has previously been shown that VL dimer binds one ligand at pH 5 and two ligands at pH 8, and that VL conformation at pH 5 is tighter. Hence, our results suggest that anti-VL tightens the conformation of VL dimer at pH 8.0 such that it can bind only one ligend. Since Fv is not affected by anti-VL, the results indicate that a combining site made of two identical chains (VL dimer) can undergo a conformational change upon interaction with its antibody. Such conformational change can indirectly affect the binding properties.     

The antibody site in Atlantic salmon; phage display and modeling of scFv with anti-hapten binding ability.

Cloning and sequencing the cDNA of around 50 VH (VDJ) and 15 VL genes in Atlantic salmon demonstrated nine VH families (above 80% identity within each family) and one dominating but relatively diverse VL family in this species. The highest variability of the VH was seen in the CDR3, but CDR2 also expressed a modest variability. The 'whole' antibody repertoire was expressed as single chain Fv (scFv) in a phage display library by combining 12 VH and two VL specific primers (FR1/microl and FR1/CL, respectively). The PCR products (VH and VL) were ligated (with a G-rich spacer) into the lambda Surf-Zap (Stratagene) vector and expressed as a surface fusion protein on the M13 phage. Anti-TNP and anti-FITC specific scFv clones were isolated by panning using hapten-coated magnetic beads and the coding DNA sequenced. The specificities of the anti-TNP and anti-FITC clones were similar to mouse monoclonal antibodies. 3D-models of the active sites (CDRs) of the anti-TNP and anti-FITC clones suggest hapten-interacting structures of the salmon antibody site similar to mammalian antibodies.     

Rabbit antisera to the variable region domains of an anti-alpha(1----6) dextran using E. coli-produced VL and VH fusion proteins as immunogens.

Bacteria were engineered for the expression of mouse immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) fusion proteins. cDNAs encoding the VL and VH of anti-alpha(1----6)dextran hybridoma protein 19.22.1 were inserted into the pATH 10 prokaryotic expression vector downstream of trp operon sequences. V domains joined to approximately 330 amino acids of the trp E gene product encoded by the expression plasmids accumulated at high levels in E. coli. In addition, the VL domain was expressed with a 15 amino acid extension at low levels in lon mutant bacteria. The trp E-VL and trp E-VH proteins were used to raise antisera in rabbits and the V specificity of the sera demonstrated.     

抗血小板膜糖蛋白单克隆抗体SZ-2单链抗体的构建、表达及功能研究

目的：降低鼠源性抗体的免疫原性及分子量，为其进一步研究、应用奠定基础。方法：应用RT—PCR技术，克隆SZ-2重链、轻链可变基因。应用基因重组技术构建SZ-2单链抗体表达载体pET22-2scFv，导入大肠杆菌BL21(DE3)Plys，诱导表达。流式细胞术、ELISA、Western blot检测SZ-2 scFv与血小板结合能力，瑞斯托霉素、凝血酶和ADP诱导血小板聚集试验对表达产物功能进行研究。结果：克隆基因序列符合小鼠轻、重链可变区基因特征；pET22—2scFv表达质粒拼接正确；表达产物以包涵体形式为主，表达量占菌体总蛋白的25％；纯化、复性后证明表达产物具有与血小板结合的活性，并可抑制瑞斯托霉素、凝血酶诱导的血小板聚集。结论：成功表达了SZ—2单链抗体，该小分子抗体具有与血小板结合的活性，并可抑制瑞斯托霉素、凝血酶诱导的血小板聚集。     

Structural aspects of human IgM antibodies expressed in chronic B lymphocytic leukemia.

BACKGROUND: Malignant B cells from patients with chronic B lymphocytic leukemia (B CLL) generally express both surface IgM and the pan T cell antigen CD5, a characteristic of the B1 population of B lymphocytes. The IgM on the surface of these B CLL cells is frequently polyreactive with respect to its capacity to recognize multiple structurally dissimilar antigens (Ag). OBJECTIVES: To understand the structural characteristics of the polyreactive binding sites of human IgM molecules expressed on B CLL cells by: (1) analyzing the nucleotide and protein sequences of the variable (V) domains of five IgM molecules expressed in cases of B CLL and; (2) utilizing these sequences to generate three-dimensional (3D) models of Fv (VL - VH) molecules. STUDY DESIGN: Peripheral blood leukocytes obtained from five cases of B CLL were tested for polyreactive binding properties by assessing their capacity to bind mouse IgG by indirect immunofluorescence. The V region genes of light and heavy chains were amplified using the polymerase chain reaction, subsequently cloned and their nucleotide sequences obtained. Translated amino acid sequences of the V domains were used to generate homology models of the Fv molecules. RESULTS: Low affinity binding of mouse IgG was demonstrated for all B CLL samples examined, confirming the polyreactive nature of the IgM expressed on these cells. There was an absence or minimal mutation within V region genes when compared to germline Ig genes. Junctional diversity was not observed for VL regions, although truncations and insertions were frequent in D minigenes of VH regions. The binding sites were predicted to form either relatively flat surfaces with occasional protrusions or cavities at the VL - VH domain interface. Aromatic side chains covered a large proportion of the potential binding surfaces in the models of B CLL Fv components. DISCUSSION: Primary DNA sequences can be categorized as germline, suggesting that the B cells involved in B CLL are germline or naive in origin. The medium to large HCDR3s provide the majority of probable contact residues for antigens. While prominent aromatic residues are likely to engage in binding patterns which are conserved (e.g. mouse Ig reactivity), the diverse binding sites predicted for B CLL-derived IgMs also have properties which are conducive to polyreactive antigen binding.     

Humanization of an anti-human TNF-alpha antibody by variable region resurfacing with the aid of molecular modeling

The murine monoclonal antibody Z12 is of therapeutic interest for its neutralizing biological activity against human tumor necrosis factor-alpha (hTNF-alpha). We attempted to humanize Z12 with variable domain resurfacing guided by computer modeling. First, the genes of heavy and light chain variable region (VH, VL) of Z12 were cloned and the whole three-dimensional structure of Fv fragment was constructed by using homology-based modeling and molecular docking methods. Then the complex model of Fv interacting with hTNF-alpha whose crystal structure derived from PDB database was gained with computer-guided docking program. Based on this model, a humanized version was designed. The humanized Fab antibody was constructed, expressed and purified in the pComb3H vector system and it showed unaltered binding affinity to the antigen as determined by ELISA and atomic force microscopy (AFM). The method described here can be used to humanize other anti-hTNF-alpha antibodies.     

A novel variable antibody fragment dimerized by the dHLX peptide with enhanced affinity against amantadine compared to its corresponding scFv antibody

Amantadine (AMA) is an illegally used antiviral drug in the poultry industry, it is necessary to establish a fast, accurate and time-saving detection method for poultry food. The antibody-based immunoassay can achieve fast and accurate requirements. We developed a recombinant antibody-based specificity immunoassay for AMA. In the recombinant antibody, the heavy chain variable region (VH) is connected covalently with the light chain variable region (VL) by the artificial linker. Here, two recombinant antibodies’ single-chain variable fragment (scFv) and scFv-dHLX were constructed and functionally expressed in the periplasm of Escherichia coli. The helix-turn-helix peptide was utilized to dimerize VH and VL similar to the IgG counterpart. The ScFv-dHLX protein showed a higher binding ability and affinity resulting in improvement of in vitro affinity activity over its corresponding scFv. Our results not only indicated scFv-dHLX as an alternative for scFv in analytical application, but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced affinity.     

Novel anti-digoxin monoclonal antibodies with different binding specificities for digoxin metabolites and other glycosides

Spleen cells of BALB/c mice immunized with a digoxin-bovine serum albumin conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Seven monoclonal antibodies (MAb) selected by indirect ELISA were produced, purified and characterized. All the MAb were IgG1 isotypes. The apparent equilibrium association constants (Ka) of four of the MAb, determined by Scatchard analysis of the RIA data, ranged from 1 x 10(9) M-1 to 5.9 x 10(9) M-1. The estimated Ka values of the three other MAb were found to be between 4.8 x 10(7) M-1 and 5.9 x 10(8) M-1. Using digoxin and eighteen structurally-related compounds, the seven MAb could be divided into five groups based on their binding specificities assessed by an inhibition immunoenzymatic test. The MAb in Groups I and II, in particular, showed very different specificity profiles: the two MAb in Group I had low cross-reactivity with cardioinactive digoxin metabolites, whereas the high affinity MAb in Group II recognized all the digoxin metabolites tested. The MAb in Group I might be useful in a digoxin immunoassay and the Group II MAb in therapeutic reversal of digoxin intoxication.