Construction, expression and characterisation of a single-chain diabody derived from a humanised anti-Lewis Y cancer targeting antibody using a heat-inducible bacterial secretion vector

 A single-chain antibody fragment (scFv) of the humanised monoclonal antibody, hu3S193, that reacts specifically with Le^y antigen expressed in numerous human epithelial carcinomas was constructed. A five-residue linker joined the C-terminus of the V_H and the N-terminus of the V_L, which prevented V-domain association into a monomeric scFv and instead directed non-covalent association of two scFvs into a dimer or diabody. The diabody was secreted into the E. coli periplasm using a heat-inducible vector, pPOW3, and recovered as a soluble, correctly processed protein, following osmotic shock or solubilised with 4M urea from the insoluble fraction. The diabody from both fractions was isolated by a rapid batch affinity chromatography procedure, using the FLAG affinity tag to minimise degradation and aggregation. The purified diabody has an M_r of ˜54 kDa, was stable and demonstrated similar binding activity as the parent monoclonal antibody, as measured by FACS and BIAcore analyses. The radiolabelled diabody showed a rapid tumour uptake, with fast blood clearance, proving it to be an excellent potential candidate as a tumour-imaging agent. Received: 16 November 2000 / Accepted: 8 March 2001     

The role of monocytes and natural killer cells in mediating antibody-dependent lysis of colorectal tumour cells

Monocytes and natural killer (NK) cells are known to be important effector cell populations in mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Purified monocyte and NK effector cell populations, from normal and colorectal cancer (CRC) patients, together with a number of murine (17-1A and 323/A3) and their chimaeric (c17-1A) or humanised (3622W94) equivalents, and chimaeric (c) SF25 were compared for their ability to mediate ADCC of colorectal tumour cells. The chimaeric and humanised antibodies were significantly better at mediating tumour lysis than their murine equivalents with all-effector populations. When effector cells from CRC patients were used the cSF25 antibody was significantly better than 3622W94 (P < 0.02) which, in turn, was significantly better than c17-1A (P < 0.03). Depletion of NK cells produced a decrease in specific tumour lysis with all antibodies. In addition a higher rate of NK cell death was observed in CRC patients during the assay than in normal controls. The chimaeric and humanised antibodies stained a similar percentage of the HT-29 target cells (>80%), but 3622W94 bound to significantly more cells from primary tumour biopsies than cSF-25 (P = 0.001). Together, the results suggest that NK cells are the most important effector cell type mediating ADCC in vitro, that there is some impairment of NK function in CRC patients, and that cSF25 is the most potent antibody. For use in vivo the anti-Ep-CAM antibody 3622W94 would appear to be the most suitable reagent for further study. Received: 3 June 1999 / Accepted: 22 July 1999     

CpG oligonucleotides enhance the tumor antigen-specific immune response of an anti-idiotype antibody-based vaccine strategy in CEA transgenic mice

A murine monoclonal anti-idiotype (Id) antibody, 3H1 has been developed and characterized previously. Anti-Id 3H1 mimics a specific epitope of carcinoembryonic antigen (CEA) and can be used as a surrogate antigen for CEA. 3H1 induced anti-CEA immunity in different species of animals as well as humans and showed promise as a potential vaccine candidate in phase I/II clinical trials for colon cancer patients. One area of interest to us has been the development of new immune adjuvants that may augment the potency of 3H1 as a tumor vaccine. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. In this study, we have evaluated the efficacy of 3H1 as a tumor vaccine when admixed with a select CpG ODN 1826 in transgenic mice that express human CEA. The vaccine potential of 3H1 was also assessed in the presence of another widely used adjuvant, QS-21. 3H1 coupled to keyhole limpet hemocyanin (KLH) and mixed with Freund’s adjuvant (FA) was used as a gold standard in this system. 3H1 vaccination with different adjuvants induced both humoral and cellular anti-3H1, as well as anti-CEA immunity in CEA transgenic mice. The immune sera could lyse CEA-transfected murine colon carcinoma cells, C15 effectively in an antibody-dependent cellular cytotoxicity assay. The anti-CEA antibody responses were somewhat comparable in each adjuvant-treated group of mice, whereas cellular immune responses were significantly greater when CpG was used as an adjuvant. Splenocytes obtained from 3H1–CpG-immunized mice showed an increased proliferative CD4⁺ Th1-type T-cell response when stimulated in vitro with 3H1 or CEA and secreted elevated levels of Th1 cytokines (IL-2, IFN-γ). This vaccine also induced MHC class I antigen-restricted CD8⁺ T-cell responses. In a solid tumor model, C15 tumor growth was significantly inhibited by 3H1 vaccinations. In 3H1–CpG-vaccinated mice, the duration of survival was, however, longer compared to the 3H1–QS21-vaccinated mice. These findings suggest that 3H1-CpG vaccinations can break peripheral tolerance to CEA and induce protective antitumor immunity in this murine model transgenic for human CEA.     

Antitumour activity of a chimeric antibody against the leucocyte antigen CD48

Preclinical studies with the murine anti-CD48 antibody, mHuLym3 (IgG2a) have shown it to be a potentially useful therapeutic reagent in the treatment of human leukaemia and lymphoma. For clinical use, humanised antibodies can have a number of advantages over their original murine version, including mediation of higher effector cell function with human cells, longer serum half-life and lower immunogenicity. In this study, we have produced a mouse/human chimeric HuLym3 antibody (cHuLym3) where the murine antibody constant regions have been replaced with human constant regions. We report the production and preclinical characterisation of the antibody, cHuLym3, with potent in vitro and in vivo antitumour activity. The genes encoding the variable heavy and light chains were amplified by the polymerase chain reaction, sequenced and cloned into eukaryotic expression vectors containing the human light- and heavy-chain constant regions (κ and IgG1). The chimeric and murine HuLym3 antibodies had similar cell-binding specificity and affinity. In the human Raji cell severe combined immunodeficient mouse model the i.v. injection of cHuLym3 and mHuLym3 produced similar antitumour responses. Doses of cHuLym3 and mHuLym3 (100 μg) on days 1, 2 and 4 after i.v. Raji cell injection produced a 40% longer time to hind-leg paralysis than when a control antibody was used. cHuLym3 had more potent activity than mHuLym3 in antibody-dependent cellular cytotoxicity (ADCC) assays in vitro, with human peripheral blood mononuclear cells as effectors. Up to 60% specific cell lysis was observed with cHuLym3 in ADCC assays. These properties suggest that anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukaemias and lymphoma. Received: 5 May 1999 / Accepted: 12 August 1999     

An amperometric enzyme-linked immunosensor for Nitrobacter

A new amperometric enzyme-linked immunoassay for specific enumeration of Nitrobacter has been developed. This assay uses an electrode made of glassy carbon, on which the immunological reaction is carried out. The method is based on a competitive immunoassay principle, utilising monoclonal primary antibody and alkaline-phosphatase-labelled secondary antibody. The enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate generates an electroactive product which is amperometrically detected. The effects of different parameters on the performance of the sensor have been studied. Quantitative detection of Nitrobacter using the immunosensor has been compared to a previously developed enzyme-linked immunosorbent assay showing compatible results. In addition, the overall assay time can be shortened with this new sensor. A detection limit of approximately 3 × 10⁶ Nitrobacter cells/ml was obtained. Received: 27 May 1998 / Received revision: 28 August 1998 / Accepted: 28 August 1998     

Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma

 The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg) and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma. A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments, K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted crossreactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity, and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating superantigens. Received: 9 September 1999 / Accepted: 21 October 1999     

Stable expression of high affinity NK1 (substance P) and NK2 (neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells.

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (Kd = 0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50 = 240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC50 = 8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.     

胃肠肿瘤雌、孕激素受体与癌胚抗原的免疫组织化学研究

利用酶联亲和组织化学法和ＡＢＣ免疫组织化学法进行８８例胃肠肿瘤的雌激素受体（ＥＲ）、孕激素受体（ＰＲ）和癌胚抗原（ＣＥＡ）的检测．结果表明：ＥＲ、ＰＲ、ＣＥＡ的阳性率分别为３７．５％（３３／８８）、２７．３（２４／８８）、８８．６４（７８／８８）。ＥＲ、ＰＲ的阳性率与肿瘤的组织学分类及病理分级有关。ＣＥＡ仅与病理分级有关。且与性激素受体水平呈负相关。因此,性激素受体与ＣＥＡ的表达提供了肿瘤组织不同的生物学特征,同时检则ＥＲ、ＰＲ及ＣＥＡ水平对内分泌治疗及判断预后更有价值。     

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 °C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents. Received: 2 November 2000 / Accepted: 11 January 2001     

The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets

A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.     

A pentavalent single-domain antibody approach to tumor antigen discovery and the development of novel proteomics reagents

Proteomics research has delivered many novel tumor targets. However, due to key limitations, it does not specifically identify targets that are most accessible for drug delivery, such as cell-surface antigens. A novel tumor antigen discovery platform based on screening a single domain antibody (sdAb) library against tumor cells and subsequently identifying the corresponding antigens of the isolated antibodies is described. An sdAb, AFAI, specific for non-small cell lung carcinoma (A549 cell line) was isolated from a phage library derived from the heavy chain antibody repertoire of a llama. The homopentamerization property of a non-toxic verotoxin B-subunit was exploited to make the ES1 pentabody, a pentameric form of AFAI. Pentamerization improved the binding of the AFAI to A549 cells dramatically and greatly facilitated antigen identification by a Western blotting/mass spectrometry approach. The antigen of ES1, which is present only in the hydrophobic, not in the hydrophilic, fraction of A549 cellular proteins, was identified as carcinoembryonic antigen-related cell adhesion molecule 6 (CEA6). CEA6 was observed to be acidic and highly glycosylated, and to exist in multiple glycoforms. The results show that the platform described here should find wide application in antigen discovery, and demonstrated that the pentabodies are very useful immunological reagents for proteomics.     

Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms.

Basic fibroblast growth factor (bFGF) was internalized at a rapid rate by Chinese hamster ovary (CHO) cells that do not express significant numbers of high affinity receptors for bFGF as well as CHO cells that have been transfected with cDNA encoding FGF receptor-1 or FGF receptor-2. Internalization of bFGF was completely blocked by the addition of 10 micrograms/ml heparin in the parental CHO cells but only partially inhibited in cells expressing transfected FGF receptors. Bovine aortic endothelial cells also exhibit heparin-sensitive and heparin-resistant internalization of bFGF. The internalization of bFGF through the heparin-resistant pathway in CHO cells was efficiently competed by addition of unlabeled bFGF, was proportional to the number of receptors expressed, and approached saturation, suggesting that the heparin-resistant internalization was due to high affinity receptors. Internalization of bFGF through the heparin-sensitive pathway was not efficiently competed by unlabeled bFGF and did not approach saturation at concentrations of bFGF up to 50 ng/ml, properties similar to the interaction of bFGF with low affinity heparan sulfate binding sites on the cell surface. Internalization of bFGF in CHO cells not expressing FGF receptors was inhibited by heparin, heparan sulfate, and dermatan sulfate, the same glycosaminoglycans that block binding to cell-surface heparin sulfates. Internalization of bFGF in the parental CHO cells was inhibited at the same concentrations of heparin that block binding to cell-surface heparan sulfates. Finally, inhibition of the sulfation of CHO cell heparan sulfates by the addition of chlorate or digestion of CHO cell heparan sulfates with heparinase inhibited bFGF internalization in the parental CHO cells. These results demonstrate that bFGF can be internalized through a direct interaction with cell-surface heparan sulfates. Thus, there are two pathways for internalization of bFGF: high affinity receptor-mediated and heparan sulfate-mediated.     

Pharmacological administration of granulocyte/macrophage-colony-stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen

Eighteen colorectal carcinoma patients without macroscopic disease after surgery were immunized using recombinant (r) human (h) carcinoembryonic antigen (CEA) with (n = 9) or without (n = 9) the addition of soluble granulocyte/macrophage-colony-stimulating factor (GM-CSF). The dose of rhCEA per immunization was 100 μg (n = 6), 316 μg (n = 6) or 1000 μg (n = 6). rhCEA was given s.c. on day 1 and 80 μg/day of GM-CSF s.c. on days 1–4. The schedule was repeated six times during a period of 9 months. All patients in the GM-CSF group developed a strong rhCEA-dose-dependent IgG antibody response while only one-third of the non-GM-CSF patients mounted a weak antibody response. All patients (9/9) in the GM-CSF group developed a strong rhCEA-specific proliferative T cell response as well as type I T cells (interferon γ secretion). In 45% of the patients also a weak type II T cell response (interleukin-4 secretion) was evoked. Both MHC-class-I- and -II restricted rhCEA-specific T cells were noted. A specific cellular response (proliferation and/or cytokine secretion) against native hCEA could be found in 8/9 patients in the GM-CSF group, although at a significantly lower level than against rhCEA. In the non-GM-CSF group a weak rhCEA-specific T cell response was induced. Three patients had a proliferative response, 4 patients type I T cells and 6 patients type II T cells. No signs of autoimmune reactions were noted. Local pharmacological administration of GM-CSF seemed to be a prerequisite for the induction of a strong immunity against baculovirus-produced hCEA protein. However, the cellular response against native CEA was of a significantly lower magnitude. Received: 13 November 1997 / Accepted: 21 May 1998     

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients. Received: 16 November 2000 / Accepted: 11 January 2001     

In vitro induction of HLA-A2402-restricted and carcinoembryonic-antigen-specific cytotoxic T lymphocytes on fixed autologous peripheral blood cells

HLA-A2402-restricted and carcinoembryonic-antigen(CEA)-specific cytotoxic T lymphocytes (CTL) were induced by culturing human peripheral blood mononuclear cells (PBMC) on formalin-fixed autologous adhesive PBMC that had been loaded with CEA-bound latex beads. The CTL killed the CEA-producing HLA-type matched cancer cells, but not the non-producers of CEA, at an effector/target ratio of 10 within 24 h. On the basis of available HLA-A24-binding peptides, we have also attempted to identify the epitope peptide recognized by the CTL. The peptide CEA652(9), TYACFVSNL, stimulated the CTL most strongly when pulsed on HLA-A2402-expressing target cells. The other nine peptides so far tested were also active, but less efficient in their effect on CTL. The CTL failed to kill target cells pulsed with the HLA-A2-binding CEA peptide, CAP-1. The CTL were also generated on the fixed adherent cells previously pulsed with the peptide CEA652(9). Cytotoxic activity of the CTL was inhibited by monoclonal antibodies against CD3, CD8, and MHC class I molecules. These results suggest that human autologous CTL will be inducible on the autologous fixed PBMC without use of the cultured target cancer cells if tumor antigenic protein is available. Received: 31 December 1997 / Accepted: 4 May 1998     

Affinity in radioimmunoassay of antibody cross-reactive with carcinoembryonic antigen (CEA) and colon carcinoma antigen-III (CCA-III).

Several antisera raised against purified carcinoembryonic antigen (CEA) were evaluated for their content of antibody cross-reactive with colon carcinoma antigen-III (CCA-III). All antisera gave a reaction of partial identity between CEA and CCA-III and demonstrated a high titer in CEA radioimmunoassay (RIA). Between 50 and 70% of the CEA RIA activity was removed, however, by absorption with soluble CCA-III or adsorption onto CCA-III-containing immunoadsorbents. Immunoadsorbent retained antibody gave a line of complete identity between CEA and CCA-III. Purified CCA-III (2 mug) only partially depressed CEA binding by this common site antibody, whereas nanogram quantities of CCA-III inhibited the reaction between specific CCA-III antibody and radioiodinated CCA-III. In addition, low levels of CEA were equally effective in depressing CEA binding by the common site or CEA-specific antibody. The higher affinity in RIA of the common site antibody for CEA over CCA-III suggests that the common determinant expressed on CEA is stereochemically different from that on CCA-III. The results further demonstrate that interference by plasma CCA-III is not a significant factor in the measurement of CEA by RIA.     

Cell adhesion activity of non-specific cross-reacting antigen (NCA) and carcinoembryonic antigen (CEA) expressed on CHO cell surface: homophilic and heterophilic adhesion

Cell adhesion activity of carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA) has been analysed by using CHO cells which had been transfected with cDNAs and are ectopically expressing each antigen on their surface. CEA expressing CHO tended to aggregate easily within 30 min after being suspended by trypsinization. Cell adhesion assay between 51Cr labelled cells and monolayered cells showed both homophilic and heterophilic interaction, the extent of which was CEA-CEA much greater than CEA-NCA greater than NCA-NCA. These reactions were completely inhibited by Fab' fragment of anti-CEA antibody. The results strongly suggested that CEA and NCA function as Ca++ independent cell adhesion molecules by homophilic and heterophilic interactions.     

Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K _d value) of 292.8 ± 53.1 nM with 47.27 ± 5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25–36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1–5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.     

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.     

抗CEA单链抗体与链亲和素融合基因的表达

克隆分泌CEA杂交瘤细胞重链可变区（V_H）和轻链可变区（V_L）,以Linker连接V_H及V_L构建抗CEA单链抗体.同时以Spacer连接单链抗体和链亲和素,构建成功单链抗体和链亲和素融合基因,克隆该融合基因至原核表达载体,pET21a(+),经IPTG诱导表达出该双特异性融合蛋白.活性鉴定表明该融合蛋白具有结合CEA及生物素的双特异性.该融合蛋白在生物领域中有较广阔的应用前景.