In vitro and in vivo antitumor activity of a mouse CTL hybridoma expressing chimeric receptors bearing the single chain Fv from HER-2/neu- specific antibody and the γ-chain from Fc(ε) RI

To broaden the applicability of adoptive cellular immunotherapy against HER-2/neu overexpressing human cancers, we constructed a chimeric scFv/gamma gene composed of the variable regions of a HER-2/neu specific monoclonal antibody (mAb) joined to the signaling gamma-chain of the Fc(epsilon)RI receptor. The scFv(anti-HER-2/neu)/gamma chimeric gene was successfully expressed as functional surface receptor in the MD.45 cytolytic T-cell (CTL) hybridoma (MD.45-HER/gamma). Expression of the chimeric protein triggered IL-2 and IFN-gamma secretion in vitro upon encountering cell surface HER-2/neu and mediated non-major-histocompatibility-complex (MHC)-restricted HER-2/neu-specific target cell lysis. We also examined the in vivo activity of the MD.45-HER/gamma transduced cells. Severe combined immunodeficiency disease (SCID) mice that were given HER-2/neu positive (+) human tumor cell lines had significantly increased survival compared to mice treated with saline only, or with MD.45 cells transduced with a control anti-trinitrophenyl (anti-TNP) chimeric receptor gene (MD.45-TNP/gamma). These results demonstrate the feasibility of redirecting MD.45 CTL to react in vitro and in vivo with a variety of HER-2/neu(+) tumor cells by our gene transduction protocol. Moreover, they open the possibility of using the same chimeric gene for transducing primary lymphocytes and thus allowing adoptive immunotherapy against HER-2/neu(+) cancers.     

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.     

Role of MHC II affinity and molecular mimicry in defining anti-HER-2/neu MAb-3 linear peptide epitope

With the aid of computational biology, we have studied the possibility of predicting the peptides able to evoke humoral immune response by using as experimental model the human HER-2/neu breast cancer-associated antigen. We already demonstrated that HER-2/neu peptides, that are the target of humoral human and mouse immune responses, correspond to those sequences having a low degree of sequence similarity to host's proteome. Here we report that the linear peptide determinant of the anti-HER-2/neu MAb-3 is characterized by a low degree of sequence similarity to mouse proteome in combination with high binding potential to specific MHC II molecule.     

Construction and functional evaluation of a single-chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector.

9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L-agarose beads yielding 1-2 mg.L(-1) of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.     

Production of human single-chain fragment antibody (ScFv) against human epidermal growth factor receptor-2 (HER-2) by phage display technology

Breast cancer with more than 1.7 million diagnoses per year has been known as one of the most prevalent cancers among women worldwide. Despite the availability of advanced treatment options, cancer progression and metastasis is observed in 20% of patients. Human epidermal growth factor receptor-2 (HER-2) is considered as an important prognostic and diagnostic tumor marker for breast cancer. While HER-2 is expressed on the surface of normal cells, its overexpression occurs in 20–25% on breast cancer tumor cells. This type of tumor which is referred to as HER-2+ is the most aggressive type of breast cancer and shows more resistance to radiotherapy. Single-chain fragment antibodies (ScFvs) offer several advantages in comparison to conventional whole antibodies due to their small size. Particularly, ScFv fragments show improved diffusion and solid tumor penetration. In this study, a human ScFv antibody library was used to isolate anti-HER-2 ScFv antibodies through cell panning and mix antigen-cell panning strategies. Analysis of the binding activity and specificity of isolated ScFv antibodies against HER-2-expressing cell lines and recombinant HER-2 antigen indicated the higher efficiency of the cell panning strategy in isolation of ScFv antibody fragments.     

Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin

Recombinant bispecific antibodies such as tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb) have shown to be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, therapeutic efficacy is hampered by a short serum half-life of these small molecules having molecule masses of 50-60 kDa. Thus, improvement of the pharmacokinetic properties of small bispecific antibody formats is required to enhance efficacy in vivo. In this study, we generated several recombinant bispecific antibody-albumin fusion proteins and analyzed these molecules for biological activity and pharmacokinetic properties. Three recombinant antibody formats were produced by fusing two different scFv molecules, bispecific scDb or taFv molecules, respectively, to human serum albumin (HSA). These constructs (scFv(2)-HSA, scDb-HSA, taFv-HSA), directed against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor complex molecule CD3, retained full binding capacity to both antigens compared with unfused scFv, scDb, and taFv molecules. Tumor antigen-specific retargeting and activation of T cells as monitored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv(2)-HSA. T cell activation could be further enhanced by a target cell-specific costimulatory signal provided by a B7-DbCEA fusion protein. Furthermore, we could demonstrate that fusion to serum albumin strongly increases circulation time of recombinant bispecific antibodies. In addition, our comparative study indicates that single chain diabody-albumin fusion proteins seem to be the most promising format for further studying cytotoxic activities in vitro and in vivo.     

An efficient route to the production of an IgG-like bispecific antibody

Production of IgG-form bispecific antibody (BsAb-IgG) by co-expressing two antibodies in transfected cells is often inefficient owing to the unwanted pairing between the component heavy and light chains. We have developed an efficient method for the production of a novel IgG-like BsAb by using the natural dimerization mechanism between IgG heavy and light chains. Two single-chain Fv (scFv) of different specificity are fused to the constant domain of human kappa chain (C(L)) and the first constant domain of human heavy chain (C(H1)), to form two polypeptides, (scFv)(1)-C(L) and (scFv)(2)-C(H1)-C(H2)-C(H3), respectively. Co-expression of the two polypeptides in mammalian cells results in the formation of a covalently linked IgG-like hetero-tetramer, Bs(scFv)(4)-IgG, with dual specificity. Our approach yields a homogeneous bispecific IgG-like antibody product with each molecule containing four antigen binding sites, two for each of its target antigens. A Bs(scFv)(4)-IgG was prepared using two scFv antibodies each directed against a different epitope of a vascular endothelial growth factor receptor, the kinase insert domain-containing receptor (KDR). The Bs(scFv)(4)-IgG is capable of simultaneously binding to the two epitopes on the receptor. Further, the Bs(scFv)(4)-IgG also retains the antigen-binding efficacy and biological activity of its component antibodies.     

Enhanced gene delivery to HER-2-overexpressing breast cancer cells by modified immunolipoplexes conjugated with the anti-HER-2 antibody

Cationic liposome-mediated gene delivery to tumors has met with only limited success due to the low transfection efficiency and lack of target specificity. We developed a gene delivery system for HER-2-overexpressing cells by adding modified anti-HER-2 Fab' fragments to liposome/DNA complexes (lipoplexes). The modified anti-HER-2-Fab' was conjugated to liposomes containing cationic lipids such as 1,2-dioleoyl-3-(trimethylammonium) propane and cholesterol (1:1 w/w) using a maleimido-polyethyleneglycol-3400-1,2-dioleoyl-3-sn-phosphatidylethanolamine linker. The specific modification constricted the sizes of these immunolipoplexes to a range of 0.3- 0.7 microm, and they remained stable for a longer duration of time compared to the lipoplex controls (0.8-3.2 microm at 4 h). In addition, a 10-fold increase in luciferase activity was achieved after transfecting human breast cancer SK-BR3 cells with immunolipoplexes as compared to the control lipoplexes. Flow cytometry analysis demonstrated that 80% of SK-BR3 cells expressed the green fluorescent protein (GFP) 48 h after being transfected with immunolipoplexes, while only 40% of those with control lipoplexes and 3% of those with naked DNA alone expressed GFP. Furthermore, the anti-HER-2 immunolipoplexes showed specific enhancement of transfection efficiency in HER-2-overexpressing SK-BR3 cells (a 6-fold increase in luciferase activity) but not in HER-2-negative MCF-7 breast cancer cells. The enhancement of gene delivery by anti-HER-2 immunoliposomes was not affected by the presence of serum. These results demonstrate the feasibility of improving target-specific gene delivery to HER-2-overexpressing cells by insertion of lipid-modified anti-HER-2-Fab' into the preformed liposomes.     

Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.     

Single-chain Fv with Fc fragment of the human IgG1 tag: construction, Pichia pastoris expression and antigen binding characterization

We report two expression vectors in Pichia pastoris that direct the synthesis of recombinant single chain antibody variable region (scFv), derived from anti-Z-DNA monoclonal antibody Z22. The first vector codes for a scFv fused to the Ig binding domain of staphylococcal Protein A. The second vector codes for the scFv fused to the Fc fragment of the human IgG1. The fusion partner simplified the detection and purification of the secreted protein. These constructs yielded high level expression of an scFv with specific binding activity toward a Z form of DNA, with binding activity comparable to that of the scFv molecule produced in an Escherichia coli expression system and the original monoclonal antibody.     

Generation of affinity matured scFv antibodies against mouse neural cell adhesion molecule L1 by phage display

The recognition molecule L1 plays important functional roles in the nervous system and in non-neural tissues. Since antibodies to L1 are of prime importance to study its functional properties, we have generated affinity matured human single chain variable fragment (scFv) antibodies against mouse L1 by introducing random mutations in the complementarity determining regions (CDRs) of a previously isolated scFv antibody heavy chain (CDR1 and CDR2) and light chain (CDR3). After biopanning the mutant library, a clone (5F7) that gave the strongest ELISA signal was expressed, purified, and characterized. The dissociation constant of 5F7 (2.86 x 10(-8)M) was decreased 60-fold compared to the wild type clone G6 (1.72 x 10(-6)M). 5F7 detected L1 by Western blot analysis in mouse brain homogenates and recognized L1 in L1 transfected cells and cryosections from mouse retina and optic nerve by immunofluorescence. Bivalent 5F7 scFv antibody (5F7-Cys) was also generated and showed a dissociation constant of 5.22 x 10(-9)M that is 5.5-fold lower than that of monomeric 5F7 antibody. The bivalent affinity matured L1 scFv antibody thus showed stronger binding by a factor of 310 compared to the wild type clone. This antibody should be useful in various biological assays.     

Production,purification and characterisation of genetically derived scFv and bifunctional antibody fragments capable of detecting illicit drug residues

We have generated a single chain antigen binding protein (scFv) recognising morphine. Variable regions of heavy (V(H)) and light (V(L)) chain antibody genes isolated from a murine immune repertoire were connected via a glycine-serine linker and cloned into the expression vector pAK 400. The scFv was produced in Escherichia coli JM83 yielding a functional protein of approximately M(r) 30000. Immunoaffinity chromatography using M3G-BSA-Sepharose column proved most effective for scFv purification. Purity was monitored by SDS-PAGE and Western blotting and the scFv characterised using ELISA and BIAcore. The scFv was capable of specifically binding free morphine in solution and was applicable to real sample analysis in saliva. In order to express a bivalent "minibody" the scFv gene was recloned into a vector containing a gene encoding a helix for dimerisation. The scFv was expressed as a protein of M(r) 75000 and retained its antibody binding capabilities. Cloning the scFv gene into a vector containing the bacterial alkaline phosphatase gene produced a bifunctional molecule, which retained the binding activity of the parental scFv along with the enzymatic activity of alkaline phosphatase.     

Improving the developability of an anti-EphA2 single-chain variable fragment for nanoparticle targeting

Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.     

Different equilibrium stability behavior of ScFv fragments: identification, classification, and improvement by protein engineering.

A classification of scFv fragments concerning their unfolding/refolding equilibria is proposed. It is based on the analysis of different mutants of the levan-binding A48 scFv fragment and the HER-2 binding 4D5 scFv fragment as well as a "hybrid" scFv carrying the VL domain of 4D5 and the VH domain of an A48 mutant. The denaturant-induced unfolding curves of the corresponding scFv fragments were measured and, if necessary for the classification, compared with the denaturation of the isolated domains. Depending on the relative intrinsic stabilities of the domains and the stability of the interface, the different scFv fragments were grouped into different classes. We also demonstrate with several examples how such a classification can be used to improve the stability of a given scFv fragment, by concentrating engineering efforts on the "weak part" of the particular molecule, which may either be the intrinsic stability of VL, of VH, or the stability of the interface. One of the scFv fragments obtained by this kind of approach is extremely stable, starting denaturation only at about 7 M urea. We believe that such extremely stable frameworks may be very suitable recipients in CDR grafting experiments. In addition, the thermodynamic equilibrium stabilities of seven related A48 scFv mutants covering a broad range of stabilities in urea unfolding were shown to be well correlated with thermal aggregation properties measured by light scattering and analytical gel filtration.     

Substitution analog peptide derived from HER-2 can efficiently induce HER-2-specific, HLA-A24 restricted CTLs

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC₅₀<1,000 nM) and substituted analog peptides (IC₅₀<50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.     

HER-2/neu oncogene expression and DNA ploidy in normal human kidney and renal cell carcinoma.

Using flow cytometry (FCM), we have investigated both the DNA content (stained with propidium iodide) and HER-2/neu oncogene expression (revealed by means of an anti-HER-2/neu monoclonal antibody) in neoplastic and non-neoplastic kidney samples from 20 patients with renal cell carcinoma. All the non-neoplastic samples and 15/20 (75%) renal cell cancers showed diploid modal DNA content while the remaining 5 neoplastic sample (25%) showed both diploid and hyperdiploid cell populations. In normal kidney the level of HER-2/neu oncoprotein was low (median fluorescence values in arbitrary units = 7.5 AU, range: 4-10 AU). In diploid renal cancers the level of HER-2/neu was slightly increased (median fluorescence values = 20 AU, range: 9.5-30 AU) (p < .005). The relationship of HER-2/neu expression to the cell cycle in these tumor samples is not clear since most of the cells express the antigen in all phases of the cell cycle. On the other hand, there is an association between HER-2/neu expression and abnormal DNA content suggesting that aneuploid pattern may be biologically related to overexpression of the HER-2/neu gene.     

Timely DNA vaccine combined with systemic IL-12 prevents parotid carcinomas before a dominant-negative p53 makes their growth independent of HER-2/neu expression

Double transgenic mice overexpressing the transforming rat HER-2/neu oncogene and the mutated p53, with both dominant-negative and a gain-of-function properties, display early aggressive and metastasizing parotid tumors. Multiple acinar and ductal hyperplasia foci overexpressing the HER-2/neu gene product are evident at wk 5 and progress to poorly differentiated carcinoma by wk 7. Mice die before wk 18 with invasive carcinomas and multiple metastases that no longer express HER-2/neu. A combination of repeated electroporations of plasmids coding for the extracellular and transmembrane domains of the rat HER-2/neu receptor with systemic IL-12 administrations started when the parotids that present diffuse hyperplasia protected all female and 50% of the male mice until the close of the experiment at wk 40. This combined treatment began when multifocal in situ carcinomas that were already present cured 33% of the females and 25% of the males. The most prominent immunologic features associated with the antitumor protection were the production of high titers of anti-HER-2/neu Abs and the nonappearance of cell-mediated cytotoxic reactivity. In conclusion, anti-HER-2/neu vaccination combined with systemic IL-12 control parotid carcinomas as far as p53 mutation makes their growth independent of HER-2/neu expression.     

一种新型的重组单链三特异抗体的构建与表达

双特异抗体由于其缺乏共刺激信号 ,激活的T细胞往往会导致凋亡。为了更有效地杀伤肿瘤细胞 ,构建了抗卵巢癌单链×抗CD3单链×抗CD2 8单域重组三特异抗体的表达载体。利用大肠杆菌中的分子伴侣FkpA与三特异抗体共表达 ,提高了三特异抗体的在BL2 1Star菌中的可溶性原核表达。ELISA结果显示 ,可溶性的重组单链三特异抗体 (sTRI)与SK OV 3细胞的膜抗原 ,Jurkat细胞上的CD3分子以及重组CD2 8抗原均有较强的结合活性。桥连试验证明该抗体能同时与SK OV 3细胞和Jurkat细胞结合。体外杀伤试验表明该抗体能激活外周血T细胞来杀伤肿瘤细胞。形态学观察也进一步说明该抗体具有良好的结合活性以及体外杀伤活性。这种新型的重组单链三特异抗体为基于T细胞的癌症免疫治疗建立了一个新的技术平台。     

Direct detection of herceptin/trastuzumab binding on breast tissue sections.

The protooncogene product HER-2/neu is the target of the humanized monoclonal antibody trastuzumab (Herceptin). Several tests are used clinically to identify patients with HER-2/neu overexpression based on evaluation by pathologists of gene amplification by fluorescence in situ hybridization or protein expression using immunohistochemistry (IHC). A simple technique has been developed for staining formalin-fixed, paraffin-embedded breast cancer tissue using unmodified Herceptin/trastuzumab as the primary antibody. Results were compared with staining with the commercial kit, HercepTest, as well as with polyclonal anti-HER-2/neu antibodies and with biotinylated trastuzumab. These procedures were tested using four breast cancer microarrays. There were 854 cores that were stained with all four antibodies, representing 325 cases. A standard 4-point scoring system (0-3) was used. A total of 156 cases (48%) were scored as 0 by all the methods used and 31 (9.5%) were positive (3+) by all methods. Of interest, three cases scored negative using polyclonal anti-HER-2/neu antibodies but were positive using unmodified trastuzumab. To clarify this discrepancy, whole sections of tumors were examined with both antibodies using double labeling. There were some tumors that demonstrated a mosaic pattern of staining with neighboring cells or groups of cells stained exclusively with one antibody or the other. These results demonstrate that unmodified humanized or human therapeutic antibodies could be used for preclinical testing or in a clinical laboratory setting for IHC-based selection of patients for treatment, and results of such selection could be different from those obtained using polyclonal antibody-based IHC procedure.     

The dipeptide, gamma-glutamylcysteine, is recognized by the anti-glutathione antibody single chain Fv fragment 20C9

The anti-glutathione antibody scFv 20C9, which we previously isolated from a human synthetic phage antibody scFv library [Hirose, M., Hayano, T., Shirai, H., Nakamura, H., and Kikuchi, M. (1998) Protein Eng. 11, 243-248], was expressed in the E. coli pET system and purified by sequential chromatography on Ni and glutathione-conjugated affinity resins. The purified scFv 20C9 antibody was characterized for its binding affinity for several glutathione derivatives by the BIACORE system. Although GSH, GSSG, and gamma-Glu-Cys could bind to the immobilized antibody, this was not the case for Cys-Gly, l-Glu, l-Cys, l-Gly, or several other glutathione derivatives such as gamma-Glu-Ser-Gly. The results suggest that a gamma-glutamic acid and sulfur atom are important for scFv 20C9 antibody recognition of glutathione. This is the first report to indicate that an scFv antibody can recognize a region as small as a dipeptide.