A humanized immunoenzyme with enhanced activity for glucuronide prodrug activation in the tumor microenvironment

Antibody-directed enzyme prodrug therapy (ADEPT) utilizing β-glucuronidase is a promising method to enhance the therapeutic index of cancer chemotherapy. In this approach, an immunoenzyme (antibody-β-glucuronidase fusion protein) is employed to selectively activate anticancer glucuronide prodrugs in the tumor microenvironment. A major roadblock to the clinical translation of this therapeutic strategy, however, is the low enzymatic activity and strong immunogenicity of the current generation of immunoenzymes. To overcome this problem, we fused a humanized single-chain antibody (scFv) of mAb CC49 to S2, a human β-glucuronidase (hβG) variant that displays enhanced catalytic activity for prodrug hydrolysis. Here, we show that hcc49-S2 displayed 100-fold greater binding avidity than hcc49 scFv, possessed greater enzymatic activity than wild-type hβG, and more effectively killed antigen-positive cancer cells exposed to an anticancer glucuronide prodrug as compared to an analogous hβG immunoenzyme. Treatment of tumor-bearing mice with hcc49-S2 followed by prodrug significantly delayed tumor growth as compared to hcc49-hβG. Our study shows that hcc49-S2 is a promising targeted enzyme for cancer treatment and demonstrates that enhancement of human enzyme catalytic activity is a powerful approach to improve immunoenzyme efficacy.     

Pharmacokinetics and biodistribution of a light-chain-shuffled CC49 single-chain Fv antibody construct

Murine monoclonal antibodies to tumor-associated glycoprotein 72 (anti-TAG-72 mAb B72.3 and CC49) are among the most extensively studied mAb for immunotherapy of adenocarcinomas. They have been used clinically to localize primary and metastatic tumor sites; however, murine mAb generally induce potent human anti-(mouse antibody) responses. The immunogenicity of murine mAb can be minimized by genetic humanization of these antibodies, where non-human regions are replaced by the corresponding human sequences or complementary determining regions are grafted into the human framework regions. We have developed a humanized CC49 single-chain antibody construct (hu/muCC49 scFv) by replacing the murine CC49 variable light chain with the human subgroup IV germline variable light chain (Hum4 V_L). The major advantages of scFv molecules are their excellent penetration into the tumor tissue, rapid clearance rate, and much lower exposure to normal organs, especially bone marrow, than occur with intact antibody. The biochemical properties of hu/muCC49 scFv were compared to those of the murine CC49 scFv (muCC49 scFv). The association constants (K _a) for hu/muCC49 and muCC49 constructs were 1.1 × 10⁶ M⁻¹ and 1.4 × 10⁶ M⁻¹ respectively. Pharmacokinetic studies in mice showed similar rapid blood and whole-body clearance with a half-life of 6 min for both scFv. The biodistribution studies demonstrated equivalent tumor targeting to human colon carcinoma xenografts for muCC49 and hu/muCC49 scFv. These results indicate that the human variable light-chain subgroup IV can be used for the development of humanized or human immunoglobulin molecules potentially useful in both diagnostic and therapeutic applications with TAG-72-positive tumors. Received: 29 December 1999 / Accepted: 4 February 2000     

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20-25% binding whereas both His6-scFv and scFv (no His-tag) showed 60-65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (KA) for His6-scFv and scFv as 1.19 x 10(6) M(-1) and 3.27 x 10(6) M(-1), respectively. No K(A) value could be calculated for scFv-His6 due to poor binding kinetics (kon and koff). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.     

Viral specific cytotoxic T cells inhibit the growth of TfR-expressing tumor cells with antibody targeted viral peptide/HLA-A2 complex

A fusion protein of single chain antibody (scFv) specific for transferrin receptor (TfR, CD71) and viral peptide/HLA-A2 complex was prepared in this study to redirect cytotoxic T cells (CTLs) of viral specificity to tumor cells by attaching the ligand of T cell receptor (TCR) to tumor cells via binding of TfR scFv to TfR. The results demonstrate that the fusion protein can attach the active virus-peptide/HLA-A2 complex to HLA class I-negative, TfR-expressing K562 cells through binding of TfR scFv to TfR, and mediate cytotoxicity of viral peptide-specific CTLs against K562 cells in vitro. In addition, the fusion protein can induce inhibition of solid tumor formation and improve survival time in tumor xenograft nude mouse with the injection of the sorted viral peptide-specific CTLs generated by co-culture of peripheral blood lymphocytes from HLA-A2 positive donors with inactivated T2 cells pulsed with the viral peptide.     

Constructs of biotin mimetic peptide with CC49 single-chain Fv designed for tumor pretargeting

Single-chain Fv constructs comprising a biotin mimetic peptide (BMP) and scFv of CC49 monoclonal antibody were produced to improve pretargeted radioimmunotherapy. BMP units that bind streptavidin were added to the carboxyl terminus of the CC49 V(H) region. An engineered scFvBMP monomer and a sc(Fv)(2)BMP dimer showed an excellent antigen recognition in vitro with a specific binding of 72+/-5 and 81+/-4%, respectively. Properties of 125I-sc(Fv)(2)BMP in mice bearing LS-174T xenografts were comparable to these of the parent 125I-sc(Fv)(2). Complexing of scFvBMPs with streptavidin increased tumor targeting and gave exceptionally high tumor-to-blood values of 63+/-7 for 125I-sc(Fv)(2)BMP-streptavidin compared with 37+/-4 for sc(Fv)(2)BMP at 72h after administration. High tumor and negligible normal tissue levels of these novel pretargeting constructs indicate a great potential for pretargeted radioimmunotherapy.     

Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance

Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.     

Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification, and characterization

Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.     

一株针对人EGFR的单链抗体克隆与哺乳细胞表达

目的:制备特异性抗人表皮生长因子受体(EGFR)的单链抗体(sc Fv),鉴定其生物学活性,为进一步研究基于单链抗体的免疫治疗奠定基础。方法:从分泌抗人EGFR单克隆抗体的杂交瘤细胞系提取总RNA,利用5'RACE技术扩增轻链和重链可变区(VL、VH)基因,构建具有VL、VH基因的单链抗体基因,并将构建的单链抗体基因克隆到真核细胞表达载体pc DNA3.1中进行表达和鉴定。ELISA鉴定单链抗体对抗原的特异性;Fortebio检测抗原抗体间的亲和力,流式细胞术检测单链抗体结合肺癌细胞系天然EGFR的功能活性。结果:获得唯一的轻重链可变区序列VL、VH,成功构建EGFR-sc Fv,特异性与天然EGFR蛋白结合,亲和力达3.22×10-9mol/L。结论:成功构建了抗人EGFR单链抗体,为肺癌免疫导向治疗研究奠定了基础。     

Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.     

Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, an anti-BAFF single-chain antibody fragment (scFv) was genetically linked to the C terminus of the enhanced green fluorescent protein (EGFP) to generate an EGFP/scFv fusion protein. The EGFP/scFv fusion protein had an apparent molecular weight of 52 kDa and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. After being purified by immobilized metal affinity chromatography, the fusion protein exhibited similar fluorescence spectra with native EGFP. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorting showed the EGFP/scFv could bind to human soluble BAFF and BAFF positive cell lines in vitro. The binding of EGFP/scFv can also be visualized under laser scanning confocal microscopy. Furthermore, the results of the competition assay indicated its antigen binding specificity. Therefore, the fusion protein EGFP/scFv has several characteristics including high sensitivity, stability and convenience for manipulation, and can be a powerful tool for the study of the underlying pathology of BAFF relevant to autoimmune diseases.     

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.     

Production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pastoris

The Pichia pastoris expression system was used to produce functionalized single-chain antibody fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis. Therefore, scFv antibody fragments recognizing the ED-B domain are potential markers for angiogenesis. We constructed four functionalized scFv antibody fragments for direct labeling with radioactive molecules or toxins or for attachment to liposomes serving as carriers for cytotoxic or antiangiogenic compounds. The C-termini of the scFv antibody fragments contain 1-3 cysteine residues that are separated by a hydrophilic linker (GGSSGGSSGS) from the binding domain and are accessible for site-specific functionalization with thiol-reactive reagents. Plasmid expression, culture conditions, and purification were optimized in 1-L cultures. The scFv antibody fragments were purified by anion exchange chromatography. The yields were 5-20 mg/L culture medium. The large-scale production of one scFv antibody fragment in a 3.7-L fermenter gave a yield of 60 mg. The reactivity of the cyteines was demonstrated by labeling with the thiol-reactive fluorescent dye ABD-F. The four scFv antibody fragments bound specifically to ED-B-modified Sepharose and binding was further confirmed by immunofluorescence on cell cultures using ED-B-positive human Caco-2 tumor cells. Furthermore, we could demonstrate specific binding of scFv-modified liposomes to ED-B-positive tumor cells. Our results indicate that the P. pastoris expression system is useful for the large-scale production of cysteine-functionalized alpha-ED-B scFv antibody fragments.     

Generation and characterization of C305, a murine neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pill protein of MI 3 filamentous phage was screened using BLyS.After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.     

Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3

Vascular endothelial growth factor C (VEGF-C) is a key mediator of lymphangiogenesis, acting via its receptors VEGF-R2 and VEGF-R3. High expression of VEGF-C in tumors correlates with increased lymphatic vessel density, lymphatic vessel invasion, sentinel lymph node metastasis and poor prognosis. Recently, we found that in a chemically induced skin carcinoma model, increased VEGF-C drainage from the tumor enhanced lymphangiogenesis in the sentinel lymph node and facilitated metastatic spread of cancer cells via the lymphatics. Hence, interference with the VEGF-C/VEGF-R3 axis holds promise to block metastatic spread, as recently shown by use of a neutralizing anti-VEGF-R3 antibody and a soluble VEGF-R3 (VEGF-C/D trap). By antibody phage-display, we have developed a human monoclonal antibody fragment (single-chain Fragment variable, scFv) that binds with high specificity and affinity to the fully processed mature form of human VEGF-C. The scFv binds to an epitope on VEGF-C that is important for receptor binding, since binding of the scFv to VEGF-C dose-dependently inhibits the binding of VEGF-C to VEGF-R2 and VEGF-R3 as shown by BIAcore and ELISA analyses. Interestingly, the variable heavy domain (V_H) of the anti-VEGF-C scFv, which contains a mutation typical for camelid heavy chain-only antibodies, is sufficient for binding VEGF-C. This reduced the size of the potentially VEGF-C-blocking antibody fragment to only 14.6 kDa. Anti-VEGF-C V_H-based immunoproteins hold promise to block the lymphangiogenic activity of VEGF-C, which would present a significant advance in inhibiting lymphatic-based metastatic spread of certain cancer types.     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs.

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.     

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。     

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     

Single chain antibodies that recognize the N-glycosylation site

We aimed to identify antibodies that can recognize the Asn-Xaa-Ser/Thr(NXS/T) N-glycosylation site that guides oligosaccharyltransferase (OT) activity. We used synthetic Asn-Cys-Ser/Thr(NCS/T) tripeptides conjugated to bovine serum albumin to isolate single chain antibody fragments of a variable region (scFv) from the Griffin 1 phage antibody library. Although Ser and Thr have different side chains, the scFv proteins thus isolated bound to both NCS and NCT with Kd values of the order of 10(-6) M and accepted the substitution of the Cys residue with various amino acids, including Ala, Gly, and Val. However, these proteins recognized neither Asn-Pro-Ser/Thr nor non-NXS/T tripeptides. The scFv proteins recognized NCS/T and N-glycosylation site of mutant yeast protein disulfide isomerase when they were in their native but not denatured state. These results indicate that antibody recognition of the NXS/T motif is conformation dependent and suggest that NXS/T spontaneously adopts a specific conformation that is necessary for antibody recognition. These features are likely to correlate with the known binding specificity of OT.     

Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv)2 bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2. ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC.     

Expression and Characterization of a Functional Single-Chain Variable Fragment (scFv) Protein Recognizing MCF7 Breast Cancer Cells in E. coli Cytoplasm

Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production.