Single chain antibody fragment display systems: a review / 生物工程学报

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Engenharia de anticorpos na busca por fragmentos funcionais para insumos de diagnóstico e aplicações terapêuticas / Antibody engineering in the search for functional fragments for diagnostic inputs and therapeutic applications

Anticorpos, agentes empregados no desenvolvimento de pesquisas biomédicas, no diagnóstico e na terapêutica, possuem elevada capacidade de interação aos mais variados ligantes, Estruturalmente são heterotetrameros constituídos por duas cadeias leves e duas cadeias pesadas com massa molecular de aproximadamente 150 kDa, Visando melhorar as características farmacocinéticas e minimizar possíveis reações adversas desencadeadas por imunoglobulinas de origem não humana, a engenharia molecular de anticorpos vem obtendo fragmentos de anticorpos como porções Fab, F(ab?)2, scFv e Fv, Em adição aos anticorpos convencionais, camelídeos produzem imunoglobulinas funcionais desprovidas de cadeia leve, onde o domínio variável da cadeia pesada, denominado VHH ou nanocorpo, é responsável pelo reconhecimento antigênico, Apresentando características adequadas ao desenvolvimento de fármacos com alta capacidade de neutralização, fragmentos VHHs vêm sendo propostos para uso em imunoterapia passiva ou em drug-delivery, No diagnóstico esses fragmentos podem ser aplicados na construção de biosensores ou na imagiologia, atuando na detecção de células cancerígenas, no monitoramento de tumores ou em alterações celulares...

Antibodies, agents employed for the development of biomedical research, diagnostic and therapeutic, have high ability to interact with different ligands. Structurally are heterotetramers constituted by two light and two heavy chains, with molecular weight of approximately 150 kDa. Aiming to improve the pharmacokinetic properties and minimize possible adverse reactions triggered by immunoglobulins of non-human origin, the molecular engineering of antibodies has been obtaining fragments of antibodies, such as Fab, F(ab?)2, Fv and scFv. In addition to the conventional antibodies, camelids produce functional immunoglobulins devoid of light chain, in which the variable domain, named VHH or nanocorpo, is able to recognize the antigen. With appropriate characteristics for the development of drugs with high neutralizing capacity, VHH fragments have been proposed for use in passive immunotherapy or drug-delivery. To the diagnosis, these fragments can be used to construct biosensors, in the imagiology , acting in the detection of cancer cells, tumor monitoring or cell changes...

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Gammapatía monoclonal de significado incierto A propósito de un caso de nefropatía por depósito de cadenas ligeras lambda / Monoclonal gammopathy of undetermined significance Apropos of a case of lambda light chain deposition nephropathy

La nefropatía asociada a las gammapatías monoclonales es debida principalmente al depósito de cadenas ligeras. Las enfermedades renales paraproteinémicas son lesiones asociadas con depósito de inmunoglobulinas intactas o fragmentos de inmunoglobulinas (cadenas pesadas y cadenas ligeras). La enfermedad por depósito de cadenas ligeras es una condición rara, caracterizada por el depósito de cadenas ligeras monoclonales en muchos órganos y a nivel renal predominantemente en glomérulos y membranas basales tubulares. La enfermedad está frecuentemente asociada con desórdenes linfoproliferativos, y la mayoría de casos son causados por depósito de cadenas ligeras kappa. Aunque se presenta sobre todo en cuadros malignos, en ocasiones no se detecta patología hematológica y se denomina idiopática o "primaria". Suele manifestarse como una insuficiencia renal severa con proteinuria nefrótica, no tiene tratamiento claramente establecido y el pronóstico es malo. Se describen las características clínicas e histológicas del segundo caso informado en Colombia de nefropatía por depósito de cadenas ligeras diagnosticado en el contexto de una enfermedad renal paraproteinémica sin datos de malignidad. (Acta Med Colomb 2014; 39: 196-201).

Nephropathy associated with monoclonal gammopathies is mainly due to light chain deposition. The paraproteinemic kidney diseases are lesions associated with deposition of intact immunoglobulins or fragments of immunoglobulins (heavy and light chains). The disease due to deposition of light chains is a rare condition characterized by deposition of monoclonal light chains in many organs and as for the kidney, predominantly in glomeruli and tubular basement membranes. The disease is frequently associated with lymphoproliferative disorders and the majority of cases are caused by deposition of kappa light chains. Although presented primarily in clinical pictures of malignancy, sometimes no hematological pathology is detected and is called idiopathic or "primary". It usually manifests as severe renal failure with nephrotic proteinuria, has not a clearly established treatment and the prognosis is poor. The clinical and histological features of the second case reported in Colombia of a light chain deposition nephropathy diagnosed in the context of a kidney paraproteinemic disease without malignancy data, is presented. (Acta Med Colomb 2014; 39: 196-201).

Preliminary radioimmunoimaging and biodistribution of ¹³¹iodine-labeled single-chain antibody fragment against progastrin-releasing peptide(₃₁₋₉₈) in small cell lung cancer xenografts / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Monoclonal antibodies (mAbs) such as DD3, raised against progastrin-releasing peptide(31-98) (ProGRP (31-98)) antigen, have been used to target small cell lung cancer (SCLC). However, as an intact mAb, DD3 is cleared slowly from the body, with an optimal radioimmunoimaging time of 72 hours. More recently, a single-chain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research. Thereby, it potentially increases the radioimmunoimaging efficacy. However, there have been few studies with this antibody fragment. The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of (131)I-anti-ProGRP(31-98) scFv in nude mice bearing SCLC xenografts.</p><p><b>METHODS</b>Anti-ProGRP(31-98) scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry. (131)I-anti-ProGRP(31-98) scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram (%ID/g) in various organs was calculated. Similarly, the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated. After injection of (131)I-anti-ProGRP(31-98) scFv, treated mice were imaged at 1, 24, and 30 hours. Then the tumor/base ratios were calculated.</p><p><b>RESULTS</b>ProGRP was highly expressed in NCI-H446 cells and xenograft tissue. The metabolism of (131)I-anti-ProGRP(31-98) scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes, respectively. The %ID/g of (131)I-anti-ProGRP(31-98) scFv in xenografts was much higher than in healthy tissues at 12 hours after injection, reaching a maximum of (5.38±0.92) %ID/g at 24 hours. Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours, with 24 hours proving optimal.</p><p><b>CONCLUSION</b>(131)I-anti-ProGRP(31-98) scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid blood clearance, indicating that it could be a promising agent for SCLC radioimmunoimaging.</p>

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfide-bond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37ºC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/l after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain the majority of the target protein in their periplasmic space (i.e. 4 hrs post induction).

Gene construction and screening of humanized single chain antibody library against VSTM1-v2 cytokine / 药学学报

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.

Secretory expression and biological activity analysis of an anti-H5 single-chain antibody from Pichia pastoris / 病毒学报

In our previous study, a panel of 52 broadly cross-reactive H5-specific monoclonal antibodies (MAbs) were generated and characterized. The 13D4, one of these MAbs, has been demonstrated to protect mice against lethal challenge by 4 strains of H5N1 avian influenza virus representing the currently prevailing genetic populations, clades 1, 2.1, 2.2, and 2.3. Here, we further cloned the gene of the 13D4 MAb and constructed a single-chain variable fragment. Then, the 13D4 single-chain antibody (scFv) was expressed in secretory maner in Pichia pastoris. The supernatant of the culture was concentrated and subjected to ammonium sulfate precipitation. The purity of the 13D4 scFv was around 90% in SDS-PAGE following ion-exchange chromatography. We further investigated its binding property using hemagglutination inhibition (HI) test and blocking ELISA. The results indicated that the 13D4 scFv shared the same binding sites and comparable HI titer with the prototype murine 13D4 Mab. In conclusion, an anti-H5 single-chain wide-spectrum neutralizing antibody is prepared successfully in yeast system.

Engineering antibody fragments: replicating the immune system and beyond: [review] / La ingeniería de fragmentos de anticuerpos: imitando y expandiendo el sistema inmune: [revisión]

Since genetic engineering of humanized murine monoclonal antibodies was first demonstrated over two decades ago, antibody engineering technologies have evolved based upon an increasing understanding of the mechanisms involved in antibody generation in vivo, and a constant search for alternative routes to evolve and exploit the characteristics of antibodies. As a result, antibody engineers have devised innovative strategies for the rapid evolution and selection of antibodies and novel antibody designs (i.e., antibody fragments). Phage display, cell display and ribosome display technologies, which comprise the core of the currently available technologies for the discovery and preparation of such antibodies, are reviewed herein. This article intends to communicate the state-of-the-art technology available for the engineering of antibodies to a general readership interested in this important field. Therefore, important immunology concepts are introduced before detailed descriptions of the three antibody engineering technologies are presented in later sections. A comparison of these methodologies suggests that despite the predominance of phage display for the engineering of antibody fragments in the past 20 years, cell display and ribosome display will likely gain importance in the selection and discovery of the antibody fragments in the future. Finally, these technologies are likely to play an important role in the production of the next generation of antibody-based therapeutics.

Las tecnologías para la ingeniería de anticuerpos han evolucionado durante las últimas dos décadas, desde la demostración de la posibilidad de humanizar anticuerpos monoclonales de ratón mediante ingeniería genética, apoyadas en el creciente entendimiento de los mecanismos involucrados en la generación de anticuerpos in vivo, y en una búsqueda constante de rutas alternativas para evolucionar y explotar sus características. Es así como los ingenieros de anticuerpos han desarrollado estrategias innovadoras para la evolución y selección de anticuerpos y de novedosos diseños de anticuerpos conocidos como fragmentos de anticuerpos. Esta revisión se enfoca en tres tecnologías que comprenden el núcleo de las tecnologías actualmente disponibles para el descubrimiento y preparación de tales anticuerpos: la presentación en fagos, la presentación en células, y la presentación en ribosomas. Este artículo busca presentar el estado del arte de estas tecnologías a un grupo general de lectores interesados en este campo, por lo que inicialmente se introducen importantes conceptos de inmunología requeridos para comprender en detalle las tecnologías discutidas. Una comparación de estas metodologías para la ingeniería de anticuerpos sugiere que a pesar del dominio de las tecnologías basadas en la presentación en fagos durante los últimos 20 años, en los próximos años la presentación en células y la presentación en ribosomas probablemente ganarán importancia para la selección y descubrimiento de fragmentos de anticuerpos. Finalmente, es probable que estas tecnologías jueguen un papel importante en la producción de la siguiente generación de terapéuticos basados en anticuerpos.

Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system.</p><p><b>METHODS</b>The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody.</p><p><b>RESULTS</b>The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII.</p><p><b>CONCLUSION</b>An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.</p>

Preparation and bioactivity of anti-human red blood cell ScFv and CSFV E2 bifunctional fusion protein / 生物工程学报

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.

Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of an Escherichia coli

BACKGROUND: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. METHODS: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize V(H) and V(L) fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. RESULTS: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of V(H) or V(L) domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of V(H) of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. CONCLUSION: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library.

We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naive phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software.The structure was refined using the molecular dynamics method.The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained.Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting.SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis.The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa,respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.

Expression purification and verification of HBscFv-IFNgamma in Pichia pastoris x33 / 生物工程学报

In order to effectively cure hepatitis B virus (HBV), we studied on fusion protein HBscFv-IFNgamma, which was connected with single-chain Fv against HBV surface antigen(HBscFv) and gamma-interferon(IFNgamma) of being used in clinic against HBV. Adopting overlap PCR, the hbscfv and the ifngamma were connected into hbscfv-ifngamma. Then the pPICZalphaA/(hbscfv-ifngamma)(1,2,4) of multi-copy recombinant plasmid were constructed and transformed into Pichia pastoris x33. The engineering strain x4 was screened from transformed x33 and could secretively express HBscFv-IFNgamma. The preliminary verification indicates that HBscFv-IFNgamma has the bioactivity of HBscFv and IFNgamma by SDS-PAGE, Western blotting and ELISA. The supernatant of culturing X4 was purified by 14F7 affinity chromatography to HBscFv-IFNgamma with purity of 95%-98%. The HBscFv-IFNgamma is able to bind 27.9% HBV surface antigen (HBsAg) in the serum of HBV transgenic mice, which shows the antibody of HBscFv-IFNgamma has bioactivity in vivo. Therefore HBscFv-IFNgamma can shed light on the development of a new promising HBV-targeted drug.

Selection of a novel single-chain variable fragment antibody specifically against a linear epitope of white spot syndrome virus / 生物工程学报

White spot syndrome virus (WSSV) is one of the most important pathogens in shrimp farm throughout the world. Many researches on WSSV have been done, but no efficient approach has been gained to protect and cure the disease. In this study, we constructed a single-chain fragment variable (scFv) antibody library displayed on phage using spleen cells from mice immunized with denatured WSSV. After several rounds of panning respectively against purified intact WSSV virions and purified VP28 expressed in Escherichia coli, five novel scFv antibodies specifically against WSSV were selected, one of which, clone P75E8, recognized a linear epitope. The location in virions of the epitopes recognized by the five scFv clones was determined by immunoelectron microscopy. This study provides a new way to obtain more different antibodies specifically binding to WSSV, and especially provides a new strategy to obtain scFvs against linear epitopes.

Construction and expression of anti-clenbuterol single chain Fv recombinant vector / 生物工程学报

To construct the recombinant vector pBV220-scFv and express anti-clenbuterol (CBL) scFv antibody in Escherichia coli, we amplified the scFv gene using plasmid pCANTABSE-CBL as a template, recombined it with pPICZalphaA, then amplified the scFv-His-tag gene from plasmid pPICZalphaA-scFv and linked it with expression plasmid pBV220. We identified the recombinant plasmid by restrictive enzyme digestion, PCR amplification and sequence analysis. Finally, we transformed the recombinant vector into E. coli DH5alpha that was temperature-induced and expressed recombinant protein. We identified the recombinant protein by SDS-PAGE, Western blotting and indirect competitive ELISA. The results show that recombinant plasmid pBV220-scFv contained the inserted fragment with highest homology about 99.8%. The expression of scFv induced by temperature show 37 kD Mw and anti-His-tag mAb recognized-activity by SDS-PAGE and Western blotting respectively, and could competitively combine with CBL, the IC50 is 4.55 ng/mL. The recombinant plasmid pBV220-scFv is constructed and expresses the scFv gene of CBL in E. coli successfully. This study suggests the corresponding immunoassay methods could be set up by the recombinant scFv.

The influence of the FR-1 in heavy chain (VH) of antibodies on antibody secretion / 病毒学报

The N-terminal segment (FR-1) of the heavy chain (VH) of antibodies may have a great impact on IgG secretion in Escherichia coli and other hosts. Decrease in secretion may be caused by a single amino acid change in the framework region. To investigate the high antibody expression in mammalian cells, we designed the site-directed mutagenesis of the FR-I of the pCMV-RV/VH gene,which expressed the immunoglobulin heavy chain of human anti-Rabies virus antibody. Mutating Glu (H6) to Gln could improve both antibody secretion and affinity. The immunofluorescence assay indicated that both the secretion-deficient antibodies and the secretion- efficient antibodies could be transcribed and translated intracellularly, and led into ER,then transferred to Golgi apparatus,and the difference in secretion may relate to the contribution of the FR-I to the folding and assembly of the antibody. In this study, we have confirmed experimentally that the nature of residues H6 in antibody heavy chains indeed determines the antibody secretion in mammalian cells. These results also provide the basis for antibody production.

Therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE).</p><p><b>METHODS</b>EAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol.</p><p><b>RESULTS</b>EAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells.</p><p><b>CONCLUSION</b>The immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.</p>

Construction and expression of an anti-GD2,ScFv-IL-2 fusion protein gene / 生物医学工程学杂志

By combining interleukin2 (IL-2) with a tumor specific antibody, immunotherapy of tumors may become more effective in the future. Anti-GD2 single chain antibody directed to the extracellular domain of GD2 disialoganglioside can result in an antitumor response in some pateins with tumors expressing GD2. In this study, the fusion protein consisting of GD2 single chain antibody (ScFv) and IL-2(Ala125) was constructed. Anti-GD2 ScFv and IL-2 genes were obtained by PCR, then the ScFv-IL-2 gene was constructed by over lap PCR. The gene was inserted into the pMD18-T easy vector. Genes from pMD18-T -vector were inserted into expression vector pSE380. Recombinant expression vector was identified by restriction enzyme-cutting and then was transformed into BL21. SDS-PAGE and Western blot analysis confirmed that the transformed E. Coli BL21 could express ScFv-IL-2 fusion-proteins and the molecular weight is 43 kDa. The fusion protein was purified by affinity chromatograph and Sephacryl S-200HR then was identified through ELISA. The results show that the fusion protein retains the activities of both antigen binding and IL-2.

Modification and identification of a vector for making a large phage antibody library / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies.</p><p><b>METHODS</b>scFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC III encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated.</p><p><b>RESULTS</b>The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression.</p><p><b>CONCLUSIONS</b>The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.</p>