Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.

THE STUDIES ON MONOCLONAL ANTIBODY SZ-39 AGAINST HUMAN GLIOMA CELLS

A hybridoma cell line SZ-39 secreting monoclonal antibody against the human glioma cell has been established by a fusion between NS-1 myeloma cells and spleen cells from mice immunized with human glioma cell lines. Monoclonal antibody (McAb) SZ-39 was analyzed by ELISA, quantitative absorption, indirect immunofluorescence and ABC immunohistology. McAb SZ-39 strongly bound to 9/10 glioma cell lines, 17/20 glioma tissues, weakly bound to one liver cancer cell line and 1/2 lung cancer line, but they did not band with other tested human cancer linse. NcAb SZ-39 have no cross-reaction with lymphocyte, ABC red blood cells, white blood cells, blood platelet, normal bone marrow cells, fibroblast cells and 12 normal human tissues.The result indicated the antigen recognized by McAb SZ-39 may be a glioma-associated antigen <GAA). This GAA was analyzed by means of Western blotting. It was a MW 180 Kd glycopro-tein. The 131I-McAb SZ-39 specifically localized in human glioma xenografted in nude mice that indicate it may be useful in radioimmunoimaging and as a target for immunotherapy on human glioma.

Identification of a gene engineering antibody against cystic echinococcosis in liver

OBJECTIVE: To identify a gene engineering antibody against cystic echinococcosis in liver. METHODS: A single chain of variable fragment of human antibodies (ScFvs) was selected from the library by using affinity selection technique with the recombinant antigen on solid surface. The positive clones were demonstrated by ELISA and their DNA sequences were also determined. RESULTS: The DNA sequence data showed that the antibody gene is composed of 768bp. In addition, a specific combination capacity with recombinant Echinococcus granulosus antigen B (r-EgB) was demonstrated by ELISA. CONCLUSION: The obtained gene engineering antibody against r-EgB may have potential implications in immunological treatment and drug targeting delivery.

A Novel Human Antibody,HF,against HER2/erb-B2 Obtained by a Computer-Aided Antibody Design Method

Fully human antibodies have minimal immunogenicity and safety profiles.At present,most potential antibody drugs in clinical trials are humanized or fully human.Human antibodies are mostly generated using the phage display method(in vitro)or by transgenic mice(in vivo);other methods include B lymphocyte immortalization,human–human hybridoma,and single-cell polymerase chain reaction.Here,we describe a structure-based computer-aided de novo design technology for human antibody generation.Based on the complex structure of human epidermal growth factor receptor 2(HER2)/Herceptin,we first designed six short peptides targeting the potential epitope of HER2 recognized by Herceptin.Next,these peptides were set as complementarity determining regions in a suitable immunoglobulin frame,giving birth to a novel anti-HER2 antibody named "HF,"which possessed higher affinity and more effective anti-tumor activity than Herceptin.Our work offers a useful tool for the quick design and selection of novel human antibodies for basic mechanical research as well as for imaging and clinical applications in immune-related diseases,such as cancer and infectious diseases.

Construction of human Fab library and screening of a single-domain antibody of amyloid-beta 42 oligomers

Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli- gomers and constructed a have human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe- cifically recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked im- munosorbent assay demonstrated this antibody bound specifically to human amyloid-beta 42 tetramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc- cessfully constructed a human phage display library and screened a single-domain antibody that specifically recognized amyloid-beta 42 oligomers.

Rabies Virus Neutralizing Activity,Safety,and Immunogenicity of Recombinant Human Rabies Antibody Compared with Human Rabies Immunoglobulin in Healthy Adults

Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.

Phase I Dose Escalation Study with the Lewis Y Carbohydrate Specific Humanized Antibody IGN311

Purpose: Investigation of safety, tolerability, pharmacokinetics, and anti-tumor activity of the Lewis Y-specific, fully humanized monoclonal antibody (mAb) IGN311 in patients with Lewis Y positive tumors in a Phase I clinical trial. Experimental Design: Twelve patients (pts) were enrolled in an open-label, uncontrolled, dose escalating Phase I study. Three pts received 50 mg, three pts 100 mg and six pts 200 mg IGN311 by i.v. infusion on days 1 and 15. Blood samples were taken immediately before infusion, and 0.5, 4, 8, 24 hours post infusion, as well as on days 3, 5 and 8 after the first and second infusion, respectively, and day 29. A final visit was scheduled for day 43. Results: No drug related adverse events were observed in the 50 mg and 100 mg dose groups. Three out of six patients in the 200 mg dose group showed drug related adverse reactions with nausea, vomiting and hypotension in one patient (NCI CTC grade 3) being the dose limiting toxicities. t1/2 of IGN311 was ~20 days after second infusion of IGN311. Sera of patients receiving IGN311 were capable of lysing Lewis Y positive tumor cells in vitro by both, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Circulating tumor cells found in the peripheral blood in two out of twelve pts prior to treatment were reduced after treatment to below the quantification limit of the detection method. None of the patients showed an increase in the number of disseminated tumor cells during treatment period. Conclusions: The good safety and PK profile, the biological activity regarding CDC and ADCC mediated tumor cell lysis, and the elimination of circulating tumor cells warrant further clinical investigation of IGN311.

SYNTHESIS OF INTERFERON-α_A MONOCLONAL ANTIBODY PACKING MATERIAL IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PURIFICATION OF RECOMBINANT HUMAN INTERFERON-α_A

A new way for the synthesis of human interferon—α;monoclonal antibody （IFN-α;-McAb） bound to silica gel packing material in high-performance affinity chromatography （HPAFC） has been developed. The high coupling efficiency and specific activity of IFN—α;-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α;（rIFN-α;） rose up to 1.03×10;IU/mg protein and the purification efficiency is appoximately 100 times.

SARS Patients-derived Human Recombinant Antibodies to S and M Proteins Efficiently Neutralize SARS-Coronavirus Infectivity

Objective To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. Methods By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. Results After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike （S） proteins, two Fab antibodies were specific for the virus membrane （M） protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect （CPE） inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus Conclusion The SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.

Recombinant Human IgG antibodies against Human Cytomegalovirus

Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus （HCMV） infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of VH and VL and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-K-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. Results SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein. Conclusion IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 μg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.

MONOCLONAL ANTIBODIES AGAINST HUMAN GASTRIC CANCER

Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) against gastric cancer, were obtained through selective culture and screening. These MoAbs have both good selectivity and a high positive rate of reaction for gastric cancer, reaching 5/5 and 84.8% to 93.5% for gastric cancer cells and tissues respectively. The reaction of MoAbs with normal cells and tissues was neglible.The corresponding antigens of the MoAbs were sensitive to digestion by trypsin and pronase and resistant to treatment with sodium periodate, indicating their nature as proteins. The antigen 3G9 could be visualized with Western blotting as two bands with molecular weights of 100KD and 70KD, however no band was found for antigens 3F4 and 3H11. There was a high expression of antigens in the majority of gastric cancer cells and tissues independent of his-topathological type of gastric cancer. A low expression of antigens was seen with other tumors and fetal gastrointestinal tissues. These could be considered as gastric cancer-associated antigens or on-cofetal antigens with a quite extensive distribution.

CD4^+ T Cell Apoptosis Induced by Anti-CD4 Antibodies

To explore the inhibitory effects of anti-CD4 human/murine chimeric antibodies on lymphocyte proliferation, CD4^+ T cell apoptosis induced by anti-CD4 antibodies was examined. Annexin- V -FITC and PI double stain method was employed to qualitatively and quantitatively determined CD4^+ T cell apoptosis induced by anti-CD4 antibodies. Our results showed that anti-CD4 chimeric antibodies could specifically induce CD4^+ T cell apoptosis. The ability of anti-CD4 chimeric antibodies to induce CD4^+ T cell apoptosis was related with the presence of monocytes. It is concluded that the further cross-linking of anti-CD4 antibodies is important for inducing CD4^+ T cell apoptosis.

A human antibody potently neutralizes RSV by targeting the conserved hydrophobic region of prefusion F

Respiratory syncytial virus(RSV) continues to pose serious threats to pediatric populations due to the lack of a vaccine and effective antiviral drugs. RSV fusion(F) glycoprotein mediates viral-host membrane fusion and is a key target for neutralizing antibodies. We generated 23 full-human monoclonal antibodies(hm Abs) against prefusion F protein(pre-F) from a healthy adult with natural RSV infection by single B cell cloning technique. A highly potent RSV-neutralizing hm Ab, named as 25-20, is selected, which targets a new site Φ-specific epitope. Site-directed mutagenesis and structural modelling analysis demonstrated that 25-20 mainly targets a highly conserved hydrophobic region located at the a4 helix and a1 helix of pre-F, indicating a site of vulnerability for drug and vaccine design. It is worth noting that 25-20 uses an unreported inferred germline(i GL) that binds very poorly to pre-F, thus high levels of somatic mutations are needed to gain high binding affinity with pre-F. Our observation helps to understand the evolution of RSV antibody during natural infection. Furthermore, by in silico prediction and experimental verification, we optimized 25-20 with KD values as low as picomolar range. Therefore, the optimized 25-20 represents an excellent candidate for passive protection against RSV infection.

THE ALTERNATIVE PATHWAY OF HUMAN T CELL ACTIVATION BY MONOCLONAL ANTIBODIES(A COMPARATIVE STUDY BETWEEN NORMAL INDIVIDUALS AND CANCER PATIENTS)

This paper described T cell proliferative response by an alternative pathway in normal subjects and In patients with malignant diseases. Two McAbs, Anti-CCTl and Lo-CD2-act recognizing two distinct epitopes on E-receptor (CD2) were used to costimulate PBMC. Proliferative responsiveness was measured by 3H-thymidine incorporation. It was found that 82% of 72 nonnal subjects gave proliferative response whereas only 23% of the 93 patients did. The average cpm±SD in patients with bladder cancer (118±2314), kidney cancer (1619±2719) or lymphoma (2518±4057) was significantly lower than that in normal subjects (4935±2314), (P<0.001). These results indicate that T cell proliferation through the alternative pathway was significantly depressed in patients with cancer, and this can be used as a new parameter to monitor the immune status of cancer patients.

The antibody preparation and expression of human Pescadillo

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigene- sis, the cDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated. Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells, and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer, colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a po- tential target in cancer diagnosis and therapy.

Generation of mouse anti-human urate anion exchanger antibody by genetic immunization and its identification

Background Human urate anion exchanger (hURAT1) as a major urate transporter expressed on renal tubular epithelial cells regulates blood urate level by reabsorbing uric acid. Antibody is an important tool to study hURAT1. This study aimed, by genetic immunization, to produce mouse anti-hURAT1 polyclonal antibody with high throughput and high specificity and to detect the location of hURAT1 in human kidney.Methods Human renal total RNA was isolated and the entire cDNA of hURAT1 was amplified by RT-PCR. The sequence of intracellular high antigenicity fragment (A280 to R349) was chosen by prediction software of protein antigenicity, and its cDNA was amplified from cDNA of hURAT1, and then cloned into pBQAP-TT vector to construct recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization. Mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-Flt3L, which helped to enhance the antibody’s generation. After four weeks, the mice were sacrificed to obtain the anti-hURAT1 antibody from serum. The antibody was identified by western blot analysis and immunohistochemistry. At the same time, rabbit anti-hURAT1 antibody was produced by protein immunization. The specificity and efficiency between the rabbit and mouse anti-hURAT1 antibody were compared by western blot analysis and immunohistochemistry. Results The entire cDNA of hURAT1 and cDNA of its intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization was confirmed by restriction digestion and sequencing. Both!the mouse anti-hURAT1 antibody and rabbit anti-hURAT1 antibody recognized 58kD hURAT1 and 64kD glycosylated hURAT1 protein bands in western blot. Immunohistochemically, hURAT1 was located at the brush border membrane of renal proximal tubular cells. In addition, the throughput and specificity of the mouse anti-hURAT1 antibody were higher than those of the rabbit anti-hURAT1 antibody.Conclusion Genetic immunization can generate anti-hURAT1 polyclonal antibody of high throughput and specificity.

Pharmacokinetics of monoclonal antibodies ]nd Fc-fusion proteins

There are many factors that can influence the pharma- cokinetics （PK） of a mAb or Fc-fusion molecule with the primary determinant being FcRn-mediatad recycling. Through Fab or Fc engineering, IgG-FcRn interaction can be used to generate a variety of therapeutic anti. bodies with significantly enhanced half-life or ability to remove unwanted antigen from circulation, Glycosyla- tion of a mAb or Fc.fusion protein can have a significant impact on the PK of these molecules, mAb charge can be important and variants with pl values of 1-2 unit difference are likely to impact PK with lower pl values being favorable for a longer half.life. Most mAbs display target mediated drug disposition （TMOO）, which can have significant consequences on the study designs of preclinical and clinical studies. The PK of mAb can also be influenced by anti-drug antibody （ADA） response and off.target binding, which require careful consideration during the discovery stage, mAbs are primarily absor- bed through the lymphatics via convection and can be conveniently administered by the subcutaneous （sc） route in large doses/volumes with co-formulation of hyaluronidase. The human PK of a mAb can be rea- sonably estimated using cynomolgus monkey data and allometric scaling methods.

Human monoclonal antibodies as candidate therapeutics against emerging viruses

The emergence of new pathogens, such as severe acute respiratory syndrome coronavirus （SARS-CoV）, Middle East respiratory syndrome coronavirus （MERS-CoV）, and Ebola virus, poses serious challenges to global public health and highlights the urgent need for novel antiviral approaches. Monoclonal antibodies （mAbs） have been successfully used to treat various diseases, particularly cancer and immunological disorders. Antigen-specific mAbs have been isolated using several different approaches, including hybridoma, transgenic mice, phage display, yeast display, and single B-celi isolation. Consequently, an increasing number of mAbs, which exhibit high potency against emerging viruses in vitro and in animal models of infection, have been developed. In this paper, we summarize historical trends and recent developments in mAb discovery, compare the advantages and disadvantages of various approaches to mAb production, and discuss the potential use of such strategies for the development of antivirals against emerging diseases. We also review the application of recently developed human mAbs against SARS-CoV, MERS-CoV, and Ebola virus and discuss prospects for the development of mAbs as therapeutic agents against emerging viral diseases.

Both structure and function of human monoclonal antibodies contribute to enhancement of Zika virus infectivity in vitro

Dear Editor,Antibody-dependent enhancement(ADE)has long been recognized for dengue virus(DENV)in vitro and in vivo.It is now clear that antibodies to DENV can also enhance Zika virus(ZIKV)infection in vitro,and vice versa(Dejnirattisai et al.,2016;Stettler et al.,2016).The characteristics of enhancing antibodies,however,remain elusive.Some have suggested that non-neutralizing antibodies or antibodies recognizing specific antigenic sites are