The role of amino acid changes in the human immunodeficiency virus type 1 transmembrane domain in antibody binding and neutralization

The detailed interactions between antibodies and the HIV-1 envelope protein that lead to neutralization are not well defined. Here, we show that several conservative substitutions in the envelope gp41 led to a ~ 100 fold increase in neutralization sensitivity to monoclonal antibodies (MAbs) that target gp41: 4E10 and 2F5. Substitution at position 675 alone did not impact neutralization susceptibility to MAbs that recognize more distal sites in gp120 (b12, VRC01, PG9). However, changes at position 675 in conjunction with Thr to Ala at position 569 increased the neutralization sensitivity to all gp41 and gp120 MAbs and plasma, in some cases by more than 1000-fold. Interestingly, the T569A change had a dramatic effect on b12 binding, but no effect on neutralization sensitivity. This finding suggests that antibody neutralization may occur through a multi-step pathway that includes distinct changes in envelope conformation that may affect binding but not neutralization susceptibility.     

人源抗HIV-1 gp120趋化蛋白受体结合位点Fab单克隆抗体基因的获得

目的：筛选人源抗HIV－1 gp120趋化蛋白受体结合位点噬菌体Fab单克隆抗体基因。方法：根据HIV－1 gp120趋化蛋白受体结合位点的氨基酸序列合成23肽，并以此为固相抗原从HIV－1噬菌体Fab抗体库筛选阳性克隆，并进行鉴定及序列测定。结果：获得了1株抗HIV－1 gp120趋化蛋白受体结合位点的人源Fab抗体克隆，具有较高的亲和力、特异性和抑制率，序列测定及分析显示该抗体属IgG I亚类，κ型，重、轻链可变区分别属VhⅢ和VκⅢ基因家族。结论：成功地获得了人源抗HIV－1 gp120趋化蛋白受体结合位点噬菌体Fab单克隆抗体基因。     

High efficiency reconstitution of a human-mouse chimeric Fab of CAb-1 antibody specific to human colon cancer

Monoclonal antibody (mAb) has been widely applied in the treatment of human diseases, especially in malignant tumours. However, most antibodies produced in mouse by hybridoma technology might induce severe human anti-mouse reactions. We had reported a murine mAb CAb-1 of therapeutic interest for its specifically binding to a cell surface glycoprotein of human colon cancer. Here, we attempted to generate a reconstituted human–mouse chimeric Fab (cFab) of CAb-1 in vitro to reduce its antigenicity and increase its capacity of penetration. First, the genes of heavy and light chain variable region (VH, VL) of CAb-1 were cloned. Then, the chimeric light chain (cL) and Fd (cFd) were constructed and expressed in Escherichia coli . Finally, the reconstituted cFab was obtained by gradient dialysis of the mixture of cFd and cL. SDS-PAGE and western blot analysis showed the reconstituted cFab with a recovery rate of 70.2% when the initial total concentration of cL and cFd proteins to be 100  μ g/ml. The reconstituted cFab maintained the affinity and specificity to colon cancer cells compared with its parental antibody as determined by immunostaining analysis, FACS and ELISA. Our results established a foundation for further application of the cFab in diagnosis and treatment of colon cancer.     

Characteristic of HIV-1 in V3 loop region based on seroreactivity and amino acid sequences in Thailand

The third variable (V3) domain of the envelop (env) protein has been used for determining genetic subtype and phenotypic characteristics of human immunodeficiency virus type 1 (HIV-1) isolates. Based on the seroreactivity of the HIV-1 subtype by V3 peptide binding enzyme immunoassay (EIA) of 351 samples obtained in 1998 from HIV-1 infected individuals and AIDS patients, we found that 283 (80.6%) were subtype E, 20 (5.7%) were subtype B, 28 (8.0%) were cross-reactive between both types and 20 (5.7%) were non-typeable. The degree of seroreactivity of HIV-1 subtype E decreased significantly when the amino acid at the crown of the V3 loop was substituted from a GPGQ motif to GPGR motif. Interestingly, AIDS patients who had V3 sequences of subtype E as GPGR motif had a stronger immunoreactivity to GPGQ motif peptides than to GPGR motif peptides, in contradiction for their proviral sequences. The results suggested that mutations in the V3 loop may lead to a changed immunoreactivity that makes HIV-1 mutants unrecognizable or allow escape from the primary immune response by means of neutralizing sensitivity. In connection with vaccine development, it should be pointed out that the combination of V3 sequencing and peptide EIA could provide a novel approach to obtain a primarily infected virus sequence as a target for a preventive AIDS vaccine.     

A human immunoglobulin G1 antibody originating from an in vitro-selected Fab phage antibody binds avidly to tumor-associated MUC1 and is efficiently internalized

We describe the engineering and characterization of a whole human antibody directed toward the tumor-associated protein core of human MUC1. The antibody PH1 originated from the in vitro selection on MUC1 of a nonimmune human Fab phage library. The PH1 variable genes were reformatted for expression as a fully human IgG1. The resulting PH1-IgG1 human antibody displays a 160-fold improved apparent kd (8.7 nmol/L) compared to the kd of the parental Fab (1.4 micromol/L). In cell-binding studies with flow cytometry and immunohistochemistry, PH1-IgG1 exhibits staining patterns typical for antibodies recognizing the tumor-associated tandem repeat region on MUC1, eg, it binds the tumor-associated glycoforms of MUC1 in breast and ovarian cancer cell lines, but not the heavily glycosylated form of MUC1 on colon carcinoma cell lines. In many tumors PH1-IgG1 binds to membranous and cytoplasmic MUC1, with often intense staining of the whole-cell membrane (eg, in adenocarcinoma). In normal tissues staining is either absent or less intense, in which case it is found mostly at the apical side of the cells. Finally, fluorescein isothiocyanate-labeled PH1-IgG1 internalizes quickly after binding to human OVCAR-3 cells, and to a lesser extent to MUC1 gene-transfected 3T3 mouse fibroblasts. The tumor-associated binding characteristics of this antibody, its efficient internalization, and its human nature, make PH1-IgG1 a valuable candidate for further studies as a cancer-targeting immunotherapeutic.     

熊果酸对胃癌细胞中cofilin-1蛋白表达的影响

目的 研究熊果酸作用胃癌细胞BGC-823后丝切蛋白cdilin-1表达的变化.方法 熊果酸作用于细胞BGC-823,双向电泳结合质谱检测胞质内cofilin-1的表达,免疫印迹检测cofilin-1蛋白和细胞色素C的表达.结果 胞质内cofilin-1蛋白表达降低(P<0.01),而线粒体表达却明显升高(P<0.01),同时细胞色素C在胞质中的表达量也明显下调(P<0.01).结论 熊果酸作用BGC-823,可能使cofilin-1蛋白从胞质转位至线粒体内,促使细胞色素C释放,激活caspase级联反应,诱导细胞凋亡.     

Partial amino acid sequences of variable regions of a human IgG1 (kappa) myeloma protein with anti-rubella activity

Amino acid sequences have been determined for the first 26 residues at the amino (N?) terminus of the light chain, the first 27 residues of the heavy chain and the first 23 residues of a large heavy chain fragment of an IgG1 (κ) myeloma protein with anti-rubella specificity. These data furnish useful information about the primary structure of anti-rubella antibody.     

Human single-chain Fv (scFv) antibody specific to human IL-6 with the inhibitory activity on IL-6-signaling

Human anti-IL-6 antibody may be useful for the immunotherapy of various inflammatory diseases, such as rheumatoid arthritis. Since IL-6 is a growth factor for B cell hybridoma, it is not easy to isolate murine B cell hybridomas producing the anti-IL-6 antibody with the IL-6-signaling inhibitory activity. In this study, the antibody library (Vgamma-Vkappa, Vgamma-Vlambda, Vmu-Vkappa or micro-Vlambda ligated into the pCANTAB 5E phagemid vector) was prepared from peripheral blood mononuclear cells of 20 healthy subjects. The phage display library was panned with an IL-6-coated plastic plate, and the binding specificity was confirmed by ELISA and BIAcore. From the antibody library (Vgamma-Vlambda), five IL-6-specific phage clones were isolated. The effects of the soluble scFvs purified from these phage clones were tested on the growth of the IL-6-dependent human cell line, KT-3. Two of these clones significantly inhibited the growth of KT-3, and three showed no inhibition.     

Comparison of amino acid sequences at the amino acid 130-160 region of VP1 polypeptide of Indian field isolates of foot-and-mouth disease virus serotype Asia 1

The nucleotide and deduced amino acid sequences in the amino acid (aa) 130-160 region of VP1 polypeptide of 65 field isolates of foot- and mouth disease virus (FMDV) serotype Asia 1 were determined and the consensus sequences were deduced. Comparison of amino acid sequences revealed conservation of NGK (130-132), TYG (134-136), RGD (142-144), and LPTSF (156-160) motifs and aa 148 (L) while variation was observed at the rest of the region (variability index (VI) of 2.06 to 16.85). Synonymous and non-synonymous mutations at the nucleotide level were well correlated with those of the corresponding amino acids. Comparison of the aa 130-160 sequence of Asia 1 serotype with those of other serotypes of FMDV revealed conservation of aa 135, 148-149, 157 and 160. Amino acids 133-138 and 148-154 were unique for Asia 1 serotype and are presumably responsible for its distinct antigenic nature. The present study revealed that the FMDV isolates of serotype Asia 1 causing outbreaks in India are very much heterogeneous in the aa 130-160 region of VP1.     

Construction of scFv derived from a tumor-associated monoclonal antibody having tumoricidal activity on human hepatocellular carcinoma

A mouse monoclonal antibody (Mab-HepTAA43), classified as an anti-tumor-associated antigen, was raised by immunizing BALB/c mice with the Thai human hepatocellular carcinoma S102 (HCC-S102) cell line cells using hybridoma techniques. The Mab-HepTAA43 reacted with and markedly inhibited the growth of human hepatocellular carcinoma cell lines as well as a tumor mass in an animal model. Human hepatoma transplanted into nude mice did not show metastasis after 20 injections amounting to a total of about 4 mg of the Mab over 1-month period. A single-chain variable fragment (scFv) molecule derived from the Mab was constructed by phage display method. DNA sequence analysis of the active variable regions of both heavy- and light-chains of the cDNA clone was subsequently performed. The scFv43 molecule contains a V(L) kappa type and a unique V(H) sequence having 88% amino acid homology to that of Mab-MAK B raised against tumor-associated antigen. Immunohistochemical staining on frozen sections of paired hepatoma (NCI-I) and normal liver tissue from the same individual showed that both scFv43 and Mab-HepTAA43 antibodies reacted with hepatoma but not with normal liver tissue. The results suggest that scFv43 may be useful in the immunotherapy of hepatocellular carcinoma.     

Cytotoxic necrotizing factor type 1-neutralizing monoclonal antibody NG8 recognizes three amino acids in a C-terminal region of the toxin and reduces toxin binding to HEp-2 cells

Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are toxins of pathogenic Escherichia coli that share 85% identity over 1,014 amino acids. Although both of these toxins modify GTPases, CNF1 is a more potent inducer of multinucleation in HEp-2 cells, binds more efficiently to HEp-2 cells, and, despite the conservation of amino acids (C866 and H881) required for enzymatic activity of the toxins, deamidates RhoA and Cdc42 better than CNF2. Here we exploited the differences between CNF1 and CNF2 to define the epitope on CNF1 to which the CNF1-specific neutralizing monoclonal antibody (MAb) (MAb NG8) binds and to determine the mechanism by which MAb NG8 neutralizes CNF1 activity on HEp-2 cells. For these purposes, we generated a panel of 21 site-directed mutants in which amino acids in CNF1 were exchanged for the amino acids in CNF2 between amino acids 546 and 869 and vice versa. This region of CNF1 not only is recognized by MAb NG8 but also is involved in binding of this toxin to HEp-2 cells. All the mutants retained the capacity to induce multinucleation of HEp-2 cells. However, the CNF1 double mutant with D591E and F593L mutations (CNF1(D591E F593L)) and the CNF1(H661Q) single mutant displayed drastically reduced reactivity with MAb NG8. A reverse chimeric triple mutant, CNF1(E591D L593F Q661H), imparted MAb NG8 reactivity to CNF2. MAb NG8 neutralized CNF2(E591D L593F Q661H) activity in a dose-dependent manner and reduced the binding of this chimeric toxin to HEp-2 cells. Taken together, these results pinpoint three amino acids in CNF1 that are key amino acids for recognition by neutralizing MAb NG8 and further help define a region in CNF1 that is critical for full toxin binding to HEp-2 cells.     

Goat antibodies to amino acid sequences of human chorionic gonadotropin (hCG)

Human chorionic gonadotropin, its two subunits and its conjugate with thyroglobulin were used for immunization. The strongest immunogens were demonstrated to be the intact hormone and beta subunit, the weakest was alpha subunit. Using three peptides corresponding to hormone subunits coupled to Sepharose 4B various antibodies were isolated from goat antiserum by immunoaffinity chromatography. Specificity of the purified antibody preparations was studied. It was demonstrated that enzyme linked immunosorbent assay with two goat antibodies--one for coating of microtiter plates and the other one conjugated with peroxidase--is suitable for sensitive assays of both human chorionic gonadotropin and luteinizing hormone. Specific and sensitive assays were performed using combination of goat antibody anti-122-145-beta hCG and monoclonal antibody anti-alpha hCG.     

Effect of Berkefeld filtration on the binding activity of human gastric juice

The intrinsic factor content of Berkefeld-filtered human gastric juice has been studied. This appears to vary with the pH at which filtration is carried out and also between individual filters. Significant losses of intrinsic factor may result from filtration and complete loss when filtration is carried out at a low pH. The most suitable pH for filtration appears to be in the range pH 7 to 8.     

熊果酸抑制人胃腺癌SGC-7901细胞系COX-2的表达

目的观察熊果酸(UA)对胃腺癌细胞SGC-7901环氧化酶-2(COX-2)表达的影响。方法采用MTT法观察细胞增殖;Western blot法检测COX-2蛋白表达;放射免疫分析法观察细胞合成PGE2。结果UA可以抑制SGC-7901细胞的增殖,并呈浓度和时间依赖性;外源性PGE2并不能逆转UA的抑制增殖作用;UA可以降低细胞COX-2蛋白的表达,并呈浓度依赖性;UA可以抑制PGE2的合成,但该作用无浓度依赖性。结论UA通过抑制COX-2的表达、减少PGE2的合成抑制胃腺癌细胞SGC-7901的增殖,可能成为一种胃癌预防剂。     

Possible use of similar framework region amino acid sequences between human and mouse immunoglobulins for humanizing mouse antibodies.

We have previously noted that a specific amino acid sequence could form the second framework region of human, mouse and rabbit immunoglobulin light chains, suggesting that this sequence has been preserved for 80 million years. Through divergent evolution, each species has acquired a different set of framework region sequences; however, these sets still share a few similar or identical amino acid sequences. In the present study, we have identified such sequences for all four framework regions between human and mouse immunoglobulin light and heavy chains. They may be useful in humanizing or reshaping mouse or rat antibodies for therapeutic applications in human patients.     

Studies on the amino and carboxyl terminal amino acid sequences of reovirus capsid polypeptides

Techniques are described for isolating several reovirus capsid polypeptides in amounts sufficient for determination of amino and carboxyl terminal amino acid sequences and fingerprinting. Among them are chromatography on CM- and DEAE-Sephadex in the presence of urea, and gel filtration on agarose A-15 m in the presence of sodium dodecyl sulfate. Using a combination of these procedures, and starting with either virions or cores, polypeptides σ3, σ2, and μ2 have been obtained essentially pure. Polypeptides λ1 and λ2 have been obtained as a mixture which has so far not been resolved.All reovirus capsid polypeptides except μ2 possess blocked amino terminal amino acid residues. The amino terminal amino acid sequence of polypeptide μ2 is H₂N-Pro-Gly-Gly-Val-Pro-. This suggests that polypeptide μ2 is derived from its precursor, polypeptide μ1, by cleavage of the amino terminal portion of the polypeptide chain.The carboxyl terminal regions of at least three of the five major reovirus capsid polypeptides are different. Polypeptide σ3 ends in -(val, val, leu)-COOH; polypeptide μ2 in -leu-(arg, tyr, tyr)-Arg-COOH; and either one or both of the two polypeptides λ1 and λ2 terminate(s) in -Arg-COOH, the adjacent amino acid sequence being different from that of μ2.     

Expression and detection strategies for an scFv fragment retaining the same high affinity than Fab and whole antibody: Implications for therapeutic use in prion diseases

Since antibodies currently constitute the most rapidly growing class of human therapeutics, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. Using as model a monoclonal antibody directed against the human prion protein that we prepared previously and tested for its therapeutic value, we describe here experimental conditions allowing the production of large quantities (up to 35 mg/l of bacterial culture) of correctly refolded and totally functional single chain fragment variable (scFv). These quantities were sufficient to characterize the binding properties of this small recombinant fragment through in vitro and ex vivo approaches. Interestingly, this scFv retains full binding capacity for its antigen, i.e. the human prion protein, when compared with the corresponding Fab or whole antibody, and recognizes soluble, solid-phase-adsorbed, and membrane-bound prion protein. This strongly suggests that from the mAb cloning step to the refolding of the recombinant fragment, each stage is well controlled, leading to almost 100% functional scFv. These results are of interest not only in view of possible immunotherapy for prion diseases, but also more generally in emphasizing the great promise of these small recombinant molecules in the context of targeted therapies.     

Generation and preliminary characterization of an antibody library with preferential reactivity to human colorectal cancer cells as compared to normal human blood cells

We are developing recombinant polyclonal antibody libraries (PCALs) reactive to human colorectal cancer cells as an anti-cancer therapeutic approach. To test the hypothesis that PCALs with preferential recognition of tumor cells as compared to normal cells could be generated, a Fab phage display library reactive to human colorectal cancer cell lines was absorbed with normal human blood cells. ELISA analysis of the absorbed Fab phage display library showed that 70% of tested clones reacted to colorectal cancer cells. Reactivity of the library to blood cells was reduced 4-10-fold, with many clones unreactive to one or more of the blood cell types. DNA fingerprint analysis of colorectal cancer-binding clones showed that the absorbed library remained polyclonal. The H and L chain V region gene pairs of the absorbed library were transferred in mass from the Fab phage display vector to a mammalian vector and expressed as IgG following transfection into Sp2/0 myeloma cells. ELISA analysis showed that 79% of transfectants expressed IgG and of those 74% were positive for binding to the SW480 human colorectal cancer cell line. A template of 95 SW480-reactive wells was assembled, designated Lib-Col2.1, and IgG purified from a consolidated mass culture. The purified Lib-Col2.1 IgG was compared to the clinically used anti-colorectal cancer mAb 17-1A, by ELISA and flow cytometry, for binding density on SW480 colorectal cancer cells and on normal human blood cells. The results showed that Lib-Col2.1 bound to SW480 cells at higher density than mAb 17-1A and that its binding to normal human blood cells was reduced 2.4-24-fold relative to an unabsorbed anti-SW480 serum. Furthermore, Lib-Col2.1 was more effective than mAb 17-1A at inhibiting the growth of SW480 cells in culture. These results suggest that a polyclonal antibody library with preferential reactivity to cancer cells as compared to normal cells could be generated.     

Generation of a neutralizing human monoclonal antibody Fab fragment to surface antigen 1 of Toxoplasma gondii tachyzoites

A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 × 10(-9) M for Tox203 and 2.01 × 10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.