scholarly journals USING OF UNIVERSAL PLASMID CONSTRUCTIONS FOR DESIGN OF RECOMBINANT ANTIBODIES WITH DEFINED SPECIFICITY IN EUKARYOTIC CELLS

2019 ◽  
Vol 1 (3) ◽  
pp. 32-39
Author(s):  
T. G. Samartseva ◽  
A. S. Oksanich ◽  
N. F. Gavrilova ◽  
I. V. Yakovleva ◽  
V. V. Sviridov ◽  
...  
Keyword(s):  
Cho Cells ◽  
Variable Region ◽  
Chimeric Antibody ◽  
Antibody Activity ◽  
Antigen Specificity ◽  
Minimum Concentration ◽  
Variable Regions ◽  
Chimeric Antibodies ◽  
Recombinant Plasmids ◽  
Heavy Chains

Aim. In this study we aimed to develop the methodology to change the antigen specificity of chimeric antibodies by replacing the variable region genes in the previously designed universal plasmid constructions pLK DT-17 and pHG DT-17 encoding the DT-17 antibody against the diphtheria toxin (DT) to the genes of antibody binding to another DT epitope — DT-22.Materials and methods. The genes of the light and heavy chain variable regions of mouse anti-DT antibodies — DT-22 were amplified from the hybridoma producing monoclonal antibodies to DT by reverse transcription and PCR methods. Genetic engineering methods were used to replace the variable regions of DT-17 antibody in the recombinant plasmids pLK DT-17 and pHG DT-17 encoding the light and heavy chains of DT-17 antibody, respectively to the relevant genes of DT-22. Subsequently, a «supervector» pSV DT-22, containing the genes of both chains of the chimeric antibody, was designed. CHO cells were transfected with a «supervector» and a highly productive clone, secreting chimeric antibodies to DT was obtained. Immunochemical and cultural methods were used to evaluate antibody activity. The affinity chromatography was used to purified preparative amounts of antibodies.Results. The yield of purified secreted chimeric DT-22 antibodies was 4 mg from per liter of culture medium. The minimum concentration of chimeric antibodies at which DT was neutralized in the CHO cells was 22 μg/mL of medium.Conclusion. Thus it has been shown how to generate new vector coding synthesis of light and heavy chains of a chimeric DT-22 antibody specific to another DT epitope using previously constructed universal recombinant plasmids pLK DT-17 and pHG DT-17 encoding, light and heavy chains of antibodies against DT DT-17, respectively.

scholarly journals PLASMID DESIGN FOR PRODUCTION OF CHIMERIC ANTIBODIES WITH DEFINED SPECIFICITY IN EUKARYOTES

2017 ◽  
pp. 56-63 ◽  
Author(s):  
A. S. Oksanich ◽  
T. G. Samartseva ◽  
E. B. Faizjuloev ◽  
N. F. Gavrilova ◽  
I. V. Yakovleva ◽  
...  
Keyword(s):  
Cho Cells ◽  
Diphtheria Toxin ◽  
Culture Medium ◽  
Variable Region ◽  
Plasmid Vector ◽  
Chimeric Antibody ◽  
Antibody Activity ◽  
Minimum Concentration ◽  
Chimeric Antibodies ◽  
Heavy Chains

Aim. In this study we aimed to design the universal genetic construction expressing the light and heavy chains of a chimeric antibody, to develop methodological approaches for the production of chimeric antibodies with defined specificity using the monoclonal antibodies to diphtheria toxin (DT) DT-17 in the CHO cells as an example and to evaluate their immunochemical and effector properties. Materials and methods. Variable region genes of the light and heavy chains of mouse antibodies DT-17 to diphtheria toxin were obtained by PCR method and cloned into pCI-neo plasmid vector. The S V DT -17neo «supervector» containing the genes of a chimeric antibody was constructed by using of genetic engineering techniques. CHO cells were transfected with «supervector» and a highly productive clone secreting chimeric antibodies to DT were collected. Immunochemical methods were used to evaluate antibody activity, and affinity chromatography was used to prepare preparative amounts of the antibodies. Results. U ni versal vectors pLK DT -17 and pHG DT-17 containing light and heavy chain genes of the chimeric antibodies DT -17 to DT were constructed. The variable and constant region genes were flanked by endonuclease restriction sites, which allows to change the specificity of the antibodies. In the future it will make possible to study the modifications of the class and species specificity of the chimeric immunoglobulins. When the CHO cell culture was transfected with the designed vectors, the accumulation of antibodies to DT in the culture medium was detected. The yield of purified DT-17 chimeric antibodies was 4 mg per 1 liter of culture medium. The minimum concentration of chimeric antibodies necessary for DT neutralization in the CHO cells was 30 pg/mL. Conclusion. Universal plasmids encoding the synthesis of light and heavy chains of chimeric DT -17 antibody have been designed. On the basis of these vectors, a «supervector» and a highly productive clone secreting specific antibodies that had neutralizing activity against DT were obtained.

scholarly journals Recombinant Mouse-Human Chimeric Antibodies as Calibrators in Immunoassays That Measure Antibodies toToxoplasma gondii

1998 ◽  
Vol 36 (5) ◽  
pp. 1277-1284 ◽  
Author(s):  
John Hackett ◽  
Jane Hoff-Velk ◽  
Alan Golden ◽  
Jeff Brashear ◽  
John Robinson ◽  
...  
Keyword(s):  
Human Plasma ◽  
High Titer ◽  
Variable Region ◽  
Immunoglobulin M ◽  
Expression Vectors ◽  
Chimeric Antibody ◽  
Chimeric Mouse ◽  
Variable Regions ◽  
Chimeric Antibodies ◽  
Igg1 Antibody

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was ≥0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

scholarly journals Expression of a public idiotype by human monoclonal IgM directed to myelin-associated glycoprotein and characterization of the variability subgroup of their heavy and light chains.

1989 ◽  
Vol 170 (5) ◽  
pp. 1551-1558 ◽  
Author(s):  
J C Brouet ◽  
K Dellagi ◽  
M C Gendron ◽  
A Chevalier ◽  
C Schmitt ◽  
...  
Keyword(s):  
Nonhuman Primate ◽  
Light Chains ◽  
Antibody Activity ◽  
Rheumatoid Factors ◽  
Variable Regions ◽  
Cold Agglutinins ◽  
Heavy Chains ◽  
Myelin Associated Glycoprotein ◽  
Kappa Light Chains

Most studies using rabbit or mouse antisera failed to detect CRI between human IgM directed to MAG. We show here that 9 of 10 such IgM express a public CRI as defined by a nonhuman primate antiserum. Shared idiotype is likely involved in (or close to) the combining site of those IgM since antiidiotypic serum inhibited the binding of IgM to MAG and reacted with IgM having different variable regions of light and heavy chains. Partial aminoterminal sequence of heavy and light chains showed that anti-MAG IgM use either lambda chains (one IgM) or kappa light chains (six IgM) of different variability subgroups (V kappa IV in three instances, V kappa I in two, and V kappa II in one), whereas heavy chains belong to the VHIII (six IgM) or to the VHII (1 IgM) subgroup. These features distinguish these IgM from other human monoclonal IgM with a defined antibody activity, such as rheumatoid factors or cold agglutinins.

Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Activated Platelet P-selectin

1997 ◽  
Vol 77 (04) ◽  
pp. 755-759 ◽  
Author(s):  
Jianming Gu ◽  
Yue Liu ◽  
Lijun Xia ◽  
Haiying Wan ◽  
Peixia Li ◽  
...  
Keyword(s):  
Large Scale ◽  
Variable Region ◽  
Expression Vectors ◽  
Chimeric Antibody ◽  
Scale Production ◽  
Fab Fragment ◽  
Variable Regions ◽  
E Coli ◽  
Large Scale Production

SummaryA murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human chimeric reagents. The E. coli expression vector. pHENl-SZ51 Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG γICHI and Cκ genes. The constructs were introduced into E. coli HB2151 for expression of soluble chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse antibody to the appropriate constant regions of human Igγl and κ. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then cotransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/1 of culture, was selected for the large scale production of chimeric antibody. Immunoblotting analysis demonstrated that both of the chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole chimeric antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 chimeric antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.

scholarly journals The amino acid sequences of the Fd fragments of two human γ 1 heavy chains

1970 ◽  
Vol 117 (4) ◽  
pp. 641-660 ◽  
Author(s):  
E. M. Press ◽  
N. M. Hogg
Keyword(s):  
Amino Acid ◽  
Aspartic Acid ◽  
Heavy Chain ◽  
Variable Region ◽  
Amino Acid Sequences ◽  
Light Chains ◽  
Aspartic Acid Residue ◽  
Variable Regions ◽  
Heavy Chain Variable Region ◽  
Heavy Chains

The amino acid sequences of the Fd fragments of two human pathological immunoglobulins of the immunoglobulin G1 class are reported. Comparison of the two sequences shows that the heavy-chain variable regions are similar in length to those of the light chains. The existence of heavy chain variable region subgroups is also deduced, from a comparison of these two sequences with those of another γ 1 chain, Eu, a μ chain, Ou, and the partial sequence of a fourth γ 1 chain, Ste. Carbohydrate has been found to be linked to an aspartic acid residue in the variable region of one of the γ 1 chains, Cor.

scholarly journals Generation and Characterization of Chimeric Antibodies against NS3, NS4, NS5, and Core Antigens of Hepatitis C Virus

2010 ◽  
Vol 17 (6) ◽  
pp. 1040-1047 ◽  
Author(s):  
Bailin Tu ◽  
Robert N. Ziemann ◽  
Bryan C. Tieman ◽  
David J. Hawksworth ◽  
Joan Tyner ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Heavy Chain ◽  
Light Chain ◽  
Cho Cells ◽  
Free Medium ◽  
Cho Cell ◽  
Alanine Scanning Mutagenesis ◽  
Variable Regions ◽  
Chimeric Antibodies

ABSTRACT Mouse-human chimeric antibodies (cAbs) against hepatitis C virus (HCV) core, NS3 (nonstructural), NS4, and NS5 antigens were developed as quality control (QC) reagents to replace the use of human sera/plasma for Abbott HCV immunoassays. The cAb retains the mouse monoclonal antibody (MAb) specificity and affinity but still reacts in the existing HCV assay format, which measures human anti-HCV immunoglobulin. Mouse heavy-chain (VH) and light-chain (VL) variable regions of anti-HCV core, NS3, NS4, and NS5 antigens were PCR amplified from hybridoma lines and then cloned with human IgG1 heavy-chain (CH) and light-chain (CL) constant regions, respectively. A single mammalian expression plasmid containing both heavy-chain and light-chain immunoglobulin genes was constructed and transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells. The transfected CHO cells were selected using hypoxanthine- and thymidine-free medium and screened by an enzyme immunoassay (EIA). The clone secreting the highest level of antibody was isolated from the CHO transfectants and further subcloned. Each cAb-expressing CHO cell line was weaned into serum-free medium, and the cAb was purified by protein A affinity chromatography. The levels of cAb production for the various CHO cell lines varied from 10 to 20 mg/liter. Purified anti-HCV cAbs were tested with Abbott HCV immunoassays and showed reactivity. Moreover, yeast surface display combined with alanine-scanning mutagenesis was used to map the epitope at the individual amino acid level. Our results suggest that these HCV cAbs are ideal controls, calibrators, and/or QC reagents for HCV assay standardization.

Genetic control of immunoglobulin synthesis

1970 ◽  
Vol 176 (1044) ◽  
pp. 329-346 ◽  
Keyword(s):  
Genetic Control ◽  
Mammalian Species ◽  
Variable Region ◽  
Lymphoid Cells ◽  
The Other ◽  
Light Chains ◽  
Common Region ◽  
Variable Regions ◽  
Immunoglobulin Synthesis ◽  
Heavy Chains

The structural features of immunoglobulins are described. This family of related proteins shows a common structural design: two identical light chains and two identical heavy chains are present in each molecule. Amino acid sequence studies have shown that light chains have one of two types of sequences in the C terminal half, whereas they differ one from the other in the N terminal half. The two halves of the sequence have been designated accordingly variable and common half. Similarly, the heavy chains have a common sequence in the C terminal three-quarters of the sequence and a variable one in the N terminal quarter. Genetic studies on the inheritance of immunoglobulin alleles have been carried out in some mammalian species. The genetic control of immunoglobulin synthesis is reviewed in man, mouse and rabbit. These studies have shown that each allele controls the inheritance of a specific common region, with the exception of one genetic system which seems to control the synthesis of the variable region of rabbit heavy chains. Immunoglobulin chains are clearly synthesized under the control of two distinct genetic elements, one of which specifies the variable region and the other the common region. The possible significance of this type of genetic control of immunoglobulin structure is discussed. It has not yet been established whether each variant of the variable region is coded for by an individual structural gene present in the genome of each individual or whether few genes for variable regions exist, which in the course of the differentiation of lymphoid cells are subject to somatic mutation processes, which generate variability. These two possibilities are discussed and elements in favour of one or the other theory are presented.

scholarly journals Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yongbing Pan ◽  
Jianhui Du ◽  
Jia Liu ◽  
Hai Wu ◽  
Fang Gui ◽  
...  
Keyword(s):  
Phage Display ◽  
Neutralizing Antibodies ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
Chain Gene ◽  
Prophylactic Efficacy ◽  
Heavy Chains ◽  
Effective Interventions ◽  
Phage Display Libraries

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11–RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

scholarly journals Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

2000 ◽  
Vol 74 (19) ◽  
pp. 9028-9038 ◽  
Author(s):  
J.-B. Nousbaum ◽  
S. J. Polyak ◽  
S. C. Ray ◽  
D. G. Sullivan ◽  
A. M. Larson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Antiviral Therapy ◽  
Variable Region ◽  
Response To Therapy ◽  
Full Length ◽  
Variable Regions ◽  
C Terminus

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

scholarly journals Characterization of Neutralizing Antibody Responses Elicited by Clade A Envelope Immunogens Derived from Early Transmitted Viruses

2008 ◽  
Vol 82 (12) ◽  
pp. 5912-5921 ◽  
Author(s):  
Zane Kraft ◽  
Katharine Strouss ◽  
William F. Sutton ◽  
Brad Cleveland ◽  
For Yue Tso ◽  
...  
Keyword(s):  
Chronic Infection ◽  
Neutralizing Antibodies ◽  
Acute Infection ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Virus Envelope ◽  
Variable Regions ◽  
Clade B ◽  
Region Length

ABSTRACT The vast majority of studies with candidate immunogens based on the human immunodeficiency virus envelope (Env) have been conducted with Env proteins derived from clade B viruses isolated during chronic infection. Whether non-clade B Env protein immunogens will elicit antibodies with epitope specificities that are similar to those of antibodies elicited by clade B Envs and whether the antibodies elicited by Envs derived from early transmitted viruses will be similar to those elicited by Envs derived from viruses isolated during chronic infection are currently unknown. Here we performed immunizations with four clade A Envs, cloned directly from the peripheral blood of infected individuals during acute infection, which differed in lengths and extents of glycosylation. The antibody responses elicited by these four Envs were compared to each other and to those elicited by a well-characterized clade B Env immunogen derived from the SF162 virus, which was isolated during chronic infection. Only one clade A Env, the one with the fewer glycosylation sites, elicited homologous neutralizing antibodies (NAbs); these did not target the V1, V2, or V3 regions. In contrast, all four clade A Envs elicited anti-V3 NAbs against “easy-to-neutralize” clade B and clade A isolates, irrespective of the variable region length and extent of glycosylation of the Env used as an immunogen. These anti-V3 NAbs did not access their epitopes on homologous and heterologous clade A, or B, neutralization-resistant viruses. The length and extent of glycosylation of the variable regions on the clade A Env immunogens tested did not affect the breadth of the elicited NAbs. Our data also indicate that the development of cross-reactive NAbs against clade A viruses faces similar hurdles to the development of cross-reactive anti-clade B NAbs.

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