排序方式: 共有17条查询结果,搜索用时 93 毫秒
1.
Peter M. Bowers Tamlyn Y. Neben Geoffery L. Tomlinson Jennifer L. Dalton Larry Altobell Xue Zhang John L. Macomber Betty F. Wu Rachelle M. Toobian Audrey D. McConnell Petra Verdino Betty Chau Robert A. Horlick David J. King 《The Journal of biological chemistry》2013,288(11):7688-7696
A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods. 相似文献
2.
Oleg V. Kovalenko Andrea Olland Nicole Piché-Nicholas Adarsh Godbole Daniel King Kristine Svenson Valerie Calabro Mischa R. Müller Caroline J. Barelle William Somers Davinder S. Gill Lidia Mosyak Lioudmila Tchistiakova 《The Journal of biological chemistry》2013,288(24):17408-17419
The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs. 相似文献
3.
The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies
(Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have
evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and
humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage
display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently
developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear
cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs. 相似文献
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酵母表达人源化糖蛋白研究进展 总被引:1,自引:0,他引:1
与人体天然复杂型糖蛋白相比,使用酵母生产的药用蛋白带有高甘露糖型N-糖链。这一差异在临床应用中产生了许多不良影响。目前,可以通过消除酵母特有的内源糖基化反应,引入哺乳动物细胞中的一系列糖基转移酶及转运蛋白对酵母糖基化路径进行改造,从而使其表达出人源化的复杂型N-聚糖。本文介绍了酵母N-糖基化特点、糖基化不均一性,综述了近年来利用基因工程改造酵母N-糖基化路径获得特定的人源N-连接糖蛋白以及使用内切糖苷酶生产人源糖蛋白的研究进展,并且对存在的问题及今后的发展前景进行了讨论。 相似文献
6.
Knapped stone tools constitute an interesting evidence of the mental abilities of their makers. Almost imperishable, they bear visible traces of the successive removals they come from, and of the flaking techniques used. On that basis, we present a few remarkable achievements along the evolution of prehistoric lithic technology, and discuss their capabilities. The tools of the oldest Palaeolithic already surpass the nut cracking from Chimpanzees. Some core reductions into flakes, as early as 2.3 My, show a repeated organization of the removals, and the capacity to prevent a problem. The regular and symmetrical hand-axes from Africa and Europe, appearing between 1 and 0.5 My, provide evidence of a mental template and hence a capacity of conceptualization. The Levalloisian core reduction, somewhat more recent, give evidence of a goal structured method: the technical actions were hierarchized and subordinate to morphological intentions. 相似文献
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Leo Borras Tea Gunde Julia Tietz Ulrich Bauer Val��rie Hulmann-Cottier John P. A. Grimshaw David M. Urech 《The Journal of biological chemistry》2010,285(12):9054-9066
Despite their favorable pharmacokinetic properties, single-chain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 × 10−9 to 1.5 × 10−11 m. Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 × 10−10 to <1.8 × 10−12 m. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86 °C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains. 相似文献
9.
Ashutosh Tiwari Durgashree Dutta Navin Khanna Subrat K. Acharya Subrata Sinha 《Molecular biotechnology》2009,43(1):29-40
5S is a mouse monoclonal IgG1 that binds to the ‘a’ epitope of the Hepatitis B surface antigen (HBsAg) and tested positive
in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment
(scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable
domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (KD = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained
antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high
affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis
and treatment for Hepatitis B infection. 相似文献
10.
Zhang Q Zhang SH Su MQ Bao GQ Liu JY Yi J Shen JJ Hao XK 《Cancer immunology, immunotherapy : CII》2007,56(4):477-489
Background The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug
therapy in vivo. In this study we have achieved humanization of the anti-γ-seminoprotein E4B7 murine mAb by guided selection.
Methods Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients.
The human Lc gene repertoire was first paired with the murine Fd gene of E4B7 mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified γ-seminoprotein antigen.
The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four
more rounds of panning, completely human Fab antibodies specific for γ-seminoprotein were selected and further identified.
Results First, using the E4B7 Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a
template, a human Fab antibody against γ-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA,
and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E4B7 mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry
analysis further confirmed that this newly constructed human anti-γ-seminoprotein Fab antibody indeed specifically bound prostate
cancer cells and tissue.
Conclusions Through guided-selection, we successfully produced a human anti-γ-seminoprotein Fab antibody. This work lays the foundation
for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody.
Zhang Qing and Zhang Si-He are co-first authors on the publication. 相似文献