排序方式: 共有11条查询结果,搜索用时 46 毫秒
1.
Le Gall F Reusch U Little M Kipriyanov SM 《Protein engineering, design & selection : PEDS》2004,17(4):357-366
Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites. 相似文献
2.
Kipriyanov Sergey M.; Little Melvyn; Kropshofer Harald; Breitling Frank; Gotter Stefanie; Dubel Stefan 《Protein engineering, design & selection : PEDS》1996,9(2):203-211
In antigenantibody interactions, the high avidity ofantibodies depends on the affinity and number of the individualbinding sites. To develop artificial antibodies with multiplevalency, we have fused the single-chain antibody Fv fragmentsto core streptavidin. The resulting fusion protein, termed scFv::strep,was found after expression in Escherichia coli in periplasmicinclusion bodies. After purification of the recombinant productby immobilized metal affinity chromatography, refolding andsize-exclusion FPLC, tetrameric complexes resembling those ofmature streptavidin were formed. The purified tetrameric scFv::strepcomplexes demonstrated both antigen- and biotin-binding activity,were stable over a wide range of pH and did not dissociate athigh temperatures (up to 70°C). Surface plasmon resonancemeasurements in a BIAlite system showed that the pure scFv::streptetramers bound immobilized antigen very tightly and no dissociationwas measurable. The association rate constant for scFv::streptetramers was higher than those for scFv monomers and dimers.This was also reflected in the apparent constants, which wasfound to be 35 times higher for pure scFv::strep tetramers thanmonomeric singlechain antibodies. We could also show that mostof biotin binding sites were accessible and not blocked by biotinylatedE.coli proteins or free biotin from the medium. These sitesshould therefore facilitate the construction of bispecific multivalentantibodies by the addition of biotinylated ligands. 相似文献
3.
4.
For the treatment of minimal residual disease in patients with leukemias and malignant lymphomas, we constructed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the T cell receptor complex. The bispecific diabody was expressed in Escherichia coli using a vector containing a dicistronic operon for co-secretion of V(H)3-V(L)19 and V(H)19-V(L)3 single-chain Fv fragments (scFv). It was purified in one step by immobilized metal affinity chromatography (IMAC) from the periplasmic extract and culture medium. Flow cytometry experiments revealed specific interactions of the diabody with both CD3 and CD19 positive cells, to which it bound with affinities close to those of the parental scFvs. It was less stable than anti-CD3 scFv but more stable than anti-CD19 scFv when incubated in human serum at 37 degrees C. In cytotoxicity tests, the diabody proved to be a potent agent for retargeting peripheral blood lymphocytes to lyse tumor cells expressing the CD19 antigen. The efficiency of cell lysis compared favorably with that obtained with a bispecific antibody (BsAb) of the same dual specificity that was prepared by the quadroma technique. 相似文献
5.
SM Kipriyanov S Dübel F Breitling RE Kontermann S Heymann M Little 《Canadian Metallurgical Quarterly》1995,26(3):187-204
Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection. 相似文献
6.
7.
Kipriyanov SM; Moldenhauer G; Martin AC; Kupriyanova OA; Little M 《Protein engineering, design & selection : PEDS》1997,10(4):445-453
Recombinant antibody fragments directed against cell surface antigens have
facilitated the development of novel therapeutic agents. As a first step in
the creation of cytotoxic immunoconjugates, we constructed a single-chain
Fv fragment derived from the murine hybridoma OKT3, that recognizes an
epitope on the epsilon-subunit of the human CD3 complex. Two amino acid
residues were identified that are critical for the high level production of
this scFv in Escherichia coli. First, the substitution of glutamic acid
encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to
a more than a 30- fold increase in the production of soluble scFv. Second,
the substitution of cysteine by a serine in the middle of CDR-H3
additionally doubled the yield of soluble antibody fragment without any
adverse effect on its affinity for the CD3 antigen. The double mutant scFv
(Q,S) proved to be very stable in vitro: no loss of activity was observed
after storage for 1 month at 4 degrees C, while the activity of scFv
containing a cysteine residue in CDR-H3 decreased by more than half. The
results of production yield, affinity, stability measurements and analysis
of three-dimensional models of the structure suggest that the sixth amino
acid influences the correct folding of the VH domain, presumably by
affecting a folding intermediate, but has no effect on antigen binding.
相似文献
8.
V. P. Bessmeltsev N. V. Goloshevsky V. V. Kasterov Ya. A. Kipriyanov K. K. Smirnov 《Optoelectronics, Instrumentation and Data Processing》2018,54(4):390-396
A method used for calibration of scanning modules on the basis of galvanometric deflectors is considered. The method is based on an iterative two-step algorithm of processing of the profilometry map of the test writing of the calibrated module. It is demonstrated that high accuracy and robustness of determining the coordinates of the reference points of the test writing can be obtained by means of finding the approximate positions of such points and identifying the fragment in the vicinity of these points at the first step and searching for the exact values of the coordinates by the correlation method at the second step. The use of this method and a simple algorithm of piecewise-planar interpolation offers a possibility of laser beam positioning with a relative error of 10?5 from the writing field. 相似文献
9.
M Welschof P Terness SM Kipriyanov D Stanescu F Breitling H D?rsam S Dübel M Little G Opelz 《Canadian Metallurgical Quarterly》1997,94(5):1902-1907
Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab')2 antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab')2 antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA was prepared from the lymphocytes of a patient with a high IgG-anti-F(ab')2 serum titer. Variable heavy and light gene segments were amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab')2 fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab')2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain. The heavy chains belonged to the V(H)3 and the light chains to the V(kappa)2 gene family. The 11 identical light-chain genes were completely homologous to a germ-line sequence (DPK-15). Binding assays showed that the single-chain antibodies bind to F(ab')2, but not to Fab, Fc, or intact IgG. This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relatively high affinity (Ka = 2.8 x 10(7) M(-1)). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-IgG antibody's interaction with antigen. The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab')2 autoantibody clones and describes the binding kinetics of the purified monomeric fragments. 相似文献
10.