共查询到20条相似文献,搜索用时 15 毫秒
1.
A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments 总被引:4,自引:0,他引:4
Huls GA Heijnen IA Cuomo ME Koningsberger JC Wiegman L Boel E van der Vuurst de Vries AR Loyson SA Helfrich W van Berge Henegouwen GP van Meijer M de Kruif J Logtenberg T 《Nature biotechnology》1999,17(3):276-281
A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen. 相似文献
2.
目的:应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш (epidermal growth factor receptor variant type Ⅲ, EGFRvIII)的单链抗体 (single chain Fv, scFv)。方法:利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv 基因,连接至噬菌粒pCANTAB 5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coli HB2151,IPTG 诱导可溶性scFv 的表达。结果:构建了库容为7.9×107 的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv 基因序列长807 bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv 可分别与纯化的EGFRvIIIex抗原以及细胞表面的EGFRvIIIex结合。结论:利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvIII scFv,为开发针对EGFRvIII的抗体药物提供了靶向载体分子。 相似文献
3.
Production and validation of the pharmacokinetics of a single-chain Fv fragment of the MGR6 antibody for targeting of tumors expressing HER-2 总被引:4,自引:0,他引:4
Turatti F Mezzanzanica D Nardini E Luison E Maffioli L Bambardieri E de Lalla C Canevari S Figini M 《Cancer immunology, immunotherapy : CII》2001,49(12):679-686
The HER-2 antigen, which is overexpressed in many breast carcinomas, is an ideal target for monoclonal antibodies due to
its low expression in normal tissue and its homogeneous distribution in the tumor mass. We have developed and characterized
the murine MAb MGR6 against HER-2, which is able to inhibit proliferation of tumor cells overexpressing HER-2. On the basis
of these preclinical results, phase I studies in breast carcinoma patients were conducted and radiolocalization data indicated
an antibody half life which directly paralleled that of other whole antibodies and thus resulting in a limited in vivo diagnostic
capacity. To obtain a smaller reagent with possibly improved in vivo properties, a single chain variable fragment (scFv) of
the original MGR6-producing hybridoma was generated by phage display technology. Biologically active MGR6 scFv was purified
rapidly and at high yield by metal affinity chromatography. Competition FACS and ELISA analyses identified an epitope on the
HER-2 extracellular domain that was shared by the scFv and the parental MAb. BIAcore analysis indicated a Koff of 9.3 × 10−4 s−1, similar to that of the intact MGR6 MAb. Distribution and elimination half-lives of MGR6 scFv, calculated from in vivo preclinical
evaluations, were much faster (13 min and 6.2 h, respectively) than previously published results for the intact MAb (mean
t1/2β of 46 h). This represents a theoretical improvement in pharmacokinetics with respect to the parental murine MAb and
points to the potential for utilizing this fragment in redirecting therapeutic agents, such as radioisotopes, to different
human carcinomas overexpressing HER-2.
Received: 10 August 2000 / Accepted: 19 October 2000 相似文献
4.
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
5.
Preclinical studies with the murine anti-CD48 antibody, mHuLym3 (IgG2a) have shown it to be a potentially useful therapeutic
reagent in the treatment of human leukaemia and lymphoma. For clinical use, humanised antibodies can have a number of advantages
over their original murine version, including mediation of higher effector cell function with human cells, longer serum half-life
and lower immunogenicity. In this study, we have produced a mouse/human chimeric HuLym3 antibody (cHuLym3) where the murine
antibody constant regions have been replaced with human constant regions. We report the production and preclinical characterisation
of the antibody, cHuLym3, with potent in vitro and in vivo antitumour activity. The genes encoding the variable heavy and
light chains were amplified by the polymerase chain reaction, sequenced and cloned into eukaryotic expression vectors containing
the human light- and heavy-chain constant regions (κ and IgG1). The chimeric and murine HuLym3 antibodies had similar cell-binding
specificity and affinity. In the human Raji cell severe combined immunodeficient mouse model the i.v. injection of cHuLym3
and mHuLym3 produced similar antitumour responses. Doses of cHuLym3 and mHuLym3 (100 μg) on days 1, 2 and 4 after i.v. Raji
cell injection produced a 40% longer time to hind-leg paralysis than when a control antibody was used. cHuLym3 had more potent
activity than mHuLym3 in antibody-dependent cellular cytotoxicity (ADCC) assays in vitro, with human peripheral blood mononuclear
cells as effectors. Up to 60% specific cell lysis was observed with cHuLym3 in ADCC assays. These properties suggest that
anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukaemias and lymphoma.
Received: 5 May 1999 / Accepted: 12 August 1999 相似文献
6.
The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors 总被引:3,自引:0,他引:3
Yoshida S Morii K Watanabe M Saito T Yamamoto K Tanaka R 《Cancer immunology, immunotherapy : CII》2001,50(6):321-327
Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses
against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear
cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma,
7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce
mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 106 cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred
in their immune responses against brain tumor via 51Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3+, CD45+, CD80+ and CD86+. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing
42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing
the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors.
Received: 2 October 2000 / Accepted: 26 April 2001 相似文献
7.
基于细胞的噬菌体抗体库筛选技术 总被引:2,自引:0,他引:2
肿瘤细胞表面抗原的表达量低,免疫原性弱,因抗原构象改变的原因,用传统的固定化抗原方法很难获得有价值的噬菌体抗体。这类抗原大多具有复杂的结构,且其转运、定位与细胞类型及所处的微环境有关。在基于细胞的筛选中,靶抗原以天然状态存在,不需纯化,甚至可以对未知抗原进行筛选,因此广泛用于对内化抗体和血管内皮抗原表位抗体的筛选。基于细胞的抗体筛选中的主要问题是因非特异结合造成的选择背景过高,迄今,已有许多旨在改善选择灵敏度和特异性的研究开展起来。随高通量流式细胞术、组合化学及蛋白质组学研究技术在细胞筛选中的运用,必将使其日趋实用和成熟。我们拟以细胞和组织筛选的技术做一概括,为了解基于细胞的筛选和优化设计提供参考。 相似文献
8.
Daniel Bedinger Llewelyn Lao Shireen Khan Steve Lee Toshihiko Takeuchi 《MABS-AUSTIN》2016,8(2):389-404
Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease. 相似文献
9.
为构建小鼠噬菌体抗体库 ,以获得对人血纤维蛋白特异的抗体 ,由小鼠脾脏提取 m RNA,经反转录 PCR扩增出抗体重链、轻链可变区基因片段 ,将二者和一段编码十五肽 (Gly4 Ser) 3的 DNA接头借助重组 PCR组装成为单链抗体 (single- chain antibody,Sc Ab)基因 .将单链抗体基因插入噬菌体展示载体 p CANTAB- 5E,通过电击法转化大肠杆菌 TG1细胞 ,用辅助噬菌体 M1 3K0 7超感染 ,构建了库容量在 1 0 8以上的噬菌体单链抗体库 .利用亲和选择方法 (淘选 ) ,从噬菌体抗体库中选得血纤维蛋白特异的单链抗体 .模拟抗体成熟过程 ,用 DNA改组 (DNA shuffling)技术使抗体基因重新组合 ,构建新的改组抗体库 ,并从中选择到提高了亲和力的噬菌体单链抗体 .抗体基因在大肠杆菌中表达 ,表达蛋白经 Sephadex G- 75柱层析分离 ,得到初步纯化的单链抗体蛋白 . 相似文献
10.
W Wels I M Harwerth N E Hynes B Groner 《The Journal of steroid biochemistry and molecular biology》1992,43(1-3):1-7
We are evaluating strategies for the inhibition of growth or the selective killing of tumor cells. Cell surface antigens which are exclusively expressed or which are enhanced in their expression in tumor cells might provide the means to target cytotoxic or cytostatic agents to these cells. Few tumor specific cell surface antigens have been found, but the enhanced expression of growth factor receptors has been described for several types of tumors. A prominent example is the overexpression of the c-erbB-2 receptor in a high percentage of primary breast and ovarian carcinomas. We have derived monoclonal antibodies against the extracellular domain of the c-erbB-2 receptor. The antibody molecules were genetically engineered to minimize their size and to allow for their functional modification. For this purpose the cDNA sequences corresponding to the variable domains of one monoclonal antibody (FRP5) were molecularly cloned and joined by a short linker. The resulting single chain antibody molecule (scFv) was expressed in bacteria and purified. We show in an immunoprecipitation experiment that this molecule retains its ability to recognize the c-erbB-2 extracellular domain. This molecule could become a valuable vehicle to specifically transport anti-tumor agents to breast cancer cells. 相似文献
11.
Kabir ME Krishnaswamy S Miyamoto M Furuichi Y Komiyama T 《Applied microbiology and biotechnology》2011,90(2):553-564
Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional
antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific
altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated
against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional
antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative
small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library
through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in
bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data
showed that the binding affinity for this single-domain antibody was 272-fold higher (K
d = 1.07 × 10−10 M) and antifungal activity was three- to fivefold more efficient (IC50 = 0.46 × 10−6 to 1.17 × 10−6 M) than that for the conventional antibody (K
d = 2.91 × 10−8 M and IC50 = 2.14 × 10−6 to 3.78 × 10−6 M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development
of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain
synthetic antibodies will find their way into a number of biotechnological or medical applications. 相似文献
12.
Barbara A. McCormack Abigail C. E. Gregory Maria E. Kerry Colin Smith G. Paul Bolwell 《Planta》1997,203(2):196-203
Membrane preparations from suspension-cultured cells of French bean (Phaseolus vulgaris L.) contained callose synthase (EC 2.4.1.34) activity which was preserved upon solubilisation. Following elicitor treatment
of cell cultures, increased activity could be extracted and this increase was maintained during purification. The enzyme was
purified by high-pressure liquid chromatography and active fractions showed a variable association of two polypeptides of
relative molecular masses (Mr) 55 000 and 65 000, the latter being in excess. The Mr-65 000 polypeptide was purified to homogeneity and an antibody raised to it. This antibody showed complex effects on callose
synthase activity when incubated with membrane and soluble extracts. In comparison with other systems, the Mr-55 000 subunit is likely to represent the catalytic subunit while the Mr-65 000 polypeptide is a possible regulatory subunit. The Mr-65 000 polypeptide was immunolocated in membranes at sites of callose synthesis in the plant, in cell plates, in sieve plates,
at the plasma membrane-wall interface of wounded cells and in papillae in infected cells.
Received: 18 January 1997 / Accepted: 8 May 1997 相似文献
13.
Roovers RC van der Linden E de Bruïne AP Arends JW Hoogenboom HR 《Cancer immunology, immunotherapy : CII》2001,50(1):51-59
Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for
therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety
of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies
to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody
fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1)
cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2)
specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity
and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody
and had an off-rate of 5 × 10−2 s−1. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity,
the genes of scFv VEL-2 were re-formatted by fusion to a human (γ1) hinge region and CH3 domain. This “minibody” was expressed
in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These
results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules
with promising characteristics for in vivo use in tumour targeting.
Received: 13 July 2000 / Accepted: 12 October 2000 相似文献
14.
Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth 总被引:4,自引:0,他引:4
Li SL Liang SJ Guo N Wu AM Fujita-Yamaguchi Y 《Cancer immunology, immunotherapy : CII》2000,49(4-5):243-252
Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic
actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate
the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies
against human IGF-IR (αIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy
chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 αIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res
Commun 196: 92–98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the
αIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble αIGF-IR scFvs, a prototype αIGF-IR scFv and its alternative type αIGF-IR scFv-Fc,
were constructed and expressed in murine myeloma cells. αIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed
in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones
by protein-A–agarose chromatography. Levels of αIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium.
Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that
αIGF-IR scFv-Fc is a dimeric antibody. αIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody
except that its binding affinity for IGF-IR was estimated to be approximately 108 M−1, which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of αIGF-IR scFv-Fc (500 μg/mouse,
twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the αIGF-IR scFv-Fc
is a first-generation recombinant αIGF-IR for the potential development of future αIGF-IR therapeutics.
Received: 21 January 2000 / Accepted: 7 March 2000 相似文献
15.
Chames P Willemsen RA Rojas G Dieckmann D Rem L Schuler G Bolhuis RL Hoogenboom HR 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(2):1110-1118
The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes. 相似文献
16.
M Enamul Kabir Senthilkumar Krishnaswamy Masahiko Miyamoto Yasuhiro Furuichi Tadazumi Komiyama 《BMC biotechnology》2009,9(1):99-16
Background
Phage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection ofrecombinant antibodies. 相似文献17.
Michael Hoffmann Mathias Schmidt Winfried Wels 《Cancer immunology, immunotherapy : CII》1998,47(3):167-175
Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological
condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3,
and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either
EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous
growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In
contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody
14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain
(sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically
to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic
mobility shift assays.
Received: 6 May 1998 / Accepted: 16 July 1998 相似文献
18.
Immunoglobulin chain recombination among antidigoxin antibodies by hybridoma-hybridoma fusion 总被引:3,自引:0,他引:3
N W Hudson M Mudgett-Hunter D J Panka M N Margolies 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(8):2715-2723
Conditions necessary for in vitro chain recombination of high affinity (10(9) to 10(12) M-1) antidigoxin monoclonal antibodies resulted in decreased affinity for both intact "native" and chain recombinant molecules. Chain recombination by somatic cell fusion was used instead to study the effects on antigen specificity and idiotypy of recombinants in which an homologous light (L) chain substituted for the parental L chain. The antidigoxin antibody 26-10 utilizes a VL sequence highly homologous to that of antibody 40-20, an antidigoxin antibody which uses a different VH gene than does 26-10 and lacks significant reactivity with an anti-26-10 idiotypic serum. The drug-marked antidigoxin cell line 26-10 (gamma 2a, kappa) and a drug-marked light chain producing variant of antidigoxin hybridoma 45-20 (lambda 1) which lacks both digoxin binding and idiotypy were fused. The fusion progeny (gamma 2a, kappa, lambda 1) which binds digoxin and is idiotype-positive, was selected for kappa loss (resulting in loss of digoxin and idiotype binding) and then fused with a heavy (H) chain loss variant of antidigoxin hybridoma 40-20 (kappa, digoxin nonbinding, idiotype negative). The resultant cell line CR-57 (gamma 2a, kappa, lambda) secretes antibodies which assemble the 26-10 H chain with both the 40-20 kappa-chain and the 45-20 lambda 1-chain. The affinity purified recombinant species consisting of 26-10 H chain and 40-20 kappa-chain expresses complete 26-10 idiotypic determinants. However, this recombinant antibody binds digoxin with decreased affinity and altered specificity relative to native 26-10. The binding specificity pattern nonetheless is most similar to the H chain donor. Amino acid and nucleotide sequence analyses of the respective light chains demonstrate six variable region differences between them, two of which are in complementarity-determining regions and the remainder in the framework. Hybridoma-hybridoma fusion provides an alternative to in vitro chain recombination for studying the contribution of chain combinational diversity to antibody diversity, antigen binding, and idiotypy. 相似文献
19.
Kim SJ Sadelain M Lee JS Seong RH Yun YS Jang YJ Chung HY 《Cancer immunology, immunotherapy : CII》1999,47(5):257-264
We show that the tumor-specific primary cytotoxic T lymphocytes (CTL) induced in vitro with the MCA205 fibrosarcoma cells
transduced with the B7.1 (CD80) gene are highly effective in adoptive-transfer therapy of the parental tumors. The MCA205
fibrosarcoma cell line was transduced with the retroviral vectors encoding the B7.1 gene and tested for their efficiency as
stimulators in short-term (5 days) mixed lymphocyte/tumor cell cultures with highly purified syngenic, unprimed T cells as
responders. The induction of the CTL required the presence of a low dose of interleukin-2 (25 U/ml). The injection of the
CTL prevented colony formation by the intravenously injected tumor cells in a lung colonization assay in which the CTL were
injected after inoculation of tumor cells. We also showed that the adoptive transfer of the same T cells was effective in
delaying the growth of the subcutaneously injected tumor cells. These results imply that the short-term mixed lymphocyte/tumor
cell culture with the tumor cells transduced with the gene for the B7.1 costimulatory molecule is potentially a good source
of CTL for adoptive-transfer therapy of tumors.
Received: 30 June 1998 / Accepted: 5 August 1998 相似文献