Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
Protein Expr Purif ; 58(1): 140-7, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17950620

ABSTRACT

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In the present study, we have constructed and expressed a recombinant immunotoxin CD25-PE38KDELKQK containing humanized anti-CD25 single-chain antibody (scFv) genetically fused to PE38KDELKQK in Escherichia coli. After washing with buffer containing 2 M urea, the purity of inclusion body was about 82%. The denatured inclusion bodies were refolded on-column in Tris buffer (pH 8.0) containing 4mM of GSH and 1 mM of GSSG using a gradient of decreasing urea. We found that the presence of GSH/GSSG (4:1) in the on-column refolding buffer was important for efficient refolding. In addition, slow flow rate was another important factor could increase refolding. Under these conditions, the activity of the refolded protein could reach about 90% of that of the native protein. The refolded proteins were purified to homogeneity ( approximately 95% purity) by a combination of His-Ni(2+) metal affinity chromatography and gel filtration chromatography. The in vitro cytotoxicity assay indicated the purified immunotoxin CD25-PE38KDELKQK had specific cytotoxicity to CD25-positive leukemic cells comparable to wild-type CD25-PE38KDEL (wt). In contrast, CD25-PE38KDELKQK was shown to be much weaker in inducing VLS in mice than wt. The protein expression, purification, and refolding system established in this paper is important for further study on immunotoxin CD25-PE38KDELKQK.


Subject(s)
ADP Ribose Transferases , Bacterial Toxins , Exotoxins , Immunoglobulin Variable Region , Immunotoxins , Interleukin-2 Receptor alpha Subunit/immunology , Virulence Factors , ADP Ribose Transferases/genetics , ADP Ribose Transferases/metabolism , Animals , Antibodies, Monoclonal/immunology , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Capillary Leak Syndrome/etiology , Cell Line, Tumor , Exotoxins/genetics , Exotoxins/metabolism , Humans , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Immunoglobulin Variable Region/metabolism , Immunoglobulin Variable Region/toxicity , Immunotoxins/genetics , Immunotoxins/immunology , Immunotoxins/isolation & purification , Immunotoxins/metabolism , Immunotoxins/toxicity , Jurkat Cells , Lung/cytology , Lung/immunology , Mice , Protein Engineering , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/toxicity , Virulence Factors/genetics , Virulence Factors/metabolism , Pseudomonas aeruginosa Exotoxin A
2.
Cancer Res ; 63(12): 3202-10, 2003 Jun 15.
Article in English | MEDLINE | ID: mdl-12810649

ABSTRACT

The potent antitumor activity of certain cytokines is often achieved at the expense of unacceptable toxicity. One avenue to improve the therapeutic index of cytokines in cancer therapy consists of fusing them to monoclonal antibodies capable of a selective localization at the tumor site. We have constructed fusion proteins of interleukin-12 (IL-12) and tumor necrosis factor (TNF-alpha) with L19, an antibody fragment specific to the extradomain B of fibronectin which has been shown to target tumors in animal models and in patients with cancer. These fusions display a potent antitumor activity in several immunocompetent murine models of cancer but do not lead to complete remissions of established aggressive tumors. In this article, we have evaluated the tumor-targeting properties and the anticancer activities of combinations of the two antibody-cytokine fusion proteins, as well as of a triple fusion protein between IL-12, L19, and TNF-alpha. Although all fusion proteins were active in vitro, the triple fusion protein failed to localize to tumors in vivo and to show significant therapeutic effects. By contrast, the combination of IL-12-L19 and L19-TNF-alpha displayed potent synergistic anticancer activity and led to the eradication of F9 teratocarcinomas grafted in immunocompetent mice. When cured mice were rechallenged with tumor cells, a delayed onset of tumor growth was observed, indicating the induction of a partial antitumor vaccination effect. Potent anticancer effects were achieved at doses of IL-12-L19 and L19-TNF-alpha (2 micro g + 2 micro g/mouse), which were at least 5-fold lower than the maximal-tolerated dose. The combined administration of the two fusion proteins showed only a modest increase in toxicity, compared with treatments performed with the individual fusion proteins. These results show that the targeted delivery of cytokines to the tumor environment strongly potentiates their antitumor activity and that the combination treatment with IL-12-L19 and L19-TNF-alpha appears to be synergistic in vivo.


Subject(s)
Immunoconjugates/therapeutic use , Immunoglobulin Variable Region/therapeutic use , Immunotherapy , Interleukin-12/therapeutic use , Teratocarcinoma/therapy , Tumor Necrosis Factor-alpha/therapeutic use , Animals , Antibodies, Monoclonal/immunology , Drug Delivery Systems , Drug Screening Assays, Antitumor , Drug Synergism , Female , Immunoconjugates/administration & dosage , Immunoconjugates/pharmacokinetics , Immunoconjugates/toxicity , Immunoglobulin Variable Region/administration & dosage , Immunoglobulin Variable Region/toxicity , Interferon-gamma/metabolism , Interleukin-12/administration & dosage , Interleukin-12/pharmacokinetics , Interleukin-12/toxicity , Mice , Neoplasm Transplantation , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/therapeutic use , Recombinant Fusion Proteins/toxicity , T-Lymphocytes/metabolism , Teratocarcinoma/pathology , Tissue Distribution , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/administration & dosage , Tumor Necrosis Factor-alpha/pharmacokinetics , Tumor Necrosis Factor-alpha/toxicity , Vaccination
3.
Cancer Res ; 61(13): 5070-7, 2001 Jul 01.
Article in English | MEDLINE | ID: mdl-11431343

ABSTRACT

Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity.


Subject(s)
ADP Ribose Transferases , Bacterial Toxins , Exotoxins/toxicity , Immunoglobulin Fragments/toxicity , Immunotoxins/toxicity , Virulence Factors , Alanine Transaminase/blood , Amino Acid Sequence , Animals , Antibodies , Disulfides/chemistry , Disulfides/toxicity , Exotoxins/chemistry , Exotoxins/pharmacokinetics , Female , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/genetics , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/toxicity , Immunotoxins/chemistry , Immunotoxins/genetics , Immunotoxins/pharmacokinetics , Isoelectric Point , Liver/drug effects , Mice , Molecular Sequence Data , Mutagenesis , Neoplasms, Experimental/immunology , Neoplasms, Experimental/therapy , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/toxicity , Pseudomonas aeruginosa Exotoxin A
4.
Clin Cancer Res ; 5(9): 2646-52, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10499644

ABSTRACT

The incidence of neoplastic meningitis is on the rise. Neoplastic meningitis can result from a direct seeding of the neuraxis by primary brain tumors or by hematogeneous spread of systemic solid tumors. A frequent genetic alteration in primary brain tumors such as gliomas is an in-frame deletion in the epidermal growth factor receptor (EGFR) gene EGFRvIII, which brings together what were normally distant polypeptide sequences in the intact receptor. A novel glycine is formed at the fusion junction, resulting in a unique and tumor-specific target. By using phage display, we have isolated a single-chain antibody specific for the EGFRvIII mutation and expressed it with a modified form of the Pseudomonas exotoxin to form the immunotoxin MR1scFvPE38KDEL (MR-1). The multiple dose toxicity and therapeutic efficacy of MR-1 immunotoxin were tested in an athymic rat model of neoplastic meningitis. The maximally tolerated doses in non-tumor-bearing rats were three doses of 3 microg each. For therapeutic studies, the target was a neoplastic meningitis induced by intrathecal inoculation of the EGFRvIII-expressing human glioma U87MG.deltaEGFR. A dose escalation study compared the survival of three equal doses of 1, 2, and 3 microg of MR-1 immunotoxin with saline or 3 microg of the control immunotoxin specific for the interleukin 2 receptor, anti-Tac. All animals treated with three doses of saline or 3 microg of anti-Tac died, with median survival of 7 and 10 days, respectively. There were 75% (six of eight) long-term survivors in the group treated with three doses of 1 microg and 57% (four of seven) long-term survivors in the groups treated with three doses of either 2 or 3 microg of MR-1 immunotoxin. None of the MR-1 immunotoxin-treated groups reached median survival by the termination of the study at 53 days. Therefore, median survival was estimated to be >53 days, resulting in an estimated increase in median survival of >657% compared with saline and 430% versus anti-Tac. Compartmental therapy with three doses of 2 microg of MR-1 immunotoxin is effective in the treatment of EGFRvIII-expressing neoplastic meningitis. This dose was found to have no clinical or histopathological effects on non-tumor-bearing animals. MR-1 immunotoxin is, therefore, considered specific and safe within its therapeutic window. Phase I clinical trials for tumors invading the intrathecal space that express the EGFRvIII target should be initiated.


Subject(s)
ADP Ribose Transferases , Bacterial Toxins , ErbB Receptors/immunology , ErbB Receptors/metabolism , Exotoxins/pharmacology , Immunoglobulin Variable Region/pharmacology , Immunotoxins/pharmacology , Meningeal Neoplasms/drug therapy , Virulence Factors , Animals , Antibody Specificity , ErbB Receptors/biosynthesis , ErbB Receptors/genetics , Exotoxins/toxicity , Female , Humans , Immunoglobulin Variable Region/toxicity , Immunotoxins/toxicity , Injections, Spinal , Meningeal Neoplasms/metabolism , Mice , Mice, Nude , Mutation , Protein Synthesis Inhibitors/pharmacology , Rats , Rats, Nude , Tumor Cells, Cultured , Pseudomonas aeruginosa Exotoxin A
5.
Proc Natl Acad Sci U S A ; 92(15): 7021-5, 1995 Jul 18.
Article in English | MEDLINE | ID: mdl-7624362

ABSTRACT

Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker. One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer. The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only. The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments. By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines. The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column. As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells.


Subject(s)
Antibody Specificity , Antineoplastic Agents/toxicity , Immunoglobulin Fragments/toxicity , Immunoglobulin Variable Region/toxicity , Amino Acid Sequence , Base Sequence , Blotting, Western , Cloning, Molecular , Escherichia coli/genetics , Immunoglobulin Fragments/genetics , Immunoglobulin Variable Region/genetics , Molecular Sequence Data , Recombinant Proteins/toxicity , Tumor Cells, Cultured
6.
Int J Cancer ; 58(1): 142-9, 1994 Jul 01.
Article in English | MEDLINE | ID: mdl-8014011

ABSTRACT

Disulfide-stabilized Fv (dsFv)-immunotoxins are recombinant immunotoxins in which the inherently unstable Fv moiety, composed of the VH-VL heterodimer, is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. Anti-Tac(dsFv)-PE38KDEL is composed of such a dsFv, directed to the alpha subunit of the IL2 receptor (IL2R), and containing a truncated form of Pseudomonas exotoxin (PE38KDEL). We have found this new type of immunotoxin to be indistinguishable in its in vitro activity and specificity from its single-chain immunotoxin counterpart, anti-Tac(Fv)-PE38KDEL. We have now examined the therapeutically relevant factors, including stability, pharmacokinetics, and antitumor activity of this new disulfide-stabilized Fv-immunotoxin. We found that anti-Tac(dsFv)-PE38KDEL was specifically cytotoxic to human activated T-lymphocytes in addition to IL2R bearing cell lines. Anti-Tac(dsFv)-PE38KDEL was considerably more stable at 37 degrees C in human serum and in buffered saline than the single-chain immunotoxin, anti-Tac(Fv)-PE38KDEL. The half-life in blood was similar for both immunotoxins (approx. 20 min). The therapeutic potential of the disulfide-stabilized immunotoxin was evaluated using an animal model of immunodeficient mice bearing subcutaneous tumor xenografts of human IL2R-bearing cells. Anti-Tac(dsFv)-PE38KDEL caused complete regression of tumors with no toxic effects in mice. Because dsFv-immunotoxins are more stable and can be produced with significantly improved yields compared to scFv-immunotoxins, dsFv-immunotoxin may be more useful for therapeutic applications.


Subject(s)
ADP Ribose Transferases , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/toxicity , Bacterial Toxins , Disulfides/pharmacology , Exotoxins/pharmacokinetics , Exotoxins/toxicity , Immunotoxins/metabolism , Immunotoxins/toxicity , Receptors, Interleukin-2/immunology , Virulence Factors , Animals , Drug Stability , Female , Humans , Immunoglobulin Fragments/immunology , Immunoglobulin Variable Region/toxicity , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/immunology , Peptide Fragments/immunology , Peptide Fragments/pharmacokinetics , Peptide Fragments/toxicity , Plasmids , Recombinant Proteins/toxicity , Pseudomonas aeruginosa Exotoxin A
SELECTION OF CITATIONS
SEARCH DETAIL
...