[Sixth congress of the European Genetic Therapy Group].
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Europe↗
Biomedical subjects
M Piechaczyk
Publications and source records attributed to M Piechaczyk.
Systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins.
Sets of short (12 residues) cellulose-bound synthetic overlapping peptides derived from the sequences of the variable regions of the heavy and light chains of three different antibodies (an anti-thyroglobulin antibody, the HyHEL-5 anti-lysozyme antibody, and an anti-angiotensin II antibody) were used to systematically assess the antigen binding capacity of peptides from the antibody paratope outside their natural molecular context. Peptides enclosing one or several of the complementarity determining region (CDR) residues had antigen binding activity, although the most active peptides were not necessarily those bearing the greatest number of CDR residues. Several residues from the framework region, preceding or following the CDR, were found to play a role in binding. Affinity constants from 4.1 x 10(-7) to 6.7 x 10(-8) M-1 for the soluble form of 9 lysozyme-binding dodecapeptides were measured by BIAcore analysis. Alanine scanning of lysozyme-binding hexapeptides from the HyHEL-5 sequence identified 38 residues important for binding, of which 22 corresponded to residues that had been shown by x-ray crystallography to be at the interface between HyHEL-5 and lysozyme. Our results could be of interest for the rational identification of biologically active peptides derived from antibody sequences and in providing an experimental basis for mutagenesis of the antibody paratope.
Amino Acid Sequence↗
In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.
In vivo production of recombinant antibodies by engineered cells may have applications for gene therapy of certain cancers and of certain severe viral diseases. It would also permit the development of new animal models of autoimmune diseases and new approaches for in vivo ablation of specific cell types for fundamental purposes. Using gene transfer of an anti-human thyroglobulin monoclonal antibody, we show here that several cell types permitting autologous grafting of genetically engineered cells are efficiently able to secrete antibodies in vitro. Those cells include skin fibroblasts, hepatocytes, and myogenic cells. We also show that the secreted antibodies display an affinity for the antigen close to that of the parental antibody, with, however, slight differences varying according to the cell type. This indicates that the foldings of antigen combining sites of antibodies produced in B cell- and non-B cell contexts are very similar. Finally, we report that, when implanted in the forelimb of a mouse, genetically modified myogenic cells are able to secrete antibodies for at least 4 months. Taken together, our observations point to the notion that genetic modification of patient cells may be used for long-term antibody-based gene therapies.
Animals↗
Decreased susceptibility to calpains of v-FosFBR but not of v-FosFBJ or v-JunASV17 retroviral proteins compared with their cellular counterparts.
The c-Fos and c-Jun transcription factors are rapidly turned over in vivo. One of the multiple pathways responsible for their breakdown is probably initiated by calpains, which are cytoplasmic calcium-dependent cysteine proteases. The c-fos gene has been transduced by two murine oncogenic retroviruses called Finkel-Biskis-Jenkins murine sarcoma virus (FBJ-MSV) and Finkel-Biskis-Reilly murine sarcoma virus (FBR-MSV); c-jun has been transduced by the chicken avian sarcoma virus 17 (ASV17) retrovirus. Using an in vitro degradation assay, we show that the mutated v-FosFBR, but not v-FosFBJ or v-JunASV17, is resistant to calpains. This property raises the interesting possibility that decreased sensitivity to calpains might contribute to the tumorigenic potential of FBR-MSV by allowing greater accumulation of the protein that it encodes in infected cells. It has also been demonstrated that resistance to cleavage by calpains does not result from mutations that have accumulated in the Fos moiety of the viral protein but rather from the addition of atypical peptide motifs at its both ends. This observation raises the interesting possibility that homologous regions in viral and cellular Fos either display slightly different conformations or are differentially accessible to interacting proteins.
Avian Sarcoma Viruses↗
Towards efficient cell targeting by recombinant retroviruses.
Efficient human gene therapy will rely on efficient gene delivery systems. To date, recombinant, engineered retroviruses are the most widely used vectors for stable clinical gene transfer. However, this technology still suffers from a number of drawbacks that limit its application. The ability to target cells of therapeutic interest, in particular, would improve both the efficiency and the safety of retroviral vectors. It might also allow the development of new animal models of human diseases and probably extend the scope of gene therapy itself.
Gene Transfer Techniques↗
Complex mechanisms for c-fos and c-jun degradation.
c-fos and c-jun proto-oncogenes have originally been found in mutated forms in murine and avian oncogenic retroviruses. They both define multigenic families of transcription factors. Both c-jun and c-fos proteins are metabolically unstable. In vivo and in vitro work by various groups suggests that multiple proteolytic machineries, including the lysosomes, the proteasome and the ubiquitous calpains, may participate in the destruction of c-fos and c-jun. The relative contribution of each pathway is far from being known and it cannot be excluded that it varies according to the cell context and/or the physiological conditions. It has been demonstrated that, in certain occurrences, the degradation of both c-fos and c-jun by the proteasome in vivo involves the ubiquitin pathway. However, the possibility that proteasomal degradation can also occur in a manner independent of the E1 enzyme of the ubiquitin cycle remains an open issue.
Animals↗
Proteolysis by calpains: a possible contribution to degradation of p53.
p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.
Animals↗
Analysis of the individual contributions of immunoglobulin heavy and light chains to the binding of antigen using cell transfection and plasmon resonance analysis.
We have cloned the Tg10 murine monoclonal antibody, which is specific for a human thyroglobulin (hTg) epitope targeted by autoantibodies in several thyroid pathologies. Transfection of COS-7 cells with plasmids expressing Tg10H and -kappa chains combined with surface plasmon resonance analysis (BIAcore) of culture supernatants showed that the entire cloned Tg10 antibody displays an affinity comparable to that of the parental antibody. This approach also permitted determination of the probable role of each chain to the recognition of the cognate epitope due to the ability of COS-7 cells to secrete independently each of the two constituting immunoglobulin chains. Tg10 heavy chain recognizes hTg in the absence of the light chain, but with a ten-fold lower affinity mainly due to an increase in kappaoff. In contrast, the light chain is unable to bind hTg on its own. This suggests that the latter is probably involved in stabilization rather than in initiating the formation of the antibody/antigen complex and that the specificity of Tg10 is mostly, if not exclusively, carried by the heavy chain. The potential applications of combined cell transfection and surface plasmon resonance to our understanding of antigen/antibody interactions are discussed.
Amino Acid Sequence↗
PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein.
Cytoplasmic degradation of c-fos protein is extremely rapid. Under certain conditions, it is a multi-step process initiated by calcium-dependent and ATP-independent proteases called calpains. PEST motifs are peptide regions rich in proline, glutamic acid/aspartic acid and serine/threonine residues, commonly assumed to constitute built-in signals for rapid recognition by intracellular proteases and particularly by calpains. Using a cell-free degradation assay and site-directed mutagenesis, we report here that the three PEST motifs of c-fos are not required for rapid cleavage by calpains. Testing the susceptibility of PEST motif-bearing and non-bearing transcription factors including GATA1, GATA3, Myo D, c-erbA, Tal-1 and Sry, demonstrates that PEST sequences are neither necessary nor sufficient for specifying degradation of other proteins by calpains. This conclusion is strengthened by the observation that certain proteins, reportedly known to be cleavable by calpains, are devoid of PEST motifs.
Base Sequence↗
Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus-derived viruses displaying single-chain antibody fragment-envelope fusion proteins.
As an approach to cell targeting by retroviruses, the lack of which constitutes one major limitation of retroviral vector technology, we engineered the Moloney murine leukemia virus ecotropic envelope glycoprotein. When inserted between amino acids 6 and 7 of the latter, a single-chain antibody fragment (ScFv) specific for human major histocompatibility complex class I molecules was shown to be able to redefine the tropism of ecotropic Moloney murine leukemia virus-derived retroviral particles by allowing infection of major histocompatibility complex class I-positive human cells. At variance with other recently described experimental systems, the type of modification adopted here allowed targeted infection in the absence of coexpressed wild-type env-encoded protein molecules. Interestingly, the chimeric ScFv-env protein also retained the ability to recognize the ecotropic receptor and allowed infection of murine cells, albeit at a reduced efficiency.
3T3 Cells↗
Isolation and characterization of c-fos-expressing murine bone marrow stromal cell lines supporting myeloid differentiation.
We have previously reported that constitutive expression of c-fos oncogene allows long-term proliferation of primary mouse bone marrow stromal cells favoring the granulocytic differentiation of myeloid precursors in an in vitro assay. Retrovirus-mediated gene transfer of the human c-fos gene was used here for immortalizing nine mouse bone marrow cell lines which were studied in detail. However, due to low expression of the ectopic c-fos gene, none of them showed characteristics of transformation as assayed by dependence upon serum for growth, the inability to form colonies in agar and contact inhibition. All of them displayed a fibroblastoid phenotype, as deduced from morphological observation and analysis of several differentiation markers. They mostly supported the granulocytic differentiation of bone marrow myeloid precursors in a GM-assay, as did c-fos-expressing primary stromal cells. Their potential for supporting myeloid progenitor proliferation was however significantly lower than that of the whole adherent layer of the Dexter-type long-term bone marrow culture they derived from (STNT cells). They showed significant variations with respect to their cytokine gene expression analyzed at the RNA level in keeping with the notion of stromal cell heterogeneity in the bone marrow. Interestingly, none of them secreted GM-CSF, SCF or IL-3, which are cytokines reputed for their ability to stimulate hematopoietic progenitors, and strikingly, only two of them were able to produce detectable levels of G-CSF in culture supernatants despite the propensity of all of them to favor granulocyte differentiation. Finally, in coculture assay, bone marrow cells were able to diminish the expression of several cytokine genes albeit at a much lower degree than in the original STNT cells.
Animals↗
Affinity and dose-dependent digoxin Na+K+ATPase dissociation by monoclonal digoxin-specific antibodies.
The effect of three monoclonal digoxin-specific antibodies and of polyclonal Digidot as reference on digoxin dissociation from rat brain Na+K+ATPase microsomes was studied to determine the role of the affinity constant (Ka) and dose of the antibody on the rate of digoxin dissociation from Na+K+ATPase. Stoichiometrical doses of 1C10, 6C9, 9F5 IgG, and Digidot (Ka = 6 10(9), 3.1 10(8), 2.5 10(7), and 8.5 10(9) M-1, respectively) resulted in digoxin dissociation related to Ka. When the IgG:digoxin molar ratio increased from 0.25 to 10, digoxin dissociation from Na+K+ATPase sites also increased according to the Hill equation, allowing comparative parameters among the three antibodies to be determined. 1C10 IgG was 2- and 10-fold more efficacious than 6C9 and 9F5, respectively. This in vitro model appears to be a useful predictive screening assay before in vivo experimentation.
Animals↗
Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26 S proteasome.
Degradation of rapidly turned over cellular proteins is commonly thought to be energy dependent, to require tagging of protein substrates by multi-ubiquitin chains, and to involve the 26 S proteasome, which is the major neutral proteolytic activity in both the cytosol and the nucleus. The c-Jun oncoprotein is very unstable in vivo. Using cell-free degradation assays, we show that ubiquitinylation, along with other types of tagging, is not an absolute prerequisite for ATP-dependent degradation of c-Jun by the 26 S proteasome. This indicates that a protein may bear intrinsic structural determinants allowing its selective recognition and breakdown by the 26 S proteasome. Moreover, taken together with observations by different groups, our data point to the notion of the existence of multiple degradation pathways operating on c-Jun.
Animals↗
Expression in Escherichia coli of soluble and M13 phage-displayed forms of a single-chain antibody fragment specific for digoxin: assessment in a novel drug immunoassay.
A high affinity anti-digoxin single-chain Fv antibody fragment (scFv) was cloned from the mouse 2C2 hybridoma cell line and was functionally expressed both in the Escherichia coli periplasm as a soluble molecule and at the surface of the filamentous M13 bacteriophage as a fusion protein with the gene III minor coat protein. The 2C2 scFv sequence significantly differs from that of all the other anti-digoxin antibodies previously described. The 2C2 scFv shares with its parental monoclonal antibody a high specificity for digoxin, a cross-reactivity with active digoxin metabolites, but none with inactive metabolites. M13 phages displaying the 2C2 scFv at their surface have a high apparent affinity constant for digoxin (6.6 x 10(8) M-1) and were directly used to set up a novel type of immunoenzymatic assay for monitoring digoxin in sera of patients treated for either congestive heart failure or cardiac arrythmias. We thus report for the first time that phages displaying scFv may constitute a large source of important new reagents in the field of immunodiagnosis.
Animals↗
Cloning and expression of a single-chain antibody fragment specific for a monomorphic determinant of class I molecules of the human major histocompatibility complex.
B9.12.1 is a monoclonal antibody specific for a monomorphic determinant of human MHC class I molecules. It is currently used for cell typing and is useful for targeting infection of human cells by murine ecotropic retroviruses. We have cloned and expressed it in the form of a single-chain variable fragment (ScFv) that recognizes the same epitope as the parental antibody. Through genetic engineering, this ScFv may be used for developing new cell-typing probes and new retroviral targeting approaches.
Amino Acid Sequence↗
Targeting of c-myc and beta-globin coding sequences to cytoskeletal-bound polysomes by c-myc 3' untranslated region.
The influence of the 3' untranslated region on mRNA localization was investigated by measuring the distribution of myc, beta-globin and hybrid myc-globin mRNAs between free, cytoskeletal-bound and membrane-bound polysomes in cells transfected with either control or chimeric gene constructs. c-myc sequences and beta-globin-coding sequences linked to the myc 3' untranslated region were present at greatest enrichment in cytoskeletal-bound polysomes. beta-Globin mRNA and myc-coding sequences linked to the beta-globin 3' untranslated region were recovered largely in the free polysomes. In situ hybridization confirmed that replacement of the c-myc 3' untranslated region by that of globin caused a relocalization of the mRNA. The results suggest that mRNA localization in differentiated eukaryotic cells depends on a mechanism that involves directional information in the 3' untranslated region of mRNAs.
Animals↗
An efficient immunization protocol for production of monoclonal antibodies against soluble human insulin.
A new immunization protocol has been developed to obtain specific monoclonal antibodies (mAb) directed against the soluble form of human insulin. Various protocols differing on the basis of the nature of immunogen, the number of injections and the route of administration of the antigen were compared. Mice with the highest anti-insulin titers were selected for cell fusion. The results showed that the immunization protocol involving 2 injections of insulin followed by a boost 2 months later mainly stimulated B lymphocytes secreting IgM mAb directed against immobilized insulin. Immunization with 2 injections of human proinsulin followed by 2 injections of a human insulin-bovine serum albumin conjugate and finally with a booster injection of this conjugate on each of the last 4 days preceding fusion was necessary to obtain a high percentage of hybridomas secreting specific IgG mAb able to recognize immobilized insulin (indirect ELISA) as well as iodinated insulin (liquid-phase radioimmunoassay).
Animals↗
c-fos proto-oncogene regulation and function.
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Amino Acid Sequence↗