Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments.

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 -/-human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 -/- human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

A tumor specific single chain antibody dependent gene expression system.

The design of conditional gene expression systems restricted to given tissues or cellular types is an important issue of gene therapy. Systems based on the targeting of molecules characteristic of the pathological state of tissues would be of interest. We have developed a synthetic transcription factor by fusing a single chain antibody (scFv) directed against p53 with the bacterial tetracycline repressor as a DNA binding domain. This hybrid protein binds to p53 and can interact with a synthetic promoter containing tetracycline-operator sequences. Gene expression can now be specifically achieved in tumor cells harboring an endogenous mutant p53 but not in a wild-type p53 containing tumor cell line or in a non-transformed cell line. Thus, a functional transactivator centered on single chain antibodies can be expressed intracellularly and induce gene expression in a scFv-mediated specific manner. This novel class of transcriptional transactivators could be referred as 'trabodies' for transcription-activating-antibodies. The trabodies technology could be useful to any cell type in which a disease related protein could be the target of specific antibodies.

Immunoglobulin variable regions usage by B-lymphocytes infiltrating a human breast medullary carcinoma.

Breast medullary carcinoma are heavily infiltrated by B-lymphocytes and associated with a good prognosis despite their high histological grade. We investigated the Ig repertoire of B-lymphocytes infiltrating one such tumour. A single cell suspension was obtained from a tumor specimen by enzymatic digestion. VH, Vkappa, and Vlambda regions were amplified by RT-PCR using mixtures of primers optimized to maximize the diversity of the PCR products. They were then cloned and sequenced. Analysis of 9 VH, 5 Vkappa, and 10 Vlambda sequences using the Kabat database indicated that several VH and VL region subgroups (I, II and III) are expressed by B-lymphocytes infiltrating this tumor. The analysis of CDR3 regions also showed a variability, although some VH and VL clones exhibited identical or nearly identical sequences. Thus, the B-cell infiltration observed in this breast medullary carcinoma does not reflect a monoclonal proliferation and represents an oligoclonal or a polyclonal B-cell proliferation.

Structurally different anthracyclines provoke different effects on cell cycle and tumor B cell differentiation.

Previously we have detected a stimulatory effect on immunoglobulin (IgG) synthesis when hybridoma cells were treated with doxorubicin. In order to determine whether this is a general property of anthracycline, we have selected three analogs--doxorubicin (DOX), pirarubicin (THP-DOX) and aclarubicin (ACR)--which differ mainly in the methylation state of their amino sugars. Cell cycle analysis by flow cytometry and drug localization by scanning confocal microscopy were also performed. The results show that when cells (UN2 hybridoma B cells), were exposed to subtoxic doses of DOX or THP (with unmethylated amino sugars), a strong increases in IgG secretion, heavy (H) and light (L) chain synthesis and the corresponding mRNA levels were induced. Furthermore these two drugs arrested the cells in the G2/M phase of the cell cycle. In contrast, exposure to ACR (with its methylated amino sugar) at similar subtoxic doses induced a blockade of cells in the G1 phase with no increase of IgG synthesis, at the subtoxic doses used, all three drugs could still be detected in the nucleus as well as in the cytoplasm, as determined by confocal laser microscopy. Thus, the relationship between cell cycle blockade, IgG stimulation and anthracycline structure is suggested by these results.

Production and characterization of mouse monoclonal antibodies to wild-type and oncogenic FLI-1 proteins.

Mouse monoclonal antibodies were raised against the C-terminal domain of human FLI-1, a member of the ETS family of transcription factors which is involved in various murine and human malignancies. This FLI-1 specific domain is included in the fusion product EWS-FLI-1, an oncogenic variant of FLI-1 expressed in Ewing tumors. Antibodies were screened first by enzyme-linked immunosorbent assay onto recombinant FLI-1-coated plates. Positive clones were then tested for their ability to immunoprecipitate over-expressed EWS-FLI-1 protein. Three monoclonal antibodies were selected and further characterized. One of them, termed 7.3 MoAb, was shown to react with FLI-1 and EWS-FLI-1 in immunoblotting, immunoprecipitation, and immunofluorescence experiments. With all three methods, this antibody not only enabled the detection of overexpressed proteins but also more interestingly, that of endogenously expressed proteins. Furthermore, the 7.3 MoAb can specifically inhibit the interaction of FLI-1 with its DNA-binding site as shown by electrophoretic mobility shift assay. The 7.3 MoAb appears to be specific for FLI-1 because it does not react with ERG, the ETS family member most closely related to FLI-1. This antibody should be a useful tool in the diagnostic evaluation of Ewing tumors and should permit biochemical analyses to study the function of the wild-type FLI-1 protein and of the EWS-FLI-1 fusion protein.

A new set of monoclonal antibodies against human Fc gamma RII (CD32) and Fc gamma RIII (CD16): characterization and use in various assays.

Four mouse anti-human Fc gamma RII (CD32) (6C4, 2B2, 3D3, 93.4) (IgG1, kappa) and one anti-human Fc gamma RIII (CD16) (7.5.4) IgG1, kappa) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, kappa), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both Fc gamma RIIa and Fc gamma RIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to Fc gamma RII. All three MAbs immunoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti Fc gamma RII MAb, 93.4, directed against the intracellular region of Fc gamma RIIa1/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both Fc gamma RIIIa and Fc gamma RIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-Fc gamma RIIa1/2)/6C4 and 3G8 (anti-Fc gamma RIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble Fc gamma RIIa1/2 and Fc gamma RIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-Fc gamma RII and of the anti-Fc gamma RIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-Fc gamma RII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM.

Bypassing tumor-specific and bispecific antibodies: triggering of antitumor immunity by expression of anti-FcgammaR scFv on cancer cell surface.

We have developed a novel immunostimulatory molecule against tumor cells, composed of an anti-FcgammaRIII (CD16) scFv fused to the platelet-derived growth factor receptor (PDGFR) transmembrane region. This fusion molecule was stably expressed on the tumor cell surface and retained the ability of the parental antibody to bind soluble CD16. Tumor cells expressing anti-CD16 scFv triggered the release of IL-2 by Jurkat-CD 16/gamma cells and of TNFalpha by monocytes when co-cultured with these cells. Furthermore, NK cells could kill scFv-transfected HLA+ class I H1299 lung carcinoma tumor cells, but not the parental cells, indicating that anti-CD16 scFv tumor expression prevents the killer inhibitory receptor (KIR)-mediated inhibition of NK cell cytotoxicity. This anti-CD16 scFv tumor expression also enhanced tumor phagocytosis by IFNgamma-activated macrophages, a mechanism known to induce a protective long-term adaptative immunity to tumors. In vivo Winn tests performed in SCID mice showed that the expression of anti-CD16 scFv on tumor cells, but not of the negative control anti-phOx scFv, prevented tumor cell growth. Thus, expression of FcR antibodies or other FcR-specific ligands on tumor cells represents a novel and potent antibody-based gene therapy approach, which may have clinical applications in cancer

Purification of soluble recombinant human FcgammaRII (CD32).

The present study describes the methodology used to purify human recombinant low-affinity FcgammaRIIa2 produced in E. coli and to evaluate its binding to surface IgG. The recombinant molecule was purified by a two-step chromatographic procedure, including affinity chromatography using IV.3 anti-FcgammaRIIa1/2 immunosorbent, followed by gel filtration chromatography. Using this method, the purified recombinant FcgammaRIIa2 was 99% pure. It exhibited an isoeletric point of 5.2. Binding studies demonstrated a specific binding of the purified recombinant molecule to surface IgG expressed by human B cells. Thus, we have set up a method which allows to purify functional human recombinant FcgammaRIIa2 for further characterization of its biological activities.

Ras and p53 intracellular targeting with recombinant single-chain Fv (scFv) fragments: a novel approach for cancer therapy?

Intracellular expression of recombinant antibodies allows one to interfere with the functions of oncogenic molecules expressed in various cell compartments and has therefore a vast clinical potential in cancer therapy. We inhibited the functions of oncogenic Ras mutant forms by intracellular expression of a neutralizing single-chain antibody (scFv). In vitro studies indicated that the scFv is expressed in the cytosol of Xenopus laevis oocytes and of tumor cells, blocks ras-mediated activation processes, and induces tumor cell death. In vivo studies performed using scFv cDNA inserted into an adenoviral vector showed that the scFv dramatically affects tumor growth. Second, intracellular expression of scFvs directed against p53 indicated that these antibody fragments can be successfully targeted to cell nucleus, bind p53, and partially restore the transcriptional activity of p53 mutants in human tumor cells. Thus, intracellular scFvs directed against oncogenic molecules may represent a new class of antitumor agents.