Corinne Venot

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.

Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains.

The wild-type protein product of the p53 tumor suppressor gene can activate transcription of genes which are involved in mediating either growth arrest, e.g. WAF1 or apoptotis, e.g. BAX and PIG3. Additionally, p53 can repress a variety of promoters, which, in turn, may be responsible for the functional activities exhibited by p53. This study shows that the Q22, S23 double mutation, which is known to inactivate a p53 trans-activation subdomain located within the initial 40 residues of the protein, while abrogating transactivation from the WAF1 promoter, only attenuates apoptosis triggering, transactivation from other p53-responsive promoters and repression of promoters by p53. The Q53, S54 double mutation, which inactivates another p53 transactivation subdomain situated between amino acids 43 and 73 results in attenuation of all of the afore-mentioned p53 activities. In contrast to the Q22, S23 double mutation, this latter mutation set does not alter mdm-2-mediated inhibition and degradation of p53. Finally, mutation of all four residues results in complete abrogation of every p53 activity mentioned above.

CD45neg but Not CD45pos Human Myeloma Cells Are Sensitive to the Inhibition of IGF-1 Signaling by a Murine Anti-IGF-1R Monoclonal Antibody, mAVE1642

Insulin-like growth factor 1 (IGF-1) is a well-known growth factor for myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for multiple myeloma treatment. In this study, we investigated the effect of the antagonistic anti-IGF-1R murineAVE1642 Ab (mAVE1642). We show that mAVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines. Since we have previously shown the functional relevance of CD45 expression in the growth of myeloma cells and the association of CD45-negative (CD45neg) status with a less favorable clinical outcome, both CD45-positive (CD45pos) and CD45neg myeloma cell lines were selected for our study. We found that mAVE1642 strongly inhibits the growth of CD45neg myeloma cell lines, leading to a G1 growth arrest, whereas it has almost no effect on the growth of CD45pos myeloma cell lines. Furthermore, mAVE1642 binding induced a significant reduction of IGF-1R expression. We next demonstrated that the overexpression of IGF-1R in the CD45pos myeloma cell line increased Akt phosphorylation but was not sufficient to sensitize these cells to mAVE1642. In contrast, we generated a stable CD45-silencing XG-1 cell line and showed that it became sensitive to mAVE1642. Thus, for the first time, we provided direct evidence that the expression of CD45 renders cells resistant to mAVE1642. Taken together, these results support that therapy directed against IGF-1R can be beneficial in treating CD45neg patients.

Anti-insulin-like growth factor 1 receptor antibody EM164 (murine AVE1642) exhibits anti-tumour activity alone and in combination with temozolomide against neuroblastoma☆

Insulin-like growth factor 1 receptor (IGF-1R) is overexpressed in many tumours and contributes to tumourigenicity, cell proliferation, metastasis and resistance, thus representing a promising therapeutic target. The human IGF-1R antagonistic monoclonal antibody EM164 (murine AVE1642) has shown activity in adult cancers and is being evaluated in patients with advanced malignancies. We investigated the EM164 for its therapeutic potential against childhood neuroblastoma. EM164 at 0.07, 0.7 and 7 μg/mL exhibited anti-proliferative activity against all nine cell lines tested in (3)H-thymidine incorporation assay in vitro. Cell proliferation after EM164 exposure ranged between 24% and 80% compared to controls. Sensitivity was independent from culture serum conditions, intensity of IGF-1R expression and IGF-II secretion, although associated with inhibition of AKT activation. In vivo, EM164 administered intravenously at 40 mg/kg twice weekly for 4 weeks yielded significant tumour growth delays (TGD) of 13.4d in advanced stage IGR-N91 and 12.9 d in SK-N-AS tumours compared to controls (p = 0.02 and p = 0.0059, respectively). Simultaneous treatment of EM164 0.7 μg/mL and temozolomide resulted in enhanced activity in vitro. In vivo, treatment with temozolomide at the maximum tolerated dose (100mg/kg/d for 5 consecutive days) and EM164 yielded a significantly greater TGD of 29.1d (p<0.01) and two complete tumour regressions (CR) compared to 18.1d (p = ns) and one CR for EM164 alone and 16.1d (p = ns) for temozolomide alone. Our results demonstrate the potential of the anti-IGF-1R antibody alone and in combination with alkylating agents and support the therapeutic development of the AVE1642 for aggressive neuroblastoma.

Discovery of the first non-ATP competitive IGF-1R kinase inhibitors: Advantages in comparison with competitive inhibitors

A new series of IGF-1R inhibitors related to hydantoins were identified from a lead originating from HTS. Their noncompetitive property as well as their slow binding characteristics provided a series of compounds with unique selectivity and excellent cellular activities.

Design of Potent IGF1‐R Inhibitors Related to Bis‐azaindoles

From an azaindole lead, identified in high throughput screen, a series of potent bis‐azaindole inhibitors of IGF1‐R have been synthesized using rational drug design and SAR based on a in silico binding mode hypothesis. Although the resulting compounds produced the expected improved potency, the model was not validated by the co‐crystallization experiments with IGF1‐R.

Grb3-3 Is Up-regulated in HIV-1-infected T-cells and Can Potentiate Cell Activation through NFATc

The MAPK pathway is required for T-cell activation; however, its role in modulating T-cell function following human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. In this report, we investigated whether Grb3-3, an isoform of the Grb2 (growth factorreceptor-bound protein-2) adaptor molecule that is associated with the MAPK pathway, could be involved. We found that Grb3-3, but not its isoform Grb2, is markedly up-regulated in CD4+ peripheral blood mononuclear cells derived from either in vitro HIV-1-infected cultures or HIV-1-infected human subjects. Analysis of HIV-1 gene products indicated that Tat and Nef, both of which have been implicated in modulating T-cell function, can independently induce expression of Grb3-3. By using NFAT/AP-1, AP-1, or NFAT reporter assays, we found that Grb3-3 can potentiate NFAT (but not AP-1) promoter activity in Jurkat T-cells upon engagement of the T-cell receptor and CD28 co-receptor. In addition, potentiation of NFAT by Grb3-3 is substantially suppressed by MEKK1, a kinase that may play an important role in retaining NFAT in the cytoplasm, and by cyclosporin A. Finally, we also found that Grb3-3 potentiates HIV-1 long terminal (LTR) repeat promoter activity following T-cell receptor stimulation, an effect that can be largely suppressed by cyclosporin A. Taken together, this study indicates that Grb3-3 is a cellular factor that can be up-regulated by HIV-1. In addition, Grb3-3 can also function as a positive factor for T-cell activation and, in doing so, may aid in establishing an intracellular environment that can optimally support HIV-1 replication.

Abstract 2785: Development program of patient tumor tissue bank to support the drug and target discovery.

Background: The humanization of mice with various tissues named Chi-mice® aimed to reproduce the human situation to be more predictive than conventional models. Despite significant progress in identifying malignancy of cancer cells, a more detailed understanding of tumor generation is needed. Xenograft of tumor cells into immunodeficient rodents has constituted the major preclinical screen for the development of new drugs. These models have identified efficacious agents, but their chemosensitivity, genetic drift and clonal selection induced by cell culture have been part of the high attrition rate observed in the clinical development. Patient-derived tumor xenograft (PDX) obtained in xenografting fresh patient tumor samples in mice are reported as being more predictive to the clinical situation in maintaining the histopathology and molecular diversity of the patient tumors. Methods: The PDXs collection has been set up under ethical agreement with informed consent of patients. The patients have been screened for absence of HIV, HBV and HCV. The anonymized patient9s clinical history and tissue banking (including normal tissue when available) are centralized in our internal biological resource center. Tumor samples were freshly implanted in nude or SCID mice. Results: Cryopreservation of the PDX is performed at early passages allowing using these PDX only at low passage. The histopathology, EGFR, PSA, AR, HER, ER and PR statute for lung, prostate and breast carcinoma and tumor growth characteristics of these PDX are being performed. Lymphoma characterization was performed using immunohistochemistry (hCD20, m/hKi67). Lymphoma detection, probably related to EBV infection, leads to switch to nude mice for xenografting.Conclusion: To create a highly diversified panel of PDX, we organized a global process from multiple centers. These PDX are currently being used in preclinical development of new therapies and clinical positioning including biomarkers identification. Citation Format: Marc Hillairet de Boisferon, Francis Bichat, Caroline Mignard, Loic Morgand, Lydia Blot, Corinne Venot, Ana Merino-Trigo, Patricia Vrignaud. Development program of patient tumor tissue bank to support the drug and target discovery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2785. doi:10.1158/1538-7445.AM2013-2785