André Pèlegrin

Cell-penetrating and cell-targeting peptides in drug delivery

During the last decade, the potential of peptides for drug delivery into cells has been highlighted by the discovery of several cell-penetrating peptides (CPPs). CPPs are very efficient in delivering various molecules into cells. However, except in some specific cases, their lack of cell specificity remains the major drawback for their clinical development. At the same time, various peptides with specific binding activity for a given cell line (cell-targeting peptides) have also been reported in the literature. One of the goals of the next years will be to optimize the tissue and cell delivery of therapeutic molecules by means of peptides which combine both targeting and internalization advantages. In this review, we describe the main strategies that are currently in use or likely to be employed in the near future to associate both targeting and delivery properties.

Single nucleotide polymorphisms, apoptosis, and the development of severe late adverse effects after radiotherapy.

Purpose: Evidence has accumulated in recent years suggestive of a genetic basis for a susceptibility to the development of radiation injury after cancer radiotherapy. The purpose of this study was to assess whether patients with severe radiation-induced sequelae (RIS; i.e., National Cancer Institute/CTCv3.0 grade, ≥3) display both a low capacity of radiation-induced CD8 lymphocyte apoptosis (RILA) in vitro and possess certain single nucleotide polymorphisms (SNP) located in candidate genes associated with the response of cells to radiation. Experimental Design: DNA was isolated from blood samples obtained from patients (n = 399) included in the Swiss prospective study evaluating the predictive effect of in vitro RILA and RIS. SNPs in the ATM, SOD2, XRCC1, XRCC3, TGFB1, and RAD21 genes were screened in patients who experienced severe RIS (group A, n = 16) and control subjects who did not manifest any evidence of RIS (group B, n = 18). Results: Overall, 13 and 21 patients were found to possess a total of <4 and ≥4 SNPs in the candidate genes. The median (range) RILA in group A was 9.4% (5.3-16.5) and 94% (95% confidence interval, 70-100) of the patients (15 of 16) had ≥4 SNPs. In group B, median (range) RILA was 25.7% (20.2-43.2) and 33% (95% confidence interval, 13-59) of patients (6 of 18) had ≥4 SNPs (P < 0.001). Conclusions: The results of this study suggest that patients with severe RIS possess 4 or more SNPs in candidate genes and low radiation-induced CD8 lymphocyte apoptosis in vitro.

Antibody–fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice

To improve the detectability of tumors by light‐induced fluorescence, the use of monoclonal antibodies (MoAb) as carriers of fluorescent molecules was studied. As a model for this approach, the biodistribution of an anticarcinoembryonic antigen (CEA) MoAb coupled to fluorescein was studied in mice bearing a human colon carcinoma xenograft. In vitro, such conjugates with fluorescein‐MoAb molar ratios ranging from four to 19, doubly labeled with 125I, showed more than 82% binding to immobilized CEA. In vivo, conjugates with a fluorescein–MoAb molar ratio of ten or less resulted in a tumor uptake of more than 30% of the injected dose of radioactivity per gram tumor at 24 hours. Tumor to liver, kidney, and muscle ratios of 20, 30 and 72, respectively, were obtained 48 hours after injection of the 125I‐MoAb–(fluorescein)10 conjugate. The highest fluorescence intensity was always obtained for the tumor with the anti‐CEA MoAb conjugate; whereas in control mice injected with fluoresceinated control immunoglobulin G1, no detectable increase in tumor fluorescence was observed. To compare these results with a classically used dye, mice bearing the same xenografts received 60 μg of Photofrin II. The intensity of the fluorescence signal of the tumor with this amount of Photofrin II was eight times lower than that obtained after an injection of 442 ng of fluorescein coupled with 20 μg of MoAb, which gave an absolute amount of fluorescein localized in the tumor of up to 125 ng/g of tumor. These results illustrate the possibility of improving the specificity of in vivo tumor localization of dyes for laser‐induced fluorescence photodetection and phototherapy by coupling them to MoAb directed against tumor markers.

Anti-HER2 vaccines: new prospects for breast cancer therapy.

Each year, breast cancer accounts for more than 400,000 new cancer cases and more than 130,000 cancer deaths in Europe. Prognosis of nonmetastatic breast cancer patients is directly related to the extent of the disease, mainly nodal spreading and tumor size, and to the molecular profile, particularly HER2 over-expression. In patients with HER2-over-expressing tumors, different studies have shown cellular and/or humoral immune responses against HER2 associated with a lower tumor development at early stages of the disease. These findings have led to the hypothesis that the generation of an anti-HER2 immune response should protect patients from HER2-over-expressing tumor growth. Taken together with the clinical efficiency of trastuzumab-based anti-HER2 passive immunotherapy, these observations allowed to envisage various vaccine strategies against HER2. The induction of a stable and strong immunity by cancer vaccines is expected to lead to establishment of immune memory, thereby preventing tumor recurrence. However, an immunological tolerance against HER2 antigen exists representing a barrier to effective vaccination against this oncoprotein. As a consequence, the current challenge for vaccines is to find the best conditions to break this immunological tolerance. In this review, we will discuss the different anti-HER2 vaccine strategies currently developed; considering the strategies having reached the clinical phases as well as those still in preclinical development. The used antigen can be either composed of tumoral allogenic cells or autologous cells, or specific to HER2. It can be delivered by dendritic cells or in a DNA, peptidic or proteic form. Another area of research concerns the use of anti-idiotypic antibodies mimicking HER2.

In vivo therapeutic synergism of anti-epidermal growth factor receptor and anti-HER2 monoclonal antibodies against pancreatic carcinomas.

Purpose: Pancreatic carcinoma is highly resistant to therapy. Epidermal growth factor receptor (EGFR) and HER2 have been reported to be both dysregulated in this cancer. To evaluate the in vivo effect of binding both EGFR and HER2 with two therapeutic humanized monoclonal antibodies (mAb), we treated human pancreatic carcinoma xenografts, expressing high EGFR and low HER2 levels. Experimental Design: Nude mice, bearing xenografts of BxPC-3 or MiaPaCa-2 human pancreatic carcinoma cell lines, were injected twice weekly for 4 weeks with different doses of anti-EGFR (matuzumab) and anti-HER2 (trastuzumab) mAbs either alone or in combination. The effect of the two mAbs, on HER receptor phosphorylation, was also studied in vitro by Western blot analysis. Results: The combined mAb treatment significantly inhibited tumor progression of the BxPC-3 xenografts compared with single mAb injection (P = 0.006) or no treatment (P = 0.0004) and specifically induced some complete remissions. The two mAbs had more antitumor effect than 4-fold greater doses of each mAb. The significant synergistic effect of the two mAbs was confirmed on the MiaPaCa-2 xenograft and on another type of carcinoma, SK-OV-3 ovarian carcinoma xenografts. In vitro, the cooperative effect of the two mAbs was associated with a decrease in EGFR and HER2 receptor phosphorylation. Conclusions: Anti-HER2 mAb has a synergistic therapeutic effect when combined with an anti-EGFR mAb on pancreatic carcinomas with low HER2 expression. These observations may open the way to the use of these two mAbs in a large panel of carcinomas expressing different levels of the two HER receptors.

Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons.

Abstract Pouget, J-P., Santoro, L., Raymond, L., Chouin, N., Bardiès, M., Bascoul-Mollevi, C., Huguet, H., Azria, D., Kotzki, P-O., Pè legrin, M., Vivès, E. and Pèlegrin, A. Cell Membrane is a More Sensitive Target than Cytoplasm to Dense Ionization Produced by Auger Electrons. Radiat. Res. 170, 192–200 (2008). To improve radioimmunotherapy with Auger electron emitters, we assessed whether the biological efficiency of 125I varied according to its localization. A-431 and SK-OV-3 carcinoma cells were incubated with increasing activities (0–4 MBq/ml) of 125I-labeled vectors targeting the cell membrane, the cytoplasm or the nucleus. We then measured cell survival by clonogenic assay and the mean radiation dose to the nucleus by assessing the cellular medical internal radiation dose (MIRD). The relationship between survival and the radiation dose delivered was investigated with a linear mixed regression model. For each cell line, we obtained dose–response curves for the three targets and the reference values (i.e., the dose leading to 75, 50 or 37% survival). When cell survival was expressed as a function of the total cumulative decays, nuclear 125I disintegrations were more harmful than disintegrations in the cytoplasm or at the cell membrane. However, when survival was expressed as a function of the mean radiation dose to the nucleus, toxicity was significantly higher when 125I was targeted to the cell membrane than to the cytoplasm. These findings indicate that the membrane is a more sensitive target than the cytoplasm for the dense ionization produced by Auger electrons. Moreover, cell membrane targeting is as cytotoxic as nuclear targeting in SK-OV-3 cells. We suggest that targeting the membrane rather than the cytoplasm may contribute to the development of more efficient radioimmunotherapies based on Auger electron radiation, also because most of the available vectors are directed against cell surface antigens.

Phase-I/II radio-immunotherapy study with Iodine-131-labeled anti-CEA monoclonal antibody F6 F(ab')2 in patients with non-resectable liver metastases from colorectal cancer.

Experimental studies in nude mice with human colon‐carcinoma grafts demonstrated the therapeutic efficiency of F(ab′)2 fragments to carcinoembryonic antigen (CEA) labeled with a high dose of 131Iodine. A phase I/II study was designed to determine the maximum tolerated dose of 131I‐labeled F(ab′)2 fragments (131I‐F(ab′)2) from anti‐CEA monoclonal antibody F6, its limiting organ toxicity and tumor uptake. Ten patients with non‐resectable liver metastases from colorectal cancer (9 detected by CT scan and 1 by laparotomy) were treated with 131I‐F(ab′)2, doses ranging from 87 mCi to 300 mCi for the first 5 patients, with a constant 300‐mCi dose for the last 5 patients. For all the patients, autologous bone marrow was harvested and stored before treatment. Circulating CEA ranged from 2 to 126 ng/ml. No severe adverse events were observed during or immediately following infusion of therapeutic doses. The 9 patients with radiologic evidence of liver metastases showed uptake of 131I‐F(ab′)2 in the metastases, as observed by single‐photon‐emission tomography. The only toxicity was hematologic, and no severe aplasia was observed when up to 250 mCi was infused. At the 300‐mCi dose, 5 out of 6 patients presented grade‐3 or ‐4 hematologic toxicity, with a nadir for neutrophiles and thrombocytes ranging from 25 to 35 days after infusion. In these 5 cases, bone marrow was re‐infused. No clinical complications were observed during aplasia. The tumor response could be evaluated in 9 out of 10 patients. One patient showed a partial response of one small liver metastasis (2 cm in diameter) and a stable evolution of the other metastases, 2 patients had stable disease, and 6 showed tumor progression at the time of evaluation (2 or 3 months after injection) by CT scan. This phase‐I/II study demonstrated that a dose of 300 mCi of 131I‐F(ab′)2 from the anti‐CEA Mab F6 is well tolerated with bone‐marrow rescue, whereas a dose of 200 mCi can be infused without severe bone‐marrow toxicity. Int. J. Cancer 75:615–619, 1998.

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA).

In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non‐overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab′ fragments from 4 different specific anti‐CEA MAbs recognizing 4 CEA epitopes (Gold 1–4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross‐inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 × 109 M−1 for the 4 parental F(ab′)2 fragments and from 1.88 to 10.14 × 109 M−1 for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab′)2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5‐ and 8.1‐fold increase, respectively, as compared with the parental F(ab′)2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab′)2 by injection of 131I‐BpAb/125I‐F(ab′)2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post‐injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab′)2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab′)2, one of the best parental F(ab′)2 fragments used in our study. Int. J. Cancer 81:285–291, 1999.

Preparation, phototoxicity and biodistribution studies of anti-carcinoembryonic antigen monoclonal antibody-phthalocyanine conjugates.

Immunophototherapy of cancer combines the specificity of a monoclonal antibody (MAb) to an overexpressed tumor marker with the phototoxic properties of a conjugated dye. Aluminum tetrasulfophthalocyanine (AlPcS4) was covalently coupled to a 35A7 MAb directed against carcinoembryonic antigen (CEA) via a five‐carbon spacer chain (A1) to yield conjugates with a molar ratio ranging from 5 to 16 mol of AlPcS4 per mol of 35A7 MAb. Conjugates were labeled with radioiodine for characterization. The immunoreactivity of the conjugates, determined in a direct binding assay on CEA coupled to sepharose, was not modified by the coupled AlPcS4A1 molecules. In vivo, these conjugates were evaluated in nude mice bearing human colon carcinoma xenografts (T380). 35A7 MAb‐(AlPcS4A1)s, 35A7 MAb‐(AlPcS4A1,)12 and 35A7 MAb‐(AIPcS4A,)16 conjugates displayed a tumor uptake of 35 ± 5.0%, 40 ± 5.7% and 32 ± 3.3% of the injected dose per gram of tumor tissue, respectively, corresponding to an uptake of 97%, 104% and 91% as compared to that of the unconjugated 35A7 MAb. In each experimental group, the tumor‐to‐normal tissue ratios obtained with the conjugates were almost identical to those obtained with unconjugated 35A7 MAb. Average values of 1.8, 7 and about 30 were obtained for blood, liver and muscle, respectively. Phototoxic efficacy of the 35A7 MAb‐(AlPcS4A,)12 conjugate was demonstrated in vitro on the LoYo cell line giving a 91 % growth inhibition for a 2.50 μ/mL AlPcS4A1 concentration. We conclude that these conjugates demonstrate clear in vivo tumor‐seeking capacity and in vitro photocytotoxic properties. Such conjugates could thus be promising candidate drugs for clinical photodynamic therapy of cancers expressing CEA.

Enhancement of 5‐fluorouracil cytotoxicity by human thymidine‐phosphorylase expression in cancer cells: In vitro and In vivo study

Transferring a gene into cancer cells in order to sensitize them to drugs is an important approach in human cancer gene‐therapy research. Thymidine phosphorylase (TP) is the first enzyme in the metabolic activation pathway of 5‐fluorouracil (5‐FU) to fluorodeoxyribonucleotides, thus, it could be used to increase the sensitivity of cancer cells to this anti‐pyrimidine agent. In this study, an expression vector containing the human TP cDNA was transfected into C26 murine colon‐carcinoma cells. Stable transfectants were selected; all showed increased TP activity, ranging from 2‐ to 10‐fold when compared with wild‐type cells. The in vitro sensitivity of transfectants to 5‐FU and 5′‐deoxy‐5‐fluorouridine (5′‐DFUR) was enhanced, in agreement with the observed increase in TP activity. Then, tumors were generated by s.c. injection of TP‐transfected or wild‐type C26 cells in syngeneic BALB/c mice. 5‐FU (25 mg/kg, i.p.) induced a growth delay of TP‐transfected C26 tumors as compared with C26 wild‐type tumors. These data suggest that TP could be transfected in tumor cells to increase the sensitivity to 5‐FU for subsequent cancer gene therapy. Int. J. Cancer 80:465–470, 1999.