[Show abstract][Hide abstract] ABSTRACT: Blockade of the human epidermal growth factor receptor 3 (HER3) and of the downstream phosphatidylinositide 3-kinase (PI3K)/AKT pathway is a prerequisite for overcoming drug resistance and to develop novel treatments for cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2 cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.
Neoplasia (New York, N.Y.) 03/2013; 15(3):335-47. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We assessed the contribution of antibody internalization in the efficacy and toxicity of intraperitoneal α-radioimmunotherapy (RIT) of small volume carcinomatosis using (212)Pb-labeled monoclonal antibodies (mAbs) that target HER2 (internalizing) or CEA (non-internalizing) receptors.
Athymic nude mice bearing 2-3 mm intraperitoneal tumor xenografts were intraperitoneally injected with similar activities (370, 740 and 1480 kBq; 37 MBq/mg) of (212)Pb-labeled 35A7 (anti-CEA), trastuzumab (anti-HER2) or PX (non-specific) mAbs, or with equivalent amounts of unlabeled mAbs, or with NaCl. Tumor volume was monitored by bioluminescence and survival was reported. Hematologic toxicity and body weight were assessed. Biodistribution of (212)Pb-labeled mAbs and absorbed dose-effect relationships using MIRD formalism were established.
Transient hematological toxicity, as revealed by white blood cells and platelets numbering, was reported in mice treated with the highest activities of (212)Pb-labeled mAbs. The median survival (MS) was significantly higher in mice injected with 1.48 MBq of (212)Pb-35A7 (non-internalizing mAbs) (MS = 94 days) than in animals treated with the same activity of (212)Pb-PX mAbs or with NaCl (MS = 18 days). MS was even not reached after 130 days when follow-up was discontinued in mice treated with 1.48 MBq of (212)Pb-trastuzumab. The later efficacy was unexpected since final absorbed dose resulting from injection of 1.48 MBq, was higher for (212)Pb-35A7 (35.5 Gy) than for (212)Pb-trastuzumab (27.6 Gy). These results also highlight the lack of absorbed dose-effect relationship when mean absorbed dose was calculated using MIRD formalism and the requirement to perform small-scale dosimetry.
These data indicate that it might be an advantage of using internalizing anti-HER2 compared with non-internalizing anti-CEA (212)Pb-labeled mAbs in the therapy of small volume xenograft tumors. They support clinical investigations of (212)Pb-mAbs RIT as an adjuvant treatment after cytoreductive surgery in patients with peritoneal carcinomatosis.
PLoS ONE 01/2013; 8(7):e69613. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Novel adjuvant therapies are needed to prevent metastatic relapses in HER2-expressing breast cancer. Here, we tested whether trastuzumab-selected single-chain Fv (scFv) could be used to develop an anti-idiotype-based vaccine to inhibit growth of HER2-positive tumor cells in vitro and in vivo through induction of long-lasting HER-specific immunity.
BALB/c mice were immunized with anti-trastuzumab anti-idiotype (anti-Id) scFv (scFv40 and scFv69), which mimic human HER2. Their sera were assessed for the presence of HER2-specific Ab1' antibodies and for their ability to reduce viability of SK-OV-3 cells, a HER2-positive cancer cell line, in nude mice. MMTV.f.huHER2(Fo5) transgenic mice were immunized with scFv40 and scFv69 and, then, growth inhibition of spontaneous HER2-positive mammary tumors, humoral response, antibody isotype as well as splenocyte secretion of IL2 and IFN-γ were evaluated.
Adoptively-transferred sera from BALB/c mice immunized with scFv40 and scFv69 contain anti-HER2 Ab1' antibodies that can efficiently inhibit growth of SK-OV-3 cell tumors in nude mice. Similarly, prophylactic vaccination with anti-Id scFv69 fully protects virgin or primiparous FVB-MMTV.f.huHER2(Fo5) females from developing spontaneous mammary tumors. Moreover, such vaccination elicits an anti-HER2 Ab1' immune response together with a scFv69-specific Th1 response with IL2 and IFN-γ cytokine secretion.
Anti-trastuzumab anti-Id scFv69, used as a therapeutic or prophylactic vaccine, protects mice from developing HER2-positive mammary tumors by inducing both anti-HER2 Ab1' antibody production and an anti-HER2 Th2-dependent immune response. These results suggest that scFv69 could be used as an anti-Id-based vaccine for adjuvant therapy of patients with HER2-positive tumors to reverse immunological tolerance to HER2.
Breast cancer research: BCR 02/2011; 13(1):R17. · 5.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We assessed the efficiency and toxicity of brief intraperitoneal radioimmunotherapy using high activities of (125)I-labeled monoclonal antibody (mAb) in the treatment of small-volume peritoneal carcinomatosis.
Brief intraperitoneal radioimmunotherapy consisted of a 185-MBq (740 MBq/mg) intraperitoneal injection of (125)I-35A7 (an anti-carcinoembryonic antigen mAb) into athymic nude mice 4 d after peritoneal tumor xenografting and, after 1 h, abundant washing of the peritoneal cavity with saline solution to remove unbound radioactivity. Another group of mice received this treatment plus a 37-MBq intravenous injection of (125)I-35A7 on day 7 or 11 after grafting. Control groups received a brief treatment followed by an additional intravenous injection on day 7 of either saline solution or irrelevant (125)I-PX. Tumor growth was monitored by bioluminescence imaging and SPECT/CT, and hematologic toxicity was evaluated by complete blood counts. Survival time was reported, and the mice were sacrificed when the bioluminescence signal reached 4.5 × 10(7) photons/s. The biodistribution of (125)I-35A7 mAb after intravenous or brief treatment was assessed, and the mean absorbed irradiation dose by organs and tumors was calculated using the MIRD formalism.
Mild, transient hematologic toxicity was observed after the brief treatment plus intravenous (125)I-mAb, with no weight loss. Median survival increased from 32 d in the control groups, to 46 d in the brief treatment group, to 66 d in the group additionally receiving intravenous treatment on day 11, to 73 d in the group additionally receiving intravenous treatment on day 7. The brief treatment alone resulted in a 3-fold higher tumor-to-blood uptake ratio than did the standard intravenous treatment, and the mean absorbed irradiation doses by tumors were 11.6 Gy for the brief treatment and 16.7 Gy for the additional intravenous treatment. For healthy tissues other than blood, the mean absorbed irradiation dose did not exceed 1 Gy after brief treatment and 4.2 Gy after intravenous treatment.
The efficiency, low toxicity, and high tumor-to-healthy tissue uptake ratio associated with brief intraperitoneal (125)I-35A7 radioimmunotherapy suggest that this method can be used in combination with radiation-synergistic drugs in the therapy of small-volume peritoneal carcinomatosis after cytoreductive surgery.
Journal of Nuclear Medicine 10/2010; 51(11):1748-55. · 5.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Various synthetic cyclopeptides bind different cellular proteins with high affinity and specificity. In this study, we designed a new series of cyclic tetrapeptides containing the RGD sequence, a ligand for the alpha(v)beta(3) integrin receptor, in which the ring closure was performed through a urea bond between the alpha-amino group of the peptide and either the alpha- or the epsilon-amino group of an additional lysine. Interestingly, we showed that the urea-closed peptide had a higher affinity for alpha(v)beta(3) receptors than a reference pentacyclopeptide. Moreover, the synthetic strategy allows coupling of the resulting cyclic tetrapeptide through the carboxylic acid moiety of its lysine residue to fluorescent molecules or drugs. In addition, this strategy could be easily adapted for the cyclization of any other peptides.
[Show abstract][Hide abstract] ABSTRACT: Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.
Proceedings of the National Academy of Sciences 02/2010; 107(6):2628-33. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that, in vitro, monoclonal antibodies (mAbs) labeled with the Auger electron emitter (125)I are more cytotoxic if they remain at the cell surface and do not internalize in the cytoplasm. Here, we assessed the in vivo biologic efficiency of internalizing and noninternalizing (125)I-labeled mAbs for the treatment of small solid tumors.
Swiss nude mice bearing intraperitoneal tumor cell xenografts were injected with 37 MBq (370 MBq/mg) of internalizing (anti-HER1) (125)I-m225 or noninternalizing (anti-CEA) (125)I-35A7 mAbs at days 4 and 7 after tumor cell grafting. Nonspecific toxicity was assessed using the irrelevant (125)I-PX mAb, and untreated controls were injected with NaCl. Tumor growth was followed by bioluminescence imaging. Mice were sacrificed when the bioluminescence signal reached 4.5 x 10(7) photons/s. Biodistribution analysis was performed to determine the activity contained in healthy organs and tumor nodules, and total cumulative decays were calculated. These values were used to calculate the irradiation dose by the MIRD formalism.
Median survival (MS) was 19 d in the NaCl-treated group. Similar values were obtained in mice treated with unlabeled PX (MS, 24 d) and 35A7 (MS, 24 d) or with (125)I-PX mAbs (MS, 17 d). Conversely, mice treated with unlabeled or labeled internalizing m225 mAb (MS, 76 and 77 d, respectively) and mice injected with (125)I-35A7 mAb (MS, 59 d) showed a significant increase in survival. Irradiation doses were comparable in all healthy organs, independently from the mAb used, whereas in tumors the irradiation dose was 7.4-fold higher with (125)I-labeled noninternalizing than with internalizing mAbs. This discrepancy might be due to iodotyrosine moiety release occurring during the catabolism of internalizing mAbs associated with high turnover rate.
This study indicates that (125)I-labeled noninternalizing mAbs could be suitable for radioimmunotherapy of small solid tumors and that the use of internalizing mAbs should not be considered as a requirement for the success of treatments with (125)I Auger electrons.
Journal of Nuclear Medicine 11/2009; 50(12):2033-41. · 5.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tight junctions have recently emerged as essential signaling regulators of proliferation and differentiation in epithelial tissues. Here, we aimed to identify the factors regulating claudin-7 expression in the colon, and analyzed the consequences of claudin-7 overexpression in colorectal carcinoma (CRC). In healthy human colonic crypts, claudin-7 expression was found to be low in the stem/progenitor cell compartment, where Tcf-4 activity is high, but strong in differentiated and postmitotic cells, where Tcf-4 is inactive. In contrast, claudin-7 was overexpressed in areas with high Tcf-4 target gene levels in CRC samples. In vitro, Tcf-4 was able to repress claudin-7 expression, and the high mobility group-box transcription factor Sox-9 was identified as an essential mediator of this effect. Claudin-7 was strongly expressed in the intestine of Sox-9-deficient mice and in CRC cells with low Sox transcriptional activity. Sox-9 overexpression in these cells reinstated claudin-7 repression, and residual claudin-7 was no longer localized along the basolateral membrane, but was instead restricted to tight junctions. Using HT-29Cl.16E CRC cell spheroids, we found that Sox-9-induced polarization was completely reversed after virus-mediated claudin-7 overexpression. Claudin-7 overexpression in this context increased Tcf-4 target gene expression, proliferation, and tumorigenicity after injection in nude mice. Our results indicate that Tcf-4 maintains low levels of claudin-7 at the bottom of colonic crypts, acting via Sox-9. This negative regulation seems to be defective in CRC, possibly due to decreased Sox-9 activity, and the resulting claudin-7 overexpression promotes a loss of tumor cell polarization and contributes to tumorigenesis.
Cancer Research 06/2008; 68(11):4258-68. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.
XenoMouse-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2kappa and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice.
Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2kappa and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2kappa and VG-IgM were determined to be 0.19 +/- 0.06 x 10(8) M(-1) and 1.30 +/- 0.06 x 10(8) M(-1), respectively, as compared with 0.61 +/- 0.05 x 10(8) M(-1) for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2kappa and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10(-7) M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10(-9) M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2kappa demonstrated a high tumor uptake (25.4 +/- 7.3%ID/g), close to that of 131I-X4 (21.7 +/- 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2kappa was still concentrated in the tumor (28.4 +/- 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 +/- 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver.
Our human anti-CEA IgG2kappa is a promising candidate for radioimmunotherapy in intact form, as F(ab')2 fragments, or as a bispecific antibody.
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor-alpha (TNF-alpha) enhances X-ray killing of human tumor cells in vitro and enhances tumor control when combined with radiotherapy (RT) in animal tumor models. In multiple Phase I studies, intravenous injection of TNF-alpha appeared to have severe systemic side effects. To overcome these limitations, we used a bispecific antibody (BAb) directed against carcinoembryonic antigen and human TNF-alpha to target this cytokine in human digestive carcinoma treated with simultaneous RT. We used human digestive carcinoma cell lines (colon cancer, LS174T, and pancreatic cancer, BxPC-3) to determine the interaction of TNF-alpha and RT on clonogenic cytotoxicity. Isobolograms were established to confirm additive or supra-additive effects between both treatments. LS174T and BxPC-3 cells were grafted subcutaneously at Day 0 into female nude mice (7-8 weeks old). When the tumors reached a volume of about 80 mm(3), the mice were randomly assigned to treatment: Group 1, normal saline i.v. injection (control group); Group 2, TNF-alpha at 1 microg/i.v. injection; Group 3, BAb at 25 microg/i.v. injection; Group 4, BAb plus TNF-alpha (ratio 25 microg to 1 microg) i.v. injection; Group 5, local RT plus normal saline (0.5 Gy. min(-1)) at a total dose of 30 Gy delivered in five fractions; Group 6, local RT plus TNF-alpha injections 3 h before RT; Group 7, local RT plus BAb plus TNF-alpha co-injected 24 h before RT. Tumor growth delay was used as the end point for all groups. In the LS174T experiments, TNF-alpha added 12 h before RT showed a statistically significant decrease in the survival fraction at 2 Gy compared with RT alone (0.23 vs. 0.42 Gy, p = 0.0017). These results were largely confirmed with the BxPC-3 cell lines (0.29 vs. 0.72, p <0.00001). Isobolograms confirmed the additivity between TNF-alpha and RT in both cell lines. At 50% survival, the data points were within the envelope of additivity. In the LS174T and BxPC-3 xenografts, RT as a single agent (Group 5) slowed tumor progression compared with Group 1 (p <0.027 and p = 0.00001, respectively). TNF-alpha alone, BAb alone, or BAb plus TNF-alpha (Groups 2, 3, and 4) had no effect. In the LS174T model, TNF-alpha plus RT enhanced the delay to reach 2000 mm(3) compared with RT alone but without statistical significance. This delay was significantly longer when BAb was added (p = 0.0033, for Group 6 vs. Group 7). In the BxPC-3 experiments, the median delay to reach 2000 mm(3) was similar between the RT and TNF-alpha plus RT groups (93 days). The use of our BAb in combination with TNF-alpha and RT dramatically enhanced this median delay (177 days, p = 0.0013). No body weight loss was observed in any group. Our data could be used as a solid preclinical rationale on which to base a clinical study of locally advanced pancreatic or rectal cancers in the near future.
International Journal of Radiation OncologyBiologyPhysics 02/2004; 58(2):580-8. · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to treat carcinoembryonic antigen (CEA)-expressing pancreatic carcinoma cells with tumour necrosis factor alpha (TNFalpha) and simultaneous radiation therapy (RT), using a bispecific antibody (BAb) anti-TNFalpha/anti-CEA. TNFalpha used alone produced a dose-dependent inhibition of the clonogenic capacity of the cultured cells. Flow cytometry analysis of cell cycle progression confirmed the accumulation of cells in G(1) phase after exposure to TNFalpha. When TNFalpha was added 12 h before RT, the surviving fraction at 2 Gy was 60% lower than that obtained with irradiation alone (0.29 vs 0.73, respectively, P<0.00001). In combination treatment, cell cycle analysis demonstrated that TNFalpha reduced the number of cells in radiation-induced G(2) arrest, blocked irreversibly the cells in G(1) phase, and showed an additive decrease of the number of cells in S phase. In mice, RT as a single agent slowed tumour progression as compared with the control group (P<0.00001). BAb+TNFalpha+RT combination enhanced the delay for the tumour to reach 1500 mm(3) as compared with RT alone or with RT+TNFalpha (P=0.0011). Median delays were 90, 93, and 142 days for RT alone, RT+TNFalpha, and RT+BAb+TNFalpha groups, respectively. These results suggest that TNFalpha in combination with BAb and RT may be beneficial for the treatment of pancreatic cancer in locally advanced or adjuvant settings.
British Journal of Cancer 11/2003; 89(10):1987-94. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carcinoembryonic antigen (CEA) and ErbB-2 are expressed in about 50 and 30% of breast cancers, respectively. We hypothesised that targeting of these two antigens by a bispecific antibody (BAb) might provide efficient tumour uptake and prolonged tumour residence time. In the present study, we first studied the expression of CEA and ErbB-2 on primary breast tumours screened by immunohistochemistry. Of 106 primary breast cancers, 69 (65%) were positive for CEA, 20 (19%) were positive for ErbB-2, and 13 (12%) expressed both antigens. We then prepared and evaluated a BAb directed against CEA and ErbB-2. Using BIACORE technology, we showed that the BAb recognised both CEA and ErbB-2 with affinities of 0.9 x 10 and 0.8 x 10 M(-1), respectively. In vivo, BAb tumour localisation was compared with that of its parental homodimeric F(ab')(2)-ORTHO-phenylene- dimaleimide (PDM) fragments. Uptake of (125)I-BAb was lower than that of (131)I-35A7F(ab')(2)-PDM in LS174T tumours, used as a model of CEA expressing tumours, and was similar to that of (131)I-FWP51 F(ab')(2)-PDM in SKOv3 tumours, used as a model of ErbB-2 expressing tumours. In a double-positive model, the SKOv3-CEA-1B9 tumour, BAb showed a similar uptake to that of 35A7 F(ab')(2)-PDM and we demonstrated that, although BAb had double specificity, it internalised as a homodimeric anti-ErbB-2 antibody. BAb showed a greater uptake than that of FWP51 F(ab')(2)-PDM and this difference was even more important 72 h after injection with an uptake of 7.3 +/- 2.1 vs. 1.4 +/- 0.5% of the injected dose per gram of tissue. The results obtained with the BAb in the double-positive tumour-bearing nude mice suggest that targeting two distinct tumour-associated antigens on the same cell could improve tumour localisation.
[Show abstract][Hide abstract] ABSTRACT: Immunophototherapy of cancer combines the specificity of a monoclonal antibody (MAb) to an overexpressed tumor marker with the phototoxic properties of the conjugated dye. To analyze the potential role of internalisation of the dye on photo-induced cytotoxicity, we compared two target antigens, carcinoembryonic antigen (CEA) that does not internalise and ErbB2 that does. Human ovarian carcinoma SKOv3 cells that express a high level of ErbB2 were transfected with the CEA cDNA. Using FACS analysis, the resulting cell line, SKOv3-CEA-1B9, demonstrated comparable levels of expression of the two target antigens. Aluminium tetrasulfophthalocyanine (AlPcS(4)) was covalently coupled to anti-CEA MAb 35A7, anti-ErbB2 MAb FSP77 and a non-specific MAb PX, via a five-carbon sulfonamide spacer chain (A(1)) at molar ratios ranging from 6 to 9 moles of AlPcS(4) per mole of MAb. The 35A7-(AlPcS(4)A(1))(8) conjugate induced 68% growth inhibition of the SKOv3-CEA-1B9 cell line after a 20 h incubation at 2.50 microg/ml (based on AlPcS(4)A(1) content) following light exposure. However, the FSP77-(AlPcS(4)A(1))(6) conjugate gave a 51% growth inhibition for an AlPcS(4)A(1) concentration as low as 0.04 microg/ml after the same incubation time and exposure to the same light dose. At a 1.25 microg/ml AlPcS(4)A(1) concentration, the FSP77-(AlPcS(4)A(1))(6) conjugate gave a 67% growth inhibition after an incubation time as short as 1 h, reaching a 96% inhibition after an 8 h incubation time. Using an unique cell line that expresses two different target antigens, we demonstrated a clear advantage of an internalising over a non-internalising MAb-dye conjugate in terms of phototoxic efficacy. In vivo evaluation of the photodynamic properties of the conjugates is in progress.
British Journal of Cancer 12/2001; 85(11):1787-93. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both experimental and clinical data show evidence of a correlation between elevated blood levels of carcinoembryonic antigen (CEA) and the development of liver metastases from colorectal carcinomas. However, a cause-effect relationship between these two observations has not been demonstrated. For this reason, we developed a new experimental model to evaluate the possible role of circulating CEA in the facilitation of liver metastases. A CEA-negative subclone from the human colon carcinoma cell line CO115 was transfected either with CEA-cDNA truncated at its 3' end by the deletion of 78 base pairs leading to the synthesis of a secreted form of CEA or with a full-length CEA-cDNA leading to the synthesis of the entire CEA molecule linked to the cell surface by a GPI anchor. Transfectants were selected either for their high CEA secretion (clone CO115-2C2 secreting up to 13 microg CEA per 10(6) cells within 72 h) or for their high CEA membrane expression (clone CO115-5F12 expressing up to 1 x 10(6) CEA molecules per cell). When grafted subcutaneously, CO115-2C2 cells gave rise to circulating CEA levels that were directly related to the tumour volume (from 100 to 1000 ng ml(-1) for tumours ranging from 100 to 1000 mm3), whereas no circulating CEA was detectable in CO115 and CO115-5F12 tumour-bearing mice. Three series of nude mice bearing a subcutaneous xenograft from either clone CO115-2C2 or the CO115-5F12 transfectant, or an untransfected CO115 xenograft, were further challenged for induction of experimental liver metastases by intrasplenic injection of three different CEA-expressing human colorectal carcinoma cell lines (LoVo, LS174T or CO112). The number and size of the liver metastases were shown to be independent of the circulating CEA levels induced by the subcutaneous CEA secreting clone (CO115-2C2), but they were directly related to the metastatic properties of the intrasplenically injected tumour cells.
British Journal of Cancer 08/1999; 80(9):1373-9. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 x 10(9) M(-1) for the 4 parental F(ab')2 fragments and from 1.88 to 10.14 x 10(9) 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.
International Journal of Cancer 05/1999; 81(2):285-91. · 6.20 Impact Factor