Targeting virulence regulation to disarm Acinetobacter baumannii pathogenesis.

The development of anti-virulence drug therapy against Acinetobacter baumannii infections would provide an alternative to traditional antibacterial therapy that are increasingly failing. Here, we demonstrate that the OmpR transcriptional regulator plays a pivotal role in the pathogenesis of diverse A. baumannii clinical strains in multiple murine and G. mellonella invertebrate infection models. We identified OmpR-regulated genes using RNA sequencing and further validated two genes whose expression can be used as robust biomarker to quantify OmpR inhibition in A. baumannii. Moreover, the determination of the structure of the OmpR DNA binding domain of A. baumannii and the development of in vitro protein-DNA binding assays enabled the identification of an OmpR small molecule inhibitor. We conclude that OmpR is a valid and unexplored target to fight A. baumannii infections and we believe that the described platform combining in silico methods, in vitro OmpR inhibitory assays and in vivo G. mellonella surrogate infection model will facilitate future drug discovery programs.

Novel Therapeutic Strategies for Ovarian Cancer Stem Cells.

Ovarian cancer (OC) is one of the most lethal gynecologic malignancies. Due to the lack of specific symptoms and screening methods, this disease is usually diagnosed only at an advanced and metastatic stage. The gold-standard treatment for OC patients consists of debulking surgery followed by taxane combined with platinum-based chemotherapy. Most patients show complete clinical remission after first-line therapy, but the majority of them ultimately relapse, developing radio- and chemoresistant tumors. It is now proposed that the cause of recurrence and reduced therapy efficacy is the presence of small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and for this reason, effective targeted therapies for the complete eradication of CSCs are urgently needed. In this review article, we highlight the mechanisms of CSC therapy resistance, epithelial-to-mesenchymal transition, stemness, and novel therapeutic strategies for ovarian CSCs.

L1 Cell Adhesion Molecule Confers Radioresistance to Ovarian Cancer and Defines a New Cancer Stem Cell Population.

Many solid tumors, including ovarian cancer, contain small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and play a role in disease recurrence. We demonstrated that the L1 cell adhesion molecule (L1CAM) is a new CSC target in ovarian cancer, triggering radioresistance. Using fluorescence-activated cell sorting, specific cell populations expressing L1CAM alone or in combination with the established CSC marker CD133 were isolated from three ovarian cancer cell lines. Double-positive L1CAM+/CD133+ cells displayed higher spherogenic and clonogenic properties in comparison to L1CAM-/CD133- cells. Furthermore, L1CAM+/CD133+ cells retained highest clonogenic capacity after irradiation and exhibited up-regulation of some CSC-specific genes, enhanced tumor-initiating capacity, self-renewal and higher tumor take rate in nude mice when compared with other cell populations. Superior radioresistance by L1CAM expression was confirmed by deletion of L1CAM using CRISPR-Cas9 technology. Moreover, we found expression signatures associated with epithelial-to-mesenchymal transition phenotype in L1CAM deleted cells. These results indicate that L1CAM in combination with CD133 defines a new cancer cell population of ovarian tumor-initiating cells with the implication of targeting L1CAM as a novel therapeutic approach for ovarian CSCs.

Combination of lutetium-177 labelled anti-L1CAM antibody chCE7 with the clinically relevant protein kinase inhibitor MK1775: a novel combination against human ovarian carcinoma.

BACKGROUND: Protein kinase inhibitors (PKIs) are currently tested in clinical studies (phase I-III) as an alternative strategy against (recurrent) ovarian cancer. Besides their anti-tumour efficacy, several PKIs have also shown radiosensitizing effects when combined with external beam radiation. Based on these results we asked if the addition of PKIs offers a therapeutic opportunity to improve radioimmunotherapy (RIT) against ovarian cancer. Five PKIs (alisertib, MK1775, MK2206, saracatinib, temsirolimus) were chosen for cytotoxicity screenings based on their current clinical trials in the treatment of ovarian cancer and their influence on cell cycle regulation and DNA damage repair pathways. We combined selected PKIs with 177Lu-labelled anti-L1CAM monoclonal antibody chCE7 for our investigations. METHODS: PKIs cytotoxicity was determined via cell colony-forming assays. Biomarker of DNA double-strand breaks (DSBs, γH2A.X) was analysed by western blot and fluorescence microscopy. Flow cytometric measurements were performed to evaluate levels of apoptosis based on mono- or combination treatments. The best combination was used for in vivo combination therapy studies in nude mice with SKOV3ip and IGROV1 human ovarian cancer xenografts. Bonferroni correction was used to determine statistical significance for multiple comparisons. RESULTS: The highest cytotoxicity against both cell lines was observed for MK1775 and alisertib. Combinations including 177Lu-labelled mAb chCE7 and MK1775 decreased 177Lu-DOTA-chCE7 IC60-values 14-fold, compared to 6-fold, when the radioimmunoconjugate was combined with alisertib. The most effective PKI MK1775 was further evaluated and demonstrated synergistic effects in combination with 177Lu-DOTA-chCE7 against IGROV1 cells. Significantly higher amounts of DSBs were detected in IGROV1 cells after combination (91%) compared to either treatment alone (MK1775: 52%; radioimmunoconjugate: 72%; p < 0.0125). Early-apoptosis was significantly enhanced in IGROV1 cells correlating with induced DSBs (177Lu-DOTA-chCE7: 8%, MK1775: 28%, 177Lu-DOTA-chCE7 + MK1775: 40%, p < 0.0125). Immunohistochemistry analysis of γH2A.X expression levels after therapy in SKOV3ip xenografts revealed a high sensitivity of the tumour cells to MK1775 and a high radioresistance. A prominent effect of tumour growth inhibition of the RIT and of the combination therapy was observed in vivo in a late stage IGROV1 xenograft model. CONCLUSIONS: Our results warrant further evaluation of combination of MK1775 and radioimmunotherapy.

Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model.

PURPOSE: The L1 cell adhesion molecule (L1CAM) is considered a valuable target for therapeutic intervention in different types of cancer. Recent studies have shown that anti-L1CAM radioimmunotherapy (RIT) with (67)Cu- and (177)Lu-labelled internalising monoclonal antibody (mAb) chCE7 was effective in the treatment of human ovarian cancer xenografts. In this study, we directly compared the therapeutic efficacy of anti-L1CAM RIT against human ovarian cancer under equitoxic conditions with the radiolanthanide (177)Lu and the potential alternative (161)Tb in an ovarian cancer therapy model. METHODS: Tb was produced by neutron bombardment of enriched (160)Gd targets. (161)Tb and (177)Lu were used for radiolabelling of DOTA-conjugated antibodies. The in vivo behaviour of the radioimmunoconjugates (RICs) was assessed in IGROV1 tumour-bearing nude mice using biodistribution experiments and SPECT/CT imaging. After ascertaining the maximal tolerated doses (MTD) the therapeutic impact of 50 % MTD of (177)Lu- and (161)Tb-DOTA-chCE7 was evaluated in groups of ten mice by monitoring the tumour size of subcutaneous IGROV1 tumours. RESULTS: The average number of DOTA ligands per antibody was 2.5 and maximum specific activities of 600 MBq/mg were achieved under identical radiolabelling conditions. RICs were stable in human plasma for at least 48 h. (177)Lu- and (161)Tb-DOTA-chCE7 showed high tumour uptake (37.8-39.0 %IA/g, 144 h p.i.) with low levels in off-target organs. SPECT/CT images confirmed the biodistribution data. (161)Tb-labelled chCE7 revealed a higher radiotoxicity in nude mice (MTD: 10 MBq) than the (177)Lu-labelled counterpart (MTD: 12 MBq). In a comparative therapy study with equitoxic doses, tumour growth inhibition was better by 82.6 % for the (161)Tb-DOTA-chCE7 than the (177)Lu-DOTA-chCE7 RIT. CONCLUSIONS: Our study is the first to show that anti-L1CAM (161)Tb RIT is more effective compared to (177)Lu RIT in ovarian cancer xenografts. These results suggest that (161)Tb is a promising candidate for future clinical applications in combination with internalising antibodies.

Paclitaxel improved anti-L1CAM lutetium-177 radioimmunotherapy in an ovarian cancer xenograft model.

BACKGROUND: Today's standard treatment of advanced-stage ovarian cancer, including surgery followed by a paclitaxel-platinum-based chemotherapy, is limited in efficacy. Recently, we could show that radioimmunotherapy (RIT) with (177)Lu-labelled anti-L1 cell adhesion molecule (L1CAM) monoclonal antibody chCE7 is effective in ovarian cancer therapy. We investigated if the efficacy of anti-L1CAM RIT can be further improved by its combination with paclitaxel (PTX). METHODS: In vitro cell viability and cell cycle arrest of human ovarian cancer cells were assessed upon different treatment conditions. For therapy studies, nude mice (n = 8) were injected subcutaneously with IGROV1 human ovarian carcinoma cells and received a single dose of 6 MBq (177)Lu-DOTA-chCE7 alone or in combination with 600 µg PTX (31.6 mg/kg). Tumour growth delay and survival were determined. To investigate whether PTX can influence the tumour uptake of the radioimmunoconjugates (RICs), a biodistribution study (n = 4) and SPECT/CT images were acquired 120 h post injections of 2 MBq (177)Lu-DOTA-chCE7 alone or in combination with 600 µg PTX. RESULTS: Lu-DOTA-chCE7 in combination with PTX revealed a significantly decreased cell viability of ovarian carcinoma cells in vitro and was effective in a synergistic manner (combination index < 1). PTX increased the RIT efficacy by arresting cells in the radiosensitive G2/M phase of the cell cycle 24 h post treatment start. In vivo combination therapy including (177)Lu-DOTA-chCE7 and PTX resulted in a significantly prolonged overall survival (55 days vs. 18 days/PTX and 29 days/RIT), without weight loss and/or signs of toxicity. Biodistribution studies revealed no significant difference in tumour uptakes of (177)Lu-DOTA-chCE7 72 h post injection regardless of an additional PTX administration. CONCLUSIONS: Combination of anti-L1CAM (177)Lu-RIT with PTX is a more effective therapy resulting in a prolonged overall survival of human ovarian carcinoma-bearing nude mice compared with either monotherapy. The combination is promising for future clinical applications.

Future prospects for SPECT imaging using the radiolanthanide terbium-155 - production and preclinical evaluation in tumor-bearing mice.

INTRODUCTION: We assessed the suitability of the radiolanthanide (155)Tb (t1/2=5.32 days, Eγ=87 keV (32%), 105keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging. METHODS: (155)Tb was produced at ISOLDE (CERN, Geneva, Switzerland) by high-energy (~1.4 GeV) proton irradiation of a tantalum target followed by ionization and on-line mass separation. (155)Tb was separated from isobar and pseudo-isobar impurities by cation exchange chromatography. Four tumor targeting molecules - a somatostatin analog (DOTATATE), a minigastrin analog (MD), a folate derivative (cm09) and an anti-L1-CAM antibody (chCE7) - were radiolabeled with (155)Tb. Imaging studies were performed in nude mice bearing AR42J, cholecystokinin-2 receptor expressing A431, KB, IGROV-1 and SKOV-3ip tumor xenografts using a dedicated small-animal SPECT/CT scanner. RESULTS: The total yield of the two-step separation process of (155)Tb was 86%. (155)Tb was obtained in a physiological l-lactate solution suitable for direct labeling processes. The (155)Tb-labeled tumor targeted biomolecules were obtained at a reasonable specific activity and high purity (>95%). (155)Tb gave high quality, high resolution tomographic images. SPECT/CT experiments allowed excellent visualization of AR42J and CCK-2 receptor-expressing A431 tumors xenografts in mice after injection of (155)Tb-DOTATATE and (155)Tb-MD, respectively. The relatively long physical half-life of (155)Tb matched in particular the biological half-lives of (155)Tb-cm09 and (155)Tb-DTPA-chCE7 allowing SPECT imaging of KB tumors, IGROV-1 and SKOV-3ip tumors even several days after administration. CONCLUSIONS: The radiolanthanide (155)Tb may be of particular interest for low-dose SPECT prior to therapy with a therapeutic match such as the ß(-)-emitting radiolanthanides (177)Lu, (161)Tb, (166)Ho, and the pseudo-radiolanthanide (90)Y.

DOTA-functionalized polylysine: a high number of DOTA chelates positively influences the biodistribution of enzymatic conjugated anti-tumor antibody chCE7agl.

Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N'-N''-N'''-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA)1-decalysine, (DOTA)3-decalysine or (DOTA)5-decalysine to the antibody heavy chain (via Gln295/297) gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with (177)Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA)-decalysine]2, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA)3-decalysine]2 and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA)5-decalysine)]2. The rapid elimination from the blood of chCE7agl-[(DOTA)-decalysine]2 (1.0±0.1% ID/g at 24 h) is associated with a high liver accumulation (23.2±4.6% ID/g at 24 h). This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.1±1.0 (DOTA)3 versus 11.7±1.4% ID/g (DOTA)5, p<0.005 at 24 h) and lower radioactivity levels in the liver (21.4±3.4 (DOTA)3 versus 5.8±0.7 (DOTA)5, p<0.005 at 24 h). We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA)5-decalysine) to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable option for radioimmunotherapy and potentially antibody-drug conjugates (ADCs).

L1-CAM-targeted antibody therapy and (177)Lu-radioimmunotherapy of disseminated ovarian cancer.

The L1-cell adhesion molecule (L1-CAM) is highly expressed in various cancer types including ovarian carcinoma but is absent from most normal tissue. A chimeric monoclonal antibody, chCE7, specifically binds to human L1-CAM and exhibits anti-proliferative effects on L1-CAM-expressing tumor cells. The goal of this study was to evaluate the efficacy of a novel (177)Lu-chCE7 radioimmunotherapeutic agent and to compare it to a treatment protocol with unlabeled, growth-inhibiting chCE7 in a mouse xenograft model of disseminated ovarian cancer. chCE7agl, an aglycosylated IgG1 variant with improved pharmacokinetics, was conjugated with 1,4,7,10-tetraazacyclododecane-N-N'-N'-N‴-tetraacetic acid (DOTA) and labeled with the low-energy ß-emitter (177)Lu. Tumor growth and survival were assessed after a single i.v. dose of 8 MBq (60 µg) radioimmunoconjugate in nude mice bearing either subcutaneous or intraperitoneal SKOV3.ip1 human ovarian cancer tumors. Therapeutic efficacy was compared with three times weekly i.p. administration of 10 mg/kg unconjugated chCE7. In vivo analysis of (177)Lu-chCE7agl biodistribution demonstrated high and specific accumulation of radioactivity at the tumor site with maximal tumor uptake of up to 48.0 ± 8.1% ID/g at 168 h postinjection. A single treatment with (177)Lu-DOTA-chCE7agl caused significant retardation of tumor growth and prolonged median survival from 33 to 71 days, while administration of a nontargeted (177)Lu-immunoconjugate had no beneficial effect. Three times weekly i.p. application of unlabeled chCE7 10 mg/kg similarly increased survival from 44 to 72 days. We conclude that a single dose of (177)Lu-DOTA-chCE7agl is as effective as repeated administration of nonradioactive chCE7 for treatment of small intraperitoneal tumors expressing L1-CAM.

The low-energy ß(-) and electron emitter (161)Tb as an alternative to (177)Lu for targeted radionuclide therapy.

INTRODUCTION: The low-energy ß(-) emitter (161)Tb is very similar to (177)Lu with respect to half-life, beta energy and chemical properties. However, (161)Tb also emits a significant amount of conversion and Auger electrons. Greater therapeutic effect can therefore be expected in comparison to (177)Lu. It also emits low-energy photons that are useful for gamma camera imaging. METHODS: The (160)Gd(n,γ)(161)Gd→(161)Tb production route was used to produce (161)Tb by neutron irradiation of massive (160)Gd targets (up to 40 mg) in nuclear reactors. A semiautomated procedure based on cation exchange chromatography was developed and applied to isolate no carrier added (n.c.a.) (161)Tb from the bulk of the (160)Gd target and from its stable decay product (161)Dy. (161)Tb was used for radiolabeling DOTA-Tyr3-octreotate; the radiolabeling profile was compared to the commercially available n.c.a. (177)Lu. A (161)Tb Derenzo phantom was imaged using a small-animal single-photon emission computed tomography camera. RESULTS: Up to 15 GBq of (161)Tb was produced by long-term irradiation of Gd targets. Using a cation exchange resin, we obtained 80%-90% of the available (161)Tb with high specific activity, radionuclide and chemical purity and in quantities sufficient for therapeutic applications. The (161)Tb obtained was of the quality required to prepare (161)Tb-DOTA-Tyr3-octreotate. CONCLUSIONS: We were able to produce (161)Tb in n.c.a. form by irradiating highly enriched (160)Gd targets; it can be obtained in the quantity and quality required for the preparation of (161)Tb-labeled therapeutic agents.

The soluble form of the cancer-associated L1 cell adhesion molecule is a pro-angiogenic factor.

A soluble form of the L1 cell adhesion molecule (sL1) is released from various tumor cells and can be found in serum and ascites fluid of uterine and ovarian carcinoma patients. sL1 is a ligand for several Arg-Gly-Asp (RGD)-binding integrins and can be deposited in the extracellular matrix. In this study we describe a novel function of this physiologically relevant form of L1 as a pro-angiogenic factor. We demonstrated that the anti-L1 monoclonal antibody (mAb) chCE7 binds near or to the sixth Ig-like domain of human L1 which contains a single RGD sequence. mAb chCE7 inhibited the RGD-dependent adhesion of ovarian carcinoma cells to sL1 and reversed the sL1-induced proliferation, matrigel invasion and tube formation of bovine aortic endothelial (BAE) cells. A combination of sL1 with vascular endothelial growth factor-A (VEGF-A(165)), which is an important angiogenic inducer in tumors, strongly potentiated VEGF receptor-2 tyrosine phosphorylation in BAE cells. Chick chorioallantoic membrane (CAM) assays revealed the pro-angiogenic potency of sL1 in vivo which could be abolished by chCE7. These results indicate an important role of released L1 in tumor angiogenesis and represent a novel function of antibody chCE7 in tumor therapy.

Antibodies directed against L1-CAM synergize with Genistein in inhibiting growth and survival pathways in SKOV3ip human ovarian cancer cells.

Antibodies directed against the L1 cell adhesion protein inhibit growth of SKOV3ip human ovarian cancer cells in vitro and in vivo. Responses of SKOV3ip cells in vitro to anti-L1 mAb chCE7 and Genistein were investigated. Genistein potentiated the anti-proliferative and pro-apoptotic effects of chCE7 in SKOV3ip cells. A combination of mAb chCE7 and Genistein strongly reduced the sensitivity of p44/42 (Erk1,2) kinase, Src kinase and Akt kinase to extracellular stimulation with serum, Epidermal Growth Factor and Hepatocyte Growth Factor. The observed synergy of antibodies directed against L1 with Genistein could lead to a new therapeutic option for ovarian cancer.

Modification of different IgG1 antibodies via glutamine and lysine using bacterial and human tissue transglutaminase.

The modification of proteins by chemical methods is well-established, however usually difficult to control. In this paper, we describe the posttranslational modification of different IgGs via the Lys or Gln side chains catalyzed by bacterial and human tissue transglutaminase (BTGase and TG2). For proof of concept, different IgG1s (commercial bovine IgG1, and L1CAM targeting chCE7 and chCE7 aglycosylated) were enzymatically functionalization with different fluorescent TGase substrates based on the CY3 analogue Dy547. The optimal reaction conditions were determined in order to assess the two enzymes. The efficiency of the enzymatic method was also compared with a standard chemical method employing a reactive NHS ester of Dy547. Three new TGase substrates were synthesized for this study including Lys-substrate 1 useful for BTGase and TG2 and two Gln-substrates tailor-made for BTGase (substrate 2) and TG2 (substrate 3). Of the two TGases tested, BTGase incorporated Lys-substrate 1 more efficiently than TG2. On the other hand, both enzymes reacted equally efficiently with the corresponding Gln-substrates 2 and 3. Reproducible labeling could be achieved in a broad concentration "window" of the substrates (up to 400 microM) without the risk of overlabeling of chCE7 or chCE7 aglycosylated. The biological activities of the functionalized antibodies were unaltered as shown by in vitro antigen affinity measurements and cell internalization experiments using confocal laser scanning microscopy. A maximum label-to-protein ratio of approximately 1 was achieved with chCE7 aglycosylated and substrate 1 using BTGase. It is important to recognize that the enzymatic activity of TGases enables the stable functionalization of proteins via the side chains of Gln, which is not possible by any chemical method available today. In addition, we could prove that the enzymatic modification of all antibodies occurred selectively at the heavy chain whereas the chemical method led to labeling of both the heavy and the light chains.

Copper-67 radioimmunotherapy and growth inhibition by anti-L1-cell adhesion molecule monoclonal antibodies in a therapy model of ovarian cancer metastasis.

PURPOSE: We examined the tumor-targeting and therapeutic effects of (67)Cu-labeled single amino acid mutant forms of anti-L1 monoclonal antibody chCE7 in nude mice with orthotopically implanted SKOV3ip human ovarian carcinoma cells. EXPERIMENTAL DESIGN: For radioimmunotherapy, chCE7 antibodies with a mutation of histidine 310 to alanine (chCE7H310A) and a mutation of asparagine 297 to glutamine (chCE7agl) were generated to achieve more rapid blood clearance. Biodistributions of (67)Cu-4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl benzoic acid tetrachloride (CPTA)-labeled mutant antibodies were measured in nude mice bearing SKOV3ip human ovarian cancer metastases. The effects of single i.v. injections of (67)Cu-chCE7agl alone on tumor reduction and survival were investigated. In addition, a combination of low-dose (67)Cu-radioimmunotherapy with unlabeled anti-L1 antibody L1-11A on survival was investigated. RESULTS: (67)Cu-CPTA-chCE7agl showed high (up to 49% ID/g) and persistent (up to 168 h) uptake in SKOV3ip metastases, with low levels in normal tissues. (67)Cu-CPTA-chCE7H310A revealed a shorter half-life in the blood and a lower tumor uptake and retention. A single low dose of 4 MBq of (67)Cu-chCE7agl reduced tumor growth but did not prolong survival significantly, whereas a single 10.5 MBq dose of (67)Cu-chCE7agl reduced tumor growth and prolonged survival significantly. The combination of unlabeled monoclonal antibody L1-11A with a subtherapeutic dose of (67)Cu-radioimmunotherapy also prolonged survival significantly. CONCLUSION: The results show improved pharmacokinetics and biodistributions as well as the therapeutic effect of the (67)Cu-labeled single amino acid mutant chCE7agl. Therapeutic data indicate, for the first time, the feasibility of combining anti-L1-directed growth inhibition and (67)Cu-radioimmunotherapy, thereby increasing the efficiency of antibody treatment of metastatic ovarian carcinoma.

Evaluation of 177Lu-DOTA-labeled aglycosylated monoclonal anti-L1-CAM antibody chCE7: influence of the number of chelators on the in vitro and in vivo properties.

INTRODUCTION: In this study, we optimized the 1,4,7,10-tetraazacyclododecane-N-N'-N"-N"'-tetraacetic acid (DOTA) chelator-to-antibody (c/a) ratio for the aglycosylated variant of the anti-L1-CAM antibody chCE7 (chCE7agl), providing high specific activity and low liver uptake in 177Lu-labeled form. METHODS: chCE7agl was substituted with increasing molar excess of DOTA-NCS. The number of chelators coupled to the antibody and the binding affinities to target tumor cells (IC50 values) of the resulting immunoconjugates were determined. The different immunoconjugates were labeled with 177Lu; specific activity was measured, and metabolic stability was analyzed in human plasma. The effect of different c/a ratios on blood clearance and liver uptake was tested in nude mice. Changes of the protein backbone structure were analyzed by circular dichroism spectroscopy. RESULTS: chCE7agl was substituted with 7, 12 or 15 DOTA ligands. The IC50 concentrations displacing radioiodinated chCE7 antibody increased with the number of chelators (1.5-fold with 7 ligands, 2.5-fold with 12 ligands and a 5-fold increase with 15 ligands). The highest specific activity for 177Lu-DOTA-chCE7agl was obtained with a c/a ratio of 12 (106 MBq/mg). Radioimmunoconjugates were stable in human plasma for at least 24 h. Blood clearance and liver uptake were measured after 24 h (c/a ratios of 12 and 15) or 48 h (c/a ratio of 7). The liver-to-blood ratios were 0.35+/-0.14 (7 ligands), 0.77+/-0.19 (12 ligands) and 17.85+/-3.44 (15 ligands). CONCLUSIONS: DOTA-chCE7agl conjugates with a c/a ratio of 12 combined high specific activity with good in vitro and in vivo properties. The rapid elimination rate from the blood and the high uptake in the liver of chCE7agl substituted with 15 DOTA ligands were found not to be due to conformational changes of the antibody backbone structure.

Efficient inhibition of intra-peritoneal tumor growth and dissemination of human ovarian carcinoma cells in nude mice by anti-L1-cell adhesion molecule monoclonal antibody treatment.

The L1 cell adhesion molecule is implicated in the control of proliferation, migration, and invasion of several tumor cell types in vitro. Recently, L1 overexpression was found to correlate with tumor progression of ovarian carcinoma, one of the most common causes of cancer-related deaths in gynecologic malignant diseases. To evaluate L1 as a potential target for ovarian cancer therapy, we investigated the effects of anti-L1 monoclonal antibodies (chCE7 and L1-11A) on proliferation and migration of L1-positive human SKOV3ip ovarian carcinoma cells in vitro and the therapeutic efficacy of L1-11A against i.p. SKOV3ip tumor growth in nude mice. In vitro, both anti-L1 antibodies efficiently inhibited the proliferation of SKOV3ip cells as well as other L1-expressing tumor cell lines (renal carcinoma, neuroblastoma, and colon carcinoma). On two cell lines, hyper-cross-linking of L1-11A with a secondary antibody was necessary for significant inhibition of proliferation, indicating that cross-linking of L1 is required for the antiproliferative effect. L1-negative prostate carcinoma cells were not influenced by antibody treatment. Biweekly treatment of ovarian carcinoma-bearing mice with L1-11A led to a dose-dependent and significant reduction of tumor burden (up to -63.5%) and ascites formation (up to -75%). This effect was associated with reduced proliferation within the tumors. L1-directed antibody-based inhibition of peritoneal growth and dissemination of human ovarian carcinoma cells represents important proof-of-principle for the development of a new therapy against one of the leading gynecologic malignant diseases.

In vivo evaluation of 177Lu- and 67/64Cu-labeled recombinant fragments of antibody chCE7 for radioimmunotherapy and PET imaging of L1-CAM-positive tumors.

PURPOSE: The L1 cell adhesion protein is overexpressed in tumors, such as neuroblastomas, renal cell carcinomas, ovarian carcinomas, and endometrial carcinomas, and represents a target for tumor diagnosis and therapy with anti-L1-CAM antibody chCE7. Divalent fragments of this internalizing antibody labeled with 67/64Cu and 177Lu were evaluated to establish a chCE7 antibody fragment for radioimmunotherapy and positron emission tomography imaging, which combines high-yield production with improved clearance and biodistribution properties. EXPERIMENTAL DESIGN: chCE7F(ab')2 fragments were produced in high amounts (0.2 g/L) in HEK-293 cells, substituted with the peptide-linked tetraazamacrocycle 3-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-triglycyl-L-p-isothiocyanato-phenylalanine, and labeled with 67Cu and 177Lu. In vivo bioevaluation involved measuring kinetics of tumor and tissue uptake in nude mice with SK-N-BE2c xenografts and NanoPET (Oxford Positron Systems, Oxford, United Kingdom) imaging with 64Cu-3-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-triglycine-chCE7F(ab')2. RESULTS: The 177Lu- and 67Cu-labeled immunoconjugates reached maximal tumor accumulation at 24 hours after injection with similar levels of 12%ID/g to 14%ID/g. Blood levels dropped to 1.0%ID/g for the 177Lu fragment and 2.3%ID/g for the 67Cu fragment at 24 hours. The most striking difference concerned radioactivity present in the kidneys, being 34.5%ID/g for the 177Lu fragment and 16.0%ID/g for the 67Cu fragment at 24 hours. Positron emission tomography imaging allowed clear visualization of s.c. xenografts and peritoneal metastases and a detailed assessment of whole-body tracer distribution. CONCLUSIONS: 67/64Cu- and 177Lu-labeled recombinant chCE7F(ab')2 revealed suitable in vivo characteristics for tumor imaging and therapy but displayed higher kidney uptake than the intact monoclonal antibody. The 67Cu- and 177Lu-labeled immunoconjugates showed different in vivo behavior, with 67/64Cu-3-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-triglycine-F(ab')2 appearing as the more favorable conjugate due to superior tumor/kidney ratios.

Hepatocyte growth factor-induced ectodomain shedding of cell adhesion molecule L1: role of the L1 cytoplasmic domain.

The L1 cell adhesion molecule and its soluble form are tumor-associated proteins and potential markers for tumor staging as well as targets for therapeutic intervention. Soluble L1 is produced by metalloprotease-mediated ectodomain shedding of L1. We investigated effects of hepatocyte growth factor (HGF), a growth factor shown to increase invasiveness of renal carcinoma cells, on ectodomain shedding of L1 from these cells. All of the tested L1-positive renal carcinoma cell lines released a 180-kDa form of L1 into the medium. In the presence of serum, addition of HGF led to a dose-dependent increase in L1 shedding with a maximum reached at 5 ng/ml. In contrast, L1 shedding was inhibited by glial cell line-derived neurotrophic factor (GDNF). The tyrosine kinase inhibitor Genistein reduced basal and HGF-stimulated L1 shedding, indicating that protein phosphorylation is involved. To investigate the role of the L1 intracellular domain, two mutants of the L1 cytoplasmic part were constructed. L1trun lacking the complete intracellular domain showed enhanced basal shedding. In a L1YH mutant, containing the mutation tyrosine 1229 to histidine that deletes the ankyrin binding motif of L1, basal shedding was reduced. Disruption of actin assembly by cytochalasin D also reduced shedding of L1. These results indicate that the cytoplasmic domain regulates basal shedding of L1, and association with the cytoskeleton through the L1 ankyrin binding site is involved. HGF stimulated L1 shedding in both mutants, indicating that receptor-mediated phosphorylation in the L1 cytoplasmic domain is not required for HGF-stimulated shedding.