Ilse Novak-Hofer

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.

Copper-67 as a therapeutic nuclide for radioimmunotherapy

Abstract. The application of the beta particle-emitting nuclide 67Cu in radioimmunotherapy is reviewed. The production of the nuclide is outlined, and different production modes are discussed with an emphasis on cyclotron production. A short survey of copper chelators currently used for antibody labelling and their impact on the pharmacokinetics of 67Cu-labelled immunoconjugates is provided. Protocols for antibody labelling with 67Cu as well as quality control procedures for 67Cu-labelled antibodies are described. Preclinical data on the biological properties of 67Cu-labelled immunoconjugates are reported and discussed. 67Cu-labelled antibodies show higher and more persistent tumour uptake than their radioiodinated counterparts due to accumulation of labelled metabolites in tumour cells. Biodistribution of 67Cu-labelled antibody fragments has been improved by selection of negatively charged chelators and peptide linkers. Pharmacokinetic analysis of the accumulated dose in tumour and critical organs such as the kidney and liver indicates that, despite this improvement, intact 67Cu-labelled antibodies achieve higher tumour uptake and better therapeutic ratios than 67Cu-labelled antibody fragments and that they are at present the logical choice for clinical studies. Clinical studies using 67Cu-labelled antibodies in lymphoma, colon carcinoma and bladder cancer patients are reviewed. Some of the advantages over radioiodinated antibodies found in the preclinical work, such as higher tumour uptake and better tumour/blood ratios, have also been found with systemic application in lymphoma and colon carcinoma. However, in both lymphoma and colon carcinoma patients, the radiation dose to the liver has been found to be higher from 67Cu- than from 131I-labelled antibodies. The intravesical application of 67Cu-labelled antibody has been shown to be a promising approach for targetting cytotoxic radiation to superficial bladder tumours, without detectable systemic absorption. Given the favourable properties of 67Cu-labelled antibodies, it is the reliable availability of the 67Cu nuclide which is the limiting factor for their more widespread evaluation in radioimmunotherapy trials.

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 67Cu-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 67Cu-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 67Cu-chCE7agl alone on tumor reduction and survival were investigated. In addition, a combination of low-dose 67Cu-radioimmunotherapy with unlabeled anti-L1 antibody L1-11A on survival was investigated. Results:67Cu-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. 67Cu-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 67Cu-chCE7agl reduced tumor growth but did not prolong survival significantly, whereas a single 10.5 MBq dose of 67Cu-chCE7agl reduced tumor growth and prolonged survival significantly. The combination of unlabeled monoclonal antibody L1-11A with a subtherapeutic dose of 67Cu-radioimmunotherapy also prolonged survival significantly. Conclusion: The results show improved pharmacokinetics and biodistributions as well as the therapeutic effect of the 67Cu-labeled single amino acid mutant chCE7agl. Therapeutic data indicate, for the first time, the feasibility of combining anti–L1-directed growth inhibition and 67Cu-radioimmunotherapy, thereby increasing the efficiency of antibody treatment of metastatic ovarian carcinoma.

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.

IRIV-adjuvanted hepatitis A vaccine : in vivo absorption and biophysical characterization

Immunopotentiating reconstituted influenza virosomes (IRIV) are 150-nm proteoliposomes composed of influenza surface glycoproteins and a mixture of natural and synthetic phospholipids. Due to size, structure and composition of the IRIVs, they serve as an antigen carrier system for efficacious vaccination, as was demonstrated for hepatitis A and influenza. This paper reviews the unique properties of IRIVs and describes the in vivo biodistribution of model antigens using 14C-labeled IRIVs and 125I-labeled streptavidin. IRIV formulated streptavidin induced a strong depot effect after intra muscular (i.m.) vaccination of mice, whereas soluble streptavidin was soon eliminated via the kidney of the animals. A mixture of antigen and IRIVs yielded higher antibody titers after i.m. inoculation than streptavidin alone. The highest immunostimulation was achieved by the binding of the antigen to the investigated adjuvant. The potential penetration of inactivated hepatitis A virions into lipid membranes was assessed by measuring the area increase of a lipid monolayer kept at a constant surface pressure corresponding to that of lipid bilayer vesicles. The monolayers were composed of phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) (75/25 mol/mol), thus resembling the lipid composition of the IRIV. The results suggested that the hepatitis A antigen may spontaneously bind to the reconstituted IRIV membranes.

Targeting of renal carcinoma with 67/64Cu-labeled anti-L1-CAM antibody chCE7: selection of copper ligands and PET imaging.

In order to optimize radiocopper labeling of anti-L1-CAM antibody chCE7, five bifunctional copper chelators were synthesized and characterized (CPTA-N-hydoxysuccinimide, DO3A-L-p-isothiocyanato-phenylalanine, DOTA-PA-L-p-isocyanato-phenylalanine, DOTA-glycyl-L-p-isocyanato-phenylalanine and DOTA-triglycyl-L-p-isocyanato-phenylalanine). Substitution with more than 11 chelators per antibody molecule was found to influence immunoreactivity and biodistributions of (67)Cu-MAb chCE7 significantly. CPTA-labeled antibody achieved the best tumor to normal tissue ratios when biodistributions of the different (67)Cu-chCE7 conjugates were assessed in tumor-bearing mice. High resolution PET imaging with (64)Cu-CPTA-labeled MAb chCE7 showed uptake in lymph nodes and heterogeneous distribution in tumor xenografts.

Anti‐neuroblastoma antibody chCE7 binds to an isoform of L1‐CAM present in renal carcinoma cells

Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti‐neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 Mr cell surface protein. The protein was partially purified by immuno‐affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH2‐terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1‐CAM), and both were characterized by the NH2‐terminal deletion of 5 amino acids, comprising exon 2 of L1‐CAM. Reverse trancription‐polymerase chain reaction (RT‐PCR) analysis of the regions spanning exon 2 and exon 27 of L1‐CAM indicated that in neuroblastoma cells both transcripts for the full‐length and exon‐deleted forms are present, whereas in the renal carcinoma cell lines only the exon‐deleted L1‐CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1‐CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1‐CAM mRNA levels are correlated with protein expression. Int. J. Cancer 83: 401–408, 1999

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.

L1-CAM-targeted antibody therapy and 177Lu-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 177Lu‐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 β‐emitter 177Lu. 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 177Lu‐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 177Lu‐DOTA‐chCE7agl caused significant retardation of tumor growth and prolonged median survival from 33 to 71 days, while administration of a nontargeted 177Lu‐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 177Lu‐DOTA‐chCE7agl is as effective as repeated administration of nonradioactive chCE7 for treatment of small intraperitoneal tumors expressing L1‐CAM.

Cleavable linkers to enhance selectivity of antibody-targeted therapy of cancer

Radioimmunotherapy of cancer utilizes anti-tumor antibodies or antibody fragments conjugated to radionuclides to deliver radiation selectively to tumors. However, radiolabeled proteins deposit radioactivity in normal organs that metabolize or conserve proteins and peptides, primarily liver and kidneys. To accelerate the clearance of radioactivity from normal tissues, linkers between the antibody or antibody fragment and the radioactive moiety have been designed for cleavage in the liver and kidneys, to liberate low molecular weight radioactive species for rapid excretion. Modest success in improving the tumor-to-liver and tumor-to-kidney radiation dose ratios have been achieved in preclinical studies. Such changes when taken to clinical studies have suggested useful impact on therapeutic work. Recent advances in the development of cleavable linkers are described.