The state of the art: immune-mediated mechanisms of monoclonal antibodies in cancer therapy.

A number of antibody products have now become accepted as effective anti-cancer therapies. Despite being mainly designed to act by inhibiting functional tumour antigens, there is increasing evidence that Fc-mediated engagement of the immune system is an important contributor to the efficacy of several of these therapies. The optimisation of this engagement offers the potential not only to augment efficacy against existing targets, but also to exploit non-functional tumour antigens. Antibodies that achieve efficacy wholly or predominantly through Fc-mediated mechanisms, represent rich opportunities for future therapeutics in oncology. This mini review summarises some of the key challenges, which need to be addressed to select the most effective molecules. These include the identification of optimal antibody characteristics and improvement of the drug discovery process, in particular, the relevance and predictive power of existing in vitro and in vivo screening methods. Advances in our understanding of tumour immunobiology and successful application of technologies designed to enhance immune system engagement will further aid this process.

Phase I evaluation of CDP791, a PEGylated di-Fab' conjugate that binds vascular endothelial growth factor receptor 2.

PURPOSE: Specific blocking of vascular endothelial growth factor receptor 2 (VEGFR-2) is a novel therapeutic approach. Here, we report the first phase I clinical trial evaluation of CDP791, a PEGylated di-Fab' conjugate that binds VEGFR-2. EXPERIMENTAL DESIGN: Cohorts of patients received CDP791 at doses between 0.3 and 30 mg/kg every 3 weeks for the initial two doses. RESULTS: The compound was well tolerated with no dose-limiting toxicity. Dose-related hypertension was observed in patients receiving CDP791 10 mg/kg or more and several patients on the higher doses developed infusion-related cutaneous hemangiomata arising 28 to 106 days after the first drug administration and resolving 3 weeks after cessation. Biopsy and histologic evaluation showed that CDP791-bound VEGFR-2 is non-phosphorylated, suggesting that the drug is biologically active. Concentrations of CDP791 considered biologically relevant were sustained for 3 weeks when doses of 10 mg/kg or more were administered. Although no reductions in vascular permeability were recorded using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), there was a significant dose level-related reduction in tumor growth. While challenging the recent dogma that active VEGF inhibitors should modulate DCE-MRI measurements of vascular permeability, this highlights the potential of serial three-dimensional tumor measurements to detect tumor growth arrest. Twelve patients received drug for more than two treatments, although no partial or complete responses were seen. CONCLUSION: The data show that CDP791 is biologically active and well tolerated, achieving appropriate plasma concentrations when administered at 10 mg/kg or more every 3 weeks.

Blockade of platelet-derived growth factor receptor-beta by CDP860, a humanized, PEGylated di-Fab', leads to fluid accumulation and is associated with increased tumor vascularized volume.

PURPOSE: CDP860 is an engineered Fab' fragment-polyethylene glycol conjugate, which binds to and blocks the activity of the beta-subunit of the platelet-derived growth factor receptor (PDGFR-beta). Studies in animals have suggested that PDGFR-beta inhibition reduces tumor interstitial fluid pressure, and thus increases the uptake of concomitantly administered drugs. The purpose of this study was to determine whether changes in tumor vascular parameters could be detected in humans, and to assess whether CDP860 would be likely to increase the uptake of a concurrently administered small molecule in future studies. PATIENTS AND METHODS: Patients with advanced ovarian or colorectal cancer and good performance status received intravenous infusions of CDP860 on days 0 and 28. Patients had serial dynamic contrast-enhanced magnetic resonance imaging studies to measure changes in tumor vascular parameters. RESULTS: Three of eight patients developed significant ascites, and seven of eight showed evidence of fluid retention. In some patients, the ratio of vascular volume to total tumor volume increased significantly (P < .001) within 24 hours following CDP860 administration, an effect suggestive of recruitment of previously non-functioning vessels. CONCLUSION: These observations suggest that inhibition of PDGFR-beta might improve delivery of a concurrently administered therapy. However, in cancer patients, further exploration of the dosing regimen of CDP860 is required to dissociate adverse effects from beneficial effects. The findings challenge the view that inhibition of PDGF alone is beneficial, and confirm that effects of PDGFR kinase inhibition mediate, to some extent, the fluid retention observed in patients treated with mixed tyrosine kinase inhibitors.

Loss of cis-diamminedichloroplatinum(II) resistance in human ovarian carcinoma cells selected for rhodamine 123 resistance.

Cisplatin (DDP)-resistant 2008 human ovarian carcinoma cells (C13*) have an elevated mitochondrial membrane potential that confers hypersensitivity to lipophilic cations. We have addressed whether this change was directly linked to the DDP-resistant phenotype or was a random alteration, unrelated to resistance. The elevated mitochondrial membrane potential and ensuing rhodamine 123 (Rh123) hypersensitivity of C13* cells provided a positive selection rationale. Cells with low mitochondrial membrane potential will have a survival advantage over those with a high mitochondrial membrane potential, when exposed to Rh123. We therefore used Rh123 to isolate revertants (RH4) from the 12-fold DDP-resistant C13* cell line that had a low mitochondrial membrane potential. RH4 cells had parental (2008) mitochondrial membrane potential levels as shown by flow cytometry analysis of Rh123 stained cells and by tetraphenylphosphonium cation uptake. The RH4 cells lost a substantial portion of their DDP resistance such that they were only 2- to 3-fold resistant to DDP. Despite this major loss of resistance, they retained a number of the phenotypic changes related to DDP resistance, observed in C13* cells. RH4 cells displayed the same DDP accumulation defect, 2-fold elevated glutathione levels and 2-fold resistance to CdCl2 as C13* cells. We conclude that although the biochemical mechanism by which an elevated mitochondrial membrane potential elicits DDP resistance is unknown, these mitochondrial changes are central to the expression of DDP resistance in C13* cells.

Mitochondrial DNA modulation of the anchorage-independent phenotype of transformed avian cells.

The progressive loss of mitochondrial DNA in the presence of ethidium bromide in immortal avian cell lines correlates with a decrease in their potential for anchorage-independent growth in soft agar. In short-term treated cells, this effect is reversible and the recovery of cloning potential parallels the recovery of control levels of mitochondrial DNA. Long-term ethidium-bromide-treated cells are permanently respiration-deficient and display anchorage-dependent growth. Anchorage-independent revertants can be selected, suggesting that the lack of a respiratory chain per se might not be responsible for the inability of mitochondrial DNA-depleted cells to grow in soft agar. Cybrids formed from the fusion of mitochondrial DNA-depleted, anchorage-dependent cells to cytoplasts from parental cells are capable of growth in soft agar. The mitochondria-specific inhibitor, rhodamine 6G, prevents the recovery of the anchorage-independent phenotype in similar hybrids. These results suggest that mitochondrial DNA is required to maintain the transformed phenotype of avian cells.

Tumor-forming ability in athymic nude mice of human cell lines devoid of mitochondrial DNA.

We have examined the contribution of the mitochondrial genome to the tumorigenic phenotype expressed by human cell lines derived from an ovarian and a cervical carcinoma and from an osteogenic sarcoma. All these continuous cell lines are anchorage-independent in soft agar and form tumors in athymic nude mice. Long-term exposure of the cells to ethidium bromide, an intercalating agent which inhibits mitochondrial DNA replication, gave rise to subclones depleted of mitochondrial DNA and RNA molecules and displaying either anchorage independence or dependence. These respiratory-deficient subclones contain disorganized and enlarged mitochondria, are auxotrophic for uridine and pyruvate, and grow in vitro at a rate nearly identical or moderately slower than their respective parent. The tumor-forming ability of both anchorage-independent and -dependent cell lines was tested by s.c. and intramuscular implantation of the cells in nude mice. We found that the tumorigenic capacity was influenced by the route of inoculation. Subcutaneously, mitochondrial DNA-less cell lines are either poorly or nontumorigenic, while all but one cell line form tumors when implanted into the hind leg muscle. The relative in vivo growth rate of the parent and the mitochondrial DNA-less subclones reflects their respective in vitro rate of growth. All intramuscular tumors introduced into culture mimic the molecular and phenotypic traits of the injected cells, with the exception of the anchorage-dependent cell lines which give rise to anchorage-independent tumor cell lines. The present observations indicate that human cells without mitochondrial DNA have the capacity to proliferate and form tumors in vivo.

Anti-major histocompatibility complex class II treatment prevents graft rejection in the hamster-to-rat cardiac xenograft.

BACKGROUND: Several groups have achieved graft acceptance in the concordant hamster to rat model by using a combination of anti-proliferative drugs and conventional immunosuppressive therapy. However, such aggressive treatment often leads to the recipient dying with a functional xenograft, as a result of opportunistic infections. This study aimed to investigate the effects of a short course of therapy with an anti-MHC class II monoclonal antibody treatment (chimeric OX6 [cOX6]) in combination with cyclosporin A (CyA) in a concordant hamster-to-rat xenograft model. METHODS: Rats receiving hamster cardiac xenografts were given CyA or cOX6 alone or in combination and were monitored daily to assess the effect of treatment on graft survival. Additional studies monitored the effect of treatment on the production of cytolytic anti-hamster antibodies by the recipient and the deposition of immunoglobulin (Ig)M and complement factors within the xenograft. RESULTS: Treatment with CyA only had no effect on graft survival, whereas treatment with cOX6 increased graft survival time by 2 days. The median graft survival time for cOX6+CyA was 76 days. cOX6 treatment of rats having undergone transplants inhibited the rise in cytotoxic anti-hamster antibodies in peripheral blood until day 5, whereas combination therapy completely inhibited anti-hamster antibody formation. Fluorescence-activated cell sorter analysis showed treatment with cOX6 significantly reduced circulating B cell numbers until day 5. Anti-MHC class II treatment also markedly reduced the deposition of both IgM and C3. Anti-MHC class II treatment with CyA gives long term survival in concordant xenografts without severe side effects. CONCLUSIONS: The mechanism of action of this combination is complex and could be caused by an initial inhibition of B cell function by the anti-MHC class II treatment and the subsequent inhibition of T cell dependent pathways by CyA.

On the tumorigenicity of mitochondrial DNA-depleted avian cells.

We have examined the tumorigenic potential of mitochondrial DNA-depleted (mtDNA-) cells derived from the tumorigenic chicken cell line DU24. The mtDNA- cells were unable to proliferate in the wing web of day-old chicks. Cytoplasmic hybrids resulting from crosses between the mtDNA- whole cells and cytoplasts from enucleated parental cells (mtDNA+) recover both mtDNA and tumorigenicity. These results are in accordance with those obtained in prior experiments where mtDNA was shown to modulate the anchorage-independent phenotype of transformed avian cells.

New locus for exopolysaccharide overproduction in Escherichia coli K-12.

A new locus for exopolysaccharide overproduction in Escherichia coli K-12 was mapped by insertion mutagenesis. A 66% linkage to serA, which is located at 62 min on the E. coli K-12 linkage map, was shown by P1 transduction. The polysaccharide produced by the mutant was isolated and was shown to be similar to colanic acid.

Development and characterization of mutant chicken cell lines for somatic cell genetics studies.

A series of stable mutants bearing nuclear genetic markers were developed from the established chicken cell line DU24. The mutants were obtained after mutagenesis of DU24 cells with ethyl methanesulfonate (EMS) or arose spontaneously when plated in the appropriate selective medium. Clones resistant to 5-bromodeoxyuridine (BrdU) were obtained following a two-step selection procedure and analyzed. The BrdUr cells were found to be deficient in thymidine kinase activity and were HAT sensitive. Molecular characterization of these mutants revealed no deletions or other rearrangements, but methylation of some cytosine residues was decreased in the mutants. A similar restriction profile was seen in a series of mutants made resistant to BrdU after cultivation of DU24 cells in increasing concentrations of the drug over a period of six months. Selection of EMS-treated BrdUr cells in 10 microM ouabain gave rise to a clone resistant to both drugs and which was still HAT sensitive. Clones resistant to 6-thioguanine were also isolated, but showed wild-type hypoxanthine phosphoribosyltransferase activity and were HAT resistant. A number of the cell lines isolated were found to be suitable for fusion experiments with both chicken cells and cells from other vertebrate species.

Development and characterization of continuous avian cell lines depleted of mitochondrial DNA.

Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy.

Transplantability of myelocytomatosis MC29 virus-producing cell lines in chicken. Lateral transformation of host cells.

The transplantability of myelocytomatosis MC29 virus-producing chicken cell lines BR-3 and OB-1 was examined in 1-day-old chicks. Both cell lines, derived from the chicken cell line DU249, harbor nuclear genetic markers for drug resistance. When these cells were inoculated subcutaneously in the wing web of chicks, tumors developed at the site of injection in 65% of the cases. Propagation in culture of cells obtained by dispase treatment of the excised tumors, followed by selection for drug resistance, revealed that only a fraction of the tumors (27% and 60% for BR-3 and OB-1, respectively) resulted from the growth of the injected cells. The other tumors derive from the proliferation of host cells transformed by MC29 virus released by the injected cells. Tumor development subsequent to similar inoculation of 1-day-old chicks with cell-free virus stock further confirmed the tumorigenic capacity of MC29 at the site of injection. These results underline the need for the determination of tumor cell origin in studies using MC29-producing cell lines and highlight the advantage of using cells with drug-resistant markers for such work. In the course of this study we determined by immunohistochemical criteria, the myogenic origin of some of the virally induced tumors. This is the first report of in vivo transformation of muscle cells by MC29-containing virus stocks in the newly hatched chick.