A strategy for antitumor vascular therapy by targeting the vascular endothelial growth factor: receptor complex.

Vascular endothelial growth factor (VEGF) is produced by cancer cells in response to hypoxia and is the primary stimulant of vascularization in solid tumors. Endothelial cells lining the blood vessels of these tumors have a high concentration of receptor-bound VEGF on their surface, providing a target for antibody- directed cancer therapy. To obtain a cloned antibody to this target when bound to its receptor on tumor endothelium, we used phage display technology to create a single-chain Fv (sFv) antibody library from mice immunized with the 165-amino acid isoform of human VEGF-A. We selected, purified, and characterized LL4, an anti-VEGF sFv that was shown to react with receptor-bound VEGF. LL4 bound selectively to blood vessel endothelium, as shown by immunohistochemistry on tissue sections of human tumors. Furthermore, using autoradiography and grain counting of histological sections, systemically administered LL4 was shown to localize selectively to the endothelial lining of tumor blood vessels in human colorectal carcinoma xenografts in vivo. This study demonstrates the feasibility of targeting tumor vasculature using recombinant antibodies to the VEGF:receptor complex.

Characterization of human IgG-binding xenoantigens expressed by porcine aortic endothelial cells.

Xenoreactive antibodies (XAb) play a major role in the rejection of xenografts. In this study, human IgG XAb that bind to xenoantigens expressed by porcine aortic endothelial cells (PAEC) were characterized, together with their corresponding xenoantigens. Using an ELISA with both fixed and unfixed confluent monolayers of PAEC, XAb of both IgG and IgM classes in pooled and individual normal human serum were identified. The binding of these IgG XAb to the endothelium is mediated by F(ab')2 and the only detectable subclasses that bind to the endothelium are IgG1 and IgG2. On the basis of direct binding experiments, inhibition and antibody adsorption studies, and enzymatic digestions, it is shown that only a minor component of the XAb binding is directed against galactose in an alpha 1,3 linkage with galactose on PAEC surfaces. There is some cross-reactivity with antigens expressed on porcine lymphocytes, but not porcine red blood cells. Histological examination of sections of porcine aortae, snap-frozen and stained using immunoperoxidase techniques, confirmed interaction with the vascular endothelium. Labeling of the PAEC with 125I, followed by cell lysis and immunoprecipitation under reducing conditions, showed binding of IgG XAb to several components on the endothelial cell surface, the most prominent of which have apparent molecular masses of 75 kDa, 110 kDa, 180 kDa, and 210 kDa. The 110-kDa component and the 180-kDa component were sensitive to digestion with endoglycosidase F, which suggests the participation of N-linked carbohydrate structures. These studies demonstrate that human IgG XAb recognize multiple determinants expressed by PAEC, a minor population of which contain alpha 1,3-linked galactose residues. Cross-reactive determinants are expressed on porcine lymphocytes but not porcine red blood cells.

Clinical applications of phage-derived sFvs and sFv fusion proteins.

Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNF alpha aims to reduce sequestration and increase tumor concentrations of systemically administered TNF alpha.

Human IgG xenoreactive antibodies mediate damage to porcine endothelial cells in vitro by both humoral and cellular mechanisms.

A major barrier to the transplantation of a porcine organ into a human recipient is the hyperacute rejection response which has been shown to be mediated by xenoreactive antibodies (XAb) and complement proteins. Here, evidence is presented that normal human sera contain IgG XAb that are able to mediate increased adhesion of polymorphonuclear leucocytes (PMN) to porcine aortic endothelial cells (PAEC) in vitro, to initiate damage to endothelial cells via antibody-dependent cellular cytotoxicity mechanisms (ADCC) along with peripheral blood mononuclear cells (PBMC) and to activate the classical complement cascade. PMN exhibit a 2.8-fold increase in adhesion to PAEC from 19.9 +/- 4% to 56 +/- 1.7% at an IgG concentration of 16 mg/ml. This increase is independent of treatment of the PMN with interferon-gamma. PAEC are lysed in the presence of complement: 42.8 +/- 0.7% lysis occurs with a 1/8 dilution of human serum as a source of immunoglobulin of all classes, while 39.3 +/- 3% lysis occurs with purified IgG at 13 mg/ml in the presence of baby rabbit complement. PAEC are also lysed by PBMC in the presence of human IgG XAb, a maximum of 52.0 +/- 5% being observed at effector-to-target (E:T) ratios of 30:1. PBMC bearing Fc gamma RIII receptors for the Fc portion of the IgG molecule mediate the endothelial cell damage since the anti-Fc gamma RIII monoclonal antibody, 3G8, can inhibit lysis by up to 77 +/- 5%. We conclude that human IgG are able to damage porcine endothelial cells using cellular and humoral mechanisms and that IgG XAb can efficiently activate the classical complement cascade in this model system.

Recombinant anti-carcinoembryonic antigen antibodies for targeting cancer.

Antibodies can be used to target cancer therapies to malignant tissue; the approach is attractive because conventional treatments such as chemo- and radiotherapy are dose limited due to toxicity in normal tissues. Effective targeting relies on appropriate pharmacokinetics of antibody-based therapeutics, ideally showing maximum uptake and retention in tumor and rapid clearance from normal tissue. We have studied the factors influencing these dynamics for antibodies against carcinoembryonic antigen (CEA). Protein engineering of anti-CEA antibodies, in vivo biodistribution models, and mathematical models have been employed to improve understanding of targeting parameters, define optimal characteristics for the antibody-based molecules employed, and develop new therapies for the clinic. Engineering antibodies to obtain the desired therapeutic characteristics is most readily achieved using recombinant antibody technology, and we have taken the approach of immunizing mice to provide high-affinity anti-CEA single-chain Fv antibodies (sFvs) from filamentous bacteriophage libraries. MFE-23, the most characterized of these sFvs, has been expressed in bacteria and purified in our laboratory for two clinical trials: a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumor deposits are detected by a hand-held probe during surgery. Both these trials showed that MFE-23 is safe and effective in localizing tumor deposits in patients with cancer. We are now developing fusion proteins that use the MFE-23 antibody to deliver a therapeutic moiety; MFE-23:: carboxypeptidase G2 (CPG2) targets the enzyme CPG2 for use in the antibody-directed enzyme prodrug therapy system and MFE::tumor necrosis factor alpha (TNFalpha) aims to reduce sequestration and increase tumor concentrations of systemically administered TNFalpha.

The role of the endothelium in systemic vasculitis.

The critical role played by the vascular endothelium in the development of vasculitic disease will be discussed within this article, attention being focused on the pathogenesis of the primary vasculitides, particularly Wegener's granulomatosis, microscopic polyarteritis and Kawasaki disease. Under normal circumstances the endothelium constitutes a barrier to the efflux of plasma proteins into the extracellular tissues, serves to maintain an anti-coagulant environment and forms a platform upon which many important biological processes take place. Alteration of the balance of these functions by inflammatory mediators and cytokines leads to the formation of a pro-coagulant environment, fluid leakage and increased adhesivity of the endothelium for neutrophils and lymphocytes. Both neutrophils and lymphocytes have the capacity to damage the endothelium directly, by the secretion of oxygen radicals, enzymes and other cytotoxic molecules, or indirectly by the secretion of cytokines which may alter the biological properties of endothelial cells, a process called endothelial activation. The role played by antibodies to the endothelium and to neutrophil cytoplasm components in mediating vascular injury in systemic vasculitis is also discussed and, finally, injury precipitated by components of the coagulation/thrombotic system.

Identification of a new isoform of human IL-12 p35 mRNA.

This study has identified an alternate mRNA isoform of the human interleukin-12 p35 gene differing from normal p35 transcripts by the deletion of exon 3. Exon 3-lacking p35 mRNA was produced by both dendritic cells and Epstein-Barr virus-transformed B cells and was detected only when transcription of normal p35 mRNA was abundant.

Viral studies in rheumatic disease.

Rheumatic disease has long been thought to represent an interaction between environmental agents on a background of genetic susceptibility. In this review herpesviruses and retroviruses are considered as possible aetiological agents in autoimmune disease with a particular emphasis on Sjögren's syndrome. A possible role for cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6) and human herpesvirus-8 (HHV-8) is reviewed. We conclude that there is no compelling evidence for the involvement of any of these herpesviruses. Retroviruses, however, are attracting increasing interest. In Man, both Human immunodeficiency virus (HIV) and human T lymphotropic virus type I (HTLV-I) infections cause autoimmune phenomena, including Sjögren's syndrome and arthritis in a minority of infected individuals. Similar reactions to retroviral infection are also seen in animal models. A possible role for the newly described human retrovirus-5 (HRV-5) is discussed, though current evidence does not support a role in Sjögren's syndrome. Other autoimmune diseases are under investigation.

Detection of human retrovirus 5 in patients with arthritis and systemic lupus erythematosus.

OBJECTIVE: To examine whether human retrovirus 5 (HRV-5) infection is associated with autoimmune rheumatic disease. METHODS: DNA from patients with various disorders including inflammatory diseases and from normal subjects was tested by nested polymerase chain reaction (PCR) for HRV-5 proviral DNA. Positive results were confirmed by DNA sequencing. RESULTS: HRV-5 proviral DNA was detected in 53% of synovial samples from arthritic joints, in 12% of blood samples from patients with rheumatoid arthritis (RA), and in 16% of blood samples from patients with systemic lupus erythematosus. In contrast, it was not detectable by PCR of affected tissues from patients with several other autoimmune diseases and was found in only 1 of >200 tissue specimens obtained at autopsy from non-RA patients. Sequence analysis of the amplified viral segment showed genetic variation between samples with maintenance of the open reading frame, typical of a replicating infectious retrovirus. CONCLUSION: This is the first report of the frequent detection of HRV-5 in any disease. We propose that the possible involvement of HRV-5 in autoimmune and rheumatic disease should be investigated further.