AIT-082 as a potential neuroprotective and regenerative agent in stroke and central nervous system injury.

The synthetic purine 4-[[3-(1,6 dihydro-6-oxo-9-purin-9-yl)-1-oxypropyl] amino] benzoic acid (AIT-082, Neotrofin, leteprinim potassium) possesses several biological properties of note: it stimulates outgrowth of neurites from PC12 cells and neurones, stimulates synthesis and/or release of neurotrophic factors from astrocytes, enhances nerve fibre regeneration in vivo and enhances of memory in animals and humans. AIT-082 also protects against glutamate neurotoxicity in vitro and in vivo, which has led to successful tests of AIT-082 in animal models of acute central nervous system injury. In such cases, AIT-082 probably functions by both acutely reducing glutamate excitotoxicity and, over a longer period, by enhancing neuronal sprouting and functional recovery.

AIT-082, a novel purine derivative with neuroregenerative properties.

Preclinical and clinical evidence support the effectiveness of neurotrophins in Alzheimer's disease (AD) and neurodegenerative diseases. Delivery of neurotrophins to target sites in the brain remains a major obstacle for their use in humans. Development of orally active agents that mimic the effects of nerve growth factor and other neurotrophins provides a promising alternative therapeutic strategy. AIT-082, a purine analogue, has been shown to reverse age-induced memory deficits in mice and is a growth factor-mimetic agent. It is orally active, rapidly penetrates the blood-brain barrier and induces the production of multiple growth factors at the appropriate target site in the central nervous system (CNS).

A chimeric Lym-1/interleukin 2 fusion protein for increasing tumor vascular permeability and enhancing antibody uptake.

A murine antihuman B-cell monoclonal antibody, Lym-1, has shown considerable promise for the treatment of human malignant lymphomas. To enhance its clinical potential, a genetically engineered fusion protein consisting of a chimeric Lym-1 (chLym-1) and interleukin 2 (IL-2) was tested for mediating cytotoxicity, increasing vasopermeability, and enhancing antibody uptake in human malignant lymphomas. The chLym-1/IL-2 fusion protein, which was expressed initially in a baculovirus system and more recently in the glutamine synthetase gene amplification system, was shown to be processed and assembled into a normal immunoglobulin monomer with two IL-2 molecules per antibody. It was found to be equivalent to the chLym-1 antibody in antigen-binding specificity and relative affinity. In addition, it maintains IL-2 cytokine activity as demonstrated by support of T-cell proliferation. Moreover, in antibody-dependent cellular cytotoxicity assays against Raji target cells, chLym-1/IL-2 had approximately 2-fold and 4-fold higher cytotoxicity than chLym-1 and murine Lym-1, respectively. Used as a pretreatment, chLym-1/IL-2 enhances the uptake of chLym-1 at the tumor site by altering the permeability of tumor vessels producing tumor:normal organ ratios of 420:1 for blood and 1708:1 for muscle at 3 days. The in vitro and in vivo activities of chLym-1/IL-2, therefore, suggest that this genetically engineered antibody fusion protein may represent a new immunotherapeutic reagent for the treatment of human malignant lymphomas.

Improved tumor localization and radioimaging with chemically modified monoclonal antibodies.

A method for the chemical modification of monoclonal antibodies using the heterobifunctional crosslinker succinimidyl 3-(2-pyridyldithio)propionate (SPDP), has been developed which dramatically alters the physiochemical properties of antibody reagents. For these studies, three murine monoclonal antibodies, B72.3, Lym-1, and TNT-1 were used to demonstrate the effects of chemical modification on clearance and biodistribution in tumor-bearing nude mice. In vitro, all three antibodies, modified to the same degree with SPDP, showed equal immunoreactivities and lower non-specific binding. Modified antibodies also were found to have lower isoelectric points compared to unmodified controls. In vivo, modified antibodies unexpectedly were found to have 2-6 times faster clearance in tumor-bearing nude mice similar to rates obtained with their F(ab')2 fragments. Paired-label in vivo biodistribution and external imaging experiments with intact antibodies and F(ab')2 fragments demonstrated that chemically modified antibodies gave 1.5-3 fold higher tumor uptake and retained less activity in normal organs thus markedly increasing the tumor to normal organ ratios. Because of these results, chemically modified antibodies produced clearer images at earlier time points by external scintigraphy. As "stealth" molecules, chemically modified monoclonal antibodies appear to have significantly improved uptake in tumors and faster clearance times compared to native molecules. These results suggest that alteration of the physicochemical properties of monoclonal antibodies may generate improved reagents for in vivo use.

A human-mouse chimeric Lym-1 monoclonal antibody with specificity for human lymphomas expressed in a baculovirus system.

A murine anti-human B-cell monoclonal antibody, Lym-1, has shown considerable promise for the treatment of human malignant lymphomas and has been utilized as a new radioimmunotherapy for refractory lymphoma. In order to enhance its clinical potential, a genetically engineered chimeric Lym-1 (chLym-1) with murine variable regions and human gamma 1 and kappa constant regions was constructed and expressed. The goal of this study was to generate a Lym-1 reagent with decreased immunogenicity and improved effector functions. Murine Lym-1 variable region cDNAs were isolated from the murine Lym-1 hybridoma cell line, fused to gamma 1 and kappa constant region cDNAs, and expressed in an insect cell expression system with the baculovirus transfer vector pAcUW31. The chLym-1 antibody expressed in this system was correctly processed and assembled into the expected immunoglobulin monomer. Chimeric Lym-1 bound to both target antigen-bearing Raji cells and a Lym-1 anti-idiotype antibody and had a similar binding affinity as murine Lym-1. The chimeric and murine versions of Lym-1 were assayed for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and to induce complement-mediated cytotoxicity (CMC) against Raji targets. Chimeric Lym-1 mediated a two-fold higher level of ADCC than murine Lym-1 and slightly lower levels of CMC than murine Lym-1. In addition, in Raji lymphoma-bearing nude mice, chLym-1 localized to the tumor with approximately equal uptake at 24 and 72 hours. Chimeric Lym-1, however, cleared from the blood of nontumor-bearing mice approximately 5 times faster than murine Lym-1 (20 h vs. 5 days), as expected for a xenogeneic protein. The improved in vitro and in vivo activities of this genetically engineered monoclonal antibody render it a new potential immunotherapeutic reagent for the treatment of human malignant lymphomas.

Adenocarcinoma-reactive human monoclonal antibody MS2B6 defines an antigen in simple glandular epithelium.

A human monoclonal antibody (MAb), MS2B6, produced from splenocytes isolated from a patient with advanced papillary serous cystadenocarcinoma of the ovary, defines a unique human tumor-associated antigen. This antigen, EA2B6 (epithelial antigen 2B6), is expressed in a tissue-restricted manner on cultured and fresh human adenocarcinomas and some normal glandular epithelial tissues. EA2B6 is a 38-48 kD protein antigen that co-fractionates with the nuclear matrix-intermediate filament scaffold of simple glandular epithelial tissues. EA2B6 is a molecule with restricted solubility, and in vitro antigen-antibody binding is dependent on the antigen being presented on a solid support. To determine if EA2B6 is a cytokeratin, competition studies were undertaken with several cytokeratin-specific murine monoclonal antibodies. None of these antibodies inhibited the binding of human MAb MS2B6 to partially purified EA2B6. Less than 1% of HT29 colon adenocarcinoma cells and fresh ovarian adenocarcinoma ascites cells express EA2B6 on their surface. The majority of EA2B6 is intracellular. Because of the restricted tissue distribution of this antigen and stability of the antibody, we believe MS2B6 is a good candidate for MAb-mediated diagnosis and therapy of human adenocarcinomas.

Human monoclonal antibody recognizing an antigen associated with ovarian and other adenocarcinomas.

MS2B6, a human monoclonal antibody derived from a patient with advanced ovarian cancer, has been used to study the distribution and characteristics of its target antigen. The MS2B6 antigen was detected by immunoperoxidase studies in 41 of 41 epithelial ovarian cancers and in the majority of nonovarian adenocarcinomas. Among normal tissues the MS2B6 antigen was found in the adult epithelia of the fallopian tube, endometrium, endocervix, colon, bronchus, breast, sweat duct, and large renal ducts. No detectable antigen was found in peritoneal epithelia, tissue stromal cells, spleen, thymus, or blood-borne cells. Immunoblotting analysis showed that the MS2B6 epitope resides on polypeptides of 38, 44, and 60 kd. The cellular location of the MS2B6 antigen was studied with immunoperoxidase and immunofluorescent staining and immunoelectronmicroscopy of ovarian cancer ascites tumor cells. The results suggest that in ascites tumor cells the MS2B6 antigen is located in a layer of the peripheral cytoplasm beginning just below the cell membrane. MS2B6 may be useful as an imaging or therapeutic agent.

Stability of specific immunoglobulin secretion by EBV-transformed lymphoblastoid cells and human-murine heterohybridomas.

We have examined variables leading to the generation of stable, antigen-specific, human immunoglobulin-secreting cell lines. Peripheral blood B lymphocytes enriched for Thomsen-Friedenreich antigen (T antigen)-specific cells were transformed with Epstein-Barr virus. Lymphoblastoid cells (LC) reactive with T antigen were either expanded without cloning or cloned at limiting dilution and then fused with murine 653 cells. Uncloned LCs from three transformations secreting polyclonal anti-T antibody (7-18 micrograms/ml/10(6) cells/24 hr total immunoglobulin) were subcultured at 100 cells/well, and T antigen-reactive cultures pooled. These cultures quickly lost specific antibody secretion, presumably due to overgrowth by clones of unknown specificity. T antigen-reactive LCs that were cloned three times at limiting dilution secreted 0.2 - 6.1 micrograms/ml/10(6) cells/24 hr but died or stopped secreting specific immunoglobulin after 77 to 155 days in culture. Pooling T antigen-reactive clones after each cloning step did not increase the long term stability compared to unpooled clones (p = 0.2). Fusions between cloned LCs and 653 cells failed to yield viable hybrids in nine of ten attempts with seven different LC lines. In contrast, fusion of uncloned LCs and 653 cells resulted in the generation of viable immunoglobulin-secreting heterohybrids in 22 of 24 fusions. The heterohybridomas produced from fusion of uncloned T antigen-reactive cultures with 653 cells secreted significantly more antibody (frequency of cell lines secreting greater than 2 micrograms/ml/10(6) cells/24 hr, p less than 0.01) and higher titers of antibody (frequency of cell lines secreting greater than four hemagglutination units of T antigen-specific antibody, p less than 0.03) than cloned lymphoblastoid cells. The hybrids maintained specific immunoglobulin secretion for longer in culture than either cloned or uncloned lymphoblastoid cell lines (p less than 0.001).