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The effects of cholesteryl ester transfer protein (CETP) inhibition on the serum lipoprotein profile in both normocholesterolemic and hypercholesterolemic hamsters has been determined following subcutaneous injection of 12.5 mg/kg of the CETP neutralizing monoclonal antibody, TP2. Inhibition of CETP activity was greater than 60% and resulted in a 30-40% increase in high density lipoprotein (HDL) in both normal and hypercholesterolemic animals. These HDL effects were observed 1 day post-injection, were maximal by 4 days, and returned to control values by 14 days. Inhibition of CETP activity resulted in a decrease in both low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol concomitant with HDL increase, and in hypercholesterolemic animals resulted in increased total serum cholesterol. In addition to the quantitative differences in LDL and HDL, there were significant increases in the size of the HDL, a shift to smaller LDL particles, and changes in apolipoprotein (apo) composition as evaluated by FPLC and Western blot analysis. Large apoA-I-poor and apoE-containing HDL became prevalent in hypercholesterolemic hamsters after CETP inhibition. In addition, the size of the CETP-containing HDL particles increased with inhibition of transfer activity. While these effects were apparent in normocholesterolemic animals, the changes in apolipoprotein distribution and HDL subspecies as detected on native gels were more significant in the hypercholesterolemic animals. The changes in the HDL profile and apolipoprotein distribution after CETP inhibition in hamsters were similar to those reported in CETP-deficient Japanese subjects, suggesting the utility of the hypercholesterolemic hamster as an in vivo model for the understanding of the lipoprotein changes associated with CETP inhibition.

Overexpression of P-glycoprotein (Pgp) by tumors results in multidrug resistance (MDR) to structurally unrelated oncolytics. MDR cells may be sensitized to these oncolytics when treated with a Pgp modulator. The present study evaluates LY335979 as a modulator both in vitro and in vivo. LY335979 (0.1 microM) fully restored sensitivity to vinblastine, doxorubicin (Dox), etoposide, and Taxol in CEM/VLB100 cells. LY335979 modulated Dox cytotoxicity even when LY335979 (0.5 microM) was removed 24 h prior to the cytotoxicity assay. LY335979 blocked [3H]azidopine photoaffinity labeling of the M(r) approximately 170,000 Pgp in CEM/VLB100 plasma membranes and competitively inhibited equilibrium binding of [3H]vinblastine to Pgp (Ki of approximately 0.06 microM). Treatment of mice bearing P388/ADR murine leukemia cells with LY335979 in combination with Dox or etoposide gave a significant increase in life span with no apparent alteration of pharmacokinetics. LY335979 also enhanced the antitumor activity of Taxol in a MDR human non-small cell lung carcinoma nude mouse xenograft model. Thus, LY335979 is an extremely potent, efficacious modulator that apparently lacks pharmacokinetic interactions with coadministered anticancer drugs and is, therefore, an exciting new agent for clinical evaluation for reversal of Pgp-associated MDR.

A conjugate of 4-desacetylvinblastine-3-carboxyhydrazide (DAVLBHY) and the glioma-reactive monoclonal antibody (mAb) 9.2.27 induced long-term suppression of tumor growth in athymic nude mice engrafted with U87MG human glioma cells. In vitro, DAVLBHY had the strongest antiproliferative activity (inhibitory concentration at which incorporation of [3H]thymidine is at 50% of untreated control is 2.0 x 10(-9) M) of seven cytotoxic drugs tested and so was chosen for conjugation to mAb 9.2.27, which reacts specifically with the core protein of chondroitin sulfate proteoglycans found in human glioblastomas. After conjugation of DAVLBHY to the carbohydrate residues of mAb 9.2.27 it retained its full binding capacity. For in vivo studies, DAVLBHY and several conjugate derivatives were evaluated by using two dosages of i.v. injections, each starting 2 days after s.c. tumor inoculation. The control tumors reached a volume of nearly 3000 mm3 within 30 days. Tumor growth was delayed by about 20 days with four i.v. injections of 0.5 mg/kg 9.2.27-DAVLBHY, which was slightly superior to the unconjugated drug. Moreover, 9.2.27-DAVLBHY produced a highly significant suppression of growth so that the average tumor volume was only 3% of that observed in untreated controls after 28 days. Four injections of this conjugate at a larger dose, 2.0 mg/kg, prevented recurrence of the tumors for 130 days in all animals tested, thus demonstrating a significant increase in the therapeutic index, since the unconjugated drug provided limited inhibition of tumor growth for only 40 days. The specificity of the antitumor effect was demonstrated in a comparison with the control conjugate, KS1/4-DAVLBHY, which despite partial tumor suppression had only a transient effect. The specific antitumor effect of 9.2.27-DAVLBHY was unexpected, since the target antigen is expressed at a relatively low density (40,000 sites/cell) on U87MG glioma cells.

Monoclonal antibody (MoAb) conjugates have been used to treat a variety of malignancies. The majority of the MoAbs which have been used therapeutically are from murine sources. The infusion of these foreign proteins into humans can be expected to elicit anti-murine antibodies and may be one of the major limitations to the clinical use of murine MoAbs. In these studies, we report on the nature and specificity of the human anti-murine antibody (HAMA) response in patients given single and multiple infusions of the two Vinca alkaloid conjugates of the MoAb KS1/4, which recognizes tumor-associated antigens in a variety of adenocarcinomas. A HAMA response was induced in a majority of the patients receiving infusions of KS1/4 conjugates, regardless of the specific conjugate used or the number of infusions. The magnitude of the response did not appear to be dose related. Antibodies directed to the drug moieties of these conjugates, anti-Vinca alkaloids, were also induced in patients with HAMA responses. The magnitude of the anti-Vinca response paralleled that of the HAMA.

UCLA-P3 human lung adenocarcinoma cells were grown in nude mice and given repetitive treatments of a monoclonal antibody-Vinca alkaloid immunoconjugate. Although this therapy resulted in a greater than 4-fold reduction in mean tumor mass of the established tumors, some animals experienced a reinitiation of tumor growth after cessation of conjugate treatment. Two such animals were treated again with high doses of monoclonal antibody-Vinca but one of the tumors was no longer regressed by the drug conjugate. The tumor was excised, enzymatically dissociated, and grown in tissue culture. Cultured cells were reimplanted in nude mice and subjected to further therapy with a monoclonal antibody-Vinca conjugate. The resulting tumors were also refractory to the immunoconjugate therapy. This cycle was repeated for a total of three times and resulted in the serial in vivo selection of three conjugate resistant variants. The mechanism responsible for the in vivo resistance of human tumor cells to the monoclonal antibody-Vinca immunoconjugate is unknown but does not appear to involve antigen modulation, altered tumor cell growth rate, or an apparent decrease in tumor targeting in vivo. The resistance was also not accompanied by any detectable elevation in multidrug resistance 1 mRNA or P-glycoprotein expression. Significantly, the resistance pattern was observed only in vivo and was not maintained by cells grown in vitro.

The activity of the conjugate of monoclonal antibody KS1/4 with 4-desacetylvinblastine-3-carboxhydrazide (KS1/4-DAVLB-HY) was explored in the OVCAR-3 human ovarian xenograft tumor model. Multiple schedules of KS1/4-DAVLB-HY administration were employed, including a comparison of i.p. and i.v. routes of treatment. When inoculates of 6 x 10(7) OVCAR-3 cells were injected i.p. into female athymic nude mice, untreated control animals had a mean survival of 18-34 days, with the development of massive ascites and large intraabdominal tumors. Significant increases in survival were observed in KS1/4-DAVLB-HY conjugate-treated animals with all schedules utilized. Parallel therapy with equivalent doses of free DAVLB-HY or a non-antigen-binding immunoconjugate did not significantly increase the survival of the animals. These data demonstrate that the immunoconjugate KS1/4-DAVLB-HY significantly increases the survival of OVCAR-3 tumor-bearing mice and indicates that this immunoconjugate may be useful in the treatment of human ovarian cancer.

Monoclonal antibody (Mab) 9.2.27 was utilized in a combination of biosynthetic and biochemical investigations as an immunological probe for the study of chondroitin sulfate proteoglycans (CSP) in human melanoma cells. Pulse-chase and long-term intrinsic labeling immunoprecipitation experiments combined with the biosynthetic inhibitors monensin, cycloheximide, and paranitrophenol-beta-D-xyloside all suggest that Mab 9.2.27 recognizes a set of glycoprotein molecules ranging to a 250-kDa glycoprotein which serves as the core glycoprotein for CSP in human melanoma cells. Peptide maps comparing the 250-kDa and CSP molecule verify that the 250-kDa glycoprotein is the CSP core protein in human melanoma cells. Further studies document that the CSP released by melanoma cells and recognized by Mab 9.2.27 contains (2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-beta-4-O-sulfo-D-galactose and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-beta-6-O-sulfo-D-galactose saccharides and this CSP can interact with hyaluronic acid-Sepharose. Topographical studies indicate that this CSP has pericellular punctuated distribution on the melanoma cell surface and may play a role in cell-substrate interactions in the biology of metastatic human melanoma.

A human adenocarcinoma-associated antigen (KSA) defined by the monoclonal antibody KS1/4 has become the focus of several site-directed strategies for tumor therapy. KSA, a 40,000 Da cell surface glycoprotein antigen, is found at a high density in all adenocarcinomas examined to date and in corresponding normal epithelial tissues. Here we describe the cloning and sequencing of overlapping complementary DNA clones which encode the entire KSA as expressed in UCLA-P3, a human lung adenocarcinoma cell line. We have deduced the 314-amino acid sequence and have compared it to the N-terminal amino acid sequence data of the affinity-purified antigen. The KSA is synthesized as a 314-residue-long preproprotein that is then processed to a 232-residue-long antigen. KSA appears to have a single transmembrane domain of 23 residues that separates the highly charged 26-residue cytoplasmic domain from the extracellular domain. The N-terminal region of the propeptide is rich in cysteines and contains three potential N-glycosylation sites. Computer-assisted analyses at both the DNA and protein levels have found no significant similarities of this protein to known sequences, but a GC-rich 5' terminus is evident. Northern blot analysis shows that transcription of KSA can be detected in RNA isolated from normal colon but not in RNA isolated from normal lung, prostate, or liver.