The oncofetal protein, 5T4, is a suitable target for antibody-guided anti-cancer chemotherapy with calicheamicin.

The oncofetal protein, 5T4, is a tumor-associated protein displayed on the cell membrane of various carcinomas. This molecule is a promising target for anti-tumor vaccine development and for targeted therapy with staphylococcus exotoxin. The potential use of 5T4 as a target for antibody-guided chemotherapy has not been demonstrated. We report oncolytic efficacy and selectivity in vitro and in vivo with immuno-conjugates of calicheamicin (CM) and the anti-5T4 antibody, H8. CM is a potent cytotoxic drug that causes double strand breaks in DNA. Conjugates of CM and H8 were constructed with acid-labile as well as acid-stabile linkers. In vitro, when applied to monolayers of 5T4(+) cells, CM-conjugates targeting 5T4 were consistently more toxic than either free drug or a non-binding control CM-conjugate. This difference was less pronounced on 5T4-deficient cells. In vivo, four 5T4-positive subcutaneous tumor models were treated with conjugates. Efficacy was demonstrated by reduction of tumor growth relative to controls treated with drug vehicle. To evidence selectivity, the efficacy of the anti-5T4 conjugates was compared to the efficacy of H8, a mixture of H8 and calicheamicin, calicheamicin alone or calicheamicin conjugated to the anti-CD33 antibody, hP67.6. In addition, the efficacy and selectivity of an acid-labile conjugate of H8 was evaluated in an orthotopic model for 5T4(+) lung cancer. Increased survival following treatment was used as a parameter of efficacy. Calicheamicin conjugates of H8 were effective and selective in all the examined tumor models. Differences in efficacy between the acid-labile and acid-stabile conjugates depended on the investigated tumor model.

Determinants of the response of neuroblastoma cells to DNA damage: the roles of pre-treatment cell morphology and chemical nature of the damage.

Chemotherapeutic agent-induced DNA cleavage gives rise to apoptosis in a subpopulation of SK-N-SH human neuroblastoma cells; the remaining cells undergo Schwann cell-like differentiation. Like other neural crest and primitive neurectodermal tumor-derived cell lines, SK-N-SH cultures contain cells of neural (N-type) and epithelial (substrate-adherent, or S-type) phenotypes. Using isolated N-type and S-type cells from neuroblastoma, medulloblastoma, melanoma and glioma cell lines, we demonstrate that the determinants of the response to DNA cleavage are intrinsic properties of the cell. Furthermore, using a series of analogues of enediyne deoxyribonucleic acid (DNA) cleaving agents, we show that the molecular target of these agents is likely to be the same in N- and S-type cells, implying that the difference in response characteristics is a function of different distal pathways that are triggered by DNA cleavage. We demonstrate that the concentration of the DNA damaging agent used, and not the specific characteristics of the damage it produces, is the trigger for production of the cellular response. Response type does not correlate with previously published values for expression of the apoptosis modulators Bcl-2, Bcl-XL, wildtype p53, or, in medulloblastoma lines, p75.

Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics.

The calicheamicin family of antitumor antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Their potency suggested that the calicheamicins would be excellent candidates for targeted delivery and a hydrazide prepared from the most potent and abundant of the naturally occurring derivative, gamma 1I, was linked to oxidized sugars on CT-M-01, an internalizing anti-polyepithelial mucin antibody. The conjugates retained the immunoreactivity of the unmodified antibody and were specifically cytotoxic toward antigen positive tumor cells in vitro and in vivo. Hydrazide analogues of less potent calicheamicin derivatives were also prepared and conjugated to CT-M-01. Comparison of the therapeutic efficacy of the conjugates against the MX-1 xenograft tumor implanted s.c. in nude mice showed that conjugates of derivatives missing the rhamnose, a sugar residue that is part of the DNA binding region of the drug, were not as promising as antitumor therapies. However, conjugates of two derivatives, alpha 3I and N-acetyl-gamma 1I, in which the rhamnose residue is present but the amino sugar residue of the parent drug is either missing or modified, significantly inhibited tumor growth over a 4-fold dose range and produced long-term tumor-free survivors. Sterically hindering methyl groups adjacent to the disulfide in the linker further increased the therapeutic window of these potent conjugates.

Matrix-assisted UV-laser desorption/ionization mass spectrometric analysis of monoclonal antibodies for the determination of carbohydrate, conjugated chelator, and conjugated drug content.

The chemically averaged molecular weights of a variety of native and conjugated monoclonal antibodies, approximately 150,000, were measured by matrix-assisted UV-laser desorption/ionization mass spectrometry. The average mass of the carbohydrate present in a monoclonal antibody was estimated from the difference between the measured mass of the monoclonal antibody and the mass of the protein present in the monoclonal antibody computed from the amino acid translation of the DNA sequence. The loading of chelators and anticancer drugs conjugated to a monoclonal antibody was quantitated from the difference in the measured masses for the conjugated and untreated monoclonal antibody relative to the expected mass change upon conjugation of 1 mol of chelator or drug. The loading results obtained by mass spectrometry were consistent in most cases with measurements obtained by radioactivity trace assay or UV spectrometry. Similar matrix-assisted UV-laser desorption/ionization mass spectrometric studies were also made after reducing untreated and conjugated monoclonal antibodies with dithiothreitol to determine the distribution of carbohydrate and chelator between the light and heavy chains of the molecules. Matrix-assisted UV-laser desorption/ionization mass spectra were used to compute loading values for covalently bound drugs and proteins, while the loading values obtained by use of gel-filtration HPLC and UV spectrometry cannot distinguish between covalently and noncovalently bound drugs and proteins.

Synthesis and structure of the platelet aggregation factor thromboxane A2.

In 1975, Hamberg et al. reported evidence for the existence of an unstable platelet-aggregating factor which they named thromboxane A2 (TXA2) and for which they proposed a novel bicyclic oxetane structure (1, below) based on the short half-life of the factor (t1/2 (37 degrees C) = 32 s at pH 7.4) and the isolation of degradation products related to thromboxane (TXB2) (2, below). As natural TXA2 has not yet been isolated and characterized as a pure compound, we have synthesized the proposed structure (1) from TXB2 and compared its biological properties with those of authentic, biologically generated material. Here we present evidence that synthetic material having structure (1) is indistinguishable from platelet-derived TXA2 in various biological assays and that the proposed structure (1) for TXA2 is correct.

Lung biopsy through the flexible fiberoptic bronchoscope.

Peripheral lung lesions are a difficult diagnostic problem. The technique of forceps lung biopsy through the flexible fiberoptic bronchoscope provides potential access to the entire lung. Experience to date documents the procedure's safety and high yield of helpful information.