Biomedical subjects

C N Ahlem

Publications and source records attributed to C N Ahlem.

5 recordsLinked to original sources

PubMed1991

Bifunctional antibody: a binary radiopharmaceutical delivery system for imaging colorectal carcinoma.

In clinical studies we have evaluated a unique monoclonal antibody-based drug delivery system, a bifunctional antibody designed to deliver imaging or therapeutic agents, such as radioisotopes, drugs, or biologics, to tumor cells, while minimizing the dose to normal tissue. The bifunctional antibody, with one specificity to a tumor-associated antigen (carcinoembryonic antigen) and another specificity to a hapten, is injected and allowed to localize at a tumor site for 4 days. A hapten, tagged with a radioisotope, is subsequently injected for delivery to and capture by the prelocalized antibody at the tumor site. In studies reported here, the sulfhydryl groups of Fab' fragments of ZCE-025 and CHA-255 were linked with bis-maleimidomethyl ether to form an F(ab')2 bifunctional antibody coupled by a stable thioether linkage. EOTUBE, a hydroxyethylthiourido derivative of benzyl EDTA, was used as the hapten carrier of 111In. Fourteen patients 62-82 years old with recurrent or metastatic adenocarcinoma of the colon were studied. Twenty of 21 known lesions were imaged, and eight of nine new lesions were confirmed. With this fundamentally new approach to drug delivery, clearance from normal tissue is rapid, and high tumor:normal tissue ratios are expeditiously achieved.

Adenocarcinoma↗

PubMed1984

Mouse primase initiation sites in the origin region of simian virus 40.

The sites of initiation of DNA synthesis by purified mouse DNA primase in the origin-of-replication region of simian virus 40 (SV40) were examined. Using as template the separated strands of a cloned fragment of SV40 approximately equal to 300 base pairs (bp) long that includes the origin, we observed specific sites of initiation on the two strands. On the early strand that is the template for early mRNA synthesis, the primary starts are at four positions within 10 nucleotides of each other around nucleotide 5215 and an additional site around nucleotide 5147 that is used at one-sixth the frequency of the major sites. The major start sites on the early strand are within the 65-bp minimal origin of replication and lie between tumor antigen binding sites I and II. On the late strand that is the template for late mRNA synthesis, six major initiation sites were observed, each within the 3' C-C-C-G-C-C 5' sequence in the template that is repeated twice within each of the three 21-bp repeats that lie adjacent to the minimal origin, on its late side. A 6-bp deletion in the 65-bp minimal origin that eliminates its function as an origin reduced the major initiations around nucleotide 5215 on the early strand by 90% but did not affect initiations at the minor start site on the early strand or initiations on the late strand. Mouse DNA primase is able to recognize specific regions on the SV40 DNA. Those on the early strand are within the minimal origin of replication and those on the late strand are within the 21-bp repeat region necessary for maximum replication.

Animals↗

PubMed1983

A DNA primase from mouse cells. Purification and partial characterization.

An enzymatic activity that synthesizes oligoribonucleotides in lengths of 9-10 nucleotides and multiples thereof has been purified over 10,000-fold from mouse hybridoma cells. The oligoribonucleotides serve as primers to initiate DNA synthesis, and the activity has properties expected of mammalian DNA primase. The most highly purified fraction has two major protein components, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, of 56,000 and 46,000 Da. These proteins co-purify in a 1:1 stoichiometry along with oligoribonucleotide synthesis activity and with an activity that initiates the synthesis of DNA by DNA polymerase alpha. The sedimentation coefficient on glycerol gradients is 5.5 S, and this is consistent with one 56,000- and one 46,000-Da subunit/native enzyme. No DNA polymerase activity was detected in the most highly purified fraction. Poly(dIT) is the most active template, whereas a variety of single-stranded DNA templates are 10-15% as active and double-stranded DNA templates are 10-15% as active and double-stranded DNA is less than 1%. rATP is an absolute requirement as is Mg2+. No ATPase activity was detected with or without addition of DNA, single- or double-stranded. (NH4)2SO4 and NaPO4 buffer, pH 7.6, are inhibitory above 20 mM, whereas KCl is inhibitory above 80 mM. beta-D-arabinose-CTP is a strong inhibitor of primase; approximately 50% inhibition is observed when present at one-fifth the concentration of rCTP.

Animals↗

PubMed1982

DNA primase activity from human lymphocytes. Synthesis of oligoribonucleotides that prime DNA synthesis.

A fraction has been prepared from extracts of a human lymphoblastoid cell line that has properties of a mammalian DNA primase and also contains a DNA polymerase activity with unusual properties. With a variety of synthetic single-stranded DNA templates using rNTPs alone, the products consist of oligoribonucleotides of a restricted size range, primarily 7 to 9 nucleotides in length. Poly(dIT) is the most active template found thus far. The activity appears to have "relaxed" substrate/template complementarity requirements similar to those described previously for mammalian primase; poly(dIT) template with rATP alone results in synthesis of oligo(rA) of the same size as oligo(rAC) made when both rATP and rCTP are present. When dNTPs are added to the reaction, DNA is synthesized by extension of the oligoribonucleotide, which acts as primer. The DNA product appears in relatively discrete sizes that differ by approximately 8 nucleotides, with a large proportion of the product around 24 and 32 nucleotides. In addition to the relatively discrete size of its product, the DNA polymerase activity that utilizes the endogenously synthesized oligoribonucleotide primer on poly(dIT) template differs from polymerase alpha in its resistance to aphidicolin and low Km for dNTP.

Cell Line↗

PubMedDate not supplied

Preparation and characterization of a beta-lactamase-Fab' conjugate for the site-specific activation of oncolytic agents.

Antibody-directed catalysis (ADC) is a two-step method for the targeted delivery of chemotherapeutic agents in which enzyme-antibody conjugates, prelocalized to antigen-bearing cells, activate prodrugs designed to be substrates for the enzyme. An enzyme-Fab' conjugate exhibiting both native beta-lactamase activity and immunoreactivity toward carcinoembryonic antigen (CEA) was constructed. Treatment of CEA-expressing LS174T cells with this conjugate imparted beta-lactamase activity to the cells; beta-lactamase activity was not imparted by treatment with unconjugated beta-lactamase and not to CEA negative cells treated with conjugate. Cephalosporin-based prodrugs, and other substrates synthesized as model compounds, were found to have wide variations in their kinetic parameters toward the conjugate, with kcat values ranging from 16 to 3300 s-1 and KM values ranging from 5 to 160 microM. The prodrug derived from desacetylvinblastine-3-carboxylic acid hydrazide (DAVLBHYD) was studied in vitro and found to be 5-fold less cytotoxic to LS174T cells than the parent DAVLBHYD. For antigen-positive cells preincubated with conjugate, however, the prodrug showed the same potency as the parent drug. Thus, the combination of conjugate and prodrug appears to provide antigen-dependent toxicity to tumor cells.

Animals↗