Characterization of a complex glycoprotein whose variable metabolic clearance in humans is dependent on terminal N-acetylglucosamine content.

Glycoproteins can be cleared from circulation if they carry oligosaccharide structures that are recognized by specific receptors. High-mannose type and asialo complex oligosaccharides are cleared by the mannose and asialoglycoprotein receptors, respectively. This paper presents the protein and terminal saccharide characterization for nine batches of a glycoprotein developed for pharmaceutical use.

Validation of a rat pheochromocytoma (PC12)-based cell survival assay for determining biological potency of recombinant human nerve growth factor.

A method has been validated, according to the Guidelines of the International Conference on Harmonization (ICH), for precise quantitation of the biological activity of recombinant human nerve growth factor (rhNGF) for lot release testing. The assay is based on the survival of a subclone of rat pheochromocytoma PC12 cells (PC12-CF) in response to rhNGF. Cell survival is measured by monitoring the reduction, by living cells, of the alamarBlue dye into a red form which is highly fluorescent.

A His19 to Ala mutant of melanoma growth-stimulating activity is a partial antagonist of the CXCR2 receptor.

Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity.

186Re-labeled antibodies to p185HER2 as HER2-targeted radioimmunopharmaceutical agents: comparison of physical and biological characteristics with 125I and 131I-labeled counterparts.

Overexpression of the HER2/neu protooncogene has been shown to correlate with poor clinical prognosis. A murine monoclonal antibody (4D5) directed against the extracellular domain (ECD) of p185HER2 has been shown to inhibit in vitro and in vivo growth of carcinomas overexpressing HER2 and has been humanized (rhuMAb HER2). The objective of the study was the identification of an agent which might be useful for in vitro studies, tumor imaging and/or radioimmunotherapy by linking beta-emitting radionuclides to these HER2-targeted antibodies.

A mutant of melanoma growth stimulating activity does not activate neutrophils but blocks erythrocyte invasion by malaria.

Alanine scanning mutagenesis of the charged amino acids of melanoma growth stimulating activity (MGSA) was used to identify specific residues that are involved in binding to the human erythrocyte Duffy antigen/chemokine receptor (DARC) and to the type B interleukin-8 receptor (IL-8RB) on neutrophils. Receptor binding and biological studies with the alanine scan mutants of MGSA demonstrate that MGSA binds to DARC and the IL-8RB through distinct binding regions. One of the MGSA mutants, E6A, binds to human erythrocytes and is able to inhibit malaria invasion as efficiently as wild type MGSA but has a severely reduced ability to bind to or signal through the IL-8RB.

Development of anti-p185HER2 immunoliposomes for cancer therapy.

The product of the HER2 protooncogene, p185HER2, represents an attractive target for cancer immunotherapies. We have prepared anti-p185HER2 immunoliposomes in which Fab' fragments of a humanized anti-p185HER2 monoclonal antibody with antiproliferative properties (rhuMAb-HER2) were conjugated to either conventional or sterically stabilized liposomes. These immunoliposomes bind specifically to p185HER2-overexpressing breast cancer cells (SK-BR-3 and BT-474).

Bispecific HER2 x CD3 antibodies enhance T-cell cytotoxicity in vitro and localize to HER2-overexpressing xenografts in nude mice.

Recently, we reported the development of fully humanized bispecific F(ab')2 antibodies with dual binding specificities to human T-lymphocytes and to tumor cells overexpressing HER2. These antibodies were shown to effectively mediate targeted HER2-overexpressing tumor cell killing by freshly isolated human T-cells. In this report we extend our studies to describe the interaction of the bispecific antibody with activated T-lymphocytes (ATL) maintained in culture for an extended period of time.

Development of a humanized disulfide-stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug.

The humanized anti-p185HER2 antibody, humAb4D5-8, has completed Phase II clinical trials for p185HER2-overexpressing breast cancer. Here, this antibody is used as a building block to engineer a disulfide-linked Fv (dsFv) beta-lactamase fusion protein for use in antibody-dependent enzyme-mediated prodrug therapy using cephalosporin-based prodrugs. Three Fv variants were designed with an interchain disulfide bond buried at the VL/VH interface and secreted from Escherichia coli.

Humanization of an anti-p185HER2 antibody for human cancer therapy.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed.

High level Escherichia coli expression and production of a bivalent humanized antibody fragment.

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli.

Production of bovine insulin-like growth factor 2 (bIGF2) in Escherichia coli.

Bovine insulin-like growth factor 2 (bIGF2) was produced in inclusion bodies in the cytoplasm of Escherichia coli and accumulated at high levels: 20-25% of total Coomassie-stained bacterial protein. The level of accumulation of bIGF2 was affected by the choice of codons in the 5' end of the coding sequence and by a rpoH mutation in the host cells. Purified recombinant bIGF2 had the native N terminus and the same mitogenic activity as that of bIGF2 purified from bovine serum.

Monoclonal antibody therapy of human cancer: taking the HER2 protooncogene to the clinic.

The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics.

The extracellular domain of HER2/neu is a potential immunogen for active specific immunotherapy of breast cancer.

The proto-oncogene HER2/neu encodes a protein tyrosine kinase (p185HER2) that is homologous to the human epidermal growth factor receptor. Amplification and/or overexpression of HER2/neu occurs in multiple human malignancies and appears to be integrally involved in progression of some breast and ovarian cancers. Because of this fact, HER2/neu is an intriguing target for specific cancer therapeutic strategies.

ELISA for quantitation of the extracellular domain of p185HER2 in biological fluids.

The HER2/neu proto-oncogene encodes a receptor that belong to the tyrosine-specific protein kinase family. Amplification of the HER2 gene in patients with breast and ovarian cancer has been shown to predict poorer survival rates. In order to understand the role of HER2 in malignant and normal cells, it is necessary to devise assays that can quantitate expression levels of the HER2 gene product (p185HER2) in production samples, biopsy specimens and biological fluids.

Food safety and pharmacokinetic studies which support a zero (0) meat and milk withdrawal time for use of sometribove in dairy cows.

Sometribove (SB) is a synthetic form of bovine somatotropin (BST) whose amino acid sequence is the same for 190 of the 191 amino acids in BST. Administration of 500 mg of SB to dairy cows every 14 d increases the efficiency of milk production. Regulatory agencies have authorized a zero (0) milk and meat withdrawal time for investigational use of SB.

Phylogenetic variations in the calcium-dependent electrophoretic shift of alpha-lactalbumin.

alpha-Lactalbumin undergoes a calcium-dependent electrophoretic shift at pH 8.3. When Ca2+ is removed by a chelator, the mobility of the protein increases, reflecting the exposure of negative electrical charges.

Fragments of bovine insulin-like growth factors I and II stimulate proliferation of rat L6 myoblast cells.

The active sites of bovine insulin-like growth factor (IGF) I and II fragments were studied. Overlapping fragments of IGF I (residues 1-25, 11-35, 21-45, 31-55, and 41-70) and of IGF II (residues 1-24, 10-34, 20-44, 30-54, and 40-67) were chemically synthesized. The activity of the fragments was measured by stimulating the proliferation of rat L6 myoblast cells.

The calcium-dependent electrophoretic shift of alpha-lactalbumin, the modifier protein of galactosyl transferase.

alpha-Lactalbumin, the modifier protein of galactosyl transferase in the synthesis of lactose by the mammary gland, has been shown to undergo a Ca2+-dependent electrophoretic shift. Such shifts, characteristic of most calcium modulated proteins, are related to gross conformational changes upon binding calcium when detected in the presence of detergent (SDS-PAGE). However, we detected the calcium shift for alpha-lactalbumin using non-denaturing PAGE (ND-PAGE) where electrical charge changes are observed upon binding calcium.

Evidence for inhibitory activity against cell proliferation in bovine liver.

Bovine calf liver was homogenized in 0.1M NH4Ac, pH 7.4, and centrifuged.

Expression of secreted insulin-like growth factor-1 in Escherichia coli.

The synthesis, processing and secretion of insulin-like growth factor-1 (IGF-1 or somatomedin-C) fused to LamB and OmpF secretion leader sequences in Escherichia coli have been investigated. Expression and secretion of IGF-1 was achieved. The major portion of this secreted IGF-1 accumulated in the periplasmic space as insoluble aggregates.

Mistranslation in IGF-1 during over-expression of the protein in Escherichia coli using a synthetic gene containing low frequency codons.

Partial misincorporation of Lys for Arg has been observed for the Arg residues of IGF-1 when the molecule is expressed in Escherichia coli using a synthetic gene with the low frequency AGA codon encoding all six Arg residues and yeast preferred codons encoding the remaining residues. The Lys for Arg substitution at these residues could not be detected when a gene containing E. coli preferred codons, with the codon CGT coding for all Arg residues, was used for the expression of the protein.

Activation of human placental 5-pregnene-3,20-dione isomerase activity by pyridine nucleotides.

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.

Stimulation of in vitro muscle cell proliferation by sera from swine injected with porcine growth hormone.

The proliferation-promoting activity of sera obtained from pigs before and after porcine growth hormone injections was tested in a muscle cell culture bioassay. For 3 d, purified porcine growth hormone (pGH) was administered by intramuscular injection to crossbred barrows. Two levels of pGH were administered: 18 micrograms pGH X kg-1 body weight X d-1 (low dose) or 143 micrograms pGH X kg-1 body weight X d-1 (high dose).

A statistically standardized muscle cell culture bioassay measuring the effect of swine serum on muscle cell proliferation.

We have developed a statistically standardized bioassay for quantifying the effect of swine serum on the proliferation rate of cultured L6 myogenic cells. The intra-assay coefficient of variation for this assay is 2.5%.

Characterization of a nonenzymatic component in the isomerization of 5-pregnene-3,20-dione catalyzed by human placental microsomes in vitro.

When human placental microsomes were heated in boiling water or exposed to trypsin, 30 to 40% of the 5-ene,3-ketosteroid isomerase activity was stable. Aqueous suspensions of chloroform:methanol extracts of microsomes also catalyzed isomerization of 5-pregnene-3,20-dione, activity being associated with the polar lipid fraction. The trypsin- and heat-stable activities, as well as that of resuspended microsomal lipids, showed a dependence on buffer composition and concentration.