Evaluation of the aggregation states of monoclonal antibodies by diverse and complementary methods.

Therapeutic monoclonal antibodies (MAbs) with high specificity and fewer adverse effects are becoming widely used for the treatment of various diseases. MAbs need to be stored and administered at high concentrations in solution, the conditions under which MAbs may aggregate. As aggregated MAbs compromise their safety and efficacy, aggregation should be prevented; thus, it is important to analyze the aggregation states of MAbs in detail. We obtained 2 MAbs against dinitrophenol (DNP) that exhibited different aggregation properties: anti-DNP1 exhibited a much higher aggregate content (dimer or trimer) than anti-DNP2 when analyzed by size-exclusion chromatography (SEC). As anti-DNP1 had a longer complementarity-determining region 3 (CDR3) light chain than anti-DNP2 by 2 amino acid residues, we hypothesized that the increased aggregation of DNP1 was due to these extra residues; therefore, we prepared mutant antibodies with shorter CDR3s to compare their aggregation properties. Anti-DNP1-ΔEI, with the same CDR3 length as anti-DNP2, exhibited no aggregates as expected. Anti-DNP1-ΔI, with 1 additional residue, exhibited a smaller peak than wild-type (WT) in SEC, whereas this mutant exhibited stronger thioflavin T fluorescence than WT, which is indicative of amyloid formation. In addition, the anti-DNP1-ΔI solution (but not others) became opalescent at 4°C and exhibited large visible particles, which are undetectable by SEC. The fragment antigen-binding region of this mutant was found to have lower thermal stability than the others by differential scanning calorimetry. These data suggest that diverse analytical methods should be applied to evaluate MAb aggregation, in addition to the commonly used SEC.

Antineoplastic effect of a single oral dose of the novel Flt3 inhibitor KRN383 on xenografted human leukemic cells harboring Flt3-activating mutations.

Activating mutations of Fms-like tyrosine kinase 3 (Flt3) are the most common genetic abnormalities found in acute myeloid leukemia (AML) and represent potential therapeutic targets. The novel Flt3 inhibitor KRN383 inhibited the autophosphorylation of Flt3 bearing internal tandem duplications (ITDs) and the Asp835Tyr (D835Y) point mutation with half-maximal inhibitory concentration (IC(50)) values of < or =5.9 and 43 nM, respectively. KRN383 also inhibited the proliferation of the ITD-positive cell lines with IC(50) values of < or =2.9 nM. A single oral administration of 80 mg/kg of KRN383 eradicated ITD-positive xenograft tumors in nude mice and prolonged the survival of SCID mice carrying ITD-positive AML cells. The effectiveness of a single oral dose of KRN383 suggests that it has the potential to be used in a wide variety of clinical regimens, including multicycle and combination therapies.

Identification of potent and selective inhibitors of PDGF receptor autophosphorylation.

We report the structure-activity relationship of quinoline and quinazoline derivatives, which include urea, thiourea, urethane, and acylthiourea groups, as inhibitors of the platelet-derived growth factor (PDGF) receptor autophosphorylation. Our previous studies showed that the quinoline and quinazoline derivatives including urea, thiourea, and carbamate groups were highly potent compounds as the PDGF receptor autophosphorylation inhibitor, but these compounds did not exhibit receptor selectivity between the PDGF receptor and the c-kit receptor. As a result of further synthesis and biological evaluation, we have found that the quinoline and quinazoline-acylthiourea derivatives showed not only good inhibitory activity for the PDGF receptor but also receptor selectivity between the PDGF receptor and the c-kit receptor. Furthermore N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}-N'-(2-methylbenzoyl)thiourea exhibited potent oral efficacy in in vivo assay using the rat carotid balloon injury model. Therefore, the quinoline and quinazoline-acylthiourea derivatives may be expected to have potential as therapeutic agents for the treatment of restenosis.

Orally active anti-proliferation agents: novel diphenylamine derivatives as FGF-R2 autophosphorylation inhibitors.

(6,7-Disubstituted-quinolin-4-yloxy-phenyl)(4-substituted-phenyl)amine derivatives were synthesized and evaluated by a cellular autophosphorylation assay for FGF-R2 in the human scirrhous gastric carcinoma cell line, OCUM-2MD3. We also performed metabolic stability studies showing that substitutions at the 7-position of quinoline affect its biological stability. In this study, we achieved a remarkable improvement in the solubility and metabolic stability of the diphenylamine derivative. The most promising compound 15e showed a significant decrease in tumor volume when orally administered.

Preparation of a whole genome phage library using fragmented Escherichia coli genome and its characterization of protein binding properties by surface plasmon resonance.

A novel phage library has been prepared using the Escherichia coli genome digested with three restriction enzymes. The resulting DNA fragments were ligated to the expression vector pCANTAB5 to obtain the library of recombinant M13 phages displaying relatively long exogenous peptides. The library was screened to isolate recombinant phages with high affinity to alkaline phosphatase (AP) from calf intestine. After four rounds of panning three phages (AP1, AP2 and AP3) were shown to have specific binding properties toward AP by enzyme-linked immunosorbent assay. The phages were further characterized by surface plasmon resonance (SPR). Among the three phages AP3 bound the AP-immobilized sensor chip most and caused the highest resonant angle shift. The sensor response decreased with the decrease of the concentration of AP3 added. Furthermore, displacement of AP3 from the AP-immobilized sensor chip was observed upon injection of AP solution to the SPR system, whereas injection of bovine serum albumin solution led to the great increase of the sensor response. This result indicates the specific binding of AP3 to AP.