Anti-TNFα domain antibody construct CEP-37247: Full antibody functionality at half the size.

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11 - 13kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g. high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising VL dAb, truncated CH1, hinge, CH2 and CH3 domains, CEP-37247 has a molecular mass of approximately 78kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF-mediated cytotoxicity assay. In a human TNFα-over-expressing mouse model of polyarthritis, CEP-37247 prevents development of disease, and is at least as effective as the marketed product etanercept. Fc functionality is intact - CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above, and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic, where other potential advantages such as enhanced efficacy due to improved tissue distribution, and beneficial immunogenicity profile, will be evaluated.

Hydrolysis of the 5'-p-nitrophenyl ester of TMP by oligoribonucleases (ORN) from Escherichia coli, Mycobacterium smegmatis, and human.

Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3'-5' exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from the beta and gamma subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E. coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (k(cat)=100-650 min(-1), K(M)=0.4-2.0 mM, at pH 8.00 and 25 degrees C, with 1 mM Mn(2+)). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn(2+) being preferred over Mg(2+) as cofactor, and was inhibited by Ni(2+). The concentration dependency of Mn(2+) was examined, giving K(Mn) values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.