High Resolution Crystal Structure of a Human Tumor Necrosis Factor-α Mutant with Low Systemic Toxicity

Abstract

A human tumor necrosis factor-␣ (TNF-␣) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-Å resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the Nterminal seven amino acids that is disordered in the structure of wild-type TNF-␣. Compared with wild-type TNF-␣, it exhibits 11-and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-␣. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29 -36 into a rigid segment as a consequence of induced formation of intra-and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe 56 projects into a hydrophobic cavity, while Ile 52 is exposed to the bulk solvent. Ile 52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-␣. It is also expected that the 1.8-Å resolution structure will serve as an accurate model for explaining the structure-function relationship of wildtype TNF-␣ and many TNF-␣ mutants reported previously and for the design of new TNF-␣ mutants.

O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques

“…Likewise, intrinsic conformational stability is expected to provide increased resistance to chemical and proteolytic degradation of therapeutic proteins in vivo (16) but is probably just one of several factors that influence drug bioavailability and in vivo half-life. Nonetheless, several studies have demonstrated that increasing the conformational stability of therapeutic enzymes, cytokines and antibodies can correlate to increased efficacy and/or half-life in vivo (17)(18)(19)(20).…”

Determination of the Limited Trypsinolysis Pathways of Tumor Necrosis Factor-α and Its Mutant by Electrospray Ionization Mass Spectrometry

“…To reduce its serious systemic toxicity and enhance its in vivo stability, a great number of mutants have been constructed and evaluated for the clinical applicability (22)(23)(24). It has been found that M3S is much more resistant to the proteolysis by trypsin than wild-type TNF-␣ (wtTNF-␣) (19,25). Two cleavage sites of wtTNF-␣, Arg 6 and Arg 44 ( Fig.…”

“…Recently, we have reported the design and X-ray structure of a human tumor necrosis factor-␣ (TNF-␣) mutant (M3S) with low systemic toxicity in vivo (18,19). The biochemical properties of TNF-␣ have been studied extensively because of its potential use as a therapeutic agent for cancer patients (20,21).…”

Development of human tumor necrosis factor-α muteins with improved therapeutic potential

“…Acute lethal toxicities of wild-type TNF-α and selected muteins in D-galactosamine-sensitized ICR mice at positions 52 and 56 on the bottom region of the TNF-α trimer. This mutein showed a high cytotoxic activity towards various human tumor cells and exhibited anticancer activities against transplanted human tumors in nude mice (16)(17)(18). The diverse activities of TNF are mediated by binding to each of the two receptors, TNF-R55 (55 kDa) and TNF-R75 (75 kDa), on the cell surface.…”