Induction of adaptive immune responses against antigens incorporated within the capsid of simian virus 40.

Simian virus 40 (SV40) is a monkey polyomavirus. The capsid structure is icosahedral and comprises VP1 units that measure 45 nm in diameter. Five SV40 VP1 molecules form one pentamer subunit, and a single icosahedral subunit comprises 72 pentamers; a single SV40 VP1 capsid comprises 360 SV40 VP1 molecules. In a previous study, we showed that an influenza A virus matrix protein 1 (M1) CTL epitope inserted within SV40 virus-like particles (VLPs) induced cytotoxic T lymphocytes (CTLs) without the need for an adjuvant. Here, to address whether SV40 VLPs induce adaptive immune responses against VLP-incorporated antigens, we prepared SV40 VLPs containing M1 or chicken ovalbumin (OVA). This was done by fusing M1 or OVA with the carboxyl terminus of SV40 VP2 and co-expressing them with SV40 VP1 in insect cells using a baculovirus vector. Intraperitoneal (i.p.) or intranasal administration of SV40 VLPs incorporating M1 induced the production of CTLs specific for the M1 epitope without the requirement for adjuvant. The production of antibodies against SV40 VLPs was also induced by i.p. administration of SV40 VLPs in the absence of adjuvant. Finally, the administration of SV40 VLPs incorporating OVA induced anti-OVA antibodies in the absence of adjuvant; in addition, the level of antibody production was comparable with that after i.p. administration of OVA plus alum adjuvant. These results suggest that the SV40 capsid incorporating foreign antigens can be used as a vaccine platform to induce adaptive immune responses without the need for adjuvant.

Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method.

Affinity maturation is one of the cardinal strategies for improving antibody function using in vitro evolutionary methods; one such well-established method is phage display. To minimise gene deletion, we previously developed an open sandwich (OS) method wherein selection was performed using only phage-displaying VH fragments after mixing with soluble VL fragments. The decrease in anti-EGFR antibody 528 affinity through humanization was successfully recovered by selecting VH mutants using this OS method. However, the affinity was not similar to that of parental 528. For further affinity maturation, we aimed to isolate VL mutants that act in synergy with VH mutants. However, the OS method could not be applied for selecting VL fragments because the preparation of soluble VH fragments was hampered by their instability and insolubility. Therefore, we initially designed a modified OS method based on domain-swapping of VH fragments, from added soluble Fv fragments to phage-displaying VL fragments. Using this novel Fv-added OS selection method, we successfully isolated VL mutants, and one of the Fv comprising VH and VL mutants showed affinity almost equivalent to that of parental 528. This method is applicable for engineering other VL fragments for affinity maturation.

Domain order of a bispecific diabody dramatically enhances its antitumor activity beyond structural format conversion: the case of the hEx3 diabody.

The domains of bispecific diabodies (BsDbs) can be ordered in four different ways; however, the influence of domain order on the cytotoxicity of BsDbs that retarget immune cells against tumor cells had not been addressed. We previously reported the marked antitumor effects of a humanized BsDb that targets epidermal growth factor receptor and CD3 (hEx3-Db). Here, we rearranged the domains of hEx3-Db to examine the influence of domain order on the function of BsDbs. We successfully prepared homogenous dimers of hEx3-Db in all four domain configurations. Interestingly, all three rearranged hEx3s inhibited cancer growth more effectively than did the original hEx3-Db, in which both components were in variable heavy domain (VH)-variable light domain (VL) order (redesignated as hEx3-HL), and the highest effects were observed with hEx3-LH (hEx3-Db with both components in VL-VH order). In addition, hEx3-LH had comparable in vitro growth inhibitory effects to those of the tandem single-chain variable fragment (scFv) format of hEx3-Db (hEx3-tandem scFv (taFv)), which we previously showed to have greater cytotoxicity than does hEx3-HL. Flow cytometry suggested that the enhanced cytotoxicity of hEx3-LH is attributable to structural superiority for cross-linking, similar to that of hEx3-taFv. Furthermore, hEx3-LH inhibited cancer growth in mice more effectively than did hEx3-taFv; this difference may be due to differences in antibody stability. Our results show that merely rearranging the domain order of BsDbs can enhance their effects beyond those with structural format conversion.

Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

Nonunion in proximal phalanx of great toe treated by grafting with precisely processed autologous bone PEG.

We report a rare case of nonunion in the proximal phalanx of the great toe treated by grafting with a precisely processed autologous bone peg. The concept of this surgical method was to fix the nonunion of the proximal phalanx of the great toe using press-fit fixation and a bone peg precisely formed to fit the medullary cavity similar to an intramedullary nail. We believe that our new technique is a viable alternative for the treatment of phalanx fractures or nonunions requiring bone grafts.

Integration of PEGylation and refolding for renaturation of recombinant proteins from insoluble aggregates produced in bacteria--application to a single-chain Fv fragment.

The conjugation of polyethylene glycol (PEGylation) with downsized compact antibodies is an effective method for overcoming the problem of rapid elimination of the compact antibodies from the body. We integrated site-specific PEGylation with the refolding of a single-chain Fv (scFv) of humanized monoclonal antibody 528 with affinity for the epidermal growth factor receptor, to prepare active PEGylated scFv from insoluble aggregates produced in an Escherichia coli expression system. The insertion of a cysteine residue at the C-terminus of scFv to serve as the conjugation site for PEG led to the formation of highly multimeric scFv during the refolding process; however, PEGylation after refolding drastically dispersed the multimer into monomeric active scFv fragments. Further, the PEGylation of partially refolded scFv during the refolding process improved the PEGylation efficiency and suppressed the formation of highly multimeric scFv; consequently, monomeric active scFv fragments were obtained directly from the insoluble aggregates in E. coli. We show that in vitro refolding of PEGylated scFv should be useful for improving downstream processing performance in the production of clinically useful small antibodies from insoluble fractions.

Dumbbell-type spinal solitary fibrous tumor with paraplegia.

Solitary fibrous tumors are rare tumors that most commonly arise in the pleura. This article describes a case of a large dumbbell-shaped solitary fibrous tumor of the thoracic spine that was causing partial paraplegia. The patient was a 75-year-old woman who presented with swelling of the upper back and weakness of the lower extremities. Magnetic resonance imaging revealed a dumbbell-shaped tumor mass compressing the spinal cord, predominantly at the level of T8. The tumor extended transversely from the bilateral foramen of T8-T10 to the back muscles. The patient experienced increasing weakness of the lower extremities, so a T7-T11 laminectomy was performed and the tumor was excised piecemeal. Microscopically, the tumor was found to be composed of a proliferation of fibroblastic spindle cells. Immunohistochemically, it was strongly positive for CD34 but negative for S100. These findings were consistent with a solitary fibrous tumor. Three months postoperatively, thoracic radiographs showed progression of spinal kyphosis. Posterior fusion with instrumentation was performed. Three years postoperatively, there was no clinical evidence of tumor recurrence. Spinal solitary fibrous tumors are rare, particularly dumbbell-shaped tumors; to our knowledge, only 2 have been reported in the literature. The correct diagnosis is aided by noting the bland fibrous histology, strong CD34 immunostaining, and absence of S100 and cytokeratin positivity. Although solitary fibrous tumors of the spine are rare, they should be considered in the differential diagnosis of intraspinal lesions, particularly those occurring near pleura.

Cloning and expression of the MutM gene from obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F).

The gene encoding a MutM from Desulfovibrio vulgaris (Miyazaki F) was cloned and expressed in Escherichia coli. A 5.9-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by XhoI and PvuII, contained a MutM gene and other open reading frames. The nucleotide sequence of the MutM gene indicated that the protein was composed of 336 amino acids. The amino-acid sequence deduced from the MutM gene was highly homologous with the MutM of other bacteria; however an additional insert consisted of 64 amino acids. An expression system for the MutM gene under the control of the T7 promoter was constructed in E. coli. From the kinetic analysis results, the purified His-tagged MutM showed 8-oxoguanine-DNA glycosylase activity comparable with that of MutM from E. coli. In this study, the amounts of mRNA and protein for MutM were scant in the D. vulgaris (Miyazaki F). MutM activity may be induced by oxidative stress. However, its induction may not be frequently generated because sulfate-reducing bacteria generally grow in anaerobic conditions. MutM might play a role in the protection against the mutagenicity of oxygen when oxygen stress exceeded the capacity of the defense systems against oxygen toxicity.