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Nectin-2 is a transmembrane glycoprotein which is involved in the process of Ca2+-independent cell-cell adhesion. In our previous study, we have demonstrated that Nectin-2 is over-expressed in breast and ovarian cancer tissues by using gene expression analysis and immunohistochemistry. Furthermore, we discovered multiple anti-Nectin-2 fully human monoclonal antibodies which inhibited tumor growth in in vivo subcutaneous xenograft models with antibody-dependent cellular cytotoxicity (ADCC) as the principal mechanism of action. In this report, we assessed the toxicity of Y-443, a fully human IgG1/kappa anti-Nectin-2 monoclonal antibody exhibiting strong in vitro ADCC and in vivo anti-tumor activity in cynomolgus monkeys (Macaca fascicularis (Cynos)). Unexpectedly, upon administration, Y-443 induced strong thrombocytopenia through Nectin-2 expressed on Cyno platelets, presumably followed by phagocytosis in the mononuclear phagocytic system. To mitigate the adverse safety profile, we mutated the Fc region of Y-443 to reduce the Fc binding activity to Fcγ receptor I, which is the primary receptor for phagocytosis on macrophages. Moreover, we further engineered the Fc through defucosylation to maintain ADCC activity. The resultant Fc engineered antibody, termed Y-634, demonstrated diminished thrombocytopenia in Cyno toxicological studies and maintained anti-tumor activity in a mouse xenograft model. These findings suggest that Y-634 may have a therapeutic potential for the treatment of Nectin-2 positive cancers, and moreover, Fc engineering is a potential mitigation strategy to ameliorate safety liabilities in antibody induced thrombocytopenia while maintaining antibody potency.

A new animal model to evaluate the long-term growth rate produced by a sustained-release formulation of recombinant human growth hormone (rhGH) over one month was developed and the usefulness of our microcapsule formulations was demonstrated in this model. Long-term pharmacological effects by subcutaneous injection of microcapsules for sustained release of rhGH were evaluated in hypophysectomized (Hpx) rats treated with immunosuppressive agent along with hormone supplement. Copoly(dl-lactic/glycolic)acid (PLGA) microcapsules for sustained release of rhGH, a two-week sustained-release formulation (rhGH-SR-2W) and a one-month sustained-release formulation (rhGH-SR-1M), were prepared by a solid-in-oil-in-water emulsion solvent evaporation technique. Body-weight gain, body-length gain and serum levels of rat insulin-like growth factor-l (rlGF-l) induced by subcutaneous injection of rhGH-SR were compared with those by daily injections of rhGH solution in Hpx rats for 35 days. Serum IGF-l levels in Hpx rats after the injection of rhGH-SR-2W microcapsules were higher than those after daily injections of rhGH solution. Body-length gain, a new parameter, after single injection of rhGH-SR-1M microcapsules demonstrated the higher growth rate than that after daily injections of rhGH solution for 35 days. Thus, single injection of rhGH-SR microcapsules demonstrated long-term pharmacological effects greater than those by daily injections of rhGH solution in a newly developed model, immunosuppressed Hpx rats.

Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI-TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N-glycans contained a Gal(β1–6)Man-unit which has not been found in glycoprotein-N-glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.

An additional methionine residue of recombinant human growth hormone (hGH) was converted into an oxoacyl form with glyoxylic acid, copper(II) sulfate and pyridine, and then cleaved from the rest of the protein with 3,4-diaminobenzoic acid in the presence of 1 M AcOH and 2 M HCO2Na. The conditions for N-terminal methionine cleavage worked better than the previous conditions with 1,2-phenylenediamine. The same protocol was also applicable to the methionylated forms of recombinant human betacellulin (BTC), neurotrophin-3 (NT-3) and human interleukin-2 (IL-2). The conversion yield for hGH, BTC, NT-3 and IL-2 increased up to approximately 80, 70, 55 and 50%, respectively. These results indicate that non-methionylated recombinant proteins could be prepared from the methionylated derivatives by chemical methods.

Sustained release of TAK-029 [4-(4-amidinobenzoylglycyl)-3-methoxycarbonyl-2-oxopiperazine-l-acetic acid], a glycoprotein (GP) IIb/IIIa antagonist, from injectable microspheres was achieved by a W/O/W emulsion solvent evaporation technique using copoly( d -lactic/glycolic)acid (PLGA). Entrapment of the drug into microspheres increased with addition of sodium chloride into an external aqueous polyvinyl alcohol solution. Addition of l -arginine to an internal water phase dose-dependently reduced initial burst of the drug from the microspheres in vitro and in vivo, probably by forming hydrophobic diffusion barriers with rigid inner structure and increased glass transition temperature (Tg). Microspheres obtained using sodium chloride and l -arginine demonstrated sustained plasma levels of TAK-029 for 3 weeks after subcutaneous injection in rats, while causing a slight increase of its plasma levels in 2–3 weeks.

We have produced a chimeric bispecific antibody that has dual specificity of human fibrin and urokinase-type plasminogen activator (u-PA). Complementary DNAs for variable regions of both anti-fibrin and anti-u-PA antibodies were cloned from two murine hybridomas secreting respective antibodies using polymerase chain reaction (PCR) techniques, and joined to cDNAs for human constant regions to form chimeric antibody genes. Both of two expression vectors for chimeric anti-fibrin and chimeric anti-u-PA antibodies were sequentially introduced into Chinese hamster ovary cells, and stable transfectants secreting the chimeric bispecific antibody were obtained. The highest producer transfectant (SULF/C2-30) secreted high level (about 40 μg ml−1) of total chimeric IgG and about 2% of the IgG had the bispecific activity of binding with both antigens. The chimeric bispecific antibody was purified by a combination of affinity chromatographies employing antigen-coupled columns and hydroxyapatite high-performance liquid chromatography. The purified chimeric bispecific antibody significantly enhanced the thrombolytic potency of single chain u-PA in an in vitro clot lysis assay as well as the original murine bispecific antibody.

LIGHT is a membrane-bound protein that belongs to the tumor necrosis factor (TNF) superfamily ligands. In this study, we established an effective strategy for producing a bioactive soluble form of LIGHT (sLIGHT), an extracellular region (Ile 84 –Val 240 ) of human LIGHT. Because sLIGHT was expressed as inclusion bodies in Escherichia coli , we investigated reagents that enhance the renaturation of sLIGHT from the inclusion bodies. Interestingly, l -cysteine in the denaturation buffer containing 3.5 M guanidine hydrochloride significantly improved the renaturation efficiency of sLIGHT. The effect of l -cysteine was synergistically enhanced by l -arginine in the refolding buffer. The optimal concentrations of l -cysteine and l -arginine in the denaturation and refolding buffers were 8 mM and 0.8 M, respectively. With these buffers, approximately 90 mg of sLIGHT was purified from 200 g of frozen E. coli cells. sLIGHT thus obtained significantly induced apoptosis in the WiDr human colon adenocarcinoma cell line at nanomolar concentrations, the same amount of sLIGHT that was produced by Sf9 insect cells. These results suggest that l -cysteine in the denaturation buffer enhances the renaturation of recombinant proteins from inclusion bodies in E. coli .

To detect in vivo activated platelets in humans as well as in animal models of thrombosis, we developed a new murine monoclonal antibody, 2T60, specific for activated human and rabbit platelets by immunizing with human thronibin-activated platelets. 2T60 (IgG(1) subclass) showed a great difference between binding to the thrombin-activated and resting human and rabbit platelets on ELISA and flow cytometer analysis. Immunoblotting analysis revealed that 2T60 reacted with a 130 or 106 kDa protein of human or rabbit platelets, respectively, only under non-reducing conditions. (125)I-labeled 2T60 inserted into the intermediate gel of CIE of solubilized human platelets was incorporated into a immunoprecipitation line. 2T60 immunoprecipitated a protein of 130 or 115 kDa from human or rabbit platelets, respectively, which had been activated and (125)I-surface-labeled. The N-terminal sequence of the affinity purified 2T60 antigen of human platelets was identical to that of GMP 140. There were differences in the carbohydrate chain content of GMP 140 between human and rabbit platelets. In experimental cerebral thrombosis of rabbits that had been injected with 2T60, the platelets adhering to the exposed subendothelium and contained in thrombi were found to bind 2T60 prominently. These results suggest that 2T60 may be a useful tool for clinical and experimental studies of thrombotic disease.

LIGHT is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. LIGHT forms a membrane-anchored homotrimeric complex on the cell surface and is often processed as a soluble protein. Recombinant soluble human LIGHT produced by mammalian cells or Escherichia coli is functional at nanomolar concentrations. However, there is little information about the biological activity of mouse LIGHT (mLIGHT) because of the difficulty in producing bioactive soluble mLIGHT. In this study, recombinant trimeric soluble mLIGHT, or Foldon-mLIGHT, was produced by fusing mLIGHT with the trimerization domain foldon from bacteriophage T4 fibritin. Foldon-mLIGHT was secreted from 293F cells as a 68-kDa trimeric protein. The recombinant protein potently inhibited the growth of the FM3A mouse mammary carcinoma cell line with an IC(50) of 77 pM; however, the monomer or dimer forms of mLIGHT produced by E. coli or mammalian cell systems showed weak or no inhibitory activity. These data clearly indicated that trimerization of soluble mLIGHT is essential for its biological activity.

An additional methionine residue of recombinant human growth hormone (hGH) was converted into an oxoacyl form with glyoxylic acid, copper(II) sulfate and pyridine, and then cleaved from the rest of the protein with 3,4-diaminobenzoic acid in the presence of 1 M AcOH and 2 M HCO2Na. The conditions for N-terminal methionine cleavage worked better than the previous conditions with 1,2-phenylenediamine. The same protocol was also applicable to the methionylated forms of recombinant human betacellulin (BTC), neurotrophin-3 (NT-3) and human interleukin-2 (IL-2). The conversion yield for hGH, BTC, NT-3 and IL-2 increased up to approximately 80, 70, 55 and 50%, respectively. These results indicate that non-methionylated recombinant proteins could be prepared from the methionylated derivatives by chemical methods.

SummaryA hybrid hybridoma (FU1-74), secreting a bispecific monoclonal antibody (bs mAb), was obtained by fusion of a murine hybridoma secreting a monoclonal antibody (mAb) specific for human fibrin with a murine hybridoma secreting a mAb against urokinase-type plasminogen activator (u-PA). The bs mAb (MA-FU1-74), purified to homogeneity from mouse ascitic fluid, migrated as a single band with apparent M r 150,000 on non-reduced SDS-PAGE and had an affinity for both human fibrin (K a = 2 × 107 M–1) and for u-PA (K a = 108 M–1) comparable to that of the mAbs obtained from the respective parental hybridomas. MA-FU1-74 did not influence the enzymatic activity of two-chain u-PA (tcu-PA) towards plasminogen or towards a chromogenic substrate. The complex of MA-FU1-74 with recombinant single chain u-PA (rscu-PA) or with tcu-PA (urokinase) enhanced the fibrinolytic potency of the plasminogen activator towards clotted human plasma 20-fold and 5-fold, respectively.In a hamster pulmonary embolism model, the rscu-PA/MA-FU1-74 complex had a 13- to 17-fold increased thrombolytic potency (percent lysis per mg/kg u-PA administered) relative to that of rscu-PA. The specific thrombolytic activity (percent lysis per εg/ml steady state plasma level of u-PA antigen) of the complex was, however, not significantly different from that of rscu-PA. The complex of rscu-PA with the parental anti-u-PA mAb (MA-UK1-3) had only a 2-fold enhanced thrombolytic potency relative to that of rscu-PA and had a 5-fold decreased specific thrombolytic activity. The plasma clearance rates of the complexes of rscu-PA with both MA-FU1-74 and MA-UK1-3 were about 10-fold lower than that of rscu-PA. In a rabbit jugular vein thrombosis model, the rscu-PA/MA-FU1-74 complex had a 4-fold enhanced thrombolytic potency, an unchanged specific thrombolytic activity and 20-fold reduced plasma clearance. In both animal models, the rscu-PA/MA-FUl-74 complex did not cause more extensive systemic activation of the fibrinolytic system than rscu-PA.It is concluded that the bispecific anti-fibrin/anti-u-PA mAb MA-FU1-74 targets u-PA to the fibrin clot, resulting in a significantly enhanced thrombolytic potency of the plasminogen activator.

Keyhole limpet hemocyanin (KLH) is known to share carbohydrate epitopes with Schistosoma mansoni. In order to define the structural basis for the observed serological cross-reactivity, KLH glycans were released either by enzyme treatment or by hydrazinolysis and probed with a rabbit hyperimmune serum directed against S. mansoni egg antigen. Both major, non-reacting oligosaccharide species as well as the minor compounds recognized were isolated by two-dimensional high performance liquid chromatography and in part by lectin affinity chromatography, and characterized by mass spectrometry. The results revealed that KLH carries predominantly high mannose-type glycans as well as short sugar chains. As a characteristic feature, a number of the latter glycans contained a Gal(beta1-6)Man-unit, which has not yet been found in glycoprotein-N-glycans. Oligosaccharides cross-reacting with schistosomal glycans comprised a terminal Fuc(alpha1-3)GalNAc-motif, which appears to represent the main carbohydrate epitope mediating cross-reactivity of KLH with glycoconjugates from S. mansoni.

The human thromboxane A2 receptor (TXA2-R)-coding gene was introduced into Chinese hamster ovary cells and a cell line (TCHO-25) stably expressing TXA2-R, at a level of 3 x 10(5)/cell, was obtained. An anti-asthmatic agent AA-2414 [(+-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7- phenylheptanoic acid] competitively inhibited the specific binding of a TXA2 mimic ([3H]U-46619) to the TCHO-25 cells, with an IC50 of 6.0 x 10(-8) M, indicating that the drug is an antagonist of human TXA2-R. The TCHO-25 cells offer a tool for the screening and characterization of human TXA2-R antagonists.

Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI-TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N-glycans contained a Gal(beta1-6)Man-unit which has not been found in glycoprotein-N-glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.

TAK-778, ((2R, 4S)-(-)-N-[(4-diethoxyphosphorylmethyl)phenyl]-1, 2, 4, 5-tetrahydro-4-methyl-7, 8-methylene-dioxy-5-oxo-3-benzothiepin-2-carboxamide), is a novel osteoblast differentiation-promoting compound, and its sustained-release formulation is expected to be clinically used for the enhancement of fracture healing. To date, TAK-778 levels in serum have been measured using conventional reverse-phase HPLC with inferior sensitivity and time-consuming procedures. We have produced polyclonal antibodies against TAK-778 by using one of its derivatives coupled with a carrier protein, and developed a one-step ELISA for the determination of TAK-778 in serum. The antibodies had minimal cross-reactivities to the biologically inactive metabolites including the oxidized-form (0.36%) and the cleavaged-form (0.00083%). The competitive ELISA was accomplished within three hours with a detection limit of 0.5ng/ml serum, and the coefficients of variations for samples ranging from 1 ng/ml to 80 ng/ml were 1.1-4.4% in an intra-assay and 3.1-9.6% in an inter-assay. The present ELISA is so rapid, sensitive and selective for TAK-778 that it could be conveniently used in a clinical field for the determination of many serum specimens.

An ex vivo antiviral assay was established which uses hepatocytes from mice given recombinant mouse interferon-beta (rmIFN-beta). Assay results were compared with results obtained with a 2',5'-oligoadenylate synthetase (2-5AS) assay. rmIFN-beta was intraperitoneally administered to C3H mice and the antiviral state of their liver parenchymal cells was evaluated in an in vitro cytopathic effect assay. In this assay, cells are infected with vesicular stomatitis virus (VSV) and surviving cells are determined colorimetrically. The antiviral state was measured as the resistance of hepatocytes to VSV infection with increasing doses of rmIFN-beta. The antiviral state correlated well with the dose-dependent increase in hepatic 2-5AS activity. This good correlation suggests that induction of 2-5AS mediates the antiviral action of interferon in liver tissue. This ex vivo assay could be a useful tool for estimating the ability of hepatocytes to resist hepatitis virus infection.

Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.

Murine hybrid hybridomas secreting bispecific monoclonal antibodies (bs mAbs) were constructed by fusing hybridomas secreting an anti-tissue plasminogen activator (tPA) or anti-urokinase (UK) mAb with hybridomas secreting a mAb which binds to human fibrin but not to fibrinogen. The bs mAbs reactive to both fibrin and PA (tPA or UK) were purified by affinity chromatography employing the respective antigen-coupled columns and characterized by fibrin-binding, amidolytic and fibrinolytic assays. Immunochemical conjugation of PAs and the bs mAbs did not impair the catalytic activity of PAs at all and made it possible to concentrate PAs on fibrin clot. Pretreatment of fibrin with the bs mAbs enhanced the fibrin-binding of PAs and the subsequent fibrinolysis.

The P1' Ser(50) at the second reactive site of soybean protease inhibitor C-II was replaced with arginine to confirm the contribution of this residue to the inhibition. The Arg derivative had less trypsin inhibitory activity (Ki = 1 X 10(-7) M) than the Ser derivative (Ki = 2 X 10(-8) M), in contrast to the results obtained from studies on peanut protease inhibitor B-III reported in the previous paper (J. Biochem. 101, 723-728 (1987)). These results suggest that each Bowman-Birk type inhibitor has an amino acid at the P1' position inherently best suited to maintaining its inhibitory activity, and that serine is not unique for the P1' amino acid in Bowman-Birk type inhibitors.

The deimination of the arginine residues in peanut trypsin-chymotrypsin inhibitor B-III caused the disappearance of its trypsin-inhibitory activity. Peanut protease inhibitor B-III was incubated with peptidylarginine deiminase, resulting in the conversion of 2.5 mol of arginine to citrulline and in the loss of its trypsin-inhibitory activity. However, the ability of the deiminated inhibitor to inhibit chymotrypsin was as strong as before. Structural analysis of the deiminated B-III indicated that the P1 arginine residues at both reactive sites, Arg(10) and Arg(38), were completely modified to citrulline by the action of peptidylarginine deiminase, and that the Arg(60) in the C-terminal region of B-III was partially deiminated. These residues seem to be exposed on the surface of the molecule. The P1' arginine residue at the first reactive site, Arg(11), was not deiminated at all.

We have constructed a murine hybrid hybridoma that secretes a bispecific monoclonal antibody (bs mAb) by fusing a hybridoma secreting an anti-tissue plasminogen activator (tPA) mAb with a hybridoma secreting a mAb that binds to human fibrin but not to fibrinogen. The bs mAb, reactive to both fibrin and tPA, was purified by affinity chromatography employing antigen-coupled columns and characterized by SDS-PAGE, a fibrin-binding assay, an amidolytic assay, a fibrinolytic assay and a thrombolytic assay. The immunochemical conjugation of tPA and the bs mAb did not impair the catalytic activity of tPA and made it possible to concentrate tPA at fibrin clots. Pretreatment of fibrin with the bs mAb enhanced the fibrin-binding of tPA and subsequent fIbrinolysis. The tPA-bs mAb immunoconjugate exhibited more thrombolytic activity than tPA alone in the rabbit jugular vein model.