Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Non-antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity.

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG4 antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non-antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG4-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non-antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.

Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity.

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model.

Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

Deficiency of the Erc/mesothelin gene ameliorates renal carcinogenesis in Tsc2 knockout mice.

Genetic crossing experiments were performed between tuberous sclerosis-2 (Tsc2) KO and expressed in renal carcinoma (Erc) KO mice to analyze the function of the Erc/mesothelin gene in renal carcinogenesis. We found the number and size of renal tumors were significantly less in Tsc2+/-;Erc-/- mice than in Tsc2+/-;Erc+/+ and Tsc2+/-;Erc+/- mice. Tumors from Tsc2+/-;Erc-/- mice exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen (Ki67) and TUNEL analysis, respectively. Adhesion to collagen-coated plates in vitro was enhanced in Erc-restored cells and decreased in Erc-suppressed cells with siRNA. Tumor formation by Tsc2-deficient cells in nude mice was remarkably suppressed by stable knockdown of Erc with shRNA. Western blot analysis showed that the phosphorylation of focal adhesion kinase, Akt and signal transducer and activator of transcription protein 3 were weaker in Erc-deficient/suppressed cells compared with Erc-expressed cells. These results indicate that deficiency of the Erc/mesothelin gene ameliorates renal carcinogenesis in Tsc2 KO mice and inhibits the phosphorylation of several kinases of cell adhesion mechanism. This suggests that Erc/mesothelin may have an important role in the promotion and/or maintenance of carcinogenesis by influencing cell-substrate adhesion via the integrin-related signal pathway.

Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization.

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra), an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.

Effective screening method of agonistic diabodies based on autocrine growth.

Agonistic diabodies that mimic the function of natural ligands are expected to increase the value of therapeutic antibodies. We have developed a method that detects agonistic diabodies based on their ability to transduce growth signals through receptors, thereby permitting cytokine-independent growth of BaF/3-derived cytokine-dependent cells. Retrovirus-mediated expression of the diabody in cytokine-dependent cells was followed by selection of clones for growth in the absence of cytokine. A diabody library derived from splenocytes of human Mpl immunized mice was constructed. Infection of cells with viral particles led to the isolation of over 500 autonomously growing clones whose cultured supernatants showed agonistic activities against Mpl. Genome-integrated diabodies were cloned; representative clone AB317 showed agonistic activities as potent as a natural ligand and cross-reactive against mouse Mpl.

Lengthening the locking loop repair for zone 2 flexor tendon laceration and partial lateral release of the tendon sheath.

The authors present the clinical outcomes of nine zone 2 flexor tendon repairs using a locking loop technique (i.e. the Modified Pennington technique). The locking loops were located approximately 10 mm away from the lacerated tendon ends to "lengthen" the locking loop repair, as experimentally and clinically recommended. The partial lateral release of the tendon sheath, including the A2 and/or A4 pulley, was performed not only to locate the sutures but also to allow a full range of motion of the repair without catching on the tendon sheath, as clinically recommended. All the patients were followed up for six months or more except for one. All digits were evaluated as excellent or good at the final follow-up by the original Strickland criteria. No rupture occurred and no bowstring of the flexor tendon was observed. The clinical outcomes of the current study indicate that "lengthening" the locking loop repair is effective for zone 2 flexor tendon repair and that the partial lateral release of the tendon sheath, including the A2 and/or A4 pulley, does not result in the bowstring of the flexor tendon.

Long-term follow-up on the use of vascularized fibular graft for the treatment of congenital pseudarthrosis of the tibia.

BACKGROUND: Congenital pseudoarthrosis of the tibia (CPT) is one of the most difficult conditions to treat. METHODS: Five girls and 3 boys with CPT were treated by vascularized fibular grafting (VFG). The average age at VFG was 7.0 years (range: 1.9-11.5 years) with an average follow-up term of 11.7 years (range: 4.9-19.6 years). Five of the children had undergone multiple operations before VFG, while the other 3 had no such history. RESULTS: Bone consolidation was obtained in all cases after an average term of 6.6 months (range: 4-10 months); this was with the first VFG in 7 cases but with the second VFG in 1 case. Complication of stress fracture and ankle pain occurred in 1 and 3 cases, respectively, only in cases undergoing multiple operations. Leg-length discrepancy was more prominent in the patients with multiple previous operations (mean: 7.5 cm), than in the cases with no prior surgery (mean: 0.7 cm). CONCLUSION: The long-term results of VFG for CPT were excellent, especially in the cases, with no prior surgery. VFG should be considered as a primary treatment option for CPT.

Diode-pumped 1 kW Q-switched Nd:YAG rod laser with high peak power and high beam quality.

We have demonstrated high-peak-power generation at 1 kW average power by applying an acousto-optic Q switch to a quasi-cw diode-pumped Nd:YAG master oscillator power amplifier. We achieved a maximum peak power of 2.3 MW by driving the Q switch in burst mode. The average repetition rate was 6 kHz. The corresponding beam quality was M2 = 9.

Novel circular-beam equalizing techniques that use graded-index fiber optics for a high-power laser diode.

We demonstrate a novel method of equalizing a laser diode (LD) beam into a circular beam. This method uses the twist effect of graded index (GI) fiber optics. An asymmetric LD beam with beam qualities of M2 = 500 in the slow axis and M2 = 4 in the fast axis is converted successfully into a symmetric circular beam with a beam quality of M2 = 175. The circular-output beam with 92% coupling efficiency from the fiber input to the fiber output is obtained with a 5-m-long GI1200 (1200 means a core diameter of 1200 microm) fiber for a 2-W LD array. We extend the experiments to a higher-power source with higher asymmetric beam qualities of M2 = 3000 and M2 = 4. By slightly bending the GI10000 (10000 means a core diameter of 10,000 microm) fiber, we have succeeded in generating a symmetric beam with a improved beam quality of M2 = 2000. The average beam quality is preserved when the asymmetric ratio is not high, and the beam quality degradation ratio is investigated up to asymmetric ratios of 750.

Aggressive active mobilization following zone II flexor tendon repair using a two-strand heavy-gauge locking loop technique.

In vitro and in vivo experimental studies have shown that a new two-strand technique increases the tensile strength of flexor tendon repair and eliminates gap formation at the healing repair site. The purpose of the current study was to clinically evaluate the new technique, followed by an aggressive active mobilization program. Seven digits with zone II flexor tendon lacerations were treated using the technique, employing a heavy (2-0) braided polyester suture. The patients were encouraged to perform active mobilization of the injured digits by themselves with almost a full range of flexion and extension after they were instructed by the surgeon for few days from the first postoperative day. All patients were followed up for at least 6 months, except for one, with whom contact was lost in 14 weeks postoperatively. Six of the seven digits were evaluated as excellent in 6 months by the original Strickland criteria, thus showing that the combination of the new repair technique and aggressive active mobilization is effective for zone II flexor tendon repair.

Automatic output-beam-collimating configuration for high-average-power multirod resonator-amplifier systems.

A novel configuration for a multirod solid-state laser system is proposed. We stabilize the thermal-lens-dependent variation in the output-beam parameters by extracting the laser beam from the collimating point of periodic beam propagation. The performance is confirmed with a 100-W class diode-pumped Nd:YAG laser-amplifier system to demonstrate tenfold stabilization of the output-beam diameter.