Koh Sato

ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST-1

Human BST‐1, a bone marrow stromal cell surface molecule, is a GPI‐anchored protein that facilitates the growth of pre‐B cells. The deduced amino acid sequences of human and mouse BST‐1 show around 30% homology with those of CD38 and Aplysia ADP ribosyl cyclase. Therefore, like CD38, BST‐1 might possess ADP ribosyl cyclase activity. Here, we report the establishment of a stable transformant CHO cell line which secretes truncated human soluble BST‐1, and show that purified soluble BST‐1 displays both ADP ribosyl cyclase cADPR hydrolase activities.

ED-71, a vitamin D analog, is a more potent inhibitor of bone resorption than alfacalcidol in an estrogen-deficient rat model of osteoporosis

Although active vitamin D is used in certain countries for the treatment of osteoporosis, the risk of causing hypercalcemia/hypercalciuria means that there is only a narrow therapeutic window, and this has precluded worldwide approval. The results of our previous animal studies have suggested that the therapeutic effect of active vitamin D on bone loss after estrogen deficiency can be dissociated at least partly from its effect of enhancing intestinal calcium absorption and suppressing parathyroid hormone (PTH) secretion. To test this, we compared the effects of ED-71, a hydroxypropoxy derivative of 1alpha,25-dihydroxyvitamin D3, with orally administered alfacalcidol, on bone mineral density (BMD) and the bone remodeling process as a function of their effects on calcium metabolism and PTH, in a rat ovariectomy (ovx) model of osteoporosis. ED-71 increased bone mass at the lumbar vertebra to a greater extent than alfacalcidol, while enhancing calcium absorption (indicated by urinary calcium excretion) and decreasing serum PTH levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers. These results suggest that active vitamin D exerts an antiosteoporotic effect by inhibiting osteoclastic bone resorption while maintaining osteoblastic function, and that these anticatabolic/anabolic effects of active vitamin D take place independently of its effects on calcium absorption and PTH. The demonstration that ED-71 is more potent in these properties than alfacalcidol makes it an attractive candidate as an antiosteoporotic drug.

Involvement of parathyroid hormone–related protein in experimental cachexia induced by a human lung cancer–derived cell line established from a bone metastasis specimen

Cachexia often causes deterioration in the quality of life in cancer patients; however, its mechanism remains poorly understood. Cachexia has often been observed in experimental animals with bone metastases, and parathyroid hormone–related protein (PTHrP) plays an important role in the formation of such metastases. We therefore investigated the possible involvement of PTHrP in an experimental cachexia model using human lung‐cancer cells (HARA‐B). HARA‐B cells produce a high amount of PTHrP but no TNF‐α, IL‐6 or leukemia inhibitory factor. The s.c. inoculation of HARA‐B cells into nude mice caused reductions in body weight, adipose tissue weight, muscle weight and serum glucose levels. Serum levels of calcium and PTHrP increased. Neutralization of PTHrP with antibody caused rapid weight gain along with a rapid decrease in serum calcium levels. Our findings suggest that PTHrP plays an important role in the development of cancer cachexia. PTHrP therefore is a possible target molecule for the treatment of cancer cachexia.

Humanization of a mouse anti-human interleukin-6 receptor antibody comparing two methods for selecting human framework regions

Mouse monoclonal antibody AUK12-20 binds to human IL-6 receptor and inhibits IL-6 functions. It has been humanized by CDR-grafting for therapeutic use. In the design of reshaped human AUK12-20 VL region, the human framework regions (FRs) from the human Bence-Jones protein REI were used. The reshaped human AUK12-20 light chain, in combination with chimeric AUK12-20 heavy chain, bound to antigen as well as chimeric AUK12-20 antibody. In the design of reshaped human AUK12-20 VH region, two sets of the human FRs were chosen and compared. One set was from the consensus amino acid sequence for human VH regions subgroup (HSG)-I and the other set was from human antibody HAX, the most similar human VH region found in a database of human immunoglobulin sequences. The HSG-I-based and the HAX-based reshaped human AUK12-20 heavy chains in combination with the reshaped human AUK12-20 light chain, showed approximately 90 and 100% antigen-binding and competition-binding activities as compared to the chimeric or mouse AUK12-20 heavy chains. Most importantly, these humanized antibodies inhibited the IL-6-dependent tumor cell growth as well as the original mouse antibody suggesting that these humanized antibodies could be efficacious in human patients. Our results show that both approaches for the design of reshaped human antibodies can be used for successful humanization. The approach based on FRs from the most similar individual human antibody, however, seemed to be best for designing a reshaped human antibody that mimicked as closely as possible the original mouse antibody.

Humanization of mouse ONS-M21 antibody with the aid of hybrid variable regions.

Mouse monoclonal antibody, ONS-M21, directed against human medulloblastoma cells, has been humanized by complementarity determining region (CDR) grafting. A humanized ONS-M21 VH region, comparable to the original mouse ONS-M21 VH region, was easily constructed based on framework regions (FRs) 1, 2 and 3 from human EU antibody and on FR4 from human ND antibody. Five alterations in the FRs were made at amino acids 27, 28, 29, 30 and 94 which are all part of the canonical structure for CDR1 (H1). The humanized ONS-M21 VL regions were constructed based on the FRs from human REI antibody. We first identified five amino acid residues in the FRs at positions 20, 21, 71, 73 and 87 as having a possible adverse influences on antigen binding. None of the versions with a variety of combinations at these five positions showed any bindings to antigen. In order to identify the mouse residues that must be retained in the human FRs, hybrid VL regions were constructed by joining the mouse ONS-M21 VL region and the first humanized version within CDR2. The hybrid VL regions revealed that residues in FR1 and/or FR2 were critical in creating a functional antigen binding site. Redesigning several versions with alterations in FR1 and FR2 revealed that the Pro-46 residue was the only critical residue for creating an antigen binding site. This approach should be helpful in identifying key residues in difficult cases of antibody humanization.

Increased Renal Calcium Reabsorption by Parathyroid Hormone–Related Protein Is a Causative Factor in the Development of Humoral Hypercalcemia of Malignancy Refractory to Osteoclastic Bone Resorption Inhibitors

Purpose: Bisphosphonate and calcitonin lower blood calcium in humoral hypercalcemia of malignancy (HHM) by suppressing osteoclastic bone resorption, but repeated administration of these drugs often leads to relapse. In this study, we examined the roles of parathyroid hormone–related protein (PTHrP) in the development of bisphosphonate- and calcitonin-refractory HHM. Experimental Design: Nude rats bearing the LC-6 JCK tumor xenograft (LC-6 rats) exhibited high bone turnover and HHM. Repeated administration of alendronate induced a sustained suppression of the bone resorption, but it caused only early and transient reduction of the blood calcium levels, leading to unresponsiveness to the drug. Because high blood levels of PTHrP were detected in the LC-6 rats, those that developed alendronate-refractory HHM were treated with an anti-PTHrP antibody. Results: Administration of anti-PTHrP antibody to animals that received repeated administration of alendronate, thereby developing alendronate-refractory HHM, resulted in an increase in fractional excretion of calcium and a marked decrease of blood calcium level. Drug-refractory HHM was also observed in animals that received another osteoclast inhibitor, an eel calcitonin analogue elcatonin. The blood calcium level decreased after the initial administration of elcatonin, but it eventually became elevated during repeated administration. Administration of the anti-PTHrP antibody, but not of alendronate, effectively reduced the blood calcium of the animals that developed elcatonin-refractory HHM. Conclusion: High levels of circulating PTHrP and the resulting augmentation of renal calcium reabsorption is one of the major causes of the emergence of osteoclast inhibitor-refractory HHM. Thus, blockage of PTHrP functions by a neutralizing antibody against PTHrP would benefit patients who develop bisphosphonate- or calcitonin-refractory HHM.

Humanization of an anti-human IL-6 mouse monoclonal antibody glycosylated in its heavy chain variable region

Interleukin-6 (IL-6) inhibitors are good potential therapeutic agents in human patients, and anti-IL-6 antibodies are among the best candidates. Here, we have successfully humanized mouse monoclonal antibody SK2, which specifically binds to IL-6 and strongly inhibits IL-6 functions. Since this antibody possesses N-linked carbohydrates on Asn-30 of VH region, which seems to be very close to an antigen-binding site, influence of these carbohydrates on antigen-binding was investigated. A biosensor study showed that the mouse SK2 Fab and its deglycosylated fragments had almost equal Kd (Kon/Koff), 26.8 nM (1.05 x 10(6)/2.81 x 10(-2)) and 24.7 nM (1.28 x 10(6)/3.15 x 10(-2)), respectively. Furthermore, a mutant chimeric SK2 antibody, in which the N-glycosylation site was removed from the VH region, showed a Kd of 11 nM, almost similar to that of the original chimeric SK2 antibody, determined by Scatchard analysis with 125I-IL-6. These data indicate the carbohydrates of mouse SK2 VH region do not significantly influence antigen-binding activity. In the next step, two versions of each humanized SK2 VL and VH regions were carefully designed based on the amino acid sequences of human REI and DAW, respectively. Only one alteration, Tyr to Phe, was made at position 71 in the two light chains, according to the canonical residue for LI. A N-glycosylation site was introduced on the two heavy chains, by changing Ser to Asn at position 30. All four combinations of humanized light and heavy chains could bind to IL-6 as well as the chimeric SK2 antibody. The light chain first version, however, could not efficiently inhibit IL-6 binding to its receptor, indicating the importance of the LI loop conformation for the inhibitory activity of SK2 antibody. In contrast, both versions of the heavy chains were comparable, in yielding good humanized SK2 antibodies, suggesting that the glycosylation of the SK2 VH region has no influence in recreating a functional antigen-binding site in this humanization.

Parathyroid hormone–related protein (PTHrP) as a causative factor of cancer-associated wasting: Possible involvement of PTHrP in the repression of locomotor activity in rats bearing human tumor xenografts

Nude rats bearing the LC‐6‐JCK human lung cancer xenograft displayed cancer‐associated wasting syndrome in addition to humoral hypercalcemia of malignancy. In these rats, not only PTHrP but also several other human proinflammatory cytokines, such as IL‐6, leukemia‐inducing factor, IL‐8, IL‐5 and IL‐11, were secreted to the bloodstream. Proinflammatory cytokines induce acute‐phase reactions, as evidenced by a decrease of serum albumin and an increase in α1‐acid glycoprotein. Tumor resection abolished the production of proinflammatory cytokines and improved acute‐phase reactions, whereas anti‐PTHrP antibody affected neither proinflammatory cytokine production nor acute‐phase reactions. Nevertheless, tumor resection and administration of anti‐PTHrP antibody similarly and markedly attenuated not only hypercalcemia but also loss of fat, muscle and body weight. Body weight gain by anti‐PTHrP antibody was associated with increased food consumption; increased body weight from anti‐PTHrP antibody was observed when animals were freely fed but not when they were given the same feeding as those that received only vehicle. Furthermore, nude rats bearing LC‐6‐JCK showed reduced locomotor activity, less eating and drinking and low blood phosphorus; and anti‐PTHrP antibody restored them. Although alendronate, a bisphosphonate drug, decreased blood calcium, it affected neither locomotor activity nor serum phosphorus level. These results indicate that PTHrP represses physical activity and energy metabolism independently of hypercalcemia and proinflammatory cytokine actions and that deregulation of such physiologic activities and functions by PTHrP is at least in part involved in PTHrP‐induced wasting syndrome.

Monoclonal antibody to parathyroid hormone-related protein induces differentiation and apoptosis of chondrosarcoma cells.

We investigated the effects of treatment with anti-parathyroid hormone-related protein (1-34) monoclonal murine antibody (anti-PTHrP MoAb) on apoptosis and the differentiation of chondrosarcoma HTB-94 cells. Treatment with anti-PTHrP MoAb accelerated apoptosis of HTB-94 cells in a dose-dependent manner, and anti-PTHrP MoAb also promoted the chondrogenic differentiation of HTB-94 cells. The induction of apoptosis by anti-PTHrP MoAb via imbalance of Bcl-2/Bax ratio and activation of caspase-3 may provide a mechanistic explanation for its potential antitumor effects. Our results suggest the possibility that anti-PTHrP MoAb may be beneficial as a new treatment for chondrosarcoma.

Structural Basis for Antibody Discrimination between Two Hormones That Recognize the Parathyroid Hormone Receptor

Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1–108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an α-helical structure extending from residues 14–29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.