Toshiaki Tsunenari

Prolonged treatment with bicalutamide induces androgen receptor overexpression and androgen hypersensitivity

Various hormone refractory prostate cancer cell models have been established with androgen depletion and have helped to clarify the mechanism for the transition into androgen‐depletion independent status. However, the mechanism of bicalutamide resistance remains unclear because few cell models have been generated.

Recombinant Human Hexamer-Dominant IgM Monoclonal Antibody to Ganglioside GM3 for Treatment of Melanoma

Purpose: L612, a human IgM monoclonal antibody produced by an EBV-transformed human B-cell line, binds to ganglioside GM3 and kills GM3-positive human melanoma cells in the presence of complement. It has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (about 20%), pentameric IgM (about 74%), and other minor IgM molecules. Because hexameric IgM activates complement more effectively than pentameric IgM, we developed and evaluated a hexamer-dominant recombinant IgM for clinical applications. Experimental Design: Chinese hamster ovary (CHO) cells were transfected with heavy- and light-chain genes of L612, with or without the joining-chain gene. Antitumor effects of the recombinant IgM secreted from CHO cells were evaluated in vitro and in vivo. Results: Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was ∼80% hexameric, whereas recombinant IgM from CHO cells transfected with heavy-, light-, and joining-chain genes (designated CJ45) was about 90% pentameric. Both CA19 and CJ45 recombinant IgMs caused complement-dependent cytotoxicity against human and mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller. I.v. injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing a GM3-positive mouse melanoma xenograft. Conclusions: A hexamer-dominant human IgM against GM3 may provide a more potent treatment option for patients with GM3-positive melanoma.

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.

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.

Design and synthesis of an androgen receptor pure antagonist (CH5137291 ) for the treatment of castration-resistant prostate cancer

A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor pure antagonistic activities for the treatment of castration-resistant prostate cancer. Since CH4933468, which we reported previously, had a problem with agonist metabolites, novel thiohydantoin derivatives were identified by applying two strategies. One was the replacement of the alkylsulfonamide moiety by a phenylsulfonamide to avoid the production of agonist metabolites. The other was the replacement of the phenyl ring with a pyridine ring to improve in vivo potency and reduce hERG affinity. Pharmacological assays indicated that CH5137291 (17b) was a potent AR pure antagonist which did not produce the agonist metabolite. Moreover, CH5137291 completely inhibited in vivo tumor growth of LNCaP-BC2, a castration-resistant prostate cancer model.

An anti–glypican 3/CD3 bispecific T cell–redirecting antibody for treatment of solid tumors

An anti–glypican 3/CD3 bispecific T cell–redirecting antibody (ERY974) is a promising therapeutic agent for solid tumors. Double trouble for solid tumors Because the endogenous immune response is not enough to clear a patient’s cancer, therapies are being designed to redirect T cells to tumor cells. This can be done by engineering the cells ex vivo, such as in CAR T cell therapy, or in vivo, such as with bispecific antibodies. Ishiguro et al. describe the development and preclinical testing of a bispecific antibody recognizing CD3 and glypican 3, a common antigen on solid tumors. This bispecific antibody was effective in a variety of mouse cancer models, even when treatment was initiated after the tumor was quite large. Treatment also appeared to be safe when administered to monkeys. These results suggest further development of this antibody for therapeutic use in multiple cancer types. Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell–redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid “on-target off-tumor” toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G–structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein–1) and CTLA-4 (cytotoxic T lymphocyte–associated protein–4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.

CH5137291, an androgen receptor nuclear translocation-inhibiting compound, inhibits the growth of castration-resistant prostate cancer cells

Resistance of prostate cancer to castration is currently an unavoidable problem. The major mechanisms underlying such resistance are androgen receptor (AR) overexpression, androgen-independent activation of AR, and AR mutation. To address this problem, we developed an AR pure antagonist, CH5137291, with AR nuclear translocation-inhibiting activity, and compared its activity and characteristics with that of bicalutamide. Cell lines corresponding to the mechanisms of castration resistance were used: LNCaP-BC2 having AR overexpression and LNCaP-CS10 having androgen-independent AR activation. VCaP and LNCaP were used as hormone-sensitive prostate cancer cells. In vitro functional assay clearly showed that CH5137291 inhibited the nuclear translocation of wild-type ARs as well as W741C- and T877A-mutant ARs. In addition, it acted as a pure antagonist on the transcriptional activity of these types of ARs. In contrast, bicalutamide did not inhibit the nuclear translocation of these ARs, and showed a partial/full agonistic effect on the transcriptional activity. CH5137291 inhibited cell growth more strongly than bicalutamide in VCaP and LNCaP cells as well as in LNCaP-BC2 and LNCaP-CS10 cells in vitro. In xenograft models, CH5137291 strongly inhibited the tumor growth of LNCaP, LNCaP-BC2, and LNCaP-CS10, whereas bicalutamide showed a weaker effect in LNCaP and almost no effect in LNCaP-BC2 and LNCaP-CS10 xenografts. Levels of prostate-specific antigen (PSA) in plasma correlated well with the antitumor effect of both agents. CH5137291 inhibited the growth of LNCaP tumors that had become resistant to bicalutamide treatment. A docking model suggested that CH5137291 intensively collided with the M895 residue of helix 12, and therefore strongly inhibited the folding of helix 12, a cause of AR agonist activity, in wild-type and W741C-mutant ARs. In cynomolgus monkeys, the serum concentration of CH5137291 increased dose-dependently and PSA level decreased 80% at 100 mg/kg. CH5137291 is expected to offer a novel therapeutic approach against major types of castration-resistant prostate cancers.

Involvement of cyclooxygenase-2 in the tumor site-dependent production of parathyroid hormone-related protein in colon 26 carcinoma

It has been shown that in the mouse colon 26 tumor model, tumors grown in the subcutis (subcutis colon 26) caused early onset of cachectic syndromes, whereas those in the liver (liver colon 26) did not. Both interleukin (IL)‐6 and parathyroid hormone‐related protein (PTHrP) were involved in the development of cachectic syndromes in this tumor model. However, whether expression of PTHrP and IL‐6 is differently regulated in the tumor microenvironment is unclear. In the present study, culturing the colon 26 cells under different conditions in vitro revealed that IL‐6 production was increased by monolayer culture under a low‐glucose condition but not by spheroid culture. In contrast, PTHrP production was increased by spheroid culture but not by monolayer culture, even under a low‐glucose condition. Gene expression profiling revealed that the expression of cyclooxygenase (COX)‐2 was up‐regulated in both subcutis colon 26 and spheroid cultures, and that COX‐2 inhibitor NS‐398 suppressed PTHrP production in spheroid cultures. Furthermore, administration of NS‐398 decreased the PTHrP level without affecting the tumor growth in mice bearing subcutis colon 26. These results demonstrate that production of PTHrP and IL‐6 largely depends on the microenvironments in which tumors are developed or metastasized and that up‐regulation of COX‐2 in a necrobiotic environment leads to PTHrP production, thereby causing cachectic syndromes. (Cancer Sci 2007; 98: 1563–1569)

Abstract DDT01-05: First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen

Immune checkpoint inhibitors such as anti-PD1 antibodies have shown promising clinical responses in several solid tumors, however there remain patients who do not show an adequate response. Recent biomarker studies have revealed that the presence of neoantigens in the tumor can determine the level of response, and thus the next challenge will be how to target tumors with a neoantigen level that is too low to be recognized by endogenous cytotoxic T cells. Hope in this area is offered by a T cell-redirecting antibody (TRAB), which bispecifically engages CD3 and a tumor antigen, even at very low expression levels, to activate the inherent cytolytic potential of T cells against target tumor cells. A TRAB is highly potent because T cells are activated only in the presence of the targeted antigens and are not restricted by the specificity of the T cell receptor. Given this very potent cytotoxicity, the key to successfully achieving strong antitumor efficacy while avoiding on-target off-tumor toxicity is to select a highly tumor-selective antigen. Our fully humanized IgG TRAB recognizes CD3 and a highly tumor-selective antigen, glypican-3 (GPC3), which is a fetal protein expressed in a wide variety of tissues during development but suppressed in most adult tissues. On the other hand, an inct101e in GPC3 expression has been reported in hepatocellular carcinoma, gastric cancer, lung squamous cell carcinoma, and other cancers. In nonclinical in vitro pharmacology studies, the anti-GPC3 TRAB elicited activation and proliferation of T cells and T cell-dependent cellular cytotoxicity against a wide variety of GPC3-expressing tumor cells, and showed long-lasting in vivo efficacy against tumor expressing very low levels of GPC3 at a few thousand molecules per cell. Furthermore, in an immunocompetent mouse model using human CD3 transgenic mice, anti-GPC3 TRAB showed strong antitumor efficacy against poorly immunogenic tumors, whereas both the immune checkpoint inhibitors and a conventional ADCC-inducing antibody recognizing GPC3 did not show significant efficacy. Pharmacokinetics and toxicology studies in nonhuman primates showed a plasma half-life comparable to a standard IgG drug, allowing a QW or Q2W regimen in humans, with toxicity which was manageable and reversible; the main observations of transient cytokine elevation and associated clinical symptoms were markedly reduced by steroid premedication. Our anti-GPC3 TRAB, which is supported by proprietary antibody engineering technology (ART-Ig) that enables large-scale GMP manufacturing, has promise as a new approach in cancer immunotherapy. Citation Format: Takahiro Ishiguro, Yasuko Kinoshita, Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Natsuki Ono, Yoko Kayukawa, Mika Kamata-Sakurai, Hirotake Shiraiwa, Akihisa Kaneko, Werner Frings, Shunichiro Komatsu, Junichi Nezu, Mika Endo. First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr DDT01-05.

Abstract 3653: Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model

Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in Phase 1 clinical trial (NCT02748837). ERY974 consists of a common light chain and two different heavy chains that respectively recognize glypican-3 (GPC3) and CD3. The Fc portion of ERY974 is modified to lose FcγR binding to prevent GPC3-independent Fc-mediated effector function. However, binding activity to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface, and induces TRAB-dependent cellular cytotoxicity mediated by the potent effector function of T cells. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head & neck, and esophageal cancer-derived xenograft tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. Cancer immunotherapy, as represented by immune checkpoint inhibitors such as PD-1, PD-L1, and CTLA-4 antibodies, has recently been demonstrating remarkable clinical benefit in various tumor types. However, the number of patients who have survival benefit is limited, and combining cancer immunotherapy with other agents is required to improve the efficacy. Although ERY974 monotherapy is expected to show clinical activity based on the preclinical data, we examined whether further improvement of ERY974-induced efficacy is attained by combination with chemotherapy. Method & Results: We evaluated the combination effect of ERY974 with chemotherapy against xenograft tumors of MKN45 (gastric cancer) or NCI-H446 (lung cancer) either in a NOD-SCID mouse model injected with human T cells or in a humanized non-obese diabetic/shi-scid/IL-2Rγnull model in which differentiated human T cells are constitutively supplied. Although ERY974 monotherapy shows only minor antitumor effect against MKN45 and NCI-H446, combination therapy remarkably enhanced efficacy. In particular, ERY974 in combination with paclitaxel or cisplatin in NCI-H446 tumors caused a tumor disappearance without regrowth for a long period. Conclusion: These preclinical data suggest the possibility that the strategy of combining ERY974 with chemotherapy may succeed in increasing the clinical benefit. Now the combination effect is being further investigated to clarify the mechanism. Citation Format: Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Kayukawa, Hironori Mutoh, Yoko Miyazaki, Takahiro Ishiguro, Shohei Kishishita, Yoshiki Kawabe, Mika Endo. Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3653. doi:10.1158/1538-7445.AM2017-3653