Targeting advanced prostate cancer with STEAP1 chimeric antigen receptor T cell and tumor-localized IL-12 immunotherapy.

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a cell surface antigen for therapeutic targeting in prostate cancer. Here, we report broad expression of STEAP1 relative to prostate-specific membrane antigen (PSMA) in lethal metastatic prostate cancers and the development of a STEAP1-directed chimeric antigen receptor (CAR) T cell therapy. STEAP1 CAR T cells demonstrate reactivity in low antigen density, antitumor activity across metastatic prostate cancer models, and safety in a human STEAP1 knock-in mouse model. STEAP1 antigen escape is a recurrent mechanism of treatment resistance and is associated with diminished tumor antigen processing and presentation. The application of tumor-localized interleukin-12 (IL-12) therapy in the form of a collagen binding domain (CBD)-IL-12 fusion protein combined with STEAP1 CAR T cell therapy enhances antitumor efficacy by remodeling the immunologically cold tumor microenvironment of prostate cancer and combating STEAP1 antigen escape through the engagement of host immunity and epitope spreading.

Emerging approaches for preventing cytokine release syndrome in CAR-T cell therapy.

Chimeric antigen receptor (CAR) T cells have demonstrated remarkable anti-tumor efficacy against hematological malignancies, such as leukemia and lymphoma. However, patients treated with CAR-T cells frequently experience cytokine release syndrome (CRS), one of the most life-threatening adverse events of the therapy induced by systemic concentrations of pro-inflammatory cytokines throughout the body. Immunosuppressants such as tocilizumab are currently administered to treat the onset and progression of CRS symptoms. In order to reduce the risk of CRS, newly designed next-generation CAR-T treatments are being developed for both hematopoietic malignancies and solid tumors. In this review, we discuss six classes of interesting approaches that control cytokine production of CAR-T cell therapy: adaptor-based strategies, orthogonal cytokine-receptor pairs, regulation of macrophage cytokine activity, autonomous neutralization of key cytokines, kill switches and methods of reversible suppression of CARs. With these strategies, future CAR-T cell therapies will be designed to preemptively inhibit CRS, minimize the patients' suffering, and maximize the number of benefiting patients.

pH-Sensitive branched ß-glucan-modified liposomes for activation of antigen presenting cells and induction of antitumor immunity.

Induction of cellular immunity is important for effective cancer immunotherapy. Although various antigen carriers for cancer immunotherapy have been developed to date, balancing efficient antigen delivery to antigen presenting cells (APCs) and their activation via innate immune receptors, both of which are crucially important for the induction of strong cellular immunity, remains challenging. For this study, branched ß-glucan was selected as an intrinsically immunity-stimulating and biocompatible material. It was engineered to develop multifunctional liposomal cancer vaccines capable of efficient interactions with APCs and subsequent activation of the cells. Hydroxy groups of branched ß-glucan (Aquaß) were modified with 3-methylglutaric acid ester and decyl groups, respectively, to provide pH-sensitivity and anchoring capability to the liposomal membrane. The modification efficiency of Aquaß derivatives to the liposomes was significantly high compared with linear ß-glucan (curdlan) derivatives. Aquaß derivative-modified liposomes released their contents in response to weakly acidic pH. As a model antigenic protein, ovalbumin (OVA)-loaded liposomes modified with Aquaß derivatives interacted efficiently with dendritic cells, and induced inflammatory cytokine secretion from the cells. Subcutaneous administration of Aquaß derivative-modified liposomes suppressed the growth of the E.G7-OVA tumor significantly compared with curdlan derivative-modified liposomes. Aquaß derivative-modified liposomes induced the increase of CD8+ T cells, and polarized macrophages to the antitumor M1-phenotype within the tumor microenvironment. Therefore, pH-sensitive Aquaß derivatives can be promising materials for liposomal antigen delivery systems to induce antitumor immune responses efficiently.

Synthesis and biological evaluation of a monocyclic Fc-binding antibody-recruiting molecule for cancer immunotherapy.

Antibody-recruiting molecules (ARMs) are bispecific molecules composed of an antibody-binding motif and a target-binding motif that redirect endogenous antibodies to target cells to elicit immune responses. To enhance the translational potential of ARMs, it is crucial to design antibody/target-binding motifs that have strong affinity and are easy to synthesize. Here, we synthesized a novel Fc-binding ARM (Fc-ARM) that targets folate receptor (FR)-positive cancer cells, Reo-3, using a recently developed monocyclic peptide 15-Lys8Leu, which binds strongly to the Fc region of an antibody. Reo-3 bound to the Fc region of the antibody with a K d of 5.8 nM, and recruited a clinically used antibody mixture to attack FR-positive IGROV-1 cells as efficiently as Fc-ARM2, in which a bicyclic Fc-binding peptide was used. These results indicate that 15-Lys8Leu, which can be synthesized readily, is suitable for various applications including the development of Fc-ARMs.

Fc-Binding Antibody-Recruiting Molecules Targeting Prostate-Specific Membrane Antigen: Defucosylation of Antibody for Efficacy Improvement*.

Synthetic small molecules that redirect endogenous antibodies to target cells are promising drug candidates because they overcome the potential shortcomings of therapeutic antibodies, such as immunogenicity and the need for intravenous delivery. Previously, we reported a novel class of bispecific molecules targeting the antibody Fc region and folate receptor, named Fc-binding antibody-recruiting molecules (Fc-ARMs). Fc-ARMs can theoretically recruit most endogenous antibodies, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) to eliminate cancer cells. Herein, we describe new Fc-ARMs that target prostate cancer (Fc-ARM-Ps). Fc-ARM-Ps recruited antibodies to cancer cells expressing prostate-specific membrane antigen but did so with lower efficiency compared with Fc-ARMs targeting the folate receptor. Upon recruitment by Fc-ARM-P, defucosylated antibodies efficiently activated natural killer cells and induced ADCC, whereas antibodies with intact N-glycans did not. The results suggest that the affinity between recruited antibodies and CD16a, a type of Fc receptor expressed on immune cells, could be a key factor controlling immune activation in the Fc-ARM strategy.

Modification of nitric oxide donors onto a monoclonal antibody boosts accumulation in solid tumors.

Although monoclonal antibodies (mAbs) have revolutionized cancer treatment, their accumulation in solid tumors is limited and requires improvement to enhance therapeutic efficacy. Here we developed a strategy to modify mAb with a donor of nitric oxide (NO) because NO functions to vasodilate as well as to enhance the permeability of vascular endothelium, which will contribute to enhancing the tumor accumulation of mAb. We selected S-nitrosothiol as a NO donor and established the procedure to modify S-nitrosothiol group on mAb under ambient conditions. The modified mAb (Ab-SNO) thus obtained released NO in a preferable speed and maintained its original properties such as binding affinity to a target antigen and efficacy to induce antibody-dependent cellular cytotoxicity. We demonstrated that Ab-SNO enhanced the tumor accumulation of co-administered proteins such as antibody and serum albumin.

Induction of ADCC by a folic acid-mAb conjugate prepared by tryptophan-selective reaction toward folate-receptor-positive cancer cells.

We developed conjugates between monoclonal antibody (mAb) and folic acid (FA) by using a tryptophan (Trp)-selective reaction, which yields relatively homogenous products compared to conventional methods. The obtained mAb-FA conjugates showed significant cellular cytotoxicity toward folate receptor-expressing cancer cells, demonstrating that the conjugates retained the Fc region's original function.

Fc-binding antibody-recruiting molecules exploit endogenous antibodies for anti-tumor immune responses.

Redirecting endogenous antibodies in the bloodstream to tumor cells using synthetic molecules is a promising approach to trigger anti-tumor immune responses. However, current molecular designs only enable the use of a small fraction of endogenous antibodies, limiting the therapeutic potential. Here, we report Fc-binding antibody-recruiting molecules (Fc-ARMs) as the first example addressing this issue. Fc-ARMs are composed of an Fc-binding peptide and a targeting ligand, enabling the exploitation of endogenous antibodies through constant affinity to the Fc region of antibodies, whose sequence is conserved in contrast to the Fab region. We show that Fc-ARM targeting folate receptor-α (FR-α) redirects a clinically used antibody mixture to FR-α+ cancer cells, resulting in cancer cell lysis by natural killer cells in vitro. Fc-ARMs successfully interacted with antibodies in vivo and accumulated in tumors. Furthermore, Fc-ARMs recruited antibodies to suppress tumor growth in a mouse model. Thus, Fc-ARMs have the potential to be a novel class of cancer immunotherapeutic agents.

Engineered collagen-binding serum albumin as a drug conjugate carrier for cancer therapy.

Serum albumin (SA) is used as a carrier to deliver cytotoxic agents to tumors via passive targeting. To further improve SA's tumor targeting capacity, we sought to develop an approach to retain SA-drug conjugates within tumors through a combination of passive and active targeting. SA was recombinantly fused with a collagen-binding domain (CBD) of von Willebrand factor to bind within the tumor stroma after extravasation due to tumor vascular permeability. Doxorubicin (Dox) was conjugated to the CBD-SA via a pH-sensitive linker. Dox-CBD-SA treatment significantly suppressed tumor growth compared to both Dox-SA and aldoxorubicin treatment in a mouse model of breast cancer. Dox-CBD-SA efficiently stimulated host antitumor immunity, resulting in the complete eradication of MC38 colon carcinoma when used in combination with anti-PD-1 checkpoint inhibitor. Dox-CBD-SA decreased adverse events compared to aldoxorubicin. Thus, engineered CBD-SA could be a versatile and clinically relevant drug conjugate carrier protein for treatment of solid tumors.

Targeted antibody and cytokine cancer immunotherapies through collagen affinity.

Cancer immunotherapy with immune checkpoint inhibitors (CPIs) and interleukin-2 (IL-2) has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T lymphocyte antigen 4 antibody (αCTLA4) + anti-programmed death ligand 1 antibody (αPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously administered CBD protein accumulated mainly in tumors. CBD conjugation or fusion decreases the systemic toxicity of both αCTLA4 + αPD-L1 combination therapy and IL-2, for example, eliminating hepatotoxicity with the CPI molecules and ameliorating pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells. In an orthotopic breast cancer model, combination treatment with CPI and IL-2 eradicated tumors in 9 of 13 animals with the CBD-modified drugs, whereas it did so in only 1 of 13 animals with the unmodified drugs. Thus, the A3 domain of VWF can be used to improve safety and efficacy of systemically administered tumor drugs with high translational promise.

The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing.

During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

A peptide inhibitor of antibody-dependent cell-mediated cytotoxicity against EGFR/folate receptor-α double positive cells.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is caused by natural killer (NK) cells upon recognition of antigen-bound IgG via FcγRIIIa. This mechanism is crucial for cytolysis of pathogen-infected cells and monoclonal antibody (mAb)-mediated elimination of cancer cells. However, there is concern that mAb-based cancer therapy induces ADCC against non-target cells expressing antigens. To date, no strategy has been reported to enhance the selectivity of ADCC to protect non-target cells expressing antigens. Here, we introduce a model inhibitor which specifically blocks ADCC of anti-EGFR mAbs towards EGFR/folate receptor α (FRα) double positive cells. This inhibitor recruits mAbs on the FRα of the cell surface independent of Fab antigen recognition. The resulting ternary and/or quaternary complexes formed on the cell surface suppress signal transduction of FcγRIIIa in NK cells, consequently leading to more specific ADCC.

Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing.

Laminin, as a key component of the basement membrane extracellular matrix (ECM), regulates tissue morphogenesis. Here, we show that multiple laminin isoforms promiscuously bind to growth factors (GFs) with high affinity, through their heparin-binding domains (HBDs) located in the α chain laminin-type G (LG) domains. These domains also bind to syndecan cell-surface receptors, promoting attachment of fibroblasts and endothelial cells. We explore the application of these multifunctional laminin HBDs in wound healing in the type-2 diabetic mouse. We demonstrate that covalent incorporation of laminin HBDs into fibrin matrices improves retention of GFs and significantly enhances the efficacy of vascular endothelial cell growth factor (VEGF-A165) and platelet-derived growth factor (PDGF-BB) in promoting wound healing in vivo, under conditions where the GFs alone in fibrin are inefficacious. This laminin HBD peptide may be clinically useful by improving biomaterial matrices as both GF reservoirs and cell scaffolds, leading to effective tissue regeneration.

Plasma pentraxin 3 (PTX3) level is associated with the disease activity of microscopic polyangiitis (MPA).

A 20-year-old woman, who was suffering from appetite loss, weight loss and livedo reticularis for one and half months, was referred to our hospital. On admission, laboratory studies demonstrated proteinuria (1.0 g/g Cr), hematuria (erythrocytes': 50 - 99/HPF), ,.enal dysfunction (Cr : 2.09 mg/dL), elevated C reactive protein (CRP: 10.82 mg/dL), elevated MPO-ANCA titer (11.6 U/mL) and elevated pentraxin3 (PTX3: 24.05 ng/mL). Her kidney and skin biopsy revealed massive crescentic necrotizing glomerulonephritis and leukocytoclastic vasculitis, respectively. She was diagnosed with microscopic polyangiitis (MPA), and treated with 500 mg/day of intravenous methyl-prednisolone (mPSL) for 3 days followed by 40 mg/day of oral PSL, rituximab (375 mg/m² once a week for a month) and plasma exchange. When PSL tapered to 30 mg/day in 4 weeks, her renal function was only partially recovered, while the CRP level had been normalized and the MPO-ANCA titer was almost negative (3.6 IU/mL). To evaluate histological activity, a second renal biopsy was conducted, which showed fibrocellular crescents in 32% of her glomeruli. The PTX3 level remained high (14.82 ng/mL) at that point. Taken together, the vasculitis was considered to be active still. Steroid pulse therapy for 3 days was administered again, followed by oral PSL 30 mg/day. Her renal function completely recovered in 70 days. The PTX3 level also normalized in 161 days. PTX3 is one of the short pentraxins, produced by a variety of cell types in response to pro-inflammatory signals such as IL-1 and TNF-α. It was reported that PTX3 reflects activity of vasculitis independently from CRP. In the presenting case, when the second renal biopsy revealed a histologically active lesion of the vasculitis, PTX3 was elevated independently from CRP and MPO-ANCA, suggesting that PTX3 may be a more sensitive marker of the disease activity than other tests.

Laparoscopy Reveals a Diversity of Peritoneal Change in Patients with Long-Term Vintage of Peritoneal Dialysis.

BACKGROUND: Although laparoscopy may provide more detailed morphological and histological information about peritoneal damage, its significance in patients with long vintage of peritoneal dialysis (PD) is not elucidated. METHODS: Findings in 12 patients with PD vintage of 7.3 (5.0-8.4) years who had undergone laparoscopy between 2007 and 2011 were reviewed. Macroscopic (peritoneal change, hypervascular change, adhesion, encapsulation) and histopathological peritoneal findings (interstitial fibrosis, microvascular change, fibrin deposition, inflammatory cell infiltration) were scored and summed as Macro-total score (Macro-TS) and Micro-total score (Micro-TS), respectively. Factors associated with these scores and the relationship between these scores were investigated. RESULTS: Neither Macro-TS nor Micro-TS were related to PD vintage (p = 0.069 and p = 0.769, respectively); moreover, Macro-TS varied from patient to patient regardless of similar PD vintage. However, Macro-TS showed a significant association with duration of acidic dialysate (p = 0.003). CONCLUSION: Macroscopic and microscopic findings via laparoscopy may help the assessment of peritoneal damage in patients with long PD vintage.

Inhibitory effect of bisphosphonate on osteoclast function contributes to improved skeletal pain in ovariectomized mice.

The aim of this study was to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of bisphosphonate (BP) on pain in an ovariectomized (OVX) mouse model. We evaluated skeletal pain in OVX mice through an examination of pain-like behavior as well as immunohistochemical findings. In addition, we assessed the effects of alendronate (ALN), a potent osteoclast inhibitor, on those parameters. The OVX mice showed a decrease in the pain threshold value, and an increase in the number of c-Fos immunoreactive neurons in laminae I-II of the dorsal horn of the spinal cord. Alendronate caused an increase in the pain threshold value and inhibited c-Fos expression. The serum level of tartrate-resistant acid phosphatase 5b, a marker of osteoclast activity, was significantly negatively correlated with the pain threshold value. Furthermore, we found that an antagonist of the transient receptor potential channel vanilloid subfamily member 1, which is an acid-sensing nociceptor, improved pain-like behavior in OVX mice. These results indicated that the inhibitory effect of BP on osteoclast function might contribute to an improvement in skeletal pain in osteoporosis patients.

Membranous nephropathy secondary to Graves' disease with deposits of thyroid peroxidase in an adult.

A 40-year-old woman was diagnosed with Graves' disease in 2001 and treated with thiamazole, but disease control was poor. She was referred to our hospital in 2011 for exacerbation of proteinuria, which had been present since 2006. Urinalysis showed a protein/creatinine ratio of 4.2 g/gCre and microhematuria, and a renal biopsy was performed. On electron microscopy, high electron density deposits were seen on the epithelial side of the glomerular basement membrane. IgG was also deposited along the glomerular loop wall, and membranous nephropathy was diagnosed. Immunostaining revealed thyroid peroxidase deposits but not thyroglobulin antigen deposits, suggesting that the membranous nephropathy was secondary to Graves' disease. The membranous nephropathy improved with 131I treatment. To our knowledge, this is the first adult case of membranous nephropathy originating from Graves' disease in which deposits of thyroid peroxidase antigens have been detected.

Rhabdomyolysis caused by peripheral T-cell lymphoma in skeletal muscle.

We report a rare case of rhabdomyolysis caused by peripheral T-cell lymphoma (PTCL) in skeletal muscle. A 62-year-old man was admitted with complaints of sudden muscle weakness. Laboratory abnormalities were identified including markedly elevated creatinine-phosphokinase, peaking at 62,640 IU/L and serum creatinine (Cr) at 5.0 mg/dL. Computed tomography scans revealed tumorous swelling of the right psoas major muscle and the obturator internus muscles. Consequently, he was diagnosed with acute renal failure caused by rhabdomyolysis and was treated with hydration and continuous hemodiafiltration, which resulted in significant improvement in renal function (Cr 1.79 mg/dL). However, the cause of the rhabdomyolysis remained unclear, and he suddenly developed a remittent fever and suffered from hemophagocytic syndrome. Serum ferritin level dramatically increased to 104,707.0 ng/mL and creatinine level to 4.09 mg/dL. We performed a biopsy of inguinal lymph nodes, leading to a diagnosis of PTCL. Finally, he was diagnosed with rhabdomyolysis caused by PTCL. Methylprednisolone pulse therapy markedly improved his general condition and renal function (Cr 1.48 mg/dL), and computed tomography scans revealed that tumorous swelling was greatly diminished. Except when the cause of rhabdomyolysis is readily apparent, such as in cases of trauma, drug and thrombophlebitis, one should consider that rhabdomyolysis may be a sequel of lymphoma.

Innervation pattern at the undersurface of the extensor carpi radialis brevis tendon in recalcitrant tennis elbow.

PURPOSE: We investigated the general, sensory, and sympathetic innervation patterns at the undersurface of the extensor carpi radialis brevis (ECRB) origin in patients with recalcitrant tennis elbow. METHODS: Eight elbows in eight consecutive patients (6 females and 2 males) with tennis elbow who underwent arthroscopic surgery were included in this study. The mean age was 45 years (38-66 years), and the mean duration of symptoms before surgery was 23 months (13-52 months). Operative treatment consisted of an arthroscopic inspection and debridement of the ECRB origin. Control tissues were obtained from biopsy of the ECRB capsule in two patients with osteochondritis dissecance of the capitellum who underwent arthroscopic resection of loose bodies. The tissue specimens were investigated immunohistochemically with antibodies delineating general (PGP9.5), sensory (SP/CGRP), and sympathetic (NPY) nerve patterns. RESULTS: In the non-tendinosis control tissue, SP/CGRP and NPY immunoreactions were heterogeneously distributed in association with blood vessels. Pathologic evaluation of the biopsy tissue showed atypical fibrous granulation containing numerous vessels and nerve structures in all eight patients. Marked reactions to PGP 9.5 took the form of nerve fibers associated with arteries and arterioles in the atypical granulation. Most of the perivascular innervation was found to express NPY. The immunoreactions for SP and CGRP were invariably weak. CONCLUSION: Increased perivascular sympathetic innervation accompanied with loss of sensory innervation at the undersurface of the ECRB tendon may play a role in chronic pain generation in recalcitrant tennis elbow. LEVEL OF EVIDENCE: Diagnostic, Level IV.

Delayed fracture healing in tetranectin-deficient mice.

Tetranectin is a plasminogen-binding protein that enhances plasminogen activation, which has been suggested to play a role in tissue remodeling. Recently, we showed that tetranectin has a role in the wound-healing process. In this study, we investigated whether tetranectin plays a role in fracture healing. The fracture-healing process was studied using a femoral osteotomy model in tetranectin-null mice, previously generated by the authors. Radiographic imaging, micro-computed tomography (µCT), and histological analysis were used to evaluate osteotomy healing. In wild-type mice, a callus was apparent from 7 days, and most samples showed marked callus formation and rebridging of the cortices at the osteotomy site at 21 days. In contrast, in the tetranectin-null mice there was no callus formation at 7 days and much less callus formation and no bridging of cortices were observed at 21 days. At 35 days, all osteotomy sites showed clear rebridging, and secondary bone formation was achieved in wild-type mice by 42 days. In contrast, no clear rebridging or secondary bone formation was observed at 42 days in the tetranectin-null mice. Analysis using µCT at 21 days after osteotomy revealed that the callus area in tetranectin-null mice was smaller than that in wild-type mice. Histological analysis also showed that soft tissue and callus formation were smaller in the tetranectin-null mice at the early stage of the healing process after drill-hole injury. These results suggested that tetranectin could have a role in the positive regulation at the early stage of the fracture-healing process, which was reflected in the delayed fracture healing in tetranectin-deficient mice.