17 results found
Ishihara A, Ishihara J, Watkins EA, et al., 2021, Prolonged residence of an albumin–IL-4 fusion protein in secondary lymphoid organs ameliorates experimental autoimmune encephalomyelitis, Nature Biomedical Engineering, Vol: 5, Pages: 387-398, ISSN: 2157-846X
Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here, we show that, in mice with EAE, the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)–IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA–IL-4 fusion protein prevents immune-cell infiltration in the spinal cord, decreases integrin expression in antigen-specific CD4+ T cells, increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells, a pathogenic cell population in EAE. In mice with chronic EAE, SA–IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA–IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis.
Yuba E, Budina E, Katsumata K, et al., 2021, Enhanced lymph node trafficking of engineered IL‐10 suppresses rheumatoid arthritis in murine models, Arthritis & Rheumatology, Vol: 73, Pages: 769-778, ISSN: 2326-5191
ObjectiveRheumatoid arthritis (RA) is a major autoimmune disease that causes synovitis and joint damage. Although clinical trials using interleukin‐10 (IL‐10), an anti‐inflammatory cytokine, have been performed as a potential treatment of RA, its therapeutic effects have been limited, potentially due to insufficient residence in lymphoid organs, where antigen recognition primarily occurs. Here, we engineered IL‐10 as a fusion with serum albumin (SA).MethodsSA‐fused IL‐10 was recombinantly expressed. After intravenous injection to mice, retention of SA‐IL‐10 at lymph node (LN), immune cell compositions at paws, and therapeutic effect on arthritis model mice were assessed.ResultsSA fusion to IL‐10 led to enhanced LN accumulation compared with unmodified IL‐10. Intravenous SA‐IL‐10 treatment restored immune cell composition in the paws to a normal status, elevated the frequency of suppressive M2 macrophages, reduced IL‐17A amount in the paw‐draining LN, and protected joint morphology. Intravenous SA‐IL‐10 treatment showed similar efficacy as treatment with an anti‐TNF‐α antibody. SA‐IL‐10 was equally effective when administered intravenously, locally or subcutaneously, which benefits clinical translation of this molecule.ConclusionSA fusion to IL‐10 is a simple but effective engineering strategy for RA therapy and holds clinical translational potential.
Mansurov A, Ishihara J, Hosseinchi P, et al., 2020, Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours, Nature Biomedical Engineering, Vol: 4, Pages: 531-543, ISSN: 2157-846X
Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in ‘immunologically cold’ tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however, the administration of IL-12 has been associated with immune-related adverse events. Here we show that, after intravenous administration of a collagen-binding domain fused to IL-12 (CBD–IL-12) in mice bearing aggressive mouse tumours, CBD–IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD–IL-12 induced sustained intratumoural levels of interferon-γ, substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD–IL-12 potently synergized with CPI to eradicate large established melanomas, induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD–IL-12 may potentiate CPI immunotherapy for immunologically cold tumours.
Katsumata K, Ishihara J, Fukunaga K, et al., 2019, Conferring extracellular matrix affinity enhances local therapeutic efficacy of anti-TNF-α antibody in a murine model of rheumatoid arthritis, Arthritis Research and Therapy, Vol: 21, Pages: 1-10, ISSN: 1478-6354
BackgroundAlthough disease in a majority of rheumatoid arthritis (RA) patients is often initially limited to one or a few joints, currently approved medications including anti-tumor necrosis factor-α antibody (α-TNF) are injected systemically. Given that α-TNF systemic injection typically does not cure RA and involves risk of treatment-related adverse events, one possible approach to enhance therapeutic efficacy and reduce α-TNF systemic exposure is to retain the antibodies in arthritic joints after local administration. The aim of this study was to evaluate the approach of conferring extracellular matrix (ECM) binding affinity to α-TNF antibodies in a RA model.Methodsα-TNF was chemically conjugated with a promiscuous ECM-binding peptide derived from placenta growth factor 2 (PlGF-2123-144). The binding activity of PlGF-2123-144-conjugated α-TNF (PlGF-2123-144-α-TNF) against ECM proteins was assessed by ELISA and by immunostaining on human cartilage specimens. The effect of conjugation on antibody function was assessed as a neutralizing activity against osteoclast differentiation. Retention at the injection site and therapeutic efficacy of PlGF-2123-144-α-TNF were tested in a collagen antibody-induced arthritis (CAIA) model in the mouse.ResultsPlGF-2123-144 peptide conjugation conferred α-TNF with affinity to ECM proteins without impairment of antigen recognition. PlGF-2123-144-α-TNF locally injected at a paw in the CAIA model was retained for at least 96 h at the injection site, whereas unmodified α-TNF was dispersed rapidly after injection. Local treatment with unmodified α-TNF did not suppress the arthritis score relative to isotype controls. By contrast, local administration of PlGF-2123-144-α-TNF suppressed arthritis development almost completely in the treated paw even at a 1000× lower dose.ConclusionThese data demonstrate that retention of α-TNF in arthritic
Williford J-M, Ishihara J, Ishihara A, et al., 2019, Recruitment of CD103+ dendritic cells via tumor-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy, Science Advances, Vol: 5, Pages: 1-15, ISSN: 2375-2548
Although a clinical breakthrough for cancer treatment, it remains that a minority of patients respond to checkpoint inhibitor (CPI) immunotherapy. The composition of tumor-infiltrating immune cells has been identified as a key factor influencing CPI therapy success. Thus, enhancing tumor immune cell infiltration is a critical challenge. A lack of the chemokine CCL4 within the tumor microenvironment leads to the absence of CD103+ dendritic cells (DCs), a crucial cell population influencing CPI responsiveness. Here, we use a tumor stroma–targeting approach to deliver CCL4; by generating a fusion protein of CCL4 and the collagen-binding domain (CBD) of von Willebrand factor, we show that CBD fusion enhances CCL4 tumor localization. Intravenous CBD-CCL4 administration recruits CD103+ DCs and CD8+ T cells and improves the antitumor effect of CPI immunotherapy in multiple tumor models, including poor responders to CPI. Thus, CBD-CCL4 holds clinical translational potential by enhancing efficacy of CPI immunotherapy.
Katsumata K, Ishihara J, Mansurov A, et al., 2019, Targeting inflammatory sites through collagen affinity enhances the therapeutic efficacy of anti-inflammatory antibodies, Science Advances, Vol: 5, Pages: 1-9, ISSN: 2375-2548
Enhancing the therapeutic efficacy of drugs for inflammatory diseases is of high demand. One possible approach is targeting drugs to the extracellular matrix of the inflamed area. Here, we target collagens in the matrix, which are inaccessible in most tissues yet are exposed to the bloodstream in the inflamed area because of vascular hyperpermeability. We conferred collagen affinity to anti–tumor necrosis factor-α (α-TNF) antibody by conjugating a collagen-binding peptide (CBP) derived from the sequence of decorin. CBP–α-TNF accumulated in the inflamed paw of the arthritis model, and arthritis development was significantly suppressed by treatment with CBP–α-TNF compared with the unmodified antibody. Similarly, CBP–anti-transforming growth factor-β (α–TGF-β) accumulated in the inflamed lung of pulmonary fibrosis model and significantly suppressed pulmonary fibrosis compared with the unmodified antibody. Together, collagen affinity enables the anticytokine antibodies to target arthritis and pulmonary fibrosis accompanied by inflammation, demonstrating a clinically translational approach to treat inflammatory diseases.
Sasaki K, Ishihara J, Ishihara A, et al., 2019, Engineered collagen-binding serum albumin as a drug conjugate carrier for cancer therapy, Science Advances, Vol: 5, Pages: 1-12, ISSN: 2375-2548
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.
Ishihara J, Ishihara A, Starke RD, et al., 2019, The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing, Blood, Vol: 133, Pages: 2559-2569, ISSN: 0006-4971
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.
Ishihara J, Ishihara A, Sasaki K, et al., 2019, Targeted antibody and cytokine cancer immunotherapies through collagen affinity, Science Translational Medicine, Vol: 11, Pages: 1-13, ISSN: 1946-6234
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.
Ishihara J, Ishihara A, Fukunaga K, et al., 2018, Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing, Nature Communications, Vol: 9, Pages: 1-14, ISSN: 2041-1723
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.
Ishihara J, Ishihara A, Potin L, et al., 2018, Improving efficacy and safety of agonistic anti-CD40 antibody through extracellular matrix affinity, Molecular Cancer Therapeutics, Vol: 17, Pages: 2399-2411, ISSN: 1535-7163
CD40 is an immune costimulatory receptor expressed by antigen-presenting cells. Agonistic anti-CD40 antibodies have demonstrated considerable antitumor effects yet can also elicit serious treatment-related adverse events, such as liver toxicity, including in man. We engineered a variant that binds extracellular matrix through a super-affinity peptide derived from placenta growth factor-2 (PlGF-2123-144) to enhance anti-CD40′s effects when administered locally. Peritumoral injection of PlGF-2123-144-anti-CD40 antibody showed prolonged tissue retention at the injection site and substantially decreased systemic exposure, resulting in decreased liver toxicity. In four mouse tumor models, PlGF-2123-144-anti-CD40 antibody demonstrated enhanced antitumor efficacy compared with its unmodified form and correlated with activated dendritic cells, B cells, and T cells in the tumor and in the tumor-draining lymph node. Moreover, in a genetically engineered BrafV600E βCatSTA melanoma model that does not respond to checkpoint inhibitors, PlGF-2123-144-anti-CD40 antibody treatment enhanced T-cell infiltration into the tumors and slowed tumor growth. Together, these results demonstrate the marked therapeutic advantages of engineering matrix-binding domains onto agonistic anti-CD40 antibody as a therapeutic given by tumori-regional injection for cancer immunotherapy.
Ishihara J, Fukunaga K, Ishihara A, et al., 2017, Matrix-binding checkpoint immunotherapies enhance antitumor efficacy and reduce adverse events, Science Translational Medicine, Vol: 9, Pages: 1-16, ISSN: 1946-6234
Immune checkpoint blockade exhibits considerable antitumor activity, but previous studies have reported instances of severe treatment-related adverse events. We sought to explore local immune checkpoint blockade, with an antibody (Ab) form that would be retained intra- or peritumorally, limiting systemic exposure. To accomplish this, we conjugated the checkpoint blockade Abs to an extracellular matrix (ECM)–super-affinity peptide derived from placenta growth factor–2 (PlGF-2123–144). We show enhanced tissue retention and lower Ab concentrations in blood plasma after PlGF-2123–144 conjugation, reducing systemic side effects such as the risk of autoimmune diabetes. Peritumoral injections of PlGF-2123–144–anti-CTLA4 (cytotoxic T lymphocyte antigen 4) and PlGF-2123–144–anti–PD-L1 (programmed death ligand 1) Abs delayed tumor growth and prolonged survival compared to the unmodified Abs in genetically engineered murine tumor models of melanoma and breast cancer. The PlGF-2123–144–Abs increased tumor-infiltrating activated CD8+ and CD4+ T cells, resulting in a delay of distant tumor growth as well. This simple and translatable approach of engineered ECM-binding Abs may present a viable and safer approach in checkpoint blockade.
Kumashiro Y, Ishihara J, Umemoto T, et al., 2015, Stripe-Patterned Thermo-responsive Cell Culture Dish for Cell Separation without Cell Labeling, Small, Vol: 11, Pages: 681-687, ISSN: 1613-6810
Suphanantachat S, Iwata T, Ishihara J, et al., 2014, A role for c-Kit in the maintenance of undifferentiated human mesenchymal stromal cells, Biomaterials, Vol: 35, Pages: 3618-3626, ISSN: 0142-9612
Ishihara J, Umemoto T, Yamato M, et al., 2014, Nov/CCN3 regulates long-term repopulating activity of murine hematopoietic stem cells via integrin αvβ3, International Journal of Hematology, Vol: 99, Pages: 393-406, ISSN: 0925-5710
Throughout life, hematopoietic stem cells (HSCs) sustain the blood cell supply through their capacities for self-renewal and multilineage differentiation. These processes are regulated within a specialized microenvironment termed the ‘niche’. Here, we show a novel mechanism for regulating HSC function that is mediated by nephroblastoma overexpressed (Nov/CCN3), a matricellular protein member of the CCN family. We found that Nov contributes to the maintenance of long-term repopulating (LTR) activity through association with integrin αvβ3 on HSCs. The resultant β3 integrin outside-in signaling is dependent on thrombopoietin (TPO), a crucial cytokine involved in HSC maintenance. TPO was required for Nov binding to integrin αvβ3, and stimulated Nov expression in HSCs. However, in the presence of IFNγ, a cytokine known to impair HSC function, not only was TPO-induced expression of Nov suppressed, but the LTR activity was conversely impaired by TPO-mediated ligation of integrin αvβ3 with exogenous ligands, including Nov, as well. Thus, Nov/integrin αvβ3-mediated maintenance of HSCs appears to be modulated by simultaneous stimulation by other cytokines. Our finding suggests that this system contributes to the regulation of HSCs within the bone marrow niche.
Yoshida T, Kumashiro Y, Iwata T, et al., 2012, Requirement of Integrin β3 for Iron Transportation during Enamel Formation, Journal of Dental Research, Vol: 91, Pages: 1154-1159, ISSN: 0022-0345
<jats:p> Rodent incisors exhibit pigmentation on their labial surfaces. Although previous studies have shown that this pigment is composed of iron, the existence of other elements has not been investigated. This study found that the lower incisors of CD61, also known as integrin β3, null mice ( CD61<jats:sup>-/-</jats:sup>) lacked pigmentation. Although ameloblasts differentiated and formed enamel normally, no ferric ion accumulation was observed in maturation-stage ameloblasts in CD61<jats:sup>-/-</jats:sup> mice. Surface elements of control and CD61-/- lower incisors were compared by x-ray photoelectron spectroscopy (XPS). XPS analysis detected C, Ca, N, O, and P on the labial surfaces of lower incisors of both mice, whereas Fe was detected only in control samples. No peak of non-ferrous metal or other element was detected in either group. Quantitative RT-PCR analysis of 18 iron-transportation-related genes with mRNA from maturation-stage ameloblasts and ALC, a pre-ameloblastic cell line, was performed. The results suggested that CD61 regulates the expressions of Slc11a2 and Slc40a1, both of which are involved in iron transportation in epithelial tissues. These results suggested that the pigment on the labial surface of mouse incisors is composed of Fe and that both anemia and reduction of iron-transporting proteins may cause the loss of pigmentation in CD61<jats:sup>-/-</jats:sup> mice. </jats:p>
Umemoto T, Yamato M, Ishihara J, et al., 2012, Integrin-αvβ3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells, Blood, Vol: 119, Pages: 83-94, ISSN: 0006-4971
<jats:title>Abstract</jats:title><jats:p>Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin “inside-out” signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent “outside-in” signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.</jats:p>
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