HMGB1 signaling phosphorylates Ku70 and impairs DNA damage repair in Alzheimer's disease pathology.

DNA damage is increased in Alzheimer's disease (AD), while the underlying mechanisms are unknown. Here, we employ comprehensive phosphoproteome analysis, and identify abnormal phosphorylation of 70 kDa subunit of Ku antigen (Ku70) at Ser77/78, which prevents Ku70-DNA interaction, in human AD postmortem brains. The abnormal phosphorylation inhibits accumulation of Ku70 to the foci of DNA double strand break (DSB), impairs DNA damage repair and eventually causes transcriptional repression-induced atypical cell death (TRIAD). Cells under TRIAD necrosis reveal senescence phenotypes. Extracellular high mobility group box 1 (HMGB1) protein, which is released from necrotic or hyper-activated neurons in AD, binds to toll-like receptor 4 (TLR4) of neighboring neurons, and activates protein kinase C alpha (PKCα) that executes Ku70 phosphorylation at Ser77/78. Administration of human monoclonal anti-HMGB1 antibody to post-symptomatic AD model mice decreases neuronal DSBs, suppresses secondary TRIAD necrosis of neurons, prevents escalation of neurodegeneration, and ameliorates cognitive symptoms. TRIAD shares multiple features with senescence. These results discover the HMGB1-Ku70 axis that accounts for the increase of neuronal DNA damage and secondary enhancement of TRIAD, the cell death phenotype of senescence, in AD.

An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies.

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line.

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.

Analyses of Nivolumab Exposure and Clinical Safety Between 3-mg/kg Dosing and 240-mg Flat Dosing in Asian Patients with Advanced Renal Cell Carcinoma in the Real-World Clinical Setting.

We aimed to identify the clinical characteristics related to increased nivolumab exposure in Japanese patients with renal cell carcinoma (RCC) in real-world clinical setting. Eleven patients were treated with the originally approved nivolumab dosing regimen of 3 mg/kg every 2 weeks (Q2W) (3-mg/kg group) and 8 patients with a flat dose of 240 mg Q2W (flat dosing group). Trough concentrations (Cmin) until the fifth cycle were measured by sandwich enzyme-linked immunosorbent assay using anti-nivolumab monoclonal antibody established by the Autonomously Diversifying Library system. Mean Cmin at four cycles of nivolumab were significantly higher in the flat dosing group than in the 3-mg/kg group. In an analysis of covariates related to nivolumab concentration, serum albumin (Alb) was significantly lower in the 3-mg/kg group than in the flat dose group. Cmin correlated significantly with serum Alb at all cycles. In conclusion, serum Alb was a potential clinically relevant covariate for nivolumab pharmacokinetics in Japanese RCC patients. Further studies should verify whether serum Alb affects nivolumab efficacy and toxicity.

Biologic Activity of Autologous, Granulocyte-Macrophage Colony-Stimulating Factor Secreting Alveolar Soft-Part Sarcoma and Clear Cell Sarcoma Vaccines.

PURPOSE: Alveolar soft-part sarcoma (ASPS) and clear cell sarcoma (CCS) are rare mesenchymal malignancies driven by chromosomal translocations that activate members of the microphthalmia transcription factor (MITF) family. However, in contrast to malignant melanoma, little is known about their immunogenicity. To learn more about the host response to ASPS and CCS, we conducted a phase I clinical trial of vaccination with irradiated, autologous sarcoma cells engineered by adenoviral-mediated gene transfer to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). EXPERIMENTAL DESIGN: Metastatic tumors from ASPS and CCS patients were resected, processed to single-cell suspensions, transduced with a replication-defective adenoviral vector encoding GM-CSF, and irradiated. Immunizations were administered subcutaneously and intradermally weekly three times and then every other week. RESULTS: Vaccines were successfully manufactured for 11 of the 12 enrolled patients. Eleven subjects received from three to 13 immunizations. Toxicities were restricted to grade 1-2 skin reactions at inoculation sites. Vaccination elicited local dendritic cell infiltrates and stimulated T cell-mediated delayed-type hypersensitivity reactions to irradiated, autologous tumor cells. Antibody responses to tissue-type plasminogen activator (tTPA) and angiopoietins-1/2 were detected. Tumor biopsies showed programmed death-1 (PD-1)-positive CD8(+) T cells in association with PD ligand-1 (PD-L1)-expressing sarcoma cells. No tumor regressions were observed. CONCLUSIONS: Vaccination with irradiated, GM-CSF-secreting autologous sarcoma cell vaccines is feasible, safe, and biologically active. Concurrent targeting of angiogenic cytokines and antagonism of the PD-1-negative regulatory pathway might intensify immune-mediated tumor destruction.

Angiogenic cytokines are antibody targets during graft-versus-leukemia reactions.

PURPOSE: The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT). EXPERIMENTAL DESIGN: A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT. RESULTS: A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival. CONCLUSIONS: These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias.

In vivo discovery of immunotherapy targets in the tumour microenvironment.

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.

Active immunotherapy induces antibody responses that target tumor angiogenesis.

The inhibition of VEGF signaling with antibodies or small molecules achieves clinical benefits in diverse solid malignancies. Nonetheless, therapeutic effects are usually not sustained, and most patients eventually succumb to progressive disease, indicating that antiangiogenic strategies require additional optimization. Vaccination with lethally irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) and antibody blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) trigger a tumor vasculopathy in some long-term responding subjects. These reactions are characterized by disrupted tumor blood vessels in association with lymphocyte and granulocyte infiltrates and zonal areas of ischemic tumor necrosis. However, the mechanisms underlying this immune-mediated destruction of the tumor vasculature remain to be clarified. Here, we show that GM-CSF-secreting tumor cell vaccines and CTLA-4 blockade elicit a functionally important humoral reaction against multiple angiogenic cytokines. Antibodies to angiopoietin-1 and angiopoietin-2 block Tie-2 binding, downstream signaling, endothelial cell tube formation, and macrophage chemotaxis. Antibodies to macrophage inhibitory factor (MIF) attenuate macrophage Tie-2 expression and matrix metalloproteinase-9 (MMP-9) production. Together, these results delineate an immunotherapy-induced host response that broadly targets the angiogenic network in the tumor microenvironment.

Milk fat globule EGF-8 promotes melanoma progression through coordinated Akt and twist signaling in the tumor microenvironment.

The pathogenesis of malignant melanoma involves the interplay of tumor cells with normal host elements, but the underlying mechanisms are incompletely understood. Here, we show that milk fat globule EGF-8 (MFG-E8), a secreted protein expressed at high levels in the vertical growth phase of melanoma, promotes disease progression through coordinated alpha(v)beta(3) integrin signaling in the tumor microenvironment. In a murine model of melanoma, MFG-E8 enhanced tumorigenicity and metastatic capacity through Akt-dependent and Twist-dependent pathways. MFG-E8 augmented melanoma cell resistance to apoptosis, triggered an epithelial-to-mesenchymal transition (EMT), and stimulated invasion and immune suppression. In human melanoma cells, MFG-E8 knockdown attenuated Akt and Twist signaling and thereby compromised tumor cell survival, EMT, and invasive ability. MFG-E8-deficient human melanoma cells also showed increased sensitivity to small molecule inhibitors of insulin-like growth factor I receptor and c-Met. Together, these findings delineate pleiotropic roles for MFG-E8 in the tumor microenvironment and raise the possibility that systemic MFG-E8 blockade might prove therapeutic for melanoma patients.

TARC and RANTES enhance antitumor immunity induced by the GM-CSF-transduced tumor vaccine in a mouse tumor model.

INTRODUCTION: Transduction of the granulocyte-macrophage colony stimulating factor (GM-CSF) gene into mouse tumor cells abrogates their tumorigenicity in vivo. Our previous report demonstrated that gene transduction of GM-CSF with either TARC or RANTES chemokines suppressed in vivo tumor formation. In this paper, we examined whether the addition of either recombinant TARC or RANTES proteins to irradiated GM-CSF-transduced tumor vaccine cells enhanced antitumor immunity against established mouse tumor models to examine its future clinical application. MATERIALS AND METHODS: Three million irradiated WEHI3B cells retrovirally transduced with murine GM-CSF cDNA in combination with either recombinant TARC or RANTES were subcutaneously inoculated into syngeneic WEHI3B-preestablished BALB/c mice. RESULTS: Vaccinations were well tolerated. Mice treated with GM-CSF-transduced cells and the chemokines demonstrated significantly longer survival than mice treated with GM-CSF-transduced cells alone. Splenocytes harvested from mice treated with the former vaccines produced higher levels of IL-4, IL-6, IFN-gamma, and TNF-alpha, suggesting enhanced innate and adaptive immunity. Immunohistochemical analysis of tumor sections after vaccination revealed a more significant contribution of CD4+ and CD8+ T cells to tumor repression in the combined vaccine groups than controls. CONCLUSIONS: TARC and RANTES enhance the immunological antitumor effect induced by GM-CSF in mouse WEHI3B tumor models and may be clinically useful.

MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances protection against tumors and infections, but GM-CSF-deficient mice develop inflammatory disease. Here we show that GM-CSF is required for the expression of milk fat globule EGF 8 (MFG-E8) in antigen-presenting cells, and that MFG-E8-mediated uptake of apoptotic cells is a key determinant of GM-CSF-triggered tolerance and immunity. Upon exposure to apoptotic cells, GM-CSF-deficient antigen-presenting cells (APCs) produce an altered cytokine profile that results in decreased Tregs and increased Th1 cells, whereas concurrent ablation of IFN-gamma promotes Th17 cells. In wild-type mice, MFG-E8 attenuates the vaccination activity of GM-CSF-secreting tumor cells through Treg induction, whereas a dominant-negative MFG-E8 mutant potentiates GM-CSF-stimulated tumor destruction through Treg inhibition. These findings clarify the immunoregulatory effects of apoptotic cells and suggest new therapeutic strategies to modulate CD4(+) T cell subsets in cancer and autoimmunity.

p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate.

BACKGROUND: p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53-responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti-cancer therapy. METHODS: In this study, we adenovirally transduced p51A cDNA into human lung, gastric and pancreatic cancer cells and analyzed the intracellular function of p51 in anti-oncogenesis in vitro and in vivo. RESULTS: Overexpression of p51A revealed an anti-proliferative effect in vitro in all the cancer cells examined in this study. The anchorage-dependent and -independent cell growth of EBC1 cells carrying mutations in both p51 and p53 was suppressed and significant apoptosis following adenoviral transduction with p51 and/or p53 was seen. This growth suppression was cooperatively enhanced by the combined infection with adenoviral vectors encoding both p51 and p53. Furthermore, p51 activated several, but not all, p53-inducible genes, indicating that the mechanisms controlling p51- and p53-mediated tumor suppression differed. CONCLUSIONS: Our observations indicate that, although p51 exhibited reduced anti-oncogenetic effects compared with p53, it cooperatively enhanced the anti-tumor effects of p53. Our results suggest that p51 functions as a tumor suppressor in human cancer cells in vitro and in vivo and may be useful as a potential tool for cancer gene therapy.

Serial analysis of gene expression in progressing and regressing mouse tumors implicates the involvement of RANTES and TARC in antitumor immune responses.

Previously we demonstrated that gene transduction of the granulocyte-macrophage colony stimulating-factor (GM-CSF) gene into mouse tumor cells eliminated tumorigenicity in vivo. The rejection process of the subcutaneous tumor was as follows: transient tumor growth peaked around 10 days after tumor injection, then the tumors were rejected within a week. In this paper, we analyzed the gene expression of the transiently established tumor masses by the serial analysis of gene expression method to identify molecules associated with the antitumor effect. We then screened those genes that were differentially expressed between the parental and the GM-CSF-transduced tumors and identified a group of genes that are suggested to have a relationship with tumor rejection, including a cytokine receptor, adhesion molecules, chemokines, cytotoxicity-related molecules, and others. Focusing on the chemokine genes TARC and RANTES, which were preferentially expressed in the GM-CSF-transduced tumors, their forced expression on mouse tumor cells showed moderate suppression of tumor formation. Transduction of GM-CSF in combination with either the TARC or the RANTES gene into tumor cells profoundly inhibited tumor establishment. Histological findings suggested the significant contribution of CD4+ T cells to tumor regression in both TARC/GM-CSF- and RANTES/GM-CSF-transduced tumor cells, in excess of that seen with GM-CSF transduction alone.

Tal1/Scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset (Callithrix jacchus) embryonic stem cells.

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.

Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus).

The successful establishment of human embryonic stem cell (hESC) lines has inaugurated a new era in regenerative medicine by facilitating the transplantation of differentiated ESCs to specific organs. However, problems with the safety and efficacy of hESC therapy in vivo remain to be resolved. Preclinical studies using animal model systems, including nonhuman primates, are essential to evaluate the safety and efficacy of hESC therapies. Previously, we demonstrated that common marmosets are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies. As this animal model is also applicable to preclinical trials of ESC therapies, we have established novel common marmoset ESC (CMESC) lines. To obtain marmoset embryos, we developed a new embryo collection system, in which blastocysts can be obtained every 3 weeks from each marmoset pair. The inner cell mass was isolated by immunosurgery and plated on a mouse embryonic feeder layer. Some of the CMESC lines were cultured continuously for more than 1 year. These CMESC lines showed alkaline phosphatase activity and expressed stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81. On the other hand, SSEA-1 was not detected. Furthermore, our novel CMESCs are pluripotent, as evidenced by in vivo teratoma formation in immunodeficient mice and in vitro differentiation experiments. Our established CMESC lines and the common marmoset provide an excellent experimental model system for understanding differentiation mechanisms, as well as the development of regenerative therapies using hESCs.

Anti-NK cell treatment induces stable mixed chimerism in MHC-mismatched, T cell-depleted, nonmyeloablative bone marrow transplantation.

OBJECTIVE: To clarify natural killer (NK) cell-mediated resistance under cytoreductive conditioning and T cell-depleted bone marrow transplantation, we investigated the effects of host NK cell depletion on engraftment and induction of stable mixed chimerism. METHODS: BALB/c mice (H-2kd) were injected intraperitoneally with anti-asialoGM1 antibody (anti-NK Ab) on day -1. On day 0, they received total body irradiation (TBI) at a dose of 500 cGy, followed by intravenous infusion of 2 x 10(7) T cell-depleted (TCD) bone marrow cells from C57BL/6 mice (H-2kb). Early engraftment and chimerism were determined by the relative ratio of peripheral blood (PB) lymphocytes expressing either H-2kd or H-2kb on day +21. Long-term engraftment and chimerism were evaluated on PB and spleen by multicolor flow cytometry. RESULTS: Although no recipients treated with TBI alone showed engraftment, all the recipients conditioned with anti-NK Ab and TBI showed successful engraftment as well as a donor-dominant pattern of mixed chimerism in both PB and spleen. Spleen cells from recipients with mixed chimerism showed specific tolerance to both host and donor strains, but not to a third party (C3H/He). None of the reconstituted mice showed signs of graft vs host disease, and all survived up to day +330. CONCLUSION: These observations indicate that host NK cell depletion may be used to reduce the intensity of conditioning regimens for engraftment of TCD grafts, and can contribute to establishment of stable mixed chimerism in major histocompatibility complex-mismatched nonmyeloablative transplantation.

Phase I study of autologous tumor vaccines transduced with the GM-CSF gene in four patients with stage IV renal cell cancer in Japan: clinical and immunological findings.

We produced lethally irradiated retrovirally GM-CSF-transduced autologous renal tumor cell vaccines (GVAX) from six Japanese patients with stage IV renal cell cancer (RCC). Four patients received GVAX ranging from 1.4 x 10(8) to 3.7 x 10(8) cells on 6-17 occasions. Throughout a total of 48 vaccinations, there were no severe adverse events. After vaccination, DTH skin tests became positive to autologous RCC (auto-RCC) in all patients. The vaccination sites showed significant infiltration by CD4(+) T cells, eosinophils, and HLA-DR-positive cells. The kinetic analyses of cellular immune responses using peripheral blood lymphocytes revealed an enhanced proliferative response against auto-RCC in four patients, and cytotoxicity against auto-RCC was augmented in three patients. T cell receptor beta-chain analysis revealed oligoclonal expansion of T cells in the peripheral blood, skin biopsy specimens from DTH sites, and tumors. Western blot analysis demonstrated the induction of a humoral immune response against auto-RCC. Two of the four patients are currently alive 58 and 40 months after the initial vaccination with low-dose interleukin-2. Our results suggest that GVAX substantially enhanced the antitumor cellular and humoral immune responses, which might have contributed to the relatively long survival times of our patients in the present study.

Hematopoietic activity of common marmoset CD34 cells isolated by a novel monoclonal antibody MA24.

OBJECTIVE: We focused on a small New World monkey, the common marmoset (Callithrix jacchus), to establish a nonhuman primate model of the treatment of hematological disorders. In this study, we developed the first monoclonal antibodies (MAbs) against marmoset CD34 and tested the in vitro and in vivo hemopoietic activity of cell populations isolated using one of these MAbs. METHODS AND RESULTS: Marmoset cDNA encoding a human CD34 homologue was cloned from bone marrow (BM)-derived RNA using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The amino acid sequence of the marmoset CD34 had 81% homology with the human sequence. Five mouse MAbs were raised against marmoset CD34 transfectant. One representative MAb, MA24 (IgM), reacted with approximately 0.5 to 1% of BM mononuclear cells (MNCs), where the colony-forming unit granulocyte/macrophage (CFU-GM) was enriched approximately 11- to 75-fold as compared with the whole BM MNCs. Multilineage differentiation of marmoset CD34+ cells in NOD/SCID mice was confirmed by flow cytometry 1 month after xenotransplantation. CONCLUSION: These results demonstrated that MA24 is useful for the analysis and enrichment of hematopoietic progenitor cells in the marmoset model for preclinical experiments.