Shoji Yokochi

Enhancement of Antitumor Radiation Efficacy and Consistent Induction of the Abscopal Effect in Mice by ECI301, an Active Variant of Macrophage Inflammatory Protein-1α

Purpose: We studied whether i.v. administration of a chemokine after local tumor site irradiation could prevent remaining, as well as distant, nonirradiated tumor cell growth by leukocyte recruitment. Experimental Design: Tumors were implanted s.c. in the right or both flanks. After local irradiation at the right flank, ECI301, a human macrophage inflammatory protein-1α variant was injected i.v. Tumor volumes were measured every 3 days after treatment. Results: In Colon26 adenocarcinoma-bearing BALB/c mice, repeated daily administration (over 3-5 consecutive days) of 2 μg per mouse ECI301 after local irradiation of 6 Gy prolonged survival without significant toxicity, and in about half of the treated mice, the tumor was completely eradicated. Three weekly administrations of ECI301 after local irradiation also led to significant, although less effective, antitumor radiation efficacy. ECI301 also inhibited growth of other syngenic tumor grafts, including MethA fibrosarcoma (BALB/c) and Lewis lung carcinoma (C57BL/6). Importantly, tumor growth at the nonirradiated site was inhibited, indicating that ECI301 potentiated the abscopal effect of radiation. This abscopal effect observed in BALB/c and C57BL/6 mice was tumor-type independent. Leukocyte depletion studies suggest that CD8+ and CD4+ lymphocytes and NK1.1 cells were involved. Conclusions: Marked inhibition of tumor growth at the irradiated site, with complete tumor eradication and consistent induction of the abscopal effect, was potentiated by i.v. administration of ECI301. The results of this study may offer a new concept for cancer therapy, namely chemokine administration after local irradiation, leading to development of novel therapeutics for the treatment of advanced metastatic cancer.

An Anti-Inflammatory Drug, Propagermanium, May Target GPI-Anchored Proteins Associated with an MCP-1 Receptor, CCR2

Monocyte chemoattractant protein-1 (MCP-1) promotes the migration and activation of monocytes and plays a pivotal role in the development of chronic inflammation. Propagermanium (3-oxygermylpropionic acid polymer) has been used as a therapeutic agent against chronic hepatitis B in Japan. We report here that propagermanium specifically inhibits in vitro chemotactic migration of monocytes by MCP-1. Propagermanium did not inhibit binding of MCP-1 to a human monocytic cell line, THP-1 cells, or affect intracellular Ca(2+) mobilization or the cAMP concentration in MCP-1-treated THP-1 cells. The effect of propagermanium seems to require glycosylphosphatidylinositol (GPI)-anchored proteins, as cleavage of GPI anchors by phosphatidylinositol-phospholipase C (PI-PLC) eliminated the inhibitory activity of propagermanium. Anti-GPI-anchored protein antibodies, such as anti-CD55 and anti-CD59, reduced staining of C-C chemokine receptor 2 (CCR2) with an anti-CCR2 antibody against the N-terminus of CCR2 in a flow cytometric analysis, and these antibodies also selectively inhibited MCP-1-induced migration of THP-1 cells. Furthermore, under fluorescence microscopy, GPI-anchored proteins colocalized with CCR2 on THP-1 cells. These results suggest that propagermanium may target GPI-anchored proteins that are closely associated with CCR2 to selectively inhibit the MCP-1-induced chemotaxis, thus providing a mechanistic basis for the anti-inflammatory effects of the drug.

Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7–8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-β), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.

Robust Antitumor Effects of Combined Anti–CD4-Depleting Antibody and Anti–PD-1/PD-L1 Immune Checkpoint Antibody Treatment in Mice

Ueha, Yokochi, Ishiwata, and colleagues show in three mouse tumor models that CD4 depletion led to tumor-specific CTL proliferation in the draining lymph node and increased tumor infiltration of PD-1+CD8+ T cells; it also synergized with PD-1/PD-L1 blockade to suppress tumor growth and prolong survival. Depletion of CD4+ cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4+ cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti–CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25+ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8+ cell depletion. CD4+ cell depletion led to the proliferation of tumor-specific CD8+ T cells in the draining lymph node and increased infiltration of PD-1+CD8+ T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti–PD-1 or anti–PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti–PD-1 or anti–PD-L1 mAb therapies. Cancer Immunol Res; 3(6); 631–40. ©2015 AACR.

SARS-CoV spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus.

Abstract A vaccine for severe acute respiratory syndrome (SARS) is being intensively pursued against its re-emergence. We generated a SARS coronavirus (SARS-CoV) spike protein-expressing recombinant vaccinia virus (RVV-S) using highly attenuated strain LC16m8. Intradermal administration of RVV-S into rabbits induced neutralizing (NT) antibodies against SARS-CoV 1 week after administration and the NT titer reached 1:1000 after boost immunization with RVV-S. Significantly, NT antibodies against SARS-CoV were induced by administration of RVV-S to rabbits that had been pre-immunized with LC16m8. RVV-S can induce NT antibodies against SARS-CoV despite the presence of NT antibodies against VV. These results suggest that RVV-S may be a powerful SARS vaccine, including in patients previously immunized with the smallpox vaccine.

Non-steroidal anti-inflammatory drug, nabumetone, prevents indometacin-induced gastric damage via inhibition of neutrophil functions

Nabumetone is a non‐steroidal anti‐inflammatory drug (NSAID). It works as a prodrug and is extensively metabolized to an active metabolite, 6‐methoxy‐2‐naphthylacetic acid (6MNA). It is well known that neutrophil infiltration and activation are critical in the pathogenesis of NSAID‐induced gastric injury, and nabumetone shows less incidence of gastrointestinal irritancy. We examined the effects of nabumetone on neutrophil activation and on indometacin‐induced gastric damage. In the indometacin‐induced gastric mucosal injury, rats were treated with indometacin and then nabumetone or 6MNA was orally administered. Nabumetone prevented gastric damage accompanied by the reduction of neutrophil infiltration into gastric mucosa, but such an effect was not observed with 6MNA. Nabumetone reduced the formyl methionyl leucyl phenylalanine (fMLP)‐induced respiratory burst of human neutrophils to 30% of the control level in‐vitro, but 6MNA did not. In addition, nabumetone prevented the fMLP‐induced migration of neutrophils. Nabumetone did not inhibit O−2 generation in the xanthine‐xanthine oxidase system. These results suggest that nabumetone prevents gastric damage induced by the active metabolite, 6MNA, via the suppression of neutrophil activation in gastric mucosa.

A combination of anti-CD4 antibody treatment and DLI ameliorates GVHD while preserving GVT effects in murine allo-HSCT

Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is not only a well‐established immunotherapy for hematologic malignancies, but is potentially useful for treating solid tumors refractory to available therapies. However, application of allo‐HSCT to solid tumors is limited, despite the beneficial antitumor effects, by the risk of graft‐versus‐host disease (GVHD). CD4+ T cells have been implicated in several aspects of GVHD, and suppress antitumor CD8+ T‐cell responses. In the present study, we investigated clinically applicable allo‐HSCT protocols designed to maximize antitumor effects while reducing the risk of GVHD. We used a mouse model of allo‐HSCT with s.c. tumors. We found that myeloablative conditioning was associated with better inhibition of tumor growth but with severe acute GVHD. Early treatment with anti‐CD4 mAb substantially ameliorated GVHD while preserving antitumor effects, leading to improved survival in myeloablative allo‐HSCT. Late treatment with anti‐CD4 mAb also ameliorated GVHD to some extent. Donor lymphocyte infusion in GVHD mice treated with anti‐CD4 mAb further suppressed tumor growth without exacerbating GVHD. Collectively, our results suggest that myeloablative allo‐HSCT followed by anti‐CD4 mAb treatment and donor lymphocyte infusion could be a potent and safe immunotherapy for patients with cancers refractory to available therapies.

Abstract 3663: An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+T cell responses in tumor-bearing mice

Administration of an anti-CD4 depleting antibody induces tumor-specific CD8+ T cell responses in tumor-bearing mice, although the mechanisms underlying this phenomenon remain unclear. CD4+ Foxp3+ regulatory T cells (Treg) impair antigen presentation by DCs through CTLA4-mediated down-regulation of CD80/CD86. In the present study, we investigated the effects of an anti-CD4 depleting monoclonal antibody (mAb) on DC responses in the B16F10 and LLC subcutaneous tumor models, and compared these effects with those of Treg targeting therapy. Administration of anti-CD4 mAb up-regulated CD80/CD86 in tumor-infiltrating DCs, and in migrating-type but not resident-type DC subsets in the tumor-draining lymph node. The upregulation of CD80/CD86 on these DC populations was correlated with increased proliferation of tumor-specific CD8+ T cells in the tumor-draining lymph node. Anti-CD4 mAb-induced CD80/CD86 up-regulation and tumor-specific CD8+ T cell responses were remarkably higher than that induced by an anti-CD25 depleting mAb or an anti-CTLA4 blocking mAb, and were equivalent to that induced by diphtheria-toxin-induced selective depletion of Tregs in Foxp3-DTR mice. Importantly, the activation of CD4+ conventional T cells and B cells in the peripheral lymph nodes that was observed following Treg depletion was not observed following anti-CD4 mAb administration. In conclusion, anti-CD4 depleting antibody therapy is an effective way to evoke anti-tumor CD8+ T cell responses by reversing regulatory T cell-induced suppression of dendritic cells, and to reduce the risk of severe autoimmunity in cancer patients. Citation Format: Satoshi Ueha, Haru Ogiwara, Shoji Yokochi, Yoshiro Ishiwata, Francis Shand, Shohei Hori, Kazuhiro Kakimi, Satoru Ito, Kouji Matsushima. An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice [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 3663. doi:10.1158/1538-7445.AM2017-3663

Abstract 4126: An anti-CD4 antibody protects mice from anti-PD-1/PDL-1 antibody-induced fatal anaphylaxis and shows potent anti-metastatic activity in the 4T1 spontaneous metastasis model

[Introduction] We previously reported that an α-CD4 depleting monoclonal antibody (mAb) has very potent anti-tumor effects in several murine tumor models, particularly when combined with α-PD-1/L1 mAbs (Cancer Immunol. Res. 3: 631-640; 2015). Here we examined the effect of this α-CD4 mAb on 4T1 murine mammary tumors, which undergo spontaneous metastasis from an orthotopically transplanted site to the lung. [Methods] 4T1 tumor cells (1 × 105) were injected into the mammary fat pad of female BALB/c mice and primary tumor volume was measured every 2-3 days. Spontaneous lung metastasis was quantitated by removing the lungs, fixing them in Bouin9s fixative, and counting the number of metastatic nodules on the surface of lung. [Results] Administration of α-CD4 mAb (clone GK1.5; days 5 and 9 after tumor inoculation) significantly inhibited 4T1 lung metastasis (n = 10) without affecting primary tumor size. α-PD-1 and α-PD-L1 mAbs had minimal effects on primary tumor growth and metastasis. However, administration of α-PD-1 or α-PD-L1 mAbs triggered an IgG1-mediated fatal anaphylactic reaction upon the third or fourth administration. Interestingly, the anaphylactic reaction was completely prevented by co-administration of the α-CD4 mAb. [Discussion] Based on these results, α-CD4 mAb may be useful for preventing metastasis associated with advanced cancer. In addition, α-CD4 mAb appeared to be protective against the IgG-mediated anaphylactic reaction and autoimmune-like side effects that are associated with α-PD-1/α-PD-L antibody administration-induced enhanced B-cell immunity. Increased myelopoiesis due to high G-CSF production by 4T1 cells may have contributed to the anaphylactic reaction. Citation Format: Satoru Ito, Shoji Yokochi, Yoshiro Ishiwata, Satoshi Ueha, Francis Shand, Kouji Matsushima. An anti-CD4 antibody protects mice from anti-PD-1/PDL-1 antibody-induced fatal anaphylaxis and shows potent anti-metastatic activity in the 4T1 spontaneous metastasis model. [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 4126.

Abstract 265: Robust anti-tumor effects of combined anti-CD4 depleting antibody and anti-PD-1/PD-L1 immune checkpoint antibody treatment in mice

Depletion of CD4+ cells in tumor-bearing mice has strong anti-tumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4+ cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the anti-tumor effects of an anti-CD4 depleting monoclonal antibody (mAb) as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26 or LLC subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong anti-tumor effects that were superior to those elicited by CD25+ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8+ cell depletion. CD4+ cell depletion led to the proliferation of tumor-specific CD8+ T cells in the draining lymph node and increased infiltration of PD-1+CD8+ T cells into the tumor, with a shift towards type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies. Citation Format: Satoru Ito, Kouji Matsushima, Satoshi Ueha, Shoji Yokochi, Yoshiro Ishiwata, Kosuke Hachiga, Haru Ogiwara, Krishant Chand, Takumi Nakajima. Robust anti-tumor effects of combined anti-CD4 depleting antibody and anti-PD-1/PD-L1 immune checkpoint antibody treatment in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 265. doi:10.1158/1538-7445.AM2015-265