Annona muricata L.-Derived Polysaccharides as a Potential Adjuvant to a Dendritic Cell-Based Vaccine in a Thymoma-Bearing Model.

Dendritic cells (DCs) are powerful antigen-presenting cells that are often used to evaluate adjuvants, particularly for adjuvant selection for various vaccines. Here, polysaccharides (named ALP) isolated from leaves of Annona muricata L., which are used in traditional medicine such as for bacterial infections and inflammatory diseases, were evaluated as an adjuvant candidate that can induce anti-tumor activity. We first confirmed the phenotypic (surface molecules, cytokines, antigen uptake, and antigen-presenting ability) and functional alterations (T cell proliferation/activation) of DCs in vitro. We also confirmed the adjuvant effect by evaluating anti-tumor activity and immunity using an ALP-treated DC-immunized mouse model. ALP functionally induced DC maturation by up-regulating the secretion of Th1-polarizing pro-inflammatory cytokines, the expression of surface molecules, and antigen-presenting ability. ALP triggered DC maturation, which is dependent on the activation of the MAPK and NF-κB signaling pathways. ALP-activated DCs showed an ample capacity to differentiate naive T cells to Th1 and activated CD8+ T cells effectively. The systemic administration of DCs that pulse ALP and ovalbumin peptides strongly increased cytotoxic T lymphocyte (CTL) activity (by 9.5% compared to that in the control vaccine groups), the generation of CD107a-producing multifunctional T cells, and Th1-mediated humoral immunity, and caused a significant reduction (increased protection by 29% over that in control vaccine groups) in tumor growth. ALP, which triggers the Th1 and CTL response, provides a basis for a new adjuvant for various vaccines.

Pembrolizumab in patients with thymic carcinoma: a single-arm, single-centre, phase 2 study.

BACKGROUND: Treatment options are limited for patients with thymic carcinoma. These aggressive tumours are not typically associated with paraneoplastic autoimmune disorders, and strong PD-L1 expression has been reported in thymic epithelial tumours. We aimed to assess the activity of pembrolizumab, a monoclonal antibody that targets PD-1, in patients with advanced thymic carcinoma. METHODS: We completed a single-arm phase 2 study of pembrolizumab in patients with recurrent thymic carcinoma who had progressed after at least one line of chemotherapy. This was a single-centre study performed at Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA. Key inclusion criteria were an Eastern Cooperative Oncology Group performance status of 0-2, no history of autoimmune disease or other malignancy requiring treatment or laboratory abnormality, and adequate organ function. Patients received 200 mg of pembrolizumab every 3 weeks for up to 2 years. The primary objective of the study was the proportion of patients who had achieved a response assessed with Response Evaluation Criteria in Solid Tumors version 1.1. Analysis was per protocol, in all eligible patients. The study is registered with ClinicalTrials.gov, number NCT02364076, and is closed to accrual; we report the final analysis. FINDINGS: 41 patients were enrolled from March 12, 2015, to Dec 16, 2016, of whom 40 were eligible and evaluable and one was excluded because of elevated liver enzymes at screening. The median follow-up was 20 months (IQR 14-26). The proportion of patients who achieved a response was 22·5% (95% CI 10·8-38·5); one (3%) patient achieved a complete response, eight (20%) patients achieved partial responses, and 21 (53%) patients achieved stable disease. The most common grade 3 or 4 adverse events were increased aspartate aminotransferase and alanine aminotransferase (five [13%] patients each). Six (15%) patients developed severe autoimmune toxicity, including two (5%) patients with myocarditis. There were 17 deaths at the time of analysis, but no deaths due to toxicity. INTERPRETATION: Pembrolizumab is a promising treatment option in patients with thymic carcinoma. Because severe autoimmune disorders are more frequent in thymic carcinoma than in other tumour types, careful monitoring is essential. FUNDING: Merck & Co.

Clinical experience of integrative cancer immunotherapy with GcMAF.

BACKGROUND: Immunotherapy has become an attractive new strategy in the treatment of cancer. The laboratory and clinical study of cancer immunotherapy is rapidly advancing. However, in the clinical setting, the results of cancer immunotherapy are mixed. We therefore contend that cancer immunotherapy should be customized to each patient individually based on their immune status and propose an integrative immunotherapy approach with second-generation group-specific component macrophage activating factor (GcMAF)-containing human serum. PATIENTS AND METHODS: The standard protocol of our integrative cancer immunotherapy is as follows: i) 0.5 ml GcMAF-containing human serum is administered intramuscularly or subcutaneously once or twice per week for the duration of cancer therapy until all cancer cells are eradicated; ii) hyper T/natural killer (NK) cell therapy is given once per week for six weeks; iii) high-dose vitamin C is administered intravenously twice per week; iv) alpha lipoic acid (600 mg) is administered orally daily; v) vitamin D3 (5,000-10,000 IU) is administered orally daily. RESULTS: By March 2013, Saisei Mirai have treated over 345 patients with GcMAF. Among them we here present the cases of three patients for whom our integrative immunotherapy was remarkably effective. CONCLUSION: The results of our integrative immunotherapy seem hopeful. We also plan to conduct a comparative clinical study.>

Clinical and immunological predictors of prognosis for Japanese patients with thymoma-associated myasthenia gravis.

There are no immunological markers to predict the prognosis of thymoma-associated myasthenia gravis (MG). Clinical and immunological factors associated with thymoma recurrence or MG relapse were examined by logistic analyses in 56 Japanese patients with thymoma-associated MG. Patients with anti-Kv1.4 antibodies showed higher frequencies of thymoma recurrence and MG relapse compared to those without. Anti-Kv1.4 antibody, Masaoka stage 4, World Health Organization type B3, and adjuvant radiotherapy were associated with thymoma recurrence. Multivariate analyses showed that anti-Kv1.4 antibody was the only independent factor associated with MG relapse. Anti-Kv1.4 antibody is a useful predictor of the prognosis of thymoma-associated MG.

Immunomodulation of curcumin on adoptive therapy with T cell functional imaging in mice.

Adoptive T-cell therapy involves the ex vivo expansion and subsequent transfusion of tumor-specific T lymphocytes to eliminate tumors. Using immune modulators to block immunosuppressive factors in the tumor microenvironment has emerged as a promising strategy to enhance T-cell-mediated tumor regression. Curcumin, a major component of turmeric, has been shown to possess antitumor and immunomodulatory effects by regulating a diverse range of molecular targets. Thus, we hypothesize that these beneficial effects of curcumin may improve the therapeutic efficacy of adoptive therapy. Here, we have shown that curcumin enhances cytotoxicity of CD8(+) T cells toward tumors via alteration of the tumor microenvironment when combined with adoptive therapy. We found that T-cell accumulation and function were increased in combined treatment due to the blockade of different immunosuppressors, including TGF-ß, indoleamine 2,3-dioxygenase, and regulatory T cells. Furthermore, bioluminescent imaging with a granzyme B promoter-conjugated optical reporter also reflected improved cytotoxicity of antigen-specific CD8(+) T cells in tumor-bearing mice during treatment. These findings suggest that combination of multitargeting drugs, such as curcumin, with adoptive therapy may have potential for clinical application. In addition, using a granzyme B-specific imaging reporter to assess T-cell function may also be applied for the development and therapeutic evaluation of new immunotherapy in preclinical studies.

Development of antiricin single domain antibodies toward detection and therapeutic reagents.

Single domain antibodies (sdAb) that bind ricin with high affinity and specificity were selected from a phage display library derived from the mRNA of heavy chain antibodies obtained from lymphocytes of immunized llamas. The sdAb were found to recognize three distinct epitopes on ricin. Representative sdAb were demonstrated to function as both capture and tracer elements in fluid array immunoassays, a limit of detection of 1.6 ng/mL was obtained. One sdAb pair in particular was found to be highly specific for ricin. While polyclonal antibodies cross react strongly with RCA120, the sdAb pair had minimal cross reactivity. In addition, the binders were found to be thermal stable, regaining their ricin binding activity following heating to 85 degrees C for an hour. Cycles of thermally induced unfolding of the sdAb and their subsequent refolding upon cooling was monitored by circular dichroism. As several of the sdAb were observed to bind to ricin's A chain, cell free translation assays were performed to monitor the ability of the sdAbs to inhibit ricin's biological activity. One of the sdAb (C8) was particularly effective and blocked ricin's biological activity with an effectiveness equal to that of a mouse antiricin antibody. These results indicate that antiricin sdAb have great potential for both diagnostic and therapeutic applications.

Hypothermia in VGKC antibody-associated limbic encephalitis.

Voltage-gated potassium channel antibody (VGKC-Ab)-associated limbic encephalitis (LE) is a recently described syndrome that broadens the spectrum of immunotherapy-responsive central nervous system disorders. Limbic encephalitis is typically characterised by a sub-acute onset of disorientation, amnesia and seizures, but the clinical spectrum is not yet fully defined and the syndrome could be under-diagnosed. We here describe the clinical profile of four patients with VGKC-Ab-associated LE who had intermittent, episodic hypothermia. One of the patients also described a prodrome of severe neuropathic pain preceding the development of limbic symptoms. Both of these novel symptoms responded well to immunosuppressive therapy, with concurrent amelioration of amnesia/seizures.

Preoperative steroid pulse therapy for invasive thymoma: clinical experience and mechanism of action.

BACKGROUND: Glucocorticoid was used in thymomas. The purpose of the study was to evaluate the efficacy of intravenous high-dose glucocorticoid (steroid pulse) therapy in patients with previously untreated advanced thymoma. Causes were also sought for a possible underlying mechanism of the effect of steroid on thymoma. METHODS: Seventeen patients with invasive thymoma who had not received previous chemotherapy or radiation therapy were enrolled in the study. All cases were treated with 2 courses of glucocorticoid therapy before surgery. Tumor response was assessed by computed tomography (CT) scan 1 week after the steroid pulse therapy. Lymphocytes associated with thymoma were analyzed for their CD4/CD8 phenotype and glucocorticoid receptor (GR). TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining was used to analyze the apoptotic lymphocytes and epithelial cells. RESULTS: The overall response rate to the steroid pulse therapy was 47.1% (8 of 17). The reduction in tumor size was most prominent in type B1 thymomas; there were significant differences between type AB and type B1 thymomas (P = .0234) and type B1 and type B3 thymomas (P = .0068). The reduction in tumor size was accompanied with a marked reduction in the CD4+8+ double-positive immature thymocytes that expressed higher levels of glucocorticoid receptor. Apoptotic changes were observed in both neoplastic epithelial cell and lymphocyte components after glucocorticoid therapy. CONCLUSIONS: The efficiency of preoperative steroid pulse therapy in type B1 thymoma was most prominent, which is probably related to the specific effect on GR-rich CD4+8+ double-positive immature lymphocytes, which are abundant in this type of thymoma.

Systemic treatment with n-6 polyunsaturated fatty acids attenuates EL4 thymoma growth and metastasis through enhancing specific and non-specific anti-tumor cytolytic activities and production of TH1 cytokines.

Recently, there has been a great interest in the effects of different types of n-6 polyunsaturated acids (n-6 PUFAs) upon the immune system and cancer development. However, the effects of n-6 PUFAs are still controversial and as yet undefined. The present study aimed to investigate the anti-tumor effects of n-6 PUFAs against EL4 thymoma and the associated immune mechanisms. To this, sesame oil, a vegetable oil enriched with n-6 PUFAs, or free linoleic acid (LA) were administered intraperitoneally into C57BL/6 mice before and after challenge with EL4 lymphoma cells. Treatment with either sesame oil or LA attenuated the growth and metastasis of EL4 lymphoma. The anti-tumor effect of LA was superior to that of sesame oil, and associated with an increase in the survival rate of the tumor-bearing mice. In addition, both sesame oil and LA showed dose-dependent anti-lymphoma growth in vitro. Treatment with LA generated significant increases in the anti-lymphoma cytolytic and cytostatic activities of T cells and macrophages, respectively, and enhanced production of IL-2 and IFN-gamma while decreased production of IL-4, IL-6 and IL-10. In summation, the results suggest that n-6 PUFAs, represented by LA, can attenuate EL4 lymphoma growth and metastasis through enhancing the specific and non-specific anti-tumor cytolytic activities and production of TH1 cytokines. These findings might be of great importance for a proper design of systemic nourishment with PUFAs emulsions for cancer patients.

Primary T-cell and activated macrophage response associated with tumor protection using peptide/poly-N-acetyl glucosamine vaccination.

The mode of peptide-based cancer vaccine administration critically affects the ability to achieve a clinically relevant tumor-specific response. We have previously shown (Cole et al., Clin. Cancer Res., 3: 867-873, 1997) that a specific formulation of the polysaccharide poly-N-acetyl glucosamine (p-GlcNAc, designated as F2 gel) is an effective vehicle for sustained cytokine and peptide delivery in vitro. The purpose of this study was to evaluate the efficacy of F2 gel/peptide vaccination in the murine EG.7-OVA tumor model and to elucidate potential mechanisms involved in the observed cell-mediated response. C57BL/6 mice were given injections of 200 microl in the base of tail/footpad using either F2 gel alone or 200 microg of: SIINFEKL minimal peptide (OVA) in PBS, OVA peptide/endoplasmic reticulum insertion signal sequence fusion (ESOVA) in PBS, OVA in F2 gel, or ESOVA in F2 gel. Splenocytes were tested 10 days later for a secondary response using a Cr51 assay as well as a primary CTL response using the lactate dehydrogenase cytotoxicity assay. Splenocytes from immunized mice were harvested at specific time points and assayed for cell surface and intracellular markers. On day 10 postvaccination, animals were challenged with EG.7-OVA murine thymoma cells. Tumor size and appearance were recorded. Vaccination with F2 gel/peptide (either OVA or ESOVA) resulted in a primary T-cell response (up to 25% tumor cell-specific lysis) and no tumor growth in 69% of the mice. By 48 h, the proportion of splenic T cells had increased 4-fold compared with B cells. Presence of an increased Th1 CD4 helper population was demonstrated by IFN-gamma production. CD4 cells were activated at 24 and 48 h as shown by IL-2 receptor alpha chain expression (from 2% basal expression to 15.4% at 48 h). Activated splenic macrophages increased from 3 to 8% within 10 h, and their level of B7-2 expression doubled. Depletion of macrophages before vaccine injection abolished any tumor-specific primary CTL response. F2 gel/peptide tumor vaccine can prime the immune system in an antigen-specific manner by generating a measurable primary T-cell response with minimal peptide; this process involves macrophage presence and activation as well as induction of Th1 CD4 cells. This is the first demonstration of a primary CTL response generated with minimal peptide vaccination using a noninfectious delivery system. These results justify additional studies to better define the mechanisms involved in F2 gel/peptide vaccination in preparation for clinical trials.

Thymic carcinoma with autoimmune syndrome: successful treatment with weekly infusional high-dose 5-fluorouracil and leucovorin.

Thymic carcinoma is known for its poor clinical outcome and unsatisfactory response to conventional chemotherapy. A 53-year-old woman was diagnosed as having metastatic thymic carcinoma in 1989. She received systemic chemotherapy containing cisplatin, doxorubicin, and cyclophosphamide, and involved-field radiotherapy. A durable complete remission was achieved and lasted for 4 years. When the disease recurred in 1995, she was found to have an autoimmune syndrome in addition to pleural effusion, a posterior mediastinal mass and a left adrenal mass. The autoimmune manifestations were seen as scleroderma, high titers of rheumatoid factor and anti-nuclear antibody. We adopted a novel HDFL regimen, which is composed of weekly 24-hour infusion of high-dose 5-fluorouracil and leucovorin, for this patient. Complete remission was achieved again, and autoimmune syndrome was well controlled.

[Immunoregulatory effect of polysaccharide of Acanthopanax senticosus (PAS). I. Immunological mechanism of PAS against cancer].

The effect of PAS against cancer and its immunoregulatory function were studied. The results indicated that PAS was able to inhibit tumor growth and prolong the survival time of tumor-bearing mice significantly at the dose of 100 mg/kg/day, ip. In addition, PAS was also able to increase the response of murine spleen cells to Con A and LPS, the amount of IgM and IgG PFC in murine spleen and the DTH response induced by BSA. These results indicate that the inhibitory effect of PAS on tumor growth is related to the enhanced immune response.

OK-432-mediated augmentation of antitumor immunity and generation of cytotoxic T lymphocytes.

Sensitization with mitomycin C-treated L1210 or EL-4 tumor cells followed by intraperitoneal injection of a streptococcal preparation OK-432 rendered histocompatible or syngeneic mice immune to the corresponding tumor cells. The antitumor immunity, which was more potent than that induced by attenuated tumor cells alone, was manifested by transplantation resistance to challenge tumor cells, and by cytotoxic activity of spleen cells from the primed mice. The former activity was closely related to the latter, which was found to be mainly due to tumor-specific cytotoxic T lymphocytes. The in vivo immunoaugmentation by OK-432 was susceptible to macrophage toxins such as trypan blue and carragheenins, and was partly dependent on the activity of noncytotoxic Ia-positive peritoneal macrophages. OK-432-mediated enhancement of Ia-positive macrophage functions was confirmed by concanavalin A-blastogenesis and T cell-dependent antibody formation. Allo-reactive cytotoxicity induced in allogeneic or semiallogeneic mice, which had been primed with clonogenic or attenuated tumor cells, was also augmented by concomitant administration of OK-432. These results suggest that OK-432 augments induction of antitumor immunity and alloreactive cytotoxicity, associated with stimulation of noncytotoxic Ia-positive accessory macrophage activity.