Mitigating the prevalence and function of myeloid-derived suppressor cells by redirecting myeloid differentiation using a novel immune modulator.

BACKGROUND: Immune suppression is common in neoplasia and a major driver is tumor-induced myeloid dysfunction. Yet, overcoming such myeloid cell defects remains an untapped strategy to reverse suppression and improve host defense. Exposure of bone marrow progenitors to heightened levels of myeloid growth factors in cancer or following certain systemic treatments promote abnormal myelopoiesis characterized by the production of myeloid-derived suppressor cells (MDSCs) and a deficiency in antigen-presenting cell function. We previously showed that a novel immune modulator, termed 'very small size particle' (VSSP), attenuates MDSC function in tumor-bearing mice, which was accompanied by an increase in dendritic cells (DCs) suggesting that VSSP exhibits myeloid differentiating properties. Therefore, here, we addressed two unresolved aspects of the mechanism of action of this unique immunomodulatory agent: (1) does VSSP alter myelopoiesis in the bone marrow to redirect MDSC differentiation toward a monocyte/macrophage or DC fate? and (2) does VSSP mitigate the frequency and suppressive function of human tumor-induced MDSCs? METHODS: To address the first question, we first used a murine model of granulocyte-colony stimulating factor-driven emergency myelopoiesis following chemotherapy-induced myeloablation, which skews myeloid output toward MDSCs, especially the polymorphonuclear (PMN)-MDSC subset. Following VSSP treatment, progenitors and their myeloid progeny were analyzed by immunophenotyping and MDSC function was evaluated by suppression assays. To strengthen rigor, we validated our findings in tumor-bearing mouse models. To address the second question, we conducted a clinical trial in patients with metastatic renal cell carcinoma, wherein 15 patients were treated with VSSP. Endpoints in this study included safety and impact on PMN-MDSC frequency and function. RESULTS: We demonstrated that VSSP diminished PMN-MDSCs by shunting granulocyte-monocyte progenitor differentiation toward monocytes/macrophages and DCs with heightened expression of the myeloid-dependent transcription factors interferon regulatory factor-8 and PU.1. This skewing was at the expense of expansion of granulocytic progenitors and rendered the remaining MDSCs less suppressive. Importantly, these effects were also demonstrated in a clinical setting wherein VSSP monotherapy significantly reduced circulating PMN-MDSCs, and their suppressive function. CONCLUSIONS: Altogether, these data revealed VSSP as a novel regulator of myeloid biology that mitigates MDSCs in cancer patients and reinstates a more normal myeloid phenotype that potentially favors immune activation over immune suppression.

Asociación entre trastorno depresivo y deterioro cognitivo en ancianos de tres ciudades del Perú / An association between the depressive disorder and cognitive deterioration in elderly subjects in three Peruvian cities

Objetivos: Determinar la asociación entre la presencia de trastorno depresivo y trastornos neurocognitivos en adultos mayores de tres ciudades del Perú. Materiales y métodos: Estudio transversal analítico, se realizó un muestreo no aleatorizado. Se consideró a aquellos adultos mayores de 60 años, provenientes de las ciudades de Ucayali, Ica y Lima. Durante los meses de marzo a mayo del 2016 se evaluó la presencia de trastorno depresivo y deterioro cognitivo mediante el uso de las escalas de Yesevage (GDS) y de Pfeiffer, respectivamente. Resultados: Se encuestaron a 267 adultos mayores, el 58,4% fueron varones; la edad media fue de 75,4 ± 7,6 años. El 36,3% presentó deterioro cognitivo y el 64% tenía depresión moderada o severa. Además, se encontró una relación entre la edad y depresión (p<0,05), y una asociación entre el deterioro cognitivo y tener depresión severa (RP (razón de prevalencias): 2,1; intervalo de confianza al 95% (IC95%): 1,4-3,2), tener entre 80 a 89 años (RP: 1,9; IC95%: 1,2-3,1) y proceder de Ucayali (RP=1,67; IC=1,21-2,31). Conclusión: En los pacientes evaluados, los índices de depresión aumentaron conforme aumentaba la edad y hubo una mayor probabilidad de tener trastorno neurocognitivo en aquellos con depresión severa

Objectives: To determine if there is an association between the occurrence of the depressive disorder and neurocognitive disorders in elderly subjects from three Peruvian cities. Materials and methods: This is an analytical cross-sectional study, and a non-randomized sample was taken. Elderly subjects more than 60 years of age from Ucayali, Ica, and Lima were included. Between March and May 2016 the presence of a depressive disorder and cognitive deterioration was assessed using the Yesevage (GDS) and the Pfeiffer scales, respectively. Results: Two hundred and sixty seven elderly subjects were interviewed; 58.4% were male; and the mean age of the whole group was 75 ± 7.6 years old. Approximately one third (36.3%) of all subjects had cognitive deterioration, and 64% had moderate or severe depression. Also, a relationship was found between age and depression (p<0.05), and there was an association between cognitive deterioration and severe depression (prevalence ratio (PR): 2.1; 95% confidence interval: 1.4-3.2); having between 80 to 89 years of age (PR: 1.9; 95% CI: 1.2-3.1), and coming from Ucayali (PR: 1.67; 95% CI: 1.21-2.31). Conclusion: In the assessed patients, depression scores increased with age, and there was a greater likelihood for developing a neurocognitive disorder in those who had severe depression

Cancer intelligence acquired (CIA): tumor glycosylation and sialylation codes dismantling antitumor defense.

Aberrant glycosylation is a key feature of malignant transformation and reflects epigenetic and genetic anomalies among the multitude of molecules involved in glycan biosynthesis. Although glycan biosynthesis is not template bound, altered tumor glycosylation is not random, but associated with common glycosylation patterns. Evidence suggests that acquisition of distinct glycosylation patterns evolves from a 'microevolutionary' process conferring advantages in terms of tumor growth, tumor dissemination, and immune escape. Such glycosylation modifications also involve xeno- and hypersialylation. Xeno-autoantigens such as Neu5Gc-gangliosides provide potential targets for immunotherapy. Hypersialylation may display 'enhanced self' to escape immunosurveillance and involves several not mutually exclusive inhibitory pathways that all rely on protein-glycan interactions. A better understanding of tumor 'glycan codes' as deciphered by lectins, such as siglecs, selectins, C-type lectins and galectins, may lead to novel treatment strategies, not only in cancer, but also in autoimmune disease or transplantation.

NGcGM3/VSSP vaccine as treatment for melanoma patients.

Gangliosides are glycosphingolipids that are present in the plasma membranes of vertebrates and are involved in multiple cellular processes. In the Center of Molecular Immunology an NGcGM3 ganglioside based vaccine has been developed and is conceptualized as a targeted therapy in cancer. NGcGM3/VSSP vaccine had been used as treatment of metastatic melanoma patients and had showed to be safe and immunogenic. The treatment improved antitumoral response or maintain the response obtained with previous onco-specific treatment as chemotherapy. The results indicate that the vaccine improved overall survival of metastatic melanoma patients after first line-chemotherapy. The clinical trial ongoing currently will allow corroborating these results.

Antitumor protection by NGcGM3/VSSP vaccine against transfected B16 mouse melanoma cells overexpressing N-glycolylated gangliosides.

BACKGROUND: Cancer vaccines are designed to modulate immunological responses against tumor cells through the presentation of tumor antigens. MATERIALS AND METHODS: The mouse mRNA of the cytidine monophospho-N-acetylneuraminic acid hydroxylase (Cmah) gene, the enzyme that catalyzes the synthesis of N-glycolylneuraminic acid (NGc), was cloned and transfected into the B16 melanoma cell line. Transfected cells (B16-H) were characterized and used as an NGcGM3-positive primary tumor model for the evaluation of the therapeutic activity of the NGcGM3/VSSP vaccine. RESULTS: The presence of NGcGM3 in B16-H cells promoted proliferation and adhesion in vitro, but resulted in reduced tumorigenicity in vivo. However, B16-H cells developed growing tumors in mice where NGcGM3/VSSP vaccination induced a therapeutic antitumor activity. NGcGM3/VSSP was ineffective in mice inoculated with parental B16 or B16-H cells that had lost NGcGM3 expression. CONCLUSION: The presence of NGcGM3 in tumor cells is critical for the antitumor activity of NGcGM3/VSSP vaccine.

Lentiviral vector followed by protein immunisation breaks tolerance against the self-antigen Her1 and results in lung cancer immunotherapy.

BACKGROUND: Lung cancer remains a leading cause of cancer mortality, and so the aim of the present study was to develop a therapeutic vaccine protocol. METHODS: We constructed a lentiviral vector (LV) expressing the extracellular domain (ECD) of murine Her1, an antigen associated with poor prognosis in lung cancer. RESULTS: A single LV injection, followed by two Her1 protein boosts, was effective in reducing the metastatic burden of Lewis lung carcinoma in mice. The Her1 LV immunisation generated CD8+ T cells that recognised Her1 ECD presented by dendritic cells, and that also homed to Her1-expressing tumours. Protein boosting further increased the CD8+ T cell response and generated anti-Her1 antibodies; in the antibody response, Her1 LV priming increased Th1-dependent immunoglobulin G2c production. CONCLUSIONS: The ability of this vaccine protocol to break both T cell and B cell tolerance to a self-antigen likely explains its effectiveness.

Induction of immunogenic apoptosis by blockade of epidermal growth factor receptor activation with a specific antibody.

Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4(+) and CD8(+) T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab')(2) suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect.

Immunogenicity and safety of a NeuGcGM3 based cancer vaccine: Results from a controlled study in metastatic breast cancer patients.

Increased levels of NeuGc-containing gangliosides have been described in human breast cancer. A controlled Phase II clinical trial was conducted in patients with metastatic breast cancer to evaluate immunogenicity, safety and to identify evidences of biological activity of a cancer vaccine composed by NeuGcGM3 in a proteoliposome of Neisseria meningitidis together with Montanide ISA 51 as adjuvant. After first line chemotherapy, 79 women were randomized 1:1 to receive the vaccine candidate or best supportive care. All patients achieved at least stable disease to the first line therapy for the metastatic condition. Treatment consisted on 5 vaccine doses every 2 weeks and then, monthly re-immunization to complete 15 doses. Vaccination with the NeuGcGM3 based vaccine was safe and the most frequent adverse events consisted on injection site reactions, fever, arthralgia and chills. The vaccine was immunogenic and a sustained increase of both IgG and IgM antibody titters against NGcGM3 was observed after the second vaccination month. Antibodies were able to recognize the NeuGcGM3(+) murine tumor cell line L1210 and the myeloma cell line P3X63. Humoral response was specific since vaccination did not result in Neu-Acetyl GM3 or GM2-antibody response. Hyperimmune sera from vaccinated patients were able to prevent the NeuGcGM3 mediated CD4 down-modulation on T lymphocytes. In the intent to treat analysis, there was a trend toward a survival advantage for the vaccine group and this effect was significant for women bearing non-visceral metastasis. Two phase III clinical studies with this vaccine candidate are ongoing.

Anti-EGFR activation, anti-proliferative and pro-apoptotic effects of polyclonal antibodies induced by EGFR-based cancer vaccine.

Up to now clinical experiences focusing EGF receptor, an attractive target for cancer therapy, have been limited to passive therapies, suggesting that therapeutic cancer vaccines inducing anti-epidermal growth factor receptor (EGFR) antibodies could also work. Here, the humoral immune response induced in mice with a vaccine formulation containing the human EGFR-extracellular domain and very small-sized proteoliposomes (VSSP), a novel nanoparticulated adjuvant was assessed. In vaccinated mice sera average of the specific polyclonal antibodies (PAb) titers was 10(-5). Anti-EGFR PAb were able to bind EGFR+ tumor cell lines, expressing different levels of the molecule. Noteworthy, the presence of Cetuximab only partially inhibited the vaccine-induced antibodies binding to H125 cells. Anti-EGFR PAb abrogated ligands-dependent EGFR phosphorylation, provoking tumor cells apoptosis. The described EGFR-based vaccine might be a superior therapeutic approach for patients with EGFR+ tumors.

Heterophilic NeuGcGM3 ganglioside cancer vaccine in advanced melanoma patients: results of a Phase Ib/IIa study.

NeuGcGM3 ganglioside is especially attractive because it is expressed on melanoma cells but it is minimally or not expressed at all on most normal human tissues. A Phase Ib/IIa clinical trial was carried out in patients with advanced cutaneous and ocular malignant melanomas, to evaluate immunogenicity and toxicity of an intramuscularly administered cancer vaccine and composed by NeuGcGM3 in a proteoliposome of Neisseria meningitides with Montanide ISA 51 as adjuvant. Twenty two patients were included, twelve at dose level of 200 microg and 10 at 400 microg. The first five doses were administered every other week and then monthly until 9 doses. 12 patients received additional immunizations. Vaccination induced specific anti-NeuGcGM3 IgM, IgG and IgA antibodies responses. Titers of IgM were greater for the highest vaccine doses. Vaccination also elicited DTH response in 45.5% of patients in the lower doses and 77.8% in the higher doses. Toxicities were mostly grade 1 or 2, according CTC-NCI criteria. Interestingly, 3 patients developed vitiligo at the lower dose (none in the highest dose) although the nominal antigen NeuGcGM3 is not present in melanocytes. Survival analysis was not the goal of this Phase I trial; nevertheless, the fact that seven patients are alive for more than 2 years after inclusion is noteworthy. Safety and immunogenicity with NeuGcGM3 vaccine treatment in advanced melanoma patients were established. The prognostic value of autoimmunity and the possibilities of dissociating anti-tumor immunity from autoimmunity deserve further research.

Active antimetastatic immunotherapy in Lewis lung carcinoma with self EGFR extracellular domain protein in VSSP adjuvant.

The epidermal growth factor receptor (EGFR) plays a central role in regulating neoplastic processes. The EGFR overexpression in many human epithelial tumors has been correlated with disease progression and bad prognosis. Passive EGFR-directed immunotherapy, but not active specific approaches, has already been introduced in medical oncology practice. Then we wonder if mice immunization with the extracellular domain of murine EGFR (mEGFR-ECD) in adjuvants can circumvent tolerance to self EGFR, by inducing an immune response with consequent antitumor effect. The present study demonstrated that despite mEGFR expression in thymus, strong DTH response was induced by inoculation of mice with the mEGFR-ECD. This self-immunization, using both CFA and very small sized proteoliposomes from Neisseria meningitidis (VSSP), promoted highly specific IgG titers, predominantly IgG2a and IgG2b. Sera from mice immunized with mEGFR-ECD/VSSP not only recognized EGFR+ tumor cell lines by FACS, but also inhibited their in vitro growth, even in the absence of complement. Noteworthy, vaccination of mice with mEGFR-ECD/VSSP stimulated a potent antimetastatic effect in the EGFR+ Lewis lung carcinoma model, while reproduction-associated side effects were absent. Curiously, mice immunized with the human EGFR-ECD (Her1-ECD) in VSSP though induced highly specific IgG antibodies with strong in vitro cytotoxic effect over EGFR+ human cell lines, showed low cross-reactivity with the mEGFR-ECD. These results further encouraged the development of the Her1-ECD/VSSP vaccine project for patients with EGFR+ tumors.

The enlargement of the hormone immune deprivation concept to the blocking of TGFalpha-autocrine loop: EGFR signaling inhibition.

Transforming growth factor alpha (TGFalpha) is a potent ligand of the epidermal growth factor receptor (EGFR). EGFR is frequently over-expressed in epithelial tumors and endogenous ligands, mostly TGFalpha, are frequently co-expressed with EGFR, potentially resulting in autocrine stimulation of tumor cell growth. Therefore, different therapeutic approaches aim for the inactivation of TGFalpha/EGF/EGFR signaling system, but no approach is based on TGFalpha as a target. The principal goal of this work was to assess the potential of an active specific immunotherapy approach to block the TGFalpha/EGFR autocrine loop. For the proof of the concept, a fusion protein between human TGFalpha (hTGFalpha) and P64k protein from Neisseria meningitidis was generated, and its immunogenicity characterized in a mouse model using different adjuvants. All immunogens were effective for the generation of specific humoral responses against hTGFalpha. The inmunodominant epitope of hTGFalpha when immunizing mice with the fusion protein involved the C-loop/C-terminal region. This region includes key residues for hTGFalpha binding to EGFR. The anti-hTGFalpha immune mice sera recognized the natural hTGFalpha precursor in A431 cells and hTGFalpha-transfected 3T3 fibroblasts as revealed by flow cytometry analysis and immunoblotting. They inhibited the binding of (125)I-TGFalpha to the EGFR, EGFR-autophosphorylation, and downstream activation of MAP kinases as well as proliferation of two EGFR-expressing human carcinoma cell lines. These data suggest that EGFR signaling activation by the hTGFalpha autocrine loop may be inhibited in vivo by induction of specifically blocking antibodies. The fusion protein reported in this paper could be a potential immunogen for the development of a new cancer vaccine.

Active specific immunotherapy of melanoma with a GM3 ganglioside-based vaccine: a report on safety and immunogenicity.

A novel cancer vaccine was obtained by combining GM3 ganglioside with Neisseria meningitidis outer membrane protein complex to obtain very-small-size proteoliposomes (GM3/VSSP). The authors report the results of a phase 1 study of intramuscular administration of GM3/VSSP/Montanide ISA 51 to patients with metastatic melanoma. Twenty-six patients were included in three dose-level cohorts of 120, 240, and 360 mug. The first five doses (induction phase) were given at 2-week intervals, and the remaining four doses were given monthly. Patients were evaluated for dose-related toxicities and antitumor effects. In addition, serum and peripheral blood mononuclear cells were obtained at baseline and throughout treatment to evaluate humoral and cellular immune responses. One episode of severe hypotension and fever was observed in a patient included at the highest dose level. Other toxicities consisted of local reactions at the site of injection and mild fever and chills. Five doses of GM3/VSSP induced an anti-GM3 IgM response in 44% of patients. Serum reactivity was also observed against melanoma cell lines and tumor biopsies. GM3/VSSP was shown to induce very strong in vitro IFNgamma secretion in all evaluated melanoma patients. Furthermore, in one patient IFNgamma secretion was shown to be GM3-specific. A 62% reduction of a mediastinal mass was documented in one patient (partial response), while a second patient benefited from initial disease stabilization followed by tumor reduction in nonmeasurable soft tissue lesions accompanied by vitiligo.

7A7 MAb: a new tool for the pre-clinical evaluation of EGFR-based therapies.

The epidermal growth factor receptor (EGFR) is highly expressed in many types of epithelial tumors. EGFR overexpression has been associated with an advanced stage of the disease, with resistance to standard therapies, and, for certain tumors, with poor patient prognosis. As a result, EGFR has been considered a meaningful target in anti-tumor strategies. Active and passive immunotherapies blocking EGFR and its ligands have been explored. But for successful pre-clinical evaluation of these approaches, well-established murine tumor models are not available and highly desirable. We described, for the first time, the generation and characterization of an anti-murine EGFR extracellular domain monoclonal antibody (7A7 MAb) (IgG1). 7A7 was generated by immunization of Balb/c mice with the recombinant extracellular domain of murine EGFR (rECD-mEGFR). 7A7 recognized an epitope present in the amino acidic core of the antigen and is cross-reactive with the human EGFR. Interestingly, this MAb was able to specifically bind EGFR at the cell surface, allowing the assessment of its differential expression in a panel of murine cells. Noteworthy, in a preliminary immunohistochemical study with 7A7 MAb, recognition of Balb/c mice skin sections and EGFR-positive tumors were observed. We concluded that 7A7 MAb is a valuable tool for EGFR-based therapeutic pre-clinical studies.

Immunotherapy of advanced breast cancer with a heterophilic ganglioside (NeuGcGM3) cancer vaccine.

PURPOSE: A heterophilic ganglioside cancer vaccine was developed by combining NeuGcGM3 with the outer membrane protein complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). A phase I clinical trial was performed to determine safety and immunogenicity of this vaccine. PATIENTS AND METHODS: Stage III to IV breast cancer patients received up to 15 (200 micro g) doses of the vaccine by intramuscular injection. The first five doses (induction phase) were given at 2-week intervals, with the remaining treatment (maintenance) administered on a monthly basis. RESULTS: Twenty-one patients, 11 of whom had metastatic disease, were included. Main toxicities included erythema and induration at the injection site, sometimes associated with mild pain, and low-grade fever (World Health Organization grades 1 and 2). All treated patients who completed the induction phase developed anti-NeuGcGM3 antibody titers between 1:1,280 and 1:164,000 immunoglobulin G (IgG), and 1:640 and 1:164,000 IgM. Noteworthy specific IgA antibodies were induced by vaccination in all stage III patients and in three stage IV patients. Serum antibody levels were higher in the stage III patients, with the larger increases observed after week 32. The antiganglioside IgG subclasses were mainly IgG1 and IgG3. Hyperimmune sera increased complement-mediated cytotoxicity versus P3X63 myeloma cells and a marked IgG differential reactivity against human mammary ductal carcinoma samples. CONCLUSION: NeuGcGM3/VSSP/Montanide ISA 51 is an unusual immunogenic ganglioside vaccine and also seems to be safe in this small trial. Immunologic surrogates of activity indicate that this reagent warrants further investigation.

In vivo and in vitro anti-tumor effect of 14F7 monoclonal antibody.

The 14F7 monoclonal antibody (MAb) is an IgG(1) antibody that reacts specifically with GM3 (NeuGc) and with tissue sections of human tumors. We demonstrated here that this MAb is agglutinin that specifically agglutinated horse erythrocytes. Additionally, the capacity of 14F7 MAb to mediate cytotoxicity against GM3 (NeuGc)-positive murine myeloma cells, in vitro and in vivo, was evaluated. High concentrations of 14F7 MAb were needed to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) against the murine myeloma cells. The most relevant finding was the ability of this MAb to directly kill the target cells without participation of complement. This cytotoxicity was dependent on the temperature and MAb concentration and the number of the target cells. In vivo, the passive treatment with 14F7 MAb produced a strong anti-tumor activity, similar to the anti-tumoral response obtained with standard chemotherapy treatment.

Estudio de la actividad biológica del coralan III: influencia sobre los leucocitos / Study of the biologic activity of coralan III: influence on leukocytes

Los polisacáridos influyen sobre el organismo provocando leucocitosis. En el trabajo se estudió la influencia del coralán administrado por la via endovenosa y se determinó que en ratas y perros provoca una leucocitosis. debida fundamentalmente al aumento del porcentaje de granulocitos