Yu-Chun Lone

Glioblastoma multiforme (GBM), a fatal malignant brain tumor, represents a challenge in terms of developing new treatments. Solutions have been proposed in connection with anti-tumor immune responses, generally T cell strategies, including techniques targeting growth factors, particularly IGF-I, the primary cancer growth factor, and its signal transduction pathways. The objective of this review is to assess the immunogene mechanism targeting IGF-I, which constitutes the basis of the described cancer gene therapy for GBM, stemming from brain neoplastic development studies. The GBM gene therapy, based on new strategies of anti-gene IGF-I techniques, including antisense or triple helix, has been combined with phytochemicals (phenolics) and nanotechnologies (theranostic nanoparticles). The three approaches have modulated IGF-I expression together, inducing common signal transduction pathways: IGF-I-R, TK/PI3K/AKT/TLR/MAPK & JAK/STAT. These signals have transformed in vitro neoplastic glioma cells into immunogenic cells. These immunogenic cells, which express MHC-1 and B7, produce a strong T CD8 anti-tumour immune response after in vivo injection. The combined strategy of anti-IGF-I/phytochemical/nanotechnology vaccines has shown promising clinical results, with patient survival of up to two and three years in some cases. The obtained results are discussed in parallel with other immunotherapies and antitumour vaccine studies that involve the immunogenicity mechanism.

The brain malignant tumor Glioblastoma multiforme (GBM) has a median survival of 14–16 months using current treatments; thus, understanding the pathology of GBM is crucial for proposing new therapies and increasing overall survival outcomes. Therefore, this study aimed to analyze different elements, particularly growth factors and the related signal transduction pathways, which play a role in brain neoplastic development, from stem cells to established solid brain tumors, and the application of current immunology techniques, molecular biology, and nanotechnology. Targeting growth factors, especially insulin-like growth factor-1 (IGF-I) (the principal neoplastic development factor) using anti-gene technologies—antisense and triple helix—has previously been shown to produce an immune anti-tumor response (CD8, CD28) through the TK/PI3K/AKT pathway. This immune response was increased using phytochemicals (phenolics), especially nanoparticles (theranostic nanoparticles), by modulating IGF-I through common pathways (IGF-I-R and TK/PI3K/AKT/TLR/MAPK and JAK/STAT). This review demonstrates how studies on central nervous system neoplastic development progressively led to establishing clinical cancer gene therapies, increasing GBM survival by 20–24 months. The presented studies compare the results of cancer gene therapy with other current immunotherapies. Moreover, this research chapter briefly describes the investigations of nanotechnology related to neurotumorigenesis and GBM therapies. The presented studies relate to nanotechnology and compare the results of cancer gene therapy with other current immunotherapies.

Introducción: el Factor Activador de células B (BAFF) está implicado como una citocina clave que juega un papel significativo en la enfermedad autoinmune precancerosa Lupus Eritematoso Sistémico, (LES). El objetivo de este trabajo fue determinar cómo las dosis diferentes de glucocorticoides aplicados en estos pacientes afectan los niveles séricos de BAFF. Métodos: se realizó un estudio observacional de tipo descriptivo en dos grupos (29 pacientes): 1 - tratamiento con Glucocorticoides (21 pacientes); 2 - tratamiento convencional (8 pacientes). El primer grupo se dividió en tres subgrupos, según las dosis de glucocorticoides: bajas (<10.0 mg/día), moderadas (10.0-49.0 mg/día) y altas (≥ 50.0 mg/día). La medición de BAFF se realizó con método de ensayo de inmunoadsorción ligado a enzimas (ELISA). Se analizaron los datos con técnicas estadísticas y la prueba de inferencia de Mann Whitney. Resultados: los valores de BAFF fueron mayores con dosis altas de Glucocorticoides (≥ 50.0 mg/día), con una media de 2,844 pg/ml ± 1,495 y con dosis más bajas (<50.0 mg/día), la media fue de 2,422 ± 1,280; la media de BAFF fue de 2,482 pg/ml, y en el grupo control de 0.990 pg/ml. La diferencia entre estos dos promedios es estadísticamente significativa, utilizando la prueba no paramétrica de Mann Whitney, con p <0.001. En pacientes tratados con mayores dosis de Glucocorticoides se observa una tendencia en el aumento de BAFF. Conclusión: El valor del nivel de BAFF en LES se convierte en el marcador diagnóstico de terapia con glucocorticoides, el estudio descrito es el primero que aborda este tema.

Introduction: Certain specific antigens, which behave as oncoproteins, are present in normal fetal/neonatal brain development, and are absent from mature brain tissues: among them, growth factors, especially insulin-like growth factor type I (IGF-I). When IGF-I reappears in the mature brain, this growth factor is over expressed in neoplastic glia, participating in the development of the most common human brain malignant tumor, glioblastoma multiforme, which is invariably fatal. Targeting the IGF-I system has emerged as a useful method to reduce glial malignant development. Methodology: In practice, in case-control study, when human glioblastoma cells, and comparatively studied primary hepatocarcinoma and colon adenocarcinoma cells derived from cancer biopsy, are transfected in vitro with vectors expressing either IGF-I antisense RNA or inducing IGF RNA-DNA triple helix, the synthesis of IGF-I is stopped on translation or transcription levels, respectively (anti – gene strategy). Three cancer groups of two patients each, cancer stage I, after surgery and radiotherapy, were injected using antisense / triple helix ‘’vaccines’’ of 1 million irradiated cells. The control groups received injected placebo. Results: Down regulation in the expression of IGF-I coincides with the reappearance of B7 and MHC class I antigens at the surface of transfected cells (immunogenicity). When injected subcutaneously, the transfected cancer cells, “vaccines”, initiate an immune reaction involving CD8+ lymphocytes, followed by tumor regression. The glioblastoma patients treated by classical surgery and radiotherapy, were “vaccinated” by three successive injections. The median survival of treated patients was 21 months (current progress in treatment of this disease involves an increase in medium survival from 8-11 months to an average of 15 months, using a chemotherapy). Using the same strategy, the patients with liver carcinoma and colon adenocarcinoma were comparatively treated. The obtained immune anti-tumor response mediated by TCD8 was similar to that of glioblastoma patients. Conclusion: The cellular immunogene therapy using anti - gene approach constitutes one of the current efficient therapies of glioblastoma and other malignancies expressing IGF-I. The described methodology applied in Europe, and previously in the USA for glioblastoma treatment, is introduced now in university hospitals of Colombia (Bogota).

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against “A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13^th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4⁺ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4⁺ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4⁺ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4⁺ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS13^1239–1253 peptide as the single immunodominant HLA-DR1-restricted CD4⁺ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4⁺ T cells from Sure-L1 mice and was recognized by CD4⁺ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS13^1239–1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4⁺ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS13^1239–1253-loaded HLA-DR tetramers.

Research on human immunology has been hindered by the lack of optimal small animal models, given that the protective immune responses of human and non-human species show significant differences. However, due to ethical constraints[1] and the high cost of clinical trials, it is urgent to improve the current animal models that can mimic faithfully human physiology, particularly the human immune system (HIS). HIS mice had been generated recently by engrafting human hematopoietic stem cells (hHSCs) or human peripheral mononuclear cells (hPBMCs) into highly immuno-deficient mice such as NSG, NOG or NRG mice. However, a major experimental drawback for studies using these models is the rapid onset of Graft-versus-Host Disease (GvHD). In the present study, we overcome this limitation by generating new immuno-deficient mice named "HUMAMICE" (HLA-A2^+/+/DR1^+/+/H-2-β₂m^-/-/IAβ^-/-/Rag2^-/-/IL2rγ^-/-/Perf^-/- mice), which expressed human HLA molecules instead of mouse MHC molecules (H-2), and whose immuno-deficient status was reversed by transferring functional HLA-matched PBMCs thus producing mice with an immuno-competent status with a functional human immune system. We showed that in this HLA-matched context, the hPBMC-transfer led to high lymphocytes engraftment rates without GvHD over three months in this novel mouse model. Furthermore, to evaluate the utility of the hPBMC-HUMAMICE, we immunized them with commercial vaccine of Hepatitis B virus (HBsAg, Hepvac@) which resulted in robust and reproducible production of high levels of HBsAg-specific antibodies, implying that both transferred T and B lymphocytes were functional in HUMAMICE. These responses are comparable to those observed in human clinical trials with this identical vaccine. In conclusion, these findings indicated that the HLA-matched-hPBMC-HUMAMICE represents a promising model for dissecting human immune responses in various human diseases, including infectious diseases, cancers and tumors, and to facilitate the development of novel vaccines and cellular therapies.

Purpose : E75, a peptide derived from the Her2/neu protein, is the most clinically advanced vaccine approach against breast cancer. In this study, we aimed to optimize the E75 vaccine using a delivery vector targeting dendritic cells - the B-subunit of Shiga toxin (STxB) - and to assess the role of various parameters (Her2/neu expression, combination with trastuzumab) in the efficacy of this cancer vaccine in a relevant preclinical model. Experimental Design : We compared the differential ability of the free E75 peptide or the STxB-E75 vaccine to elicit CD8+T cells, and the impact of the vaccine on murine HLA-A2-tumors expressing low or high levels of Her2/neu. Results : STxB-E75 synergized with GM-CSF and CpG and proved to be more efficient than the free E75 peptide in the induction of multi-functional and high avidity E75-specific anti-CD8+T cells resulting in a potent tumor protection in HLA-A2 transgenic mice. High expression of HER2/neu inhibited the expression of HLA-class I molecules, leading to a poor recognition of human or murine tumors by E75-specific cytotoxic CD8+ T cells. In line with these results, STxB-E75 preferentially inhibited the growth of HLA-A2 tumors expressing low levels of Her2/neu. Co-administration of anti-Her2/neu mAb potentiated this effect. Conclusions : STxB-E75 vaccine is a potent candidate to be tested in patients with low Her2/neu-expressing tumors. It could also be indicated in patients expressing high levels of Her2/neu and low intratumoral T cell infiltration to boost the T cell recruitment, a key parameter in the efficacy of anti-Her2/neu mAb therapy.

The rapid occurrence of emerging infectious diseases demonstrates an urgent need for a new preclinical experimental model that reliably replicates human immune responses. Here, a new homozygous humanized human leukocyte antigen (HLA)-A11/DR1 transgenic mouse (HLA-A11^+/+/DR01^+/+/H-2-β_2m^−/−/IAβ^−/−) was generated by crossing HLA-A11 transgenic (Tg) mice with HLA-A2^+/+/DR01^+/+/H-2-β_2m^−/−/IAβ^−/− mice. The HLA-A11-restricted immune response of this mouse model was then examined. HLA-A11 Tg mice expressing a chimeric major histocompatibility complex (MHC) molecule comprising the α1, α2, and β₂m domains of human HLA-A11 and the α3 transmembrane and cytoplasmic domains of murine H-2D^b were generated. The correct integration of HLA-A11 and HLA-DR1 into the genome of the HLA-A11/DR1 Tg mice (which lacked the expression of endogenous H-2-I/II molecules) was then confirmed. Immunizing mice with a recombinant HBV vaccine or a recombinant HIV-1 protein resulted in the generation of IFN-γ-producing cytotoxic T lymphocyte (CTL) and antigen-specific antibodies. The HLA-A11-restricted CTL response was directed at HLA immunodominant epitopes. These mice represent a versatile animal model for studying the immunogenicity of HLA CTL epitopes in the absence of a murine MHC response. The established animal model will also be useful for evaluating and optimizing T cell-based vaccines and for studying differences in antigen processing between mice and humans.