In vitro assessment of antitumor immune responses using tumor antigen proteins produced by transgenic silkworms.
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ABSTRACT:
SUBMITTER: Yamada K
PROVIDER: S-EPMC8128804 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recent clinical trials with the aim of developing tumor antigen (TA)-specific cancer vaccines against a number of malignancies have focused on the identification of TAs presented by tumor cells and recognized by T cells. In the present study, the TA melanoma antigen family A4 (MAGE-A4) protein was produced using a transgenic (TG) silkworm system. Using in vitro stimulation, it was subsequently determined whether MAGE-A4 protein induced MAGE-A4-specific T cells from peripheral blood mononuclear cells of healthy donors. TG silkworm lines expressing a MAGE-A4 gene under an upstream activating sequence (UAS) were mated with those expressing a yeast transcription activator protein (GAL4) at the middle silk glands (MSGs) and embryos that harbored both the GAL4 and UAS constructs were selected. Recombinant MAGE-A4 protein was extracted from the MSGs of TG silkworms and evaluated using SDS-PAGE and western blot analysis. It was observed that MAGE-A4 produced by the TG silkworm system successfully induced MAGE-A4-specific CD4+ T cell responses. Furthermore, MAGE-A4-specific CD4+ T cells recognized antigen-presenting cells when pulsed with a MAGE-A4+ tumor cell lysate. The present data suggests that recombinant tumor antigen production using the TG silkworm system may be a novel tool in the preparation of cancer vaccines.
| S-EPMC5854940 | biostudies-literature
Project description:In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO- and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.
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Project description:High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4+ and CD8+ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHHCD11b) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8+ T-cell responses against HPV+ tumors. Mice immunized with VHHCD11b conjugated to an H-2Db-restricted immunodominant E7 epitope (E749-57) had more E7-specific CD8+ T cells compared with those immunized with E749-57 peptide alone. These CD8+ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHHCD11b-E749-57 vaccination resulted in greater numbers of CD8+ tumor-infiltrating lymphocytes compared with mice receiving E749-57 peptide alone in HPV+ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers. Cancer Immunol Res; 6(7); 870-80. ©2018 AACR.
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Project description:Therapeutic proteins expressed using transgenic animals have been of great interest for several years. Especially, transgenic silkworm has been studied intensively because of its ease in handling, low-cost, high-yield and unique glycosylation patterns. However, the physicochemical property of the therapeutic protein expressed in transgenic silkworm remains elusive. Here, we constructed an expression system for the TNFR-Fc fusion protein (Etanercept) using transgenic silkworm. The TNFR-Fc fusion protein was employed to N-glycan analysis, which revealed an increased amount of afucosylated protein. Evidence from surface plasmon resonance analysis showed that the TNFR-Fc fusion protein exhibit increased binding affinity for Fcγ receptor IIIa and FcRn compared to the commercial Etanercept, emphasizing the profit of expression system using transgenic silkworm. We have further discussed the comparison of higher order structure, thermal stability and aggregation of the TNFR-Fc fusion protein.
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Project description:We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μ g DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μ g of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.
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Project description:The immune-modulating effects of radiotherapy (XRT) have gained considerable interest recently, and there have been multiple reports of synergy between XRT and immunotherapy. However, additional preclinical studies are needed to demonstrate the antigen-specific nature of radiation-induced immune responses and elucidate potential mechanisms of synergy with immunotherapy. Here, we demonstrate the ability of stereotactic XRT to induce endogenous antigen-specific immune responses when it is combined with anti-PD-1 checkpoint blockade immunotherapy. Using the small animal radiation research platform (SARRP), image-guided stereotactic XRT delivered to B16-OVA melanoma or 4T1-HA breast carcinoma tumors resulted in the development of antigen-specific T cell- and B cell-mediated immune responses. These immune-stimulating effects of XRT were significantly increased when XRT was combined with either anti-PD-1 therapy or regulatory T cell (Treg) depletion, resulting in improved local tumor control. Phenotypic analyses of antigen-specific CD8 T cells revealed that XRT increased the percentage of antigen-experienced T cells and effector memory T cells. Mechanistically, we found that XRT upregulates tumor-associated antigen-MHC complexes, enhances antigen cross-presentation in the draining lymph node, and increases T-cell infiltration into tumors. These findings demonstrate the ability of XRT to prime an endogenous antigen-specific immune response and provide an additional mechanistic rationale for combining radiation with PD-1 blockade in the clinic.
| S-EPMC4390444 | biostudies-literature
Project description:The creation of functional materials from renewable resources has attracted much interest. We previously reported on the genetic code expansion of the domesticated silkworm Bombyx mori to functionalize silk fiber with synthetic amino acids such as 4-azido-L-phenylalanine (AzPhe). The azido groups act as selective handles for biorthogonal chemical reactions. Here we report the characterization and scaled-up production of azido-functionalized silk fiber for textile, healthcare, and medical applications. To increase the productivity of azido-functionalized silk fiber, the original transgenic line was hybridized with a high silk-producing strain. The F₁ hybrid produced circa 1.5 times more silk fibroin than the original transgenic line. The incorporation efficiency of AzPhe into silk fibroin was retained after hybridization. The tensile properties of the azido-functionalized silk fiber were equal to those of normal silk fiber. Scaled-up production of the azido-functionalized silk fiber was demonstrated by rearing circa 1000 transgenic silkworms. Differently-colored fluorescent silk fibers were successfully prepared by click chemistry reactions, demonstrating the utility of the azido-functionalized silk fiber for developing silk-based materials with desired functions.
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Project description:BackgroundFc-fusion proteins have been successfully developed for therapeutic purposes, but are also a promising platform for the fast generation and purification of immunogens capable of inducing strong humoral immune responses in preclinical immunization studies. As the Fc-portion of immunoglobulins fused to an antigen confers functional properties of the parental antibody, such as dimerization, binding to Fc-receptors and complement activation, several studies reported that Fc-fusion proteins elicit stronger antigen-specific antibody responses than the unfused antigen. However, dimerization or half-life extension of an antigen have also been described to enhance immunogenicity.MethodsTo explore the role of Fc-effector functions for the immunogenicity of fusions proteins of viral glycoproteins and Fc fragments, the HIV-1 gp120 and the RBD of SARS-CoV-2 were fused to the wild type muIgG2a Fc fragment or mutants with impaired (LALA-PG) or improved (GASDIE) Fc-effector functions.ResultsImmunization of BALB/c mice with DNA vaccines encoding gp120 - Fc LALA-PG induced significantly higher antigen-specific antibody responses than gp120 - Fc WT and GASDIE. In contrast, immunization with DNA vaccines encoding the RBD fused to the same Fc mutants, resulted in comparable anti-RBD antibody levels and similar neutralization activity against several SARS-CoV-2 variants.ConclusionDepending on the antigen, Fc-effector functions either do not modulate or suppress the immunogenicity of DNA vaccines encoding Fc-antigen fusion proteins.
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Project description:The extent to which effector CD8+ T cells infiltrate into tumors is one of the major predictors of clinical outcome for patients with epithelial ovarian cancer (EOC). Immune cell infiltration into EOC is a complex process that could be affected by the epigenetic makeup of the tumor. Here, we have demonstrated that a lysine 4 histone H3 (H3K4) demethylase, (lysine-specific demethylase 5A; KDM5A) impairs EOC infiltration by immune cells and inhibits antitumor immune responses. Mechanistically, we found that KDM5A silenced genes involved in the antigen processing and presentation pathway. KDM5A inhibition restored the expression of genes involved in the antigen-presentation pathway in vitro and promoted antitumor immune responses mediated by CD8+ T cells in vivo in a syngeneic EOC mouse model. A negative correlation between expression of KDM5A and genes involved in the antigen processing and presentation pathway such as HLA-A and HLA-B was observed in the majority of cancer types. In summary, our results establish KDM5A as a regulator of CD8+ T-cell infiltration of tumors and demonstrate that KDM5A inhibition may provide a novel therapeutic strategy to boost antitumor immune responses.
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Project description:We have previously reported that tumor antigen-specific DNA vaccination in mice led to an increase in IFNγ-secreting T cells and an increase in tumor expression of PD-L1. Further, we demonstrated that increasing the encoded antigen's MHC-binding affinity led to increased PD-1 expression on antigen-specific CD8(+) T cells. Together these phenomena provided resistance to antitumor immunization that was abrogated with PD-1/PD-L1 blockade. We consequently sought to determine whether similar regulation occurred in human patients following antitumor immunization. Using clinical samples from prostate cancer patients who were previously immunized with a DNA vaccine, we analyzed changes in checkpoint receptor expression on antigen-specific CD8(+) T cells, the effect of PD-1 blockade on elicited immune responses, and for changes in checkpoint ligand expression on patients' circulating tumor cells (CTCs). We observed no significant changes in T-cell expression of PD-1 or other checkpoint receptors, but antigen-specific immune responses were detected and/or augmented with PD-1 blockade as detected by IFNγ and granzyme B secretion or trans vivo DTH testing. Moreover, PD-L1 expression was increased on CTCs following vaccination, and this PD-L1 upregulation was associated with the development of sustained T-cell immunity and longer progression-free survival. Finally, similar results were observed with patients treated with sipuleucel-T, another vaccine targeting the same prostate antigen. These findings provide in-human rationale for combining anticancer vaccines with PD-1 blocking antibodies, particularly for the treatment of prostate cancer, a disease for which vaccines have demonstrated benefit and yet PD-1 inhibitors have shown little clinical benefit to date as monotherapies.
| S-EPMC4938302 | biostudies-literature