Enhanced Activation of T Lymphocytes by Urease-Deficient Recombinant Bacillus Calmette-Guérin Producing Heat Shock Protein 70-Major Membrane Protein-II Fusion Protein

Mukai, T.; Maeda, Yosuke; Tamura, Toshiki; Matsuoka, Masao; Tsukamoto, Yumiko; Makino, Masahiko

doi:10.4049/jimmunol.1000198

Cited by 4 publications

(2 citation statements)

References 38 publications

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“…BCG-DHTM was produced by introducing the HSP70-MMP-II fusion gene into urease-deficient BCG-⌬UT-11-3. Previously, we reported that urease-deficient rBCG that secretes a fusion protein composed of HSP70 and MMP-II from M. leprae was superior to urease-deficient BCG-⌬UT-11-3 and normal BCG that secretes the fusion protein in the activation of APCs and naive T cells and the production of memory-type T cells in mice (40). Therefore, we used only vector control BCG-261H as a control BCG in this study.…”

Section: Discussionmentioning

confidence: 99%

Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

Mukai

Tsukamoto

Maeda

et al. 2013

Clin. Vaccine Immunol.

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Section: Discussionmentioning

confidence: 99%

Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

Mukai

Tsukamoto

Maeda

et al. 2013

Clin. Vaccine Immunol.

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“…Cross-presentation involves distinct, well-defined pathways that utilize the proteasome and ER/Golgi. [63][64][65] The proteasome plays an important role in antigen cross-presentation by cleaving intracellular proteins into small, 8-11…”

Section: Cross-presentation By Multiple Myeloma Involves the Proteasomentioning

confidence: 99%

Cross-Presentation Is a Source of Tumor Antigens for Multiple Myeloma Immunotherapy

Perakis

Peters

Roszik

et al. 2016

Blood

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Introduction Multiple myeloma (MM) remains incurable and accounts for 20% of deaths from hematologic malignancies. Immune modulating drugs and immunotherapy have been shown to be beneficial in MM patients, highlighting the importance of the immune system in MM. The success of these interventions relies on the unique ability of the immune system to specifically distinguish between MM and normal cells. One example involves the use of cytotoxic T-lymphocytes (CTL) that eliminate cancer cells by recognizing tumor specific antigens (TSA) that are presented on human leukocyte antigen (HLA) class I molecules on the tumor cells. TSA are presented on HLA class I via the canonical antigen-processing pathway, which causes the tumor cells to become targets for killing by CTL. On the other hand, exogenous antigens are typically presented on HLA class II molecules. Through cross-presentation, exogenous antigens are taken up and presented on HLA class I molecules. We have recently identified cross-presentation as a mechanism by which tumor cells, including MM, present exogenous antigens, leading to an expanded repertoire of possible tumor antigens that can be targeted. Here we demonstrate that targeting cross-presented antigens is a feasible immunotherapeutic strategy in MM. To this end, we used the anti-PR1/HLA-A2 antibody, 8F4, which effectively eliminates PR1/HLA-A2-expressing tumor cells, and identified novel cross-presented antigens. Experimental design NSG mice were sublethally irradiated with 200 Rad. After 24 hours, 2x106 U266 cells transduced with luciferase/GFP were IV injected. Engraftment was confirmed by measuring human IgE in the mouse serum by ELISA and bioluminescent imaging (BLI). Mice were treated with 8F4 (10 mg/kg), IgG isotype control (10 mg/kg) or PBS. Mice were sacrificed on week 9, bone marrow was harvested and analyzed by flow cytometry. To identify novel cross-presented antigens, polymorphonuclear cells (PMN) were isolated from HLA-A2- healthy donors. PMN were lysed and cultured with U266 cells for 24 hours at a 2:1 PMN:U266 cell ratio. U266 cells were lysed, incubated with anti-HLA-ABC antibody and protein-A/G-sepharose beads. Immunoprecipitated proteins were recovered by mild acid elution and peptides were discovered using tandem mass spectrometry. HLA-A2 specific peptides with strong predicted in silico binding were analyzed using the HLArestrictor 1.2 algorithm. Antigen processing machinery (APM) of primary patient samples were analyzed using western blots. Results 8F4 treated mice showed a 2 fold decrease in the percent of U266 in the bone marrow compared to isotype and untreated mice (P<0.05) (Figure 1). ELISA demonstrated approximately a 3-fold decrease in human IgE ng/mL in serum of mice treated with 8F4 (P<0.001). BLI quantification showed approximately a 0.5 fold decrease in total flux (p/s) in mice treated with 8F4 compared to isotype and untreated mice. In addition to illustrating the therapeutic potential of targeting PR1, we also sought to identify other cross-presented MM peptides. Initially, over 4x103 total peptides were discovered by mass spectrometry (Figure 2). Using our reality score indexing and bioinformatics we identified approximately 5x102 unique, high confidence, cross-presented peptides. Our top candidate peptides have predicted HLA-A2 binding affinities of <18 nM, ion scores >21, retention time >30.42 minutes, and are rank 1 for their spectral matches. Since we have demonstrated the capability of MM to cross-present antigens, we investigated whether the APM was dysregulated in MM patients. Patients show dysregulation in tapasin, LMP2/7, and calnexin by western blot analysis. Conclusion Collectively, our data demonstrate that the MM antigen repertoire is much larger than previously appreciated, and that there is a new catalogue of potential immunotherapeutic targets in MM derived from exogenous antigens. Disclosures Sergeeva: Astellas Pharma: Patents & Royalties. Molldrem:Astellas Pharma: Patents & Royalties.

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Enhanced protective efficacy against tuberculosis provided by a recombinant urease deficient BCG expressing heat shock protein 70-major membrane protein-II having PEST sequence

Tsukamoto

Maeda

Tamura

et al. 2016

Vaccine

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Enhanced Activation of T Lymphocytes by Urease-Deficient Recombinant Bacillus Calmette-Guérin Producing Heat Shock Protein 70-Major Membrane Protein-II Fusion Protein

Cited by 4 publications

References 38 publications

Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

Cross-Presentation Is a Source of Tumor Antigens for Multiple Myeloma Immunotherapy

Enhanced protective efficacy against tuberculosis provided by a recombinant urease deficient BCG expressing heat shock protein 70-major membrane protein-II having PEST sequence

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