Therapeutic and diagnostic methods for cancer
A cancer, bladder cancer technology, applied in the field of cancer treatment and diagnosis, can solve problems such as detection and treatment difficulties
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[0065] The term "pharmaceutical formulation" refers to a preparation in such a form as to allow the biological activity of the active ingredient to be effective and free of other ingredients which are unacceptably toxic to a subject to whom the formulation will be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those employed which can reasonably be administered to a subject mammal to provide an effective dosage of the active ingredient.
[0066] "Pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical formulation other than the active ingredient that is nontoxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
[0067] As used herein, the term "treatment / treatment" refers to a clinical intervention designed to alter the natural course of a treated individual or cell during the course of a clinical path...
Embodiment 1
[0402] Example 1: CD4 in the mMLR Assay + T cells and allogeneic DC
[0403] To reveal the underlying mechanism of PD-1 blockade, we developed a CD4-focused + In vitro functional assay of T cells in which CD4 + T cells recognize allogeneic MHCII and mount a detectable immune response. For this assay, called minimal MLR (mMLR), we co-cultured sorted CD4 T cells obtained from healthy donors with monocyte-derived mature dendritic cells from unrelated donors.
[0404] minimal mixed lymphocyte reaction
[0405] We developed an assay in which freshly purified CD4 + T cells were co-cultured for 5 days in the presence of monocyte-derived allogeneic mature dendritic cells (mDC). Monocytes were isolated from fresh PBMCs 1 week prior to co-culture via plastic adhesion, followed by removal of non-adherent cells. We then generated immature DCs (iDCs) from monocytes by culturing them in medium containing GM-CSF (50 ng / ml) and IL-4 (100 ng / ml) for 5 days. To induce iDC maturation,...
Embodiment 2
[0409] Example 2: PD-1 blockade improves CD4 + IFN-γ secretion and granzyme B production by T cells
[0410] We next determined the effect of anti-PD-1 blocking antibodies added to the mMLR cultures as described in Example 1.
[0411] Cytokine intracellular staining and ELISA
[0412] On day 5 from the mMLR co-culture, we collected cell culture supernatants for measuring IFN-γ levels by ELISA (R&Dsystems, following the manufacturer's instructions), and incubated the cells at 37°C in a Golgi plug (Brayffy). Dermectin A, BD Bioscience) and a Golgi terminator (monensin, BD Bioscience) were left for an additional 5 hours. Cells were then washed and stained on the surface with anti-human CD4 antibody and the live / dead fixable dye Aqua (Invitrogen) before fixation / permeabilization with fixation / permeabilization buffer (BD Bioscience). We then performed intracellular staining for Granzyme B (BD Bioscience), IFN-γ and IL-2 (both antibodies were from eBioscience). By adding an ant...
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