受験番号：32IPB003

問 1．
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Silkworms are classified into a univoltine strain, a bivoltine strain, and amultivoltine strain based on their life cycle. The univoltine strain develops to theadult stage once a year when grown under natural conditions.

Many strains ofsilkworm are univoltine. The univoltine silkworms are known to diapause in the egg
stage, and this nature is maintained in microinjected eggs. To generate a geneticallymodified silkworm in a short time without causing the diapause of eggs, nondiapausing
mutant strains or multivoltine, nondiapausing strains are typically used. However,these strains, which are experimental strains, have problems in measurable traits andother traits, requiring the production of nondiapausing eggs from practical diapausing
strains to industrially use silkworms as hosts for mass production.
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In the method of the present invention for preparing an extract for cell-free protein
synthesis, the middle silk gland of a silkworm strain that does not specifically produce
fibroin can be easily extracted even using a conventional method of extracting themiddle silk gland of a wild-type silkworm strain (see Japanese Patent Laid-Open No.
2007-210902), compared with a conventional method of preparing the extract using theposterior silk gland and fat body. The present inventors have developed a method ofextracting the middle silk gland of the silkworm strain that does not specifically
produce fibroin in a short time compared with the conventional method, as mentioned
below.
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After cultured with Gnetin C for 24 hours, AML-MT cells were stained withannexin-FITC and analyzed using flow cytometry or were adsorbed on a slide glass with

a Cytospin, stained with Giemsa, and assessed for cell morphology under a lightmicroscope.

AML-MT cells were also cultured with Gnetin C at an indicatedconcentration for 12 hours, stained with a Mitocapture reagent, and then observed
under a fluorescence microscope. In the annexin V staining assay and the Giemsa
stain, the cell proliferation inhibitory effect of Gnetin C on AML cells induced typicalmorphological features of apoptosis including cell membrane protrusions, aggregatedchromatin, nuclear fragmentation, and condensed basophilic cytoplasm.

Thesefeatures were similar to those observed in apoptosis induction of patient-derived AMLcells.

Interestingly, Gnetin C-treated cells significantly decreased their membranepotentials.

This indicates that cell apoptosis induced by Gnetin C is caused by

mitochondrial membrane dysfunction in AML cells.
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Claim 1.
A function improving agent for an immunologically exhausted CD8+ T cell,comprising a biguanide antidiabetic drug selected from the group consisting ofphenformin, buformin, and metformin.
Claim 2.
The function improving agent according to claim 1, wherein the function
improving agent restores a cytokine production capacity of the immunologicallyexhausted CD8+ T cell.
Claim 3.
Thefunction

improvingagent

accordingtoclaim2,

whereintheimmunologically exhausted T cell expresses an exhaustion marker, and the function
improving agent restores a cytokine production capacity of the immunologicallyexhausted CD8+ T cell expressing the exhaustion marker.
Claim 4.
The function improving agent according to any of claims 1 to 4, wherein thefunction improving agent suppresses apoptosis of a CD8+ T cell.

Claim 5.
A therapeutic agent for cancer in a cancer patient with immunologicalexhaustion, wherein the therapeutic agent comprises the function improving agent
according to any of claims 1 to 4 as an active ingredient.
Claim 6.
The therapeutic agent according to claim 5, wherein the immunologicalexhaustion is caused by expression of an exhaustion marker on a CD8+ T cell.
Claim 7.
The therapeutic agent according to claim 6, wherein the exhaustion marker isselected from the group consisting of Program cell death protein 1 (PD-1) and T cell
membrane protein 3 (Tim-3).
Claim 8.
The therapeutic agent according to any of claims 5 to 7, wherein the active
ingredient is administered to a cancer patient who has not been experienced
chemotherapy and radiotherapy for cancer.
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