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WO2016074580A1 - Réactif pour l'amélioration du taux de survie de lymphocytes t cd4 positifs et son application - Google Patents

Réactif pour l'amélioration du taux de survie de lymphocytes t cd4 positifs et son application Download PDF

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WO2016074580A1
WO2016074580A1 PCT/CN2015/093754 CN2015093754W WO2016074580A1 WO 2016074580 A1 WO2016074580 A1 WO 2016074580A1 CN 2015093754 W CN2015093754 W CN 2015093754W WO 2016074580 A1 WO2016074580 A1 WO 2016074580A1
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lymphocytes
tsa
positive
cells
tumor
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时玉舫
曹楷
王莹
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid

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  • the present invention pertains to the fields of biomedicine and immunology, and more particularly, to an agent for increasing the survival rate of CD4-positive T lymphocytes and uses thereof.
  • Tumor tissue is composed of a variety of cells, of which immune cells play a crucial role in anti-tumor.
  • immune cells infiltrated in tumors, particularly T lymphocytes, can secrete large numbers of different cytokines to kill tumor cells directly or indirectly. Therefore, immunotherapy of tumors is considered to be a very promising anti-tumor strategy.
  • adoptive transplantation of tumor-infiltrating T lymphocytes has been found to significantly inhibit tumor growth.
  • antibodies against CTLA-4, PD-1, and PD-L1 have also entered clinical trials and have shown significant therapeutic effects.
  • AICD activation-induced apoptosis
  • FasL-mediated AICD strictly controls the extent and activity of the immune response. Since the immune response is very important for tumor treatment, the regulation of AICD of T lymphocytes in the tumor environment will become an important strategy for new tumor treatment. This field needs to be further studied in order to develop drugs based on the regulation of immune cells.
  • a histone deacetylase inhibitor for the preparation of a CD4 positive T lymphocyte upregulator.
  • upregulation of CD4-positive T lymphocytes includes, but is not limited to, up-regulation of CD4-positive T lymphocytes and up-regulation of activity.
  • the CD4 positive T lymphocyte up-regulation includes, but is not limited to, a preparation for promoting survival of CD4-positive T lymphocytes, a preparation for increasing the activity of CD4-positive T lymphocytes, and a CD4-positive T lymphocyte.
  • a preparation for promoting survival of CD4-positive T lymphocytes a preparation for increasing the activity of CD4-positive T lymphocytes
  • a CD4-positive T lymphocyte a preparation that increases the effective duration of CD4-positive T lymphocytes
  • a preparation that increases the proportion of CD4-positive T lymphocytes in tumor-infiltrating cells and/or a preparation that reduces apoptosis of CD4-positive T lymphocytes.
  • the CD4-positive T lymphocytes are CD62L-negative cells.
  • the CD4-positive T lymphocytes are CD4-positive T lymphocytes present in tumor tissues.
  • the histone deacetylase inhibitor comprises: trichostatin A, sodium butyrate.
  • the histone deacetylase inhibitor is further used to: increase the content of T lymphocyte-associated cytokines IL-2, IFN ⁇ , TNF ⁇ and/or GM-CSF.
  • the histone deacetylase inhibitor is further used to inhibit expression of FasL or increase expression of Fas.
  • the histone deacetylase inhibitor is further used to inhibit activation-induced apoptosis (AICD) of CD4-positive T lymphocytes.
  • a histone deacetylase inhibitor for the preparation of a potentiating CD4 positive T lymphocyte preparation, said synergistic CD4 positive T lymphocyte preparation, said CD4 Positive T lymphocytes have an anti-tumor effect higher than that of CD4-positive T lymphocytes that have not been treated with histone deacetylase inhibitors.
  • kits for elevating CD4-positive T lymphocytes or for inhibiting tumors comprising:
  • the HDAC inhibitor and the anti-CTLA-4 antibody are in an amount effective.
  • the HDAC inhibitor is contained in a pharmaceutically acceptable carrier.
  • the anti-CTLA-4 antibody is contained in a pharmaceutically acceptable carrier.
  • the histone deacetylase inhibitor comprises: trichostatin A, sodium butyrate.
  • the tumor comprises: melanoma, breast cancer, lymphoma.
  • kits for raising CD4 positive T lymphocytes or for inhibiting tumors in another aspect of the invention, there is provided the use of the kit for raising CD4 positive T lymphocytes or for inhibiting tumors.
  • TSA inhibits the growth of B16F0 melanoma by promoting survival of CD4-positive T lymphocytes.
  • HDAC inhibitors can inhibit AICD of T lymphocytes, thereby promoting their survival.
  • A1.1T lymphoma cells were co-cultured with different concentrations of TSA for 24 hours in 24-well plates pre-plated with CD3 antibodies. The cells were then collected and subjected to PI staining to detect the proportion of apoptosis.
  • A1.1 cell AICD was induced with CD3 antibody and treated with 100 nM TSA for 20 hours, and then cell activity was measured using CCK-8.
  • A1.1 cells were co-cultured with 100 nM TSA with anti-CD3 (10 ⁇ g/ml), actinomycin D (ActD, 5 ⁇ g/ml), hydrogen peroxide (H 2 O 2 , 80 mM) or UV irradiation (80J/m2) treatment. After 24 hours, PI staining was performed to detect the proportion of apoptosis.
  • B-C A1.1 cell AICD was induced with CD3 antibody and treated with 100 nMTSA, and cells and supernatant were collected to detect FasL messenger RNA or protein expression.
  • A1.1 cell AICD was induced and treated with 100 nMTSA, cells were harvested and stained with Fas flow antibody to detect Fas protein expression.
  • TSA inhibits AICD of CD4 + T cells by inhibiting the expression of FasL.
  • CD4 + T lymphocytes (2.5 x 10 5 ) were treated with CD3 antibody and different concentrations of TSA for 12 hours, and then the proportion of apoptosis was measured by PI staining.
  • CD The cells in A were collected to detect the expression of Fas and FasL in CD4 + T lymphocytes during AICD.
  • TSA inhibits FasL expression by inhibiting the NFAT1-Egr2 pathway.
  • A1.1 cells were treated with CD3 antibody and varying concentrations of TSA. Collect cells at different time points and isolate cytoplasmic and cytosolic proteins, and detect NFAT1, p53, GAPDH and laminB1 proteins Distribution.
  • A1.1 cells were treated with CD3 antibody and 100 nM TSA. Cells were harvested at different time points and the expression levels of mRNA for FasL, Egr2 and Egr3 were examined.
  • TSA exerts an anti-tumor effect by inhibiting the FasL-mediated AICD process of activated T lymphocytes in the tumor microenvironment.
  • mice were injected with 5 ⁇ 10 5 B16F0 cells (7 mice per group), and intraperitoneally administered with 1 ⁇ M/kg TSA or 200 ⁇ g of anti-CTLA4 or both, once every two days. After 14 days, the mice were sacrificed and the tumors were weighed.
  • AICD of 2.5 ⁇ 10 5 A1.1 cells was induced with CD3 antibody, and different concentrations of sodium butyrate were added, and apoptosis was detected by PI 24 hours later.
  • (C) A1.1 cells were stimulated with CD3 antibody and 3 ⁇ M sodium butyrate, and the degree of apoptosis was measured by PI staining at different times, and statistics were made.
  • TSA also inhibits the growth of EL4 lymphoma by the same mechanism.
  • EL4 lymphoma cells were injected in the same manner. After 14 days, the tumor was weighed (A), and then the lymphocyte suspension in the tumor was isolated and counted, and then the ratio and number (BC) of CD4 + CD62L - cells were analyzed by flow cytometry.
  • FIG. 10 In the EL4 lymphoma and 4T1 breast cancer models, simultaneous administration of TSA and CTLA-4 antibodies may further enhance the anti-tumor effect of TSA alone.
  • mice On day 0, mice were given intramuscular injection of 5 ⁇ 10 5 EL4 cells (A) or injected 2 ⁇ 10 5 4T1 cells in the mammary fat pad. From day 6, 1 ⁇ M/kg TSA or 200 ⁇ g CTLA-4 antibody was injected every other day until day 22. Mice were sacrificed after the tumor size exceeded 1,000 mm 3 or was large enough to affect the action.
  • Statistical survival curves were calculated, with each curve representing 10 mice per group. Significant differences were statistically calculated using the Log-Rank test.
  • FIG. 11 Adoptive transplantation of CD4 + T cells from TSA-treated mice is more effective than DMSO-treated mice.
  • B16F0 cells (5 ⁇ 10 5 ) were injected into the left thigh muscle of C57BL/6 mice on day 0. From the 6th day, 1 ⁇ M/kg TSA was intraperitoneally injected every other day, and the control group was injected with DMSO (50 mice per group). On day 14, the mice were sacrificed, tumors were harvested and ground, and then magnetic beads were sorted to obtain CD4 + CD62L - lymphocytes. These cells (2 ⁇ 10 6 ) were intravenously injected into Rag1 -/- immunodeficient mice simultaneously injected with B16 tumor cells. After 14 days, the mice were sacrificed and the tumors weighed. Significant differences were statistically analyzed using Student's t-test, *p ⁇ 0.05, **p ⁇ 0.01.
  • histone deacetylase HDAC
  • a histone deacetylase inhibitor can be used as a drug for raising T lymphocytes.
  • the histone deacetylase inhibitor can be administered in combination with an anti-CTLA-4 antibody, and has a synergistic antitumor effect.
  • the present invention has been completed on this basis.
  • Histone deace- lysing enzyme is an important enzyme that strictly regulates the deacetylation process of histones and non-histone proteins. This enzyme removes acetyl groups from tyrosine, altering the structure of chromatin and proteins, which in turn regulates many important cellular activities, including gene transcription, signal transduction, and more. Therefore, histone deacetylase is very important for maintaining the homeostasis of acetylation. If their activity is abnormal, it will lead to abnormal cell activities such as gene expression, and this phenomenon is found in various diseases. Inhibitors of positive histone deacetylases (HDAC Inhibitors, HDIs) are a class of small molecules that are extracted from natural sources or artificially synthesized.
  • Trichostatin A is the first histone deacetylase inhibitor extracted from nature.
  • TSA Trichostatin A
  • trichostatin A and other small molecule inhibitors have been found to have potential therapeutic effects in a number of diseases, including neurodegenerative diseases, tumors, autoimmune diseases, acute graft-versus-host diseases, etc.
  • the specific treatment mechanism is not fully explained.
  • HDIs have been reported to induce apoptosis in a variety of cells, their effect on AICD is unclear. The inventors first revealed after intensive research that HDIs exert anti-tumor effects by modulating AICD.
  • TSA inhibits the occurrence of AICD by inhibiting the cascade signal initiated by T lymphocyte receptors, thereby promoting the survival of infiltrating CD4-positive T lymphocytes in tumors, thereby promoting an anti-tumor immune response.
  • TSA significantly inhibits the growth of B16F0 melanoma, which is dependent on CD4-positive lymphocytes. Not only that, but also found that the proportion and absolute number of activated CD4-positive T lymphocytes infiltrated in the tumor were significantly improved. Therefore, TSA can significantly increase the number of CD4-positive T lymphocytes infiltrated in tumors. However, the mechanism by which TSA increases the number of T lymphocytes remains unclear.
  • the present inventors next investigated whether TSA has an effect on AICD of T lymphocytes, and surprisingly found that TSA can completely suppress the AICD process of T lymphocytes, thereby increasing the survival of T lymphocytes, and this effect is by inhibiting FasL.
  • the expression is achieved. Since FasL plays an important role in this process, the inventors established the same tumor model in FasL-deficient mice and found that injection of TSA did not inhibit tumor growth in FasL-deficient mice, and the number of infiltrating T lymphocytes was not obvious change. This finding confirms that TSA enhances lymphocyte effector infiltration in tumors by inhibiting FasL-mediated AICD.
  • the present invention provides the use of a histone deacetylase inhibitor for the preparation of a CD4-positive T lymphocyte upregulator.
  • histone deacetylase inhibitors can be used in the present invention to increase CD4 positive T Lymphocyte reagents.
  • the histone deacetylase inhibitor comprises: trichostatin A, sodium butyrate.
  • the histone deacetylase inhibitor further comprises: increasing the content of T lymphocyte-associated cytokines IL-2, IFN ⁇ , TNF ⁇ and/or GM-CSF; inhibiting the expression of FasL or Increase the expression of Fas; inhibit the activation of apoptosis induced by CD4-positive T lymphocytes (AICD) and other functions.
  • the histone deacetylase inhibitor can promote up-regulation of CD4-positive T lymphocytes in vivo or ex vivo.
  • One way is, for example, to directly administer a histone deacetylase inhibitor to a subject in need of treatment, thereby allowing it to act on a specific site in the body, increasing the number or activity of CD4-positive T lymphocytes at that site;
  • the mode of administration is such that the histone deacetylase inhibitor is enriched to a specific site in the body.
  • Another method is, for example, isolating CD4-positive T lymphocytes from a patient to be treated, and treating the CD4-positive T lymphocytes with a histone deacetylase inhibitor under ex vivo conditions to promote an increase in activity or an increase in the number of cells. And then return. It will be understood that other modes of administration contemplated by those skilled in the art are also encompassed by the present invention based on the novel findings of the present invention.
  • the present inventors have also found that after adoptive transplantation of CD4-positive T lymphocytes treated with histone deacetylase inhibitors into tumor-bearing mice, the anti-tumor effect of the cells on tumor-bearing mice is significantly better than that of the tumor-bearing mice.
  • the present invention also provides the use of a histone deacetylase inhibitor for the preparation of a potentiating CD4-positive T lymphocyte preparation.
  • the synergistic CD4-positive T lymphocyte preparation may be a cytosol preparation containing CD4-positive T lymphocytes treated with a deacetylase inhibitor.
  • the deacetylase inhibitor treatment can be in vivo treatment or ex vivo treatment.
  • CTLA-4 cytotoxic T-lymphocyte antigen 4
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a histone deacetylase inhibitor and an anti-CTLA-4 antibody, and a pharmaceutically acceptable carrier.
  • the histone deacetylase inhibitor and the anti-CTLA-4 antibody can be formulated in a non-toxic, In a medium which is inert and compatible with the cells, wherein the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8.
  • the term "contains” means that the various ingredients can be used together in the mixture or composition of the invention.
  • the term "effective amount” or “effective amount” refers to an amount that can produce a function or activity to a cancer patient and that is acceptable to a cancer patient.
  • a “pharmaceutically acceptable” ingredient is one that is suitable for use in a mammal without excessive adverse side effects (such as toxicity, irritation, and allergies), i.e., a material having a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable carrier” means a carrier for the administration of a therapeutic agent, including various excipients and diluents, that is, a pharmaceutically acceptable carrier. The term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full description of "pharmaceutically acceptable” can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).
  • the composition may contain a liquid such as water, saline, glycerol and ethanol in the composition with the above-mentioned acceptable carrier.
  • auxiliary substances such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • compositions of the present invention comprise a safe and effective amount of said cells and a carrier compatible with said cells.
  • carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • compositions of the invention a safe and effective amount of a cell of the invention is administered to a mammal.
  • the specific dose and number of administrations should also take into account factors such as the weight, age, and health of the patient to whom it is administered, which are within the skill of those in the art.
  • compositions of the invention can be used directly to elevate CD4-positive T lymphocytes or to inhibit tumors.
  • it can be used in combination with other therapeutic agents or adjuvants.
  • the invention also provides a kit for raising CD4 positive T lymphocytes or for inhibiting tumors, comprising:
  • Container 1 comprising: an effective amount (eg, 0.1-2000 ⁇ M) of a histone deacetylase inhibitor, and a pharmaceutically acceptable carrier;
  • Container 2 comprising: an effective amount (eg, 0.1-1000 mg) of an anti-CTLA-4 antibody, and pharmaceutically acceptable Accepted by the carrier.
  • kit is convenient for those skilled in the art, particularly clinicians.
  • the kit further includes instructions for use to guide a person in the art to take a suitable method of administration.
  • TSA inhibits tumor growth by promoting infiltration of CD4-positive T lymphocytes
  • HDAC inhibitors are believed to have potential anti-tumor effects. However, its specific treatment mechanism is not clear.
  • the present inventors intramuscularly injected B16F0 cells (purchased from Jackson) into C57BL/6 mice and nude mice lacking mature T lymphocytes, and established a melanoma model of B16F0.
  • B16F0 cells purchased from Jackson
  • a melanoma model was established on C57BL/6 mice and nude mice lacking mature T lymphocytes, and two concentrations of TSA were administered. One is a lower concentration of 1 ⁇ M/kg and the other is a higher concentration of 3 ⁇ M/kg. It was found that both concentrations of TSA significantly inhibited tumor growth in normal mice.
  • the inventors also used different concentrations of TSA to stimulate B16F0 tumor cells in vitro, and found that TSA can induce tumor cell apoptosis in a concentration-dependent manner, suggesting that high concentration of TSA can exert anti-tumor effect by directly killing tumor cells.
  • TSA tumor suppressing effect
  • T lymphocytes can be divided into two subpopulations, CD4 positive T lymphocytes and CD8 positive T lymphocytes, the inventors then studied which subpopulations played a more important role in the antitumor effect of low dose TSA.
  • the inventors utilized two knockout mice, ⁇ 2m-deficient mice (purchased from Jackson) were unable to produce CD8-positive T lymphocytes, whereas CIITA-deficient mice (purchased from Jackson) did not have mature CD4-positive T lymphocytes, An animal model of melanoma was prepared as before and TSA (1 ⁇ M/kg) was administered.
  • TSA is still capable of inhibiting tumor growth in mice deficient in ⁇ 2m, however, in CIITA-deficient mice, TSA has no therapeutic effect at all, as shown in Fig. 1C. This suggests that CD4-positive T lymphocytes, but not CD8-positive T lymphocytes, play a crucial role in the anti-tumor process of TSA.
  • the present inventors examined the infiltration of CD4-positive T lymphocytes in tumors in normal C57BL/6 mice, and isolated tumors in the DMSO group and the TSA group (1 ⁇ M/kg). The infiltrated lymphocytes were counted, and then the ratio of CD4 + CD62L - T lymphocytes was detected by flow, and the absolute number of CD4 + CD62L - T lymphocytes per gram of tumor was counted. It was found that the treatment of TSA significantly increased the proportion of CD4-positive T lymphocytes in tumor-infiltrating cells, and the absolute number was also significantly different from that of the solvent-treated group, as shown in Fig. 1D-E.
  • TSA T lymphocyte-associated cytokines IL-2, IFN ⁇ , TNF ⁇ , and GM-CSF in the serum of mice treated with TSA were also significantly increased, as shown in Figure 1F.
  • the administration of TSA can significantly increase the infiltration of CD4-positive T lymphocytes in tumors, and promote anti-tumor immunity, thereby inhibiting tumor growth.
  • the present inventors next studied the specific mechanism of the increase of CD4-positive T lymphocytes infiltrated in tumors under TSA administration.
  • the present inventors examined the apoptosis of these cells, and it was found by staining of 7-AAD that the apoptosis of tumor-infiltrating CD4-positive T lymphocytes in the TSA-administered group was significantly reduced, as shown in Fig. 1G. Therefore, it is suggested that TSA exerts an anti-tumor effect by inhibiting the apoptosis of such cells and increasing the number thereof.
  • Example 2 HDAC inhibitors inhibit activation-induced cell death, thereby promoting survival of T lymphocyte hybridomas
  • TSA treats tumors by increasing T lymphocyte infiltration
  • HDAC inhibitors have been reported to promote apoptosis
  • the inventors subsequently tested whether HDAC inhibitors have an effect on apoptosis of activated CD4-positive T lymphocytes in vitro.
  • Activated T lymphocytes primarily undergo activation-induced apoptosis (AICD).
  • A1.1 cells BALB/cCr mice were immunized with poly-18/complete Freund's adjuvant, lymph node T cells were isolated and activated to produce T cell blasts. These cells were fused with BW5147 thymoma cells, and the fused CD4 positive cells were selected to obtain A1.1 cells.
  • the present inventors first induced AICD by activating A1.1 cells with a CD3 antibody (anti-CD3), and simultaneously added different concentrations of TSA, and detected the apoptosis level of A1.1 cells 24 hours later. Surprisingly, higher concentrations of TSA completely inhibited apoptosis of A1.1 cells, as shown in Figures 2A-B. Moreover, it was further found that TSA inhibited apoptosis at the initial stage of AICD, as shown in Figure 2C. The inventors also used another HDAC inhibitor, sodium butyrate, to repeat the above experiment with TSA instead, and found the same result, as shown in FIG.
  • TSA is able to inhibit apoptosis of A1.1 cells
  • the inventors speculate that it can promote the survival of A1.1 cells.
  • trypan blue staining to detect the survival of A1.1 cells. It was found that after TSA treatment, trypan blue was negative, that is, the number of surviving A1.1 cells was significantly higher than that of the untreated group, as shown in Fig. 2E.
  • the inventors then also used Cell Counting Kit-8 (CCK-8) to detect cell viability, and found that TSA can promote the activity of A1.1 cells, as shown in Fig. 2D.
  • CCK-8 Cell Counting Kit-8
  • HDAC inhibitors can promote T lymphocyte survival by inhibiting apoptosis.
  • HDAC inhibitors can inhibit the expression of FasL on T lymphocyte hybridomas
  • HDAC inhibitors are capable of inhibiting AICD in T lymphocytes.
  • T lymphocyte apoptosis there are many pathways for T lymphocyte apoptosis, and different pathways are regulated by different mechanisms.
  • the inventors examined the inhibition of TSA on other types of apoptosis. Apoptosis of A1.1 cells was induced with actinomycin D, hydrogen peroxide or ultraviolet light, respectively.
  • TSA treatment did not inhibit these stimuli-induced apoptosis, but it was also promoted, as shown in Figure 3A. This result suggests that TSA is specific for the inhibition of AICD and that the specific mechanism of inhibition should also be related to the AICD mechanism.
  • TSA may have the following two effects: one is that TSA can affect the expression of FasL at an early stage, and the other is that TSA can inhibit the FasL-Fas apoptotic signaling pathway.
  • TSA inhibits apoptosis the inventors first tested whether TSA had an effect on FasL expression of A1.1 cells in AICD.
  • TSA completely inhibits the expression of FasL not only from the level of messenger RNA but also at the protein level, as shown in Figures 3B-D. Similar results were found using sodium butyrate in place of TSA.
  • the present inventors used CD90 antibody to induce FasL expression on A1.1 cells, and found that TSA still inhibits the expression of FasL, thereby inhibiting apoptosis induced by CD90 antibody.
  • HDAC inhibitors can inhibit the expression of FasL and exert its anti-apoptotic effect.
  • the inventors added soluble FasL to the system, and as a result, it was found that the inhibitory effect of TSA was reversed, as shown in Fig. 3E. More interestingly, after the addition of soluble FasL, TSA can significantly promote apoptosis. Since this apoptosis requires the binding of Fas-FasL, the present inventors examined the expression of Fas molecules on A1.1 cells, and found that TSA can promote the expression of Fas on the surface of A1.1 cells in a dose-dependent manner.
  • TSA can completely block the expression of FasL, and thus can completely inhibit the occurrence of AICD.
  • TSA inhibits AICD of primary CD4-positive T lymphocytes by inhibiting FasL expression
  • HDAC inhibitors are able to inhibit the development of AICD by inhibiting the expression of FasL in A1.1 cells.
  • A1.1 cells used by the present inventors were artificially produced, and it is still unclear whether or not such a condition exists in primary cells. Therefore, the present inventors isolated CD4-positive T lymphocytes in the spleen of mice and stimulated the production of T cell blasts. In this state, AICD occurs if T lymphocytes are stimulated again.
  • TSA can also completely inhibit AICD of primary cells.
  • primary cells are more sensitive to TSA, showing that lower concentrations of 25 nM TSA can completely inhibit AICD, and lower concentrations of 10 nM TSA also have a certain inhibitory effect, and this concentration of TSA does not inhibit A1.1 cells.
  • Apoptotic as shown in Figures 4A-B.
  • the inventors also found that the expression forms of Fas and FasL of primary cells in AICD are identical to those of A1.1 cells, as shown in Figures 4C-D.
  • TSA anti-apoptotic effect of TSA is also present in primary T lymphocytes and is also accomplished by regulation of FasL.
  • TSA inhibits FasL expression by regulating Egr2 and NFAT1
  • CD3 antibodies activate two signaling pathways in AICD, one is Ca2+-NFAT1 (nuclear factor activated T-cells1)-Egr2/3 (early growth factors 2 and 3, earlyly Growth factor 2 and 3) pathway, one is PKC path.
  • the expression of FasL is widely thought to be regulated by both pathways, while the expression of Fas is only controlled by PKC. Since TSA only inhibited the expression of FasL without significantly affecting the expression of Fas, the inventors then isolated the cytoplasmic and nuclear proteins and detected that NFAT1 and p53 proteins are in the cytoplasmic nucleus after activation of A1.1 cells. The distribution of quality.
  • the inventors then examined the expression of NFAT1 regulated Egr2 and Egr3 in A1.1 cells, as shown in Figure 5A. The inventors have found that both factors are rapidly produced during the occurrence of AICD. However, TSA only inhibited the expression of Egr2, but had no effect on the expression of Egr3, as shown in Fig. 5B, indicating that Egr2 is an important factor regulating the expression of FasL in A1.1 cells.
  • FasL plays a crucial role in the anti-apoptosis of HDAC inhibitors.
  • the inventors first examined the FasL expression level of tumor infiltrating lymphocytes. It was found that, consistent with the in vitro results, the expression of FasL in activated CD4-positive T lymphocytes did decrease after TSA administration, as shown in Fig. 6A. The expression of FasL in activated CD4-positive T lymphocytes infiltrated in the tumor was then examined, and it was found that TSA administration did reduce the proportion of FasL-positive fractions in such cells, as shown in Fig. 6B. Therefore, TSA treatment can reduce the expression of FasL in CD4-positive T lymphocytes, thereby inhibiting the proportion of apoptosis in this group of cells during tumorigenesis and development.
  • the inventors then also examined the infiltration of CD4-positive cells in tumors and found that in gld/gld mice, TSA could not increase the proportion of lymphocytes in this group and the number of cells per gram of tumor, ie in the absence of FasL. TSA does not promote the infiltration of this group of lymphocytes, as shown in Figure 6D-E. Meanwhile, the inventors have also found that the content of cytokines in the serum of TSL-treated gld/gld mice has not increased, as shown in Fig. 6F. Therefore, the effect of TSA on increasing CD4-positive cell infiltration is dependent on FasL. Moreover, even in the absence of TSA administration, in gld/gld mice Among them, T lymphocyte infiltration was also significantly more than that of wild-type mice, which confirmed this conclusion, as shown in Fig. 6G.
  • the present inventors injected Gld/gld mice in the same manner as EL4 lymphoma cells, thereby repeating this result in the EL4 lymphoma system, as shown in FIG. It can be seen that TSA also inhibits the growth of EL4 lymphoma through the same mechanism.
  • Example 7 the anti-tumor effect of co-administration of TSA and CTLA-4 antibodies is more powerful
  • the present inventors immunized mice with TSA and CTLA-4 antibodies (anti-CTLA-4, three times with CTLA-4 antigen, and detected antibody titers by ELISA. After the last booster The spleen cells of the mouse with higher ELISA titer were isolated and fused with myeloma cells. Then, positive cell clones were selected by ELISA, and two subclones were selected to screen 100% positive cell lines to produce secreted cells. Hybridoma cells of CTLA-4 antibody.
  • the supernatant of the cultured hybridoma cells was collected, precipitated by ammonium sulfate, and the centrifuged product was dissolved in PBS, dialyzed against a dialysis membrane, and finally quantified to obtain a CTLA-4 antibody which can be used for the experiment)
  • the two drugs were used together to treat melanoma and the results of their co-administration were observed.
  • the present inventors have found that the use of TSA (1 ⁇ M/kg body weight) and CTLA-4 antibody (200 ⁇ g/mouse) significantly prolongs the lifespan of mice, and an extremely significant synergistic effect occurs compared with single administration. Significantly inhibited the growth of mouse tumors and prolonged the survival time of mice, as shown in Figure 7A-B. Not only that, CD4 positive T lymphocytes infiltrated in tumors also increased significantly, as shown in Figure 7C-D.
  • TSA and CTLA-4 antibodies can significantly increase the infiltration of CD4-positive T lymphocytes, thereby showing a more significant therapeutic effect than single administration.
  • the inventors believe that this finding can provide a new avenue for the clinical use of HDAC inhibitors.
  • Example 8 simultaneous administration of TSA and CTLA-4 antibodies can further promote the anti-tumor effect of TSA alone.
  • mice were given intramuscular injection of 5 ⁇ 10 5 EL4 cells injected or 2 ⁇ 10 5 4T1 cells in the mammary fat pad. From day 6, every day, 1 ⁇ M/kg TSA or 200 ⁇ g anti-CTLA-4 antibody or both were co-administered, and mice injected with DMSO were used as controls until day 22. Mice will be sacrificed if the tumor size exceeds 1,000 mm 3 or is large enough to affect the action. Statistical survival curves were calculated, with each curve representing 10 mice per group. Significant differences were statistically calculated using the Log-Rank test.
  • B16F0 melanoma cells (5 ⁇ 10 5 ) were injected into the left thigh muscle of C57BL/6 mice on day 0. From the 6th day, 1 ⁇ M/kg TSA was intraperitoneally injected every other day, and the control group was injected with DMSO (50 mice per group). On day 14, the mice were sacrificed, tumors were harvested and ground, and then CD4 + CD62L - lymphocytes were obtained by flow cytometry. These cells (2 ⁇ 10 6 ) were intravenously injected into Rag1 ⁇ / ⁇ immunodeficient mice injected with B16F0 melanoma cells simultaneously. After 14 days, the mice were sacrificed and the tumors weighed. Significant differences were statistically analyzed using Student's t-test, *p ⁇ 0.05, **p ⁇ 0.01.

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Abstract

La présente invention concerne un réactif permettant d'améliorer le taux de survie de lymphocytes T CD4 positifs et son application. Un inhibiteur de l'histone désacétylase peut atténuer un processus de mort cellulaire induite par l'activation (AICD) de lymphocytes T. L'inhibiteur de l'histone désacétylase comprend la trichostatine A et le butyrate de sodium. Une combinaison de l'inhibiteur de l'histone désacétylase et un anticorps anti-CTLA-4 possède un effet anti-tumoral synergique.
PCT/CN2015/093754 2014-11-14 2015-11-04 Réactif pour l'amélioration du taux de survie de lymphocytes t cd4 positifs et son application Ceased WO2016074580A1 (fr)

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CN102321580A (zh) * 2011-08-23 2012-01-18 郑颂国 一种治疗自体自身免疫性疾病的调节t细胞及其制备方法

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AU2007269836A1 (en) * 2006-06-30 2008-01-10 Albany Molecular Research, Inc. Substituted piperidines that increase P53 activity and the uses thereof
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WO2009126877A2 (fr) * 2008-04-11 2009-10-15 University Of Southern California Procédés et compositions pour accélérer la génération de cellules t régulatrices ex vivo
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