Compositions and methods for inhibiting ythdf1

Abstract

Provided are compositions and methods for attenuating YTHDF1 activity, as well as compositions and methods for promoting an immune response. For example, provided is a YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) attenuating agent, wherein the attenuating agent comprises a compound that, when bound to YTHDF1, binds to at least one residue of YTHDF1.

Description

Background Technology

[0001] Spontaneous T-cell sensitization against tumor neoantigens is crucial for the clinical efficacy of immunotherapy. However, in many patients, neoantigen recognition is insufficient to induce the sustained T-cell response required for complete tumor rejection. Identifying molecular pathways influencing immune responsiveness to tumor neoantigens could provide targets for improving immunotherapeutic responses. For example, m 6 A is the most abundant internal mRNA modification, responsible for post-transcriptional regulation of mRNAs in various cell types. Furthermore, m... 6 A can be accessed via m 6 A-binding proteins, including YTH domain-containing family protein 1 (YTHDF1), affect mRNA translation efficiency. Previous studies have shown that attenuating YTHDF1 activity in various cells of the immune system (e.g., antigen-presenting cells) can help induce adequate and sustained anti-tumor immune responses. However, effective compositions and methods for attenuating YTHDF1 activity remain urgently needed. Summary of the Invention

[0002] This application provides compositions and methods for attenuating YTHDF1 activity. This application also provides modified antigen-presenting cells (mAPCs), such as modified dendritic cells, with enhanced activity. The compositions and mAPCs of this application can be used for one or more of the following purposes: activating APCs (such as DCs); generating immune cells with enhanced antitumor activity; preventing and / or reversing the depletion of immune cells (such as T cells); treating diseases, conditions, or illnesses associated with antigen expression in subjects of need; treating cancer in subjects of need; stimulating T cell-mediated immune responses against cancer cells and / or tumor antigens in subjects of need; providing antitumor immunity in subjects of need; increasing and / or improving the proliferation and / or activity of tumor-infiltrating T cells; increasing and / or improving the proliferation and / or activity of tumor-specific T cells; enhancing the production of cytokines by T cells; 12) enhancing the antitumor response to tumor immunotherapy; 13) inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells. This application also provides methods and compositions for enhancing immune responses, the compositions combining the YTHDF1 attenuator of this application and a second active agent, such as an immune checkpoint inhibitor.

[0003] YTHDF1 is a member of the YTH structural domain family, and is derived from m 6YTHDF1 is a modified "reader." For example, by interacting with translation initiation factors, YTHDF1 helps improve the translation efficiency of mRNA. Furthermore, dysregulation of YTHDF1 can disrupt the expression balance between proto-oncogenes and tumor suppressor factors, suggesting a link between YTHDF1 and tumorigenesis. Overexpression of YTHDF1 has been reported to be associated with some malignancies such as colorectal cancer (CRC) and hepatocellular carcinoma (HCC). In addition, Ythdf1 deficiency (Ythdf1...) has been found... - / - Mice exhibited enhanced anti-tumor immune responses, suggesting that YTHDF1 is a novel potential therapeutic target. YTHDF1 was also found to be associated with the expression of T cell exhaustion marker genes. Mice lacking YTHDF1 in T cells showed better anti-tumor immunity against lymphoma, solid tumors (such as melanoma and colon cancer), and other types of cancer. Tumor-infiltrating T cell function was enhanced in YTHDF1-deficient mice. Furthermore, rescue T cell exhaustion differentiation was directed towards memory-like or stem cell-like CD8+. + The fate of T cells.

[0004] On the one hand, this application provides a YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) attenuator, which comprises a compound that, when bound to YTHDF1, binds at least one residue corresponding to amino acid residues 372-392, 479-494 and 526-535 selected from SEQ ID NO:1.

[0005] In some embodiments, when bound to YTHDF1, the compound containing the YTHDF1 attenuator binds at least one residue corresponding to the following residues: N378, F382, W384, F480 and H528 of SEQ ID NO:1.

[0006] In some implementations, compounds containing YTHDF1 depressants can block the interaction between YTHDF1 and m 6 The combination of A.

[0007] In some implementations, compounds containing the YTHDF1 depressant are substantially non-reactive with m 6 A competitive combination YTHDF1.

[0008] In some embodiments, the YTHDF1 attenuator comprises a compound of formula I, a prodrug, metabolite, derivative of a compound of formula I, or a pharmaceutically acceptable salt, ester, or amide of any of the above: (Equation 1), where R1 is selected from C 1-50 hydrocarbon group, C 1-50 Substituted hydrocarbon group, C 1-50 heterohydrocarbons and C 1-50 Substituted heterohydrocarbon groups.

[0009] In some embodiments, R1 in Formula I is (CO)-R2, and R2 is an optionally substituted alkenyl group. In some embodiments, R2 is CH=CH-R3, and R3 is an optionally substituted aryl group. In some embodiments, R3 is a group of Formula II. Wherein A is an optional substituted furan or R6 is a hydroxyl group, and R5 is an optional substituted alkenyl group.

[0010] In some implementations, in Equation II, A is And R4 is

[0011] In some implementations, A in Equation II is R6 is a hydroxyl group, R5 is CH=CH-R7, and R7 is...

[0012] In some embodiments, the compound containing the YTHDF1 depressant of this application comprises at least two dihydroxyphenyl moieties.

[0013] In some embodiments, the compound containing the YTHDF1 depressant of this application comprises at least three dihydroxyphenyl moieties.

[0014] In some embodiments, the YTHDF1 attenuator comprises a compound of formula III, a prodrug, metabolite, derivative of a compound of formula III, or a pharmaceutically acceptable salt, ester, or amide of any of the above:

[0015] (Formula III), where A is an optionally substituted furan or R6 is a hydroxyl group, and R5 is an optional substituted alkenyl group.

[0016] In some implementations, in Equation III, A is And R4 is

[0017] In some implementations, in Equation III, A is R6 is a hydroxyl group, R5 is CH=CH-R7, and R7 is...

[0018] In some embodiments, the compound comprises any of the following compounds, a prodrug, metabolite, derivative of any of the following compounds, or a pharmaceutically acceptable salt, ester, or amide of any of the above:

[0019]

[0020] In some embodiments, the compound comprises any of the following compounds, a prodrug, metabolite, derivative of any of the following compounds, or a pharmaceutically acceptable salt, ester, or amide of any of the above:

[0021]

[0022] In some embodiments, the compound includes any of the following compounds, prodrugs, metabolites, derivatives of any of the following compounds, or pharmaceutically acceptable salts, esters, or amides of any of the above:

[0023]

[0024] In some implementations, the compound containing the YTHDF1 depressant is derived from plants.

[0025] In some embodiments, a compound containing the YTHDF1 attenuator is provided in the plant extract. In some embodiments, the plant is a species of the genus *Salvia*. In some embodiments, the plant is *Danshen*.

[0026] On one hand, this application provides a modified antigen-presenting cell (mAPC), wherein the mAPC has been treated with and / or contains the YTHDF1 attenuator of this application. In some embodiments, the mAPC is a modified dendritic cell (mDC).

[0027] On one hand, this application provides a composition comprising the YTHDF1 attenuator of this application and / or the mAPC of this application. In some embodiments, the composition of this application comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is a vaccine composition.

[0028] In some embodiments, the composition further comprises a second active ingredient. In some embodiments, the second active ingredient is an anticancer agent.

[0029] In some embodiments, the second active ingredient comprises cancer immunotherapy. In some embodiments, the second active ingredient comprises an immune checkpoint inhibitor. In some embodiments, the second active ingredient comprises an agent selected from: an anti-PD-L1 antibody or its antigen-binding portion, an anti-PD-1 antibody or its antigen-binding portion, an anti-CTLA-4 antibody or its antigen-binding portion, and an IDO inhibitor.

[0030] In some embodiments, the second active ingredient includes pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, and / or an antigen-binding fragment or derivative of any of the above.

[0031] In some implementations, the second active ingredient is able to induce an increase in one or more tumor antigens in the subject receiving it.

[0032] In some implementations, the tumor antigen is selected from CEA, gp100, MAGE family proteins, DAGE, GAGE, RAGE, NY-ESO 1, Melan-A / MART 1, TRP-1, TRP-2, tyrosinase, HER-2 / neu, MUC-1, p53, KSA, PSA, PSMA, and their fragments and modified forms.

[0033] In some embodiments, the second active ingredient is contained in a separate container and is not mixed with mAPC or YTHDF1 depressant.

[0034] On the one hand, this application provides a method for reducing YTHDF1 activity, including applying an effective amount of the YTHDF1 reducer of this application.

[0035] In some implementations, the method is an in vivo method. In some implementations, the method is an in vitro method. In some implementations, the method is an ex vivo method.

[0036] On one hand, this application provides a method for determining whether a candidate drug is a YTHDF1 attenuator, comprising: contacting the candidate drug with a YTHDF1 mutant, wherein the YTHDF1 mutant includes one or more amino acid substitutions, deletions and / or additions at one or more residues, the residues corresponding to residues selected from residues 372-392, 479-494 and 526-535 of SEQ ID NO:1.

[0037] In some embodiments, the YTHDF1 mutant includes one or more amino acid substitutions, deletions, and / or additions at one or more residues, which correspond to residues selected from residues N378, F382, W384, F480, and H528 of SEQ ID NO:1.

[0038] In some implementations, the method further includes determining whether the candidate agent specifically binds to the YTHDF1 mutant.

[0039] On the one hand, this application provides a kit containing the YTHDF1 mutant of this application.

[0040] On the one hand, this application provides the use of the compound in the preparation of YTHDF1 attenuator, wherein, when bound to YTHDF1, the compound is bound to at least one residue corresponding to amino acid residues 372-392, 479-494 and 526-535 selected from SEQ ID NO:1.

[0041] In some embodiments, when bound to YTHDF1, the compound binds to at least one residue corresponding to the following residues: N378, F382, W384, F480 and H528 of SEQ ID NO:1.

[0042] In some implementations, the compound is able to block YTHDF1 from interacting with m 6 The combination of A.

[0043] In some implementations, the compound is essentially unrelated to m 6 A competitive combination YTHDF1.

[0044] In some embodiments, the compound comprises a compound of formula I, a prodrug, metabolite, derivative of a compound of formula I, or a pharmaceutically acceptable salt, ester, or amide of any of the above:

[0045] (Equation I), where R1 is selected from C 1-50 hydrocarbon group, C 1-50 Substituted hydrocarbon group, C 1-50 heterohydrocarbons and C 1-50 Substituted heterohydrocarbon groups.

[0046] In some embodiments, in the compound of formula I, R1 is (CO)-R2, and R2 is an optionally substituted alkenyl group. In some embodiments, R2 is CH=CH-R3, and R3 is an optionally substituted aryl group.

[0047] In some implementations, R3 is Equation II Where A is an optional substituted furan or R6 is a hydroxyl group, and R5 is an optional substituted alkenyl group.

[0048] In some implementations, A in Equation II is And R4 is

[0049] In some implementations, A in Equation II is R6 is a hydroxyl group, R5 is CH=CH-R7, and R7 is...

[0050] In some embodiments, the compound comprises at least two dihydroxyphenyl moieties.

[0051] In some embodiments, the compound contains at least three dihydroxyphenyl moieties.

[0052] In some embodiments, the compound comprises a compound of formula III, a prodrug, metabolite, derivative of a compound of formula III, or a pharmaceutically acceptable salt, ester, or amide of any of the above:

[0053] (Formula III), where A is an optionally substituted furan or R6 is a hydroxyl group, and R5 is an optional substituted alkenyl group.

[0054] In some implementations, in Equation III, A is And R4 is

[0055] In some implementation schemes, A is R6 is a hydroxyl group, R5 is CH=CH-R7, and R7 is...

[0056] In some embodiments, the compound comprises any one of the following compounds, or a prodrug of any one of the following compounds:

[0057] In some embodiments, the compound is derived from a plant. In some embodiments, the compound is provided in a plant extract. In some embodiments, the plant is a species of the genus *Salvia*. In some embodiments, the plant is *Salvia miltiorrhiza*.

[0058] On the one hand, this application provides a method for activating an APC, the method comprising applying the YTHDF1 depressant of this application to the APC.

[0059] On the one hand, this application provides a method for activating a DC, the method comprising applying the YTHDF1 attenuator of this application to the DC.

[0060] On the one hand, this application provides a method for treating a disease, condition or illness related to antigen expression in a subject in need, including administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or the composition of this application.

[0061] In some implementations of this method, the antigen is a tumor antigen.

[0062] In some embodiments of this method, the antigen is selected from the following tumor antigens: CEA, gp100, MAGE family proteins, DAGE, GAGE, RAGE, NY-ESO 1, Melan-A / MART 1, TRP-1, TRP-2, tyrosinase, HER-2 / neu, MUC-1, p53, KSA, PSA, PSMA, and their fragments and modified forms.

[0063] In some implementations of this method, the disease, symptom, or condition is cancer.

[0064] In some implementations, the cancer is selected from hematologic malignancies, lymphomas, and solid tumors.

[0065] In some implementations, the cancer is selected from melanoma, breast cancer, lung cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, colorectal cancer, kidney cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphoma, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, and leukemia.

[0066] On one hand, this application provides a method of treating cancer in a subject in need, comprising administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application. In some embodiments, the cancer is selected from hematologic malignancies, lymphomas, and solid tumors. In some embodiments, the cancer is selected from melanoma, breast cancer, lung cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, colorectal cancer, kidney cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphoma, gastric cancer, pancreatic cancer, testicular cancer, lymphoma, and leukemia.

[0067] On one hand, this application provides a method for stimulating T cell-mediated immune responses against cancer cells and / or tumor antigens in a subject in need, comprising administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application. In some embodiments, the tumor antigen is selected from CEA, gp100, MAGE family proteins, DAGE, GAGE, RAGE, NY-ESO 1, Melan-A / MART 1, TRP-1, TRP-2, tyrosinase, HER-2 / neu, MUC-1, p53, KSA, PSA, PSMA, and fragments and modified forms thereof.

[0068] On the one hand, this application provides a method for antitumor immunization in subjects in need, including administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or the composition of this application.

[0069] On the one hand, this application provides a method for preventing and / or reversing T cell exhaustion in a subject in need, comprising administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application.

[0070] On one hand, this application provides a method for enhancing T cell activity in a subject in need, comprising administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application. In some embodiments, the T cells comprise tumor-infiltrating T cells. In some embodiments, the T cells comprise tumor-specific T cells.

[0071] In some embodiments of the method of this application, the subject is a cancer patient. In some embodiments, the cancer is selected from hematologic malignancies, lymphomas, and solid tumors. In some embodiments, the cancer is selected from melanoma, breast cancer, lung cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, colorectal cancer, kidney cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphoma, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, and leukemia.

[0072] In some embodiments, the subject has received, is receiving, and / or will receive anticancer treatment. In some embodiments, the anticancer treatment includes cancer immunotherapy. In some embodiments, the anticancer treatment includes immune checkpoint inhibitors. In some embodiments, the anticancer treatment includes agents selected from: antiPD-L1 antibodies or their antigen-binding portions, antiPD-1 antibodies or their antigen-binding portions, antiCTLA-4 antibodies or their antigen-binding portions, and IDO inhibitors. In some embodiments, the anticancer treatment includes pembrolizumab, nivolumab, cimipril, atezolizumab, avelumab, durvalumab, ipilimumab, and / or antigen-binding fragments or derivatives of any of the above. In some embodiments, the anticancer treatment is capable of inducing an increase in one or more tumor antigens in the subject. In some implementations, the tumor antigen is selected from CEA, gp100, MAGE family proteins, DAGE, GAGE, RAGE, NY-ESO 1, Melan-A / MART 1, TRP-1, TRP-2, tyrosinase, HER-2 / neu, MUC-1, p53, KSA, PSA, PSMA, and their fragments and modified forms.

[0073] In some embodiments, the method further includes administering one or more additional anticancer treatments to the subject. In some embodiments, the additional anticancer treatment includes cancer immunotherapy. In some embodiments, the additional anticancer treatment includes immune checkpoint inhibitors. In some embodiments, the additional anticancer treatment includes agents selected from: anti-PD-L1 antibodies or their antigen-binding portions, anti-PD-1 antibodies or their antigen-binding portions, anti-CTLA-4 antibodies or their antigen-binding portions, and IDO inhibitors. In some embodiments, the additional anticancer treatment includes pembrolizumab, nivolumab, cimipril, atezolizumab, avelumab, durvalumab, ipilimumab, and / or antigen-binding fragments or derivatives of any of the above. In some embodiments, the additional anticancer treatment is capable of inducing an increase in one or more tumor antigens in the subject. In some implementations, the tumor antigen is selected from CEA, gp100, MAGE family proteins, DAGE, GAGE, RAGE, NY-ESO 1, Melan-A / MART 1, TRP-1, TRP-2, tyrosinase, HER-2 / neu, MUC-1, p53, KSA, PSA, PSMA, and their fragments and modified forms.

[0074] On one hand, this application provides the use of the YTHDF1 attenuator of this application, the mAPC of this application, and / or the composition of this application in the preparation of compositions and / or medicaments for one or more of the following purposes: 1) activating APCs; 2) activating DCs; 3) generating immune cells with enhanced antitumor activity; 4) preventing and / or reversing the depletion of immune cells (such as T cells); 5) treating diseases, symptoms, or conditions related to antigen expression in subjects in need; 6) treating cancer in subjects in need; 7) stimulating T cell-mediated immune responses against cancer cells and / or tumor antigens in subjects in need; 8) providing antitumor immunity in subjects in need; 9) increasing and / or improving the proliferation and / or activity of tumor-infiltrating T cells; 10) increasing and / or improving the proliferation and / or activity of tumor-specific T cells; 11) enhancing cytokine production by T cells; 12) enhancing the antitumor response to tumor immunotherapy; and 13) inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells.

[0075] In some implementations, the cancer or tumor is selected from hematologic malignancies, lymphomas, and solid tumors. In some implementations, the cancer or tumor is selected from melanoma, breast cancer, lung cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, colorectal cancer, kidney cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphoma, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, and leukemia.

[0076] On one hand, this application provides the use of the YTHDF1 attenuator of this application, the mAPC of this application, and / or the composition of this application, combined with additional active ingredients, in the preparation of a medicament for use in: 1) activating APCs; 2) activating DCs; 3) generating immune cells with enhanced antitumor activity; 4) preventing and / or reversing the depletion of immune cells (such as T cells); 5) treating diseases, conditions, or illnesses related to antigen expression in subjects in need; 6) treating cancer in subjects in need; 7) stimulating T cell-mediated immune responses against cancer cells and / or tumor antigens in subjects in need; 8) providing antitumor immunity in subjects in need; 9) increasing and / or improving the proliferation and / or activity of tumor-infiltrating T cells; 10) increasing and / or improving the proliferation and / or activity of tumor-specific T cells; 11) enhancing cytokine production by T cells; 12) enhancing the antitumor response to tumor immunotherapy; and 13) inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells.

[0077] In some embodiments, the additional active ingredient includes cancer immunotherapy. In some embodiments, the additional active ingredient includes immune checkpoint inhibitors. In some embodiments, the additional active ingredient includes agents selected from: anti-PD-L1 antibodies or their antigen-binding portions, anti-PD-1 antibodies or their antigen-binding portions, anti-CTLA-4 antibodies or their antigen-binding portions, and IDO inhibitors. In some embodiments, the additional active ingredient includes pembrolizumab, nivolumab, cimipril, atezolizumab, avelumab, durvalumab, ipilimumab, and / or antigen-binding fragments or derivatives of any of the above. In some embodiments, the additional active ingredient is capable of inducing an increase in one or more tumor antigens in the subject receiving it. In some implementations, the tumor antigen is selected from CEA, gp100, MAGE family proteins, DAGE, GAGE, RAGE, NY-ESO 1, Melan-A / MART 1, TRP-1, TRP-2, tyrosinase, HER-2 / neu, MUC-1, p53, KSA, PSA, PSMA, and their fragments and modified forms.

[0078] Other aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the disclosure are shown and described. It will be appreciated that other and different embodiments of the disclosure are possible, and that certain details thereof can be modified in various obvious respects without departing from the disclosure in whole or in part. Therefore, the drawings and descriptions are to be regarded as illustrative rather than restrictive.

[0079] By incorporating via reference

[0080] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

[0081] Brief description of the attached diagram

[0082] The novel features of this application are specifically described in the appended claims. A better understanding of the features and advantages of this application will be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as “Figures”), which set forth illustrative embodiments employing the principles of this application, wherein:

[0083] Figure 1 a-1b indicates the inhibitory activity of SAA. Figure 1 c indicates the inhibitory activity of SAC. Figure 1 d illustrates the binding of SAA with YTHDF1.

[0084] Figure 2 a-2c illustrates the ITC binding curve of SAA and YTHDF1.

[0085] Figure 3 a illustrates the SPR binding analysis results between SAA and YTHDF1. Figure 3 b-3c illustrates the MST binding analysis results between SAA and YTHDF1.

[0086] Figure 4 a-4b explains SAA and substances containing m 6 Results of competitive binding analysis between A mRNAs.

[0087] Figure 5 a illustrates the residual plot of the HDX MS experimental results. Figure 5 b illustrates the butterfly diagram of the HDX MS experimental results.

[0088] Figure 6 A heatmap illustrating the HDX MS experimental results is provided.

[0089] Figure 7 The local deuterium uptake kinetics of YTHDF1 are illustrated. On the right, the vertical axis represents the percentage of deuterium uptake, and the horizontal axis indicates the duration of the HDX process. Paired t-tests were used to compare changes in deuterium uptake, and P < 0.05 (*) was considered statistically significant.

[0090] Figure 8 a-8i illustrates the local exchange kinetics of the relevant peptides. The vertical axis represents the percentage of deuterium uptake, and the horizontal axis indicates the duration of the HDX process.

[0091] Figure 9This demonstrated the inhibitory activity of SAA against the YTHDF1 mutant and truncated variant. The IC50 of SAA was determined by FP analysis. 50 Values. Data are expressed as mean ± sem.

[0092] Figure 10 a-10j shows the IC50 values ​​of the YTHDF1 mutant and C-terminal truncated variant as determined by FP analysis. SAA was diluted from 100M using a two-fold serial dilution.

[0093] Figure 11 a-11g describes the K-type of YTHDF1 and its mutants or C-terminal truncated variants as determined by FP analysis. d Value. The protein was diluted from 200M using a two-fold serial dilution.

[0094] Figure 12 a-12b illustrates the binding of SAA to YTHDF1 in 293T cells. CETSA analysis was performed in the 293T cell line at temperatures ranging from 39.0°C to 59.0°C as indicated. YTHDF1 expression was detected by Western blotting using GAPDH as an internal control. Furthermore, according to the Western blotting method (… Figure 12 a) Relative expression levels are shown in Figure 12 b in.

[0095] Figure 13 a-13b illustrates the antitumor effects of SAAs that depend on T cells and DCs.

[0096] Figure 14 a-14b describes the effect of SAA on tumor cell growth in vitro.

[0097] Figure 15 a explains SAA's effect on Rag1 - / - The role of mice. Figure 15 b-15c demonstrates the ability of SAA to enhance T cell cross-sensitization via DCs.

[0098] Figure 16 a-16b demonstrates the ability of SAA to enhance the direct sensitization of T cells through DCs.

[0099] Figure 17 This indicates that SAA targets DCs to inhibit tumor growth. Figure 17 b-17c indicates that SAA can enhance the activity of tumor-infiltrating T cells.

[0100] Figure 18 a describes the PD-1 of the group treated with SAA. low The cell population expressed more CXCR5 than the DMSO group. Figure 18b illustrates the tumor-invasive terminal stage exhaustion T cells (PD-1) in mice treated with SAA. + Tim-3 + The percentage of ) decreased. Figure 18 c illustrates the anti-tumor effect of SAA combined with anti-PD-L1 antibody.

[0101] Figure 19 This demonstrates the antitumor effects of SAA and SAC.

[0102] Figure 20 This demonstrates the antitumor effect of SAA combined with PD-1 blockade.

[0103] Figure 21 This indicates that adoptive transfer of SAA-treated FLT3L DCs exhibits sustained antitumor function. Detailed Implementation

[0104] Although various embodiments of this application have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example. Various modifications, alterations, and substitutions can be made by those skilled in the art without departing from this application. It should be understood that various alternatives may be adopted to the embodiments of this application described herein.

[0105] On one hand, this application provides a YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) attenuator. The YTHDF1 attenuator may comprise a compound that, when bound to YTHDF1, binds at least one residue corresponding to amino acid residues 372-392, 479-494, and 526-535 selected from SEQ ID NO:1.

[0106] On one hand, this application provides a modified antigen-presenting cell (mAPC). The mAPC may have been treated with the YTHDF1 attenuator of this application. In some embodiments, the mAPC may contain the YTHDF1 attenuator of this application. The mAPC may be a modified dendritic cell (mDC).

[0107] On one hand, this application provides a composition (such as a pharmaceutical composition). The composition may contain the YTHDF1 attenuator of this application. Alternatively, or additionally, the composition may contain the mAPC of this application. The composition may contain a pharmaceutically acceptable carrier. In some cases, the composition may be a vaccine composition.

[0108] In some cases, the composition may contain additional or second active ingredients. In this application, the terms "additional active ingredient" and "second active ingredient" are used interchangeably.

[0109] The additional second active ingredient may be contained in a separate container and is not mixed with the mAPC or YTHDF1 depressant of this application.

[0110] In some cases, the additional active ingredient may be contained in the same package or container as the mAPC and / or YTHDF1 depressant of this application. In some cases, the additional active ingredient may be contained in a separate container; for example, the additional active ingredient may be contained in a different container than the mAPC and / or YTHDF1 depressant of this application. In some cases, the additional active ingredient does not come into direct contact with the mAPC and / or YTHDF1 depressant of this application (e.g., it is not mixed), even if they may be present in the same container or the same package.

[0111] On the one hand, this application provides a method for reducing YTHDF1 activity, which may include applying an effective amount of the YTHDF1 reducer of this application.

[0112] On one hand, this application provides a method for determining whether a candidate drug is a YTHDF1 attenuator. The method may include contacting the candidate drug with a YTHDF1 mutant. The YTHDF1 mutant may include one or more amino acid substitutions, deletions, and / or additions at one or more residues, corresponding to residues selected from residues 372-392, 479-494, and 526-535 of SEQ ID NO:1.

[0113] On one hand, this application provides a kit. The kit may contain the YTHDF1 mutant of this application.

[0114] On one hand, this application provides the use of a compound in the preparation of a YTHDF1 attenuator. When bound to YTHDF1, the compound can bind to at least one residue corresponding to amino acid residues 372-392, 479-494, and 526-535 selected from SEQ ID NO:1.

[0115] On one hand, this application provides a method for activating APC. This method may include applying the YTHDF1 depressant of this application to the APC.

[0116] On one hand, this application provides a method for activating a DC. This method may include applying the YTHDF1 depressant of this application to the DC.

[0117] On one hand, this application provides a method for treating a disease, condition, or illness related to antigen expression in a subject in need. This method may include administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application.

[0118] On one hand, this application provides methods for inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells. This method may include administering to the tumor and / or tumor cells: the YTHDF1 attenuator of this application; the mAPC of this application; and / or the composition of this application.

[0119] On one hand, this application provides a method for treating cancer in a subject in need. The method may include administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application.

[0120] On one hand, this application provides (e.g., in subjects with need) a method for stimulating T cell-mediated immune responses against cancer cells and / or tumor antigens. This method may include administering to a subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application.

[0121] On one hand, this application provides a method for delivering antitumor immunity in subjects in need. This method may include administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application.

[0122] On one hand, this application provides a method for preventing and / or reversing the depletion of immune cells (such as immune effector cells, e.g., T cells) in subjects in need. This method may include administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application.

[0123] On one hand, this application provides a method for enhancing the activity of immune cells, such as immune effector cells (e.g., T cells), in a subject in need. This method may include administering to the subject: the YTHDF1 attenuator of this application; the mAPC of this application; and / or a composition of this application. In some embodiments, the immune cells include tumor-infiltrating T cells. In some embodiments, the immune cells include tumor-specific T cells.

[0124] On the one hand, this application provides the use of the YTHDF1 attenuator of this application, the mAPC of this application, and / or the composition of this application in the preparation of compositions and / or pharmaceuticals for one or more of the following purposes: 1) activating APCs; 2) activating DCs; 3) generating immune cells with enhanced antitumor activity; 4) preventing and / or reversing the depletion of immune cells (such as immune effector cells, e.g., T cells); 5) treating diseases, conditions, or illnesses related to antigen expression in subjects in need; 6) treating cancer in subjects in need; 7) stimulating immune cells (e.g., immune effector cells) in subjects in need. 8) To provide antitumor immunity in subjects in need; 9) To increase and / or improve the proliferation and / or activity of immune cells (e.g., immune effector cells, such as T cells, e.g., tumor-infiltrating T cells); 10) To increase and / or improve the proliferation and / or activity of tumor-specific immune cells (e.g., immune effector cells, such as T cells); 11) To enhance cytokine production by T cells; 12) To enhance the antitumor response to tumor immunotherapy; and 13) To inhibit tumor growth, inhibit the proliferation of tumor cells, and / or kill tumor cells.

[0125] On one hand, this application provides the use of the YTHDF1 attenuator of this application, the mAPC of this application, and / or the composition of this application, combined with additional active ingredients, in the preparation of a medicament for one or more of the following purposes: 1) activating APCs; 2) activating DCs; 3) generating immune cells with enhanced antitumor activity; 4) preventing and / or reversing the depletion of immune cells (such as immune effector cells, e.g., T cells); 5) treating diseases, conditions, or illnesses related to antigen expression in subjects in need; 6) treating cancer in subjects in need; 7) stimulating immune cells (e.g., immune cells) in subjects in need. 8) mediated immune responses against cancer cells and / or tumor antigens by effector cells (such as T cells); 9) providing anti-tumor immunity in subjects in need; 10) increasing and / or improving the proliferation and / or activity of immune cells (e.g., immune effector cells, such as T cells, such as tumor-infiltrating T cells); 11) increasing and / or improving the proliferation and / or activity of tumor-specific immune cells (e.g., immune effector cells, such as T cells); 12) enhancing cytokine production by T cells; 13) enhancing the anti-tumor response to tumor immunotherapy; and 14) inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells.

[0126] As used herein, the terms “comprising,” “including,” “having,” “has,” “may,” “containing,” and their variations generally refer to open-ended transitional phrases, terms, or words that do not exclude the possibility of other actions or structures. The singular forms “a / an” and “described” include plural references.

[0127] The description of numerical ranges in this document explicitly covers every intermediate number with the same precision. For example, for the range of 6-9, 7 and 8 are included in addition to 6 and 9, while for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly included. Therefore, the description of the range format is for convenience and simplicity only and should not be construed as a rigid limitation on the scope of the invention of this application. The description of the range should be understood as specifically disclosing all possible sub-ranges and individual numerical values ​​within that range. For example, describing a range such as 1 to 6 should be understood as specifically disclosing sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. For example, a range such as 95-100% (e.g., 95-96%, 95-97%, 95-98%, 95-99%, 95-99.5% or higher) identity includes certain things with 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the range.

[0128] The modifier “about” used in relation to quantity includes a specified value and has a context-indicative meaning (e.g., it includes at least the degree of error associated with a particular quantity of measurement). The modifier “about” should also be understood to disclose a range defined by the absolute values ​​of the two endpoints. For example, the expression “about 2 to about 4” also discloses a range of “2 to 4.” When referring to measurable values ​​such as quantity or duration, the term “about” means to cover a difference of ±20% of the specified value, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1%, as these differences are appropriate.

[0129] As used herein, the term "subject" generally refers to a human or an animal. For example, it can refer to any vertebrate, including (but not limited to) mammals (e.g., cows, pigs, camels, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys, such as cynomolgus monkeys, chimpanzees, etc.) and humans). In some respects, the subject is a human.

[0130] The terms “treat / treated / treating” are used interchangeably herein and generally refer to a treatment method intended to alleviate (reduce) an undesirable physical condition, symptom, or disease, or to achieve a beneficial or desired clinical outcome. In some aspects of this disclosure, beneficial or desired clinical outcomes include (but are not limited to) relief of symptoms; reduction of the severity of a condition, symptom, or disease; stabilization of the condition, symptom, or disease (without worsening); delay of the onset of a condition, symptom, or disease or slowing of its progression; improvement of the condition, symptom, or disease state; and relief (whether partial or complete, detectable or undetectable) or enhancement or improvement of a condition, symptom, or disease. Treatment also includes prolonging survival compared to expected survival without treatment.

[0131] The terms “modified / modify / modification” are used interchangeably herein and generally refer to the introduction or production of changes or alterations. When used in the context of genes, the modification may include any conventional method of producing changes in cellular activity and / or function. For example, by exposing cells (e.g., antigen-presenting cells) to agents capable of modulating cellular activity and / or function.

[0132] The terms “attenuating” and “attenuation” are used interchangeably and, as used herein, can refer to inhibiting or reducing the amount of a target gene or protein (such as YTHDF1 or a target of YTHDF1), or inhibiting or reducing the activity of a target gene or protein (such as YTHDF1 or a target of YTHDF1). This attenuation can be achieved by using, for example, antibodies or derivatives thereof, antibody-drug conjugates, fusion proteins, small molecules, antisense molecules, dsRNA, siRNA, shRNA, aptamers, and / or gRNA (e.g., in combination with gene editing systems such as CRIPSR / Cas9). Alternatively, for example, YTHDF1 binding and / or recognition via m-type antigens can be inhibited / blocked by contacting antigen-presenting cells (e.g., dendritic cells) with an inhibitor of YTHDF1 (such as the compounds of this application). 6 A-modified mRNA, thereby weakening YTHDF1.

[0133] As used in this article, the term "small molecule" generally refers to molecules other than polypeptides and nucleic acids that can influence biological processes, especially regulating m-molecules. 6A small molecule is any chemical or other part of an mRNA modified (e.g., the activity of YTHDF1). Small molecules can include any number of currently known and used therapeutic agents, or therapeutic agents that can be synthesized in such molecular libraries for screening biological functions. The difference between small molecules and macromolecules lies in size. Small molecules can have a molecular weight of less than about 5,000 Daltons (Da), such as less than about 2,500 Da, less than about 1,000 Da, or less than about 500 Da. Small molecules can include (but are not limited to) organic compounds and their peptide mimics and conjugates.

[0134] The term "amino acid" as used herein broadly refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, amino acids have the universal structure H₂N-C(H)(R)-COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are synthetic amino acids; in some embodiments, amino acids are d-amino acids; and in some embodiments, amino acids are 1-amino acids. "Standard amino acid" refers to any of the 20 standard 1-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. The term "synthetic amino acid" as used herein encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids (including carboxyl and / or amino-terminal amino acids in peptides) can be modified by methylation, amidation, acetylation, substitution of protecting groups and / or other chemical groups, which can alter the cyclic half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. Amino acids may contain one or more post-translational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to free amino acids and / or amino acid residues of peptides.

[0135] The term "YTHDF1" as used in this article generally refers to YTH N6-methyladenosine RNA-binding protein 1 or a functional fragment thereof, which specifically recognizes and binds to RNA containing N6-methyladenosine (m6A) and regulates mRNA stability. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequence of human YTHDF1 can be found at accession number NP_060268.2, and the mRNA sequence encoding it can be found at accession number NM_017798.4.

[0136] The term “YTHDF1 mutant” as used herein generally refers to a nucleic acid molecule or YTHDF1 protein that encodes YTHDF1 and has one or more mutations compared to the corresponding parental or reference (e.g., wild-type) nucleic acid molecule or the corresponding parental or reference (e.g., wild-type) YTHDF1 protein.

[0137] In the case of the YTHDF1 mutant protein, the mutant protein has at least one amino acid residue that differs from the amino acid sequence of the parent or reference polypeptide (including, but not limited to, the wild-type YTHDF1 polypeptide). Mutations in the mutant protein can include the deletion, substitution, and / or addition of one or more amino acids. The size of the mutation can range from a single amino acid to a large fragment of the polypeptide. In some embodiments, insertion alters the number of amino acids in the polypeptide by adding a fragment of the polypeptide. In some embodiments, deletion alters the number of amino acids by removing a fragment of the polypeptide. In some embodiments, small deletions may remove one or more amino acids from the polypeptide. In some embodiments, substitution replaces one amino acid in the polypeptide with a different amino acid. Substitution can be a conserved amino acid substitution or a non-conserved amino acid substitution. A "conserved amino acid substitution" refers to an amino acid normally present in the sequence being replaced by a different amino acid having similar size, charge, polarity, and / or chemical properties. Examples of conserved substitution include the substitution of nonpolar (hydrophobic) residues such as isoleucine, valine, and leucine with another nonpolar residue. Similarly, examples of conserved substitutions include arginine and lysine, glutamine and asparagine, and the substitution of a polar (hydrophilic) residue such as glycine or serine for another polar residue. Furthermore, the substitution of a basic residue such as lysine, arginine, or histidine for another basic residue, or the substitution of an acidic residue such as aspartic acid or glutamic acid for another acidic residue, are conserved substitutions. Examples of “non-conserved substitutions” may include the substitution of nonpolar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, and methionine for polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of polar residues for nonpolar residues.

[0138] The terms “cancer” and “tumor” are used interchangeably in this document and generally refer to diseases characterized by the uncontrolled growth of abnormal cells. Both terms include solid tumors and fluid-filled tumors, such as diffuse or circulating tumors. This includes both pre-malignant and malignant cancers and tumors.

[0139] The phrase “disease, condition, or illness associated with antigen expression” as used herein generally includes (but is not limited to) diseases associated with antigen expression or illnesses associated with cells that express antigens, such as proliferative disorders like cancer or malignancies; or precancerous lesions such as spinal dysplasia, myelodysplastic syndromes, or preleukemia; or non-cancer-related indications associated with cells that express or overexpress an antigen (such as an antigen present in bacteria, viruses, or cells, e.g., non-cancer cells). Non-cancer-related indications associated with antigen expression as used herein include (but are not limited to) autoimmune diseases, inflammatory conditions, and transplantation.

[0140] The phrase “disease, condition, or symptom associated with tumor antigen expression” as used herein generally includes (but is not limited to) diseases associated with tumor antigen expression or conditions associated with cells expressing tumor antigens, such as proliferative disorders like cancer or malignancies, or precancerous lesions like spinal dysplasia, myelodysplastic syndromes, or preleukemia; or non-cancer-related indications associated with cells expressing tumor antigens. In one embodiment, the cancer associated with tumor antigen expression as described herein is a hematologic malignancy. In one embodiment, the cancer associated with tumor antigen expression as described herein is a solid tumor. Other diseases associated with tumor antigen expression as described herein include (but are not limited to) atypical and / or nonclassical cancers, malignancies, precancerous lesions, or proliferative disorders associated with tumor antigen expression as described herein. Non-cancer-related indications associated with tumor antigen expression as described herein include (but are not limited to) autoimmune diseases, inflammatory conditions, and transplantation. In some embodiments, tumor antigen-expressing cells express or have expressed mRNA encoding tumor antigens at any time. In one embodiment, tumor antigen-expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), and may be present in normal, increased, or decreased amounts of tumor antigen proteins.

[0141] As used herein, the terms “activity” and “activation” generally refer to specific functions of a cell. The activity of T cells can be, for example, cytolytic activity or helper cell activity, including the secretion of cytokines. The activity of antigen-presenting cells can be, for example, processing and / or presenting antigens for recognition by certain lymphocytes (such as T cells).

[0142] As used herein, the term "immune effector cell" generally refers to cells involved in an immune response, such as those that promote an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes. As used herein, "immune effector function or immune effector response" generally refers to, for example, the function or response of immune effector cells to enhance or promote the immune attack of target cells. For example, an immune effector function or response refers to the property of T or NK cells to promote the killing of target cells or to inhibit the growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0143] As used herein, the term "antigen-presenting cell" or "APC" generally refers to a cell (e.g., an immune cell) or a group of cells (e.g., a group of immune cells) capable of displaying antigens on or on its surface. The displayed antigen may complex with the major histocompatibility complex (MHC) and may be processed prior to its display. Examples of APCs include, but are not limited to, macrophages, B cells, and dendritic cells (such as Langerhans cells). Cellular immune responses can be initiated or enhanced upon recognition of antigens presented by APCs by lymphocytes (e.g., T cells). APCs can break down large molecular weight antigens into 10 to 30 amino acid fragments for loading onto HLA class I and II molecules.

[0144] The term “dendritic cell” or “DC” as used in this article generally refers to an antigen-presenting cell. DCs act as messengers between the innate and adaptive immune systems. For example, DCs can be found in tissues that come into contact with the external environment, such as the skin, the inner lining of the nose, the lungs, the stomach, and the intestines. They can also be found in the blood in an immature state. Once activated, they can migrate to lymph nodes and interact with other immune cells, such as T cells and B cells, to initiate and shape adaptive immune responses. Immature dendritic cells are also known as cryptic cells. DCs can be leukocytes derived from bone marrow (BM). They can also be derived from BM and blood and multiply in vitro using various combinations of growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and Flt3 ligand. DCs can specifically capture and process antigens, converting proteins into peptides presented on major histocompatibility complex (MHC) molecules that are recognized by other immune cells, such as T cells. Dendritic cells (DCs) can be heterogeneous, such as myeloid and plasmacytic DCs. While all DCs are capable of antigen uptake, processing, and presentation to naive T cells, DC subtypes can have different markers and differ in their location, migration pathways, detailed immune functions, and dependence on infection or inflammatory stimuli depending on their generation. In the development of adaptive immune responses, the phenotype and function of DCs play a crucial role in initiating tolerance, memory, and / or polarization differentiation of helper T cells 1 (Th1), Th2, and Th17.

[0145] In the context of this application, the following abbreviations for common nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0146] As used herein, the term "nucleic acid" or "polynucleotide" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations thereof, and polymers thereof in single-stranded or double-stranded form. The term "nucleic acid" includes genes, cDNA, or mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless specifically limited, the term covers nucleic acids containing analogues or derivatives of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly covers its conserved variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0147] The terms “cancer-associated antigen” and “tumor antigen” are used interchangeably herein and generally refer to molecules (typically proteins, glycosides, or lipids) that are preferentially expressed intact or in fragment form (e.g., MHC / peptides) on the surface of cancer cells compared to normal cells and are suitable for preferentially targeting cancer cells with agents. In some embodiments, a tumor antigen is a marker expressed by both normal cells and cancer cells. In some embodiments, a cancer-associated antigen is a cell surface molecule overexpressed in cancer cells compared to normal cells, for example, overexpressed 1-fold, 2-fold, 3-fold, or more than 3-fold compared to normal cells. In some embodiments, a cancer-associated antigen is a cell surface molecule improperly synthesized in cancer cells, for example, a molecule containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, a cancer-associated antigen will be expressed intact or in fragment form (e.g., MHC / peptides) only on the cell surface of cancer cells and not synthesized or expressed on the surface of normal cells.

[0148] As used herein, the term "specific binding" generally refers to a molecule (e.g., a small molecule, antibody, or ligand) that recognizes and binds to a homologous binding ligand protein present in a sample, but substantially does not recognize or bind to other molecules in the sample. In some embodiments, the molecules of the present invention may be smaller than about 10 -5 M (for example, less than about 9 × 10) -6 M, less than approximately 8 × 10-6 M, less than approximately 7 × 10 -6 M, less than approximately 6 × 10 -6 M, less than approximately 5 × 10 -6 M, less than approximately 4 × 10 -6 M, less than approximately 3.5 × 10 -6 M, less than approximately 3 × 10 -6 M, less than approximately 2.5 × 10 -6 M, less than approximately 2 × 10 -6 M, less than approximately 1×10 -6 M, less than approximately 5 × 10 -7 M, less than approximately 2 × 10 -7 M, less than approximately 10 -7 M, less than approximately 5 × 10 -8 M, less than approximately 2 × 10 -8 M, less than approximately 10 -8 M, less than approximately 5 × 10 -9 M, less than approximately 4 × 10 -9 M, less than approximately 3 × 10 -9 M, less than approximately 2 × 10 -9 M or less than approximately 10 -9 Binding affinity of M) (K) d It specifically binds to target molecules.

[0149] K d It usually refers to the ratio of dissociation rate to binding rate (k off / k on The K can be determined using any conventional method known in the art, including (but not limited to) surface plasmon resonance, microthermophoresis, HPLC-MS, and flow cytometry (such as FACS). In some embodiments, K d The value can be appropriately determined using flow cytometry.

[0150] As used in this article, the term "anticancer agent" generally refers to a drug that can inhibit and / or prevent the growth of tumors or cancer cells.

[0151] As used herein, the term "CTLA-4" generally refers to cytotoxic T-lymphocyte-associated protein 4 and its functional fragments derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). Exemplary sequences of human CTLA-4 include the Homo sapiens (human) CTLA-4 protein (NCBI reference sequence number AAL07473.1). Exemplary sequences of CTLA-4 include the cynomolgus monkey (monkey) CTLA-4 protein (NCBI reference sequence number XP_005574071.1). As used herein, the term “CTLA-4” is intended to encompass any form of CTLA-4, such as 1) the naturally occurring, unprocessed CTLA-4 molecule, the “full-length” CTLA-4 chain, or naturally occurring CTLA-4 variants, including, for example, splice variants or allele variants; 2) any form of CTLA-4 produced by processing in cells; or 3) full-length, fragmented (e.g., truncated, extracellular / transmembrane domain), or modified (e.g., mutant, glycosylated / pegylated, His-tagged / immunofluorescence fusion) CTLA-4 subunits produced by recombination methods.

[0152] The terms “anti-CTLA-4 antibody,” “anti-CTLA-4 binding domain,” or “CTLA-4 binding domain” refer to an antibody or antigen-binding domain that specifically binds to CTLA-4 (e.g., human or monkey CTLA-4).

[0153] As used herein, the term "PD-1" generally refers to a programmed cell death protein, belonging to the immunoglobulin superfamily and acting as a co-inhibitory receptor that negatively regulates the immune system. PD-1 is a member of the CD28 / CTLA-4 family and has two known ligands, PD-L1 and PD-L2. The representative amino acid sequence of human PD-1 is disclosed under NCBI accession number NP_005009.2, and the representative nucleic acid sequence encoding human PD-1 is shown under NCBI accession number NM_005018.2.

[0154] As used herein, the term “PD-L1” generally refers to programmed cell death ligand 1 (PD-L1, see, for example, Freeman et al., (2000) J.Exp.Med.192:1027). A representative amino acid sequence of human PD-L1 is disclosed with NCBI accession number NP_054862.1, and a representative nucleic acid sequence encoding human PD-L1 is shown with NCBI accession number NM_014143.3. PD-L1 binds to its receptors PD-1 or B7-1, which are expressed on activated T cells, B cells, and bone marrow cells. The binding of PD-L1 to its receptor induces signal transduction, thereby inhibiting TCR-mediated activation of cytokine production and T cell proliferation. Therefore, PD-L1 plays a crucial role in suppressing the immune system during specific events such as pregnancy, autoimmune diseases, and tissue allogeneic transplantation, and is thought to allow tumors or cancer cells to bypass immune checkpoints and evade immune responses.

[0155] As used herein, the terms “anti-PD-1 antibody,” “anti-PD-1 binding domain,” or “PD-1 binding domain” generally refer to an antibody or antigen-binding domain that can specifically bind to PD-1 (e.g., human or monkey PD-1) with an affinity sufficient to provide diagnostic and / or therapeutic use.

[0156] As used in this article, the term "anti-tumor immunity" generally refers to the immune response induced when immune cells recognize cancer antigens.

[0157] As used herein, the term "cancer immunotherapy" generally refers to any therapy designed to stimulate or enhance a patient's immune response to cancer cells. For example, cancer immunotherapy includes (but is not limited to) active immunotherapy specific to cancer antigens, treatment with immunomodulators (e.g., activators or inhibitors of immunosuppressants, or inhibitors of checkpoint inhibitors), or treatment with cancer cells or a mixture of antigens derived from cancer cells (treatment with antigens derived from cancer cell lines). Cancer immunotherapy includes therapeutic treatments that stimulate or restore the immune system's ability to fight cancer by inducing, enhancing, or suppressing an immune response. Cancer immunotherapy leads to the targeting of immune activity against disease-specific antigens by increasing the recognition of target immune cells or by reducing disease-associated immunosuppression.

[0158] As used herein, the term "tumor-infiltrating T cells" generally refers to T cells that infiltrate tumors. Tumor-infiltrating T cells exhibit a natural reactivity to autologous tumor antigens. These cells can be found in the tumor stroma and / or the tumor itself.

[0159] As used herein, the term "IDO inhibitor" generally refers to an agent that inhibits the activity of indoleamine 2,3-dioxygenase (IDO) and thereby reverses IDO-mediated immunosuppression. IDO inhibitors can inhibit IDO1 and / or IDO2 (INDOL1). IDO inhibitors can be reversible or irreversible IDO inhibitors. A "reversible IDO inhibitor" is a compound that reversibly inhibits IDO enzyme activity at a catalytic or non-catalytic site, and an "irreversible IDO inhibitor" is a compound that irreversibly destroys IDO enzyme activity by forming a covalent bond with the enzyme.

[0160] As used herein, the term "immune checkpoint inhibitor" generally refers to any molecule that directly or indirectly, partially or completely inhibits immune checkpoint pathways. Immune checkpoint pathways are generally considered to function by turning various aspects of the immune system on or off, particularly T cells, but also including, for example, bone marrow cells, NK cells, and B cells. Following T cell activation, numerous inhibitory receptors can be upregulated and present on the surface of T cells to suppress the immune response at appropriate times. Examples of immune checkpoint pathways include (but are not limited to) PD-1 / PD-L1, CTLA-4 / B7-1, TIM-3, LAG3, B7-H1, H4, HAVCR2, IDO1, CD276 and VTCN1, B7-H3, B7-H4, CD47, and KIR. For example, non-limiting examples of immune checkpoint inhibitors or modulators include fully human monoclonal antibodies such as BMS-936558 / MDX-1106, BMS-s936559 / MDX-1105, ipilimumab and / or antigen-binding fragments or derivatives of any of the foregoing / Yervoy, tremelimumab, BMS-986016, durvalumab, MEDI4736, urelumab, CDX-1127 and avelumab; humanized antibodies such as CT-011, IV1K-3475, Hu5F9-G4, CC-90002, MBG453, TSR-022 and atezolizumab; and fusion proteins such as AMP-224 and TTI-621, and others. Other non-limiting examples of immune checkpoint modulators (agonists) include antibodies against, for example, CD40, OX40, GITR, CD137 (4-1BB), CD27, ICOS, and TRAIL. According to the invention, one or more immune checkpoint modulators may independently be polypeptides or nucleic acid molecules encoding polypeptides; said polypeptides contain domains capable of binding to the target immune checkpoint and / or inhibiting ligands to the target immune checkpoint to exert antagonistic function (i.e., antagonizing immune checkpoint-mediated inhibitory signals) or agonistic function (i.e., enhancing immune checkpoint-mediated stimulatory signals). The one or more immune checkpoint modulators may independently be selected from peptides (e.g., peptide ligands), soluble domains of natural receptors, RNAi, antisense molecules, antibodies, and protein scaffolds. For example, an immune checkpoint modulator may be an antibody. In the context of this invention, immune checkpoint modulator antibodies are used in the broadest sense and include, for example, naturally occurring and artificially engineered full-length antibodies or functional fragments or analogs thereof capable of binding to target immune checkpoints or epitopes (thus retaining the target-binding portion). The antibody can be of any origin, such as human antibodies, humanized antibodies, animal antibodies (e.g., rodent or camel antibodies), or chimeric antibodies. The antibody can be of any isotype, with IgG1 or IgG4 isotypes being particularly preferred.Additionally, the antibody may be glycosylated or non-glycosylated. Standard analytical methods known in the art for assessing the binding ability of antibodies to immune checkpoints include, for example, ELISA, Western blot, RIA, and flow cytometry. The binding kinetics of the antibody (e.g., binding affinity) can also be assessed using standard analytical methods known in the art, such as Biacore analysis. In applications referring to immune checkpoint inhibitors, immune checkpoint modulators may also be used, except in cases where the context clearly indicates otherwise.

[0161] As used herein, the term "exhaustion" generally refers to T cell exhaustion, a state of T cell dysfunction that occurs in many chronic infections and cancers. T cell exhaustion is characterized by impaired T cell effector function, persistent expression of inhibitory receptors, and / or a transcriptional state different from that of functional effector or memory T cells. Exhaustion hinders optimal control of infections and tumors. T cell exhaustion can manifest as a gradual and progressive loss of T cell function. As used herein, "reversing exhaustion" generally refers to the activity or ability to restore at least some of the weakened or diminished antitumor activity of exhausted T cells. Reversing exhaustion can also include preventing T cell exhaustion in the first place.

[0162] As used herein, the term "T cell-mediated immune response" generally refers to an immune response regulated by T cell co-stimulation. Exemplary immune responses include T cell responses such as cytokine production and cytotoxicity. Additionally, T cell-mediated immune responses also include immune responses indirectly influenced by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0163] The term "tumor-specific T cells" as used in this article generally refers to T lymphocytes capable of specifically attacking and / or destroying tumor cells. For example, they may be conferred specific receptors (e.g., T cell receptors) that bind to antigens present on the surface of tumor cells, such as tumor-associated antigens. Each tumor-specific T cell may recognize a single tumor antigen, and a group of tumor-specific T cells may be conferred with diverse receptors targeting multiple tumor antigens.

[0164] The term “substantially does not compete with” as used herein generally means that the binding of one molecule or agent to the target does not in any significant way affect the binding of another molecule or agent to the same target (e.g., to a degree less than about 50%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.5%, less than about 0.5%, or less), for example, in analyses commonly used to determine such binding (e.g., in the analyses described in the examples herein).

[0165] As used herein, the term "antigen" or "Ag" generally refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune-active cells, or both. Those skilled in the art will understand that any macromolecule (including virtually all proteins or peptides) can act as an antigen. Furthermore, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that when the term "antigen" is used herein, any DNA containing a nucleotide sequence or a portion of a protein that elicits an immune response thus encodes that "antigen." Furthermore, those skilled in the art will understand that an antigen does not necessarily consist entirely of the full-length nucleotide sequence of a gene. An antigen does not necessarily consist of a "gene." Antigens can be synthetic, or can be derived from a biological sample, or may be macromolecules other than peptides. Such biological samples may include (but are not limited to) tissue samples, tumor samples, cells, or fluids having other biological components.

[0166] As used in this article, the terms "anti-cancer" or "anti-tumor" generally refer to biological effects that can be manifested in a variety of ways, including (but not limited to) reductions in tumor volume, number of cancer cells, number of metastases, life expectancy, cancer cell proliferation, cancer cell survival, or improvement in various physiological symptoms associated with cancer. "Anti-cancer" or "anti-tumor" effects can also manifest as the ability to initially prevent the development of cancer.

[0167] As used herein, the term "hydrocarbon group" generally refers to a portion consisting entirely of hydrogen and carbon atoms; such portions may include aliphatic and / or aromatic portions. This portion may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more carbon atoms. Examples of hydrocarbon groups include, but are not limited to, alkyl groups, such as C... 1-6Alkyl groups (e.g., C1, C2, C3, or C4 alkyl groups, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, or tert-butyl); C groups substituted with aryl (e.g., benzyl) or cycloalkyl (e.g., cyclopropylmethyl). 1-6 Alkyl; cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl); aryl (e.g., phenyl, naphthyl, or fluorenyl), etc.

[0168] As used herein, the term "heteroalkyl group" generally refers to a hydrocarbon group that optionally includes one or more heteroatoms. Heteroatoms can be any atom other than carbon, such as O, S, or N.

[0169] As used herein, the term "alkenyl" generally refers to a straight-chain or branched alkyl moiety having 2, 3, 4, 5, 6 or more carbon atoms and, where applicable, additionally having at least one E or Z stereochemical double bond. This term includes references to the group consisting of vinyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl, etc.

[0170] As used herein, the term "aryl" generally refers to an aromatic ring system containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more ring carbon atoms. Aryl groups are often phenyl, but can be polycyclic systems having two or more rings, at least one of which is an aromatic ring. The term includes references to groups such as phenyl, naphthyl, fluorenyl, azulel, indene, anthracene, etc.

[0171] The term "prodrug" as used in this article generally refers to a compound that is rapidly converted into its parent compound in the body, for example, through hydrolysis in the blood.

[0172] As used herein, the term "vaccine" generally refers to a preparation that provides active acquired immunity against a specific antigen (such as a tumor antigen or a microbial antigen) or a tissue, cell, or organism containing said antigen. Vaccines can be prophylactic (to prevent or mitigate the effects of future diseases or conditions) or therapeutic (to treat diseases or conditions that have already occurred, such as cancer).

[0173] YTHDF1 weakening agent

[0174] The YTHDF1 depressant of this application may include compounds.

[0175] Such compounds can be macromolecules. Macromolecules can be naturally occurring or chemically synthesized organic or inorganic molecules with a mass greater than or equal to about 1,000 Daltons to about or greater than 1,2,3,5,7,10 or more trillion Daltons. Macromolecules may contain two or more monomeric subunits or derivatives thereof, which are linked by covalent bonds, ionic bonds or other chemical interactions (such as hydrogen bonding, ion pairing, base pairing or pairing between charges formed by charge polarization). These monomeric subunits may be different from each other or the same as each other, and in some embodiments, may form polymers. Macromolecules may also be molecules capable of forming tertiary and / or quaternary structures, regardless of whether they have more than one subunit and / or polymers. Examples of macromolecules include polynucleotides, nucleic acid molecules including DNA, RNA (including siRNA, snRNA, tRNA, antisense RNA and ribozymes), peptide nucleic acids (PNA), polypeptides, glycopeptides, proteins, carbohydrates or lipids, or derivatives or combinations thereof, for example, nucleic acid molecules containing peptide nucleic acid moieties or glycoproteins, respectively. Examples of macromolecules further include macromolecular assemblies, such as viruses, viral particles, bacteriophages, viroids, prions, and combinations and conjugates thereof.

[0176] Such compounds can be small molecules. Small molecules can be naturally occurring or chemically synthesized organic or inorganic molecules with a Dalton value of less than about 1000 Daltons, ranging from about 1000 Daltons to about 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or smaller. Small molecules can be any molecule that is not a macromolecule, such as proteins or nucleic acids. “Small molecule” can include molecules containing two or more monomeric subunits, such as dipeptides or dinucleotides.

[0177] Such compounds may contain or be polypeptides. In some cases, such compounds may contain or be nucleic acid molecules. For example, such compounds may contain antibodies or their derivatives, antibody-drug conjugates, and / or fusion proteins.

[0178] For example, the compound can attenuate the activity of the YTHDF1 protein. For example, the compound can bind directly or indirectly (e.g., through other molecules) to one or more residues of the YTHDF1 protein. This binding can cause conformational changes in the structure and / or function of the YTHDF1 protein.

[0179] The compound can specifically bind to YTHDF1 (e.g., human YTHDF1), fragments thereof, or derivatives thereof. The YTHDF1 protein may comprise the amino acid sequence shown in SEQ ID NO:1. In some cases, YTHDF1, its fragments, or derivatives may comprise at least the amino acid residues corresponding to residues N378, F382, W384, F480, and / or H528 of SEQ ID NO:1. In some cases, YTHDF1, its fragments, or derivatives may comprise at least the amino acid residues corresponding to residues 372-392, 479-494, and / or 526-535 of SEQ ID NO:1. In some cases, the compound can bind (e.g., specifically bind) to YTHDF1 or its fragments or derivatives, wherein the YTHDF1, its fragments, or derivatives may comprise the amino acid sequence shown in any of SEQ ID NO:1-3, 9-13, and 16-18. In some cases, the compound does not specifically bind (or, substantially does not bind) to YTHDF1 or fragments or derivatives thereof containing the amino acid sequences shown in any of SEQ ID NO:4-8.

[0180] In certain circumstances, the compounds of this application may bind to YTHDF1, fragments thereof, or derivatives comprising the amino acid sequences shown in any of SEQ ID NO:4-8, with a Kd value greater than about 10. -6 M (for example, higher than approximately 5 × 10) -6 M, higher than approximately 10 -5 M, higher than approximately 5×10 -5 M, higher than approximately 10 -4 M, higher than approximately 5×10 -4 M, higher than approximately 10 -3 M, higher than approximately 5×10 -3 (M or higher). The Kd value can be determined using any method commonly used in the art, such as isothermal titration calorimetry (ITC) analysis, surface plasmon resonance (SPR) analysis, and / or micro thermophoresis (MST) analysis.

[0181] In certain circumstances, the compounds of this application may bind to YTHDF1 and its fragments or derivatives (e.g., those described in this application that contain / have the amino acid sequences shown in any of SEQ ID NO: 1-3, 9-13, and 16-18) with a Kd value less than about 10. -5 M (for example, less than approximately 9x10) -6 M, less than approximately 8x10 -6 M, less than approximately 7x10 -6 M, less than approximately 6x10 -6 M, less than approximately 5x10 -6 M, less than approximately 4x10 -6M, less than approximately 3.5 x 10 -6 M, less than approximately 3x10 -6 M, less than approximately 2.5 x 10 -6 M, less than approximately 2x10 -6 M, less than approximately 1x10 -6 M, less than approximately 5x10 -7 M, less than approximately 2x10 -7 M, less than approximately 10 -7 M, less than approximately 5x10 -8 M, less than approximately 2x10 -8 M, less than approximately 10 -8 M, less than approximately 5x10 -9 M, less than approximately 4x10 -9 M, less than approximately 3x10 -9 M, less than approximately 2x10 -9 M, or less than approximately 10 -9 The Kd value can be determined using any method commonly used in the art, such as isothermal titration calorimetry (ITC) analysis, surface plasmon resonance (SPR) analysis, and / or micro thermophoresis (MST) analysis.

[0182] In some cases, when bound to YTHDF1, the compound binds (e.g., specifically binds) to at least one residue (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 or more residues) corresponding to amino acid residues 372-392, 479-494 and 526-535 selected from SEQ ID NO: 1. In some cases, when the compound binds to YTHDF1, the compound can bind to multiple residues, and at least one of the bound residues (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 or more residues) can correspond to amino acid residues 372-392, 479-494 and 526-535 selected from SEQ ID NO:1.

[0183] In some cases, when bound to YTHDF1, the compound binds to at least one residue (e.g., at least two, at least three, at least four, or at least five) that corresponds to residues selected from N378, F382, W384, F480, and H528 of SEQ ID NO:1.

[0184] The compound contains a YTHDF1 depressant that can block YTHDF1 (e.g., human YTHDF1) and its fragments or derivatives from interacting with m 6 The binding of A. The YTHDF1 protein may contain the amino acid sequence shown in SEQ ID NO:1. In some cases, YTHDF1 and its fragments or derivatives may contain at least the amino acid residues corresponding to residues N378, F382, W384, F480 and / or H528 of SEQ ID NO:1. In some cases, YTHDF1 and its fragments or derivatives may contain at least the amino acid residues corresponding to residues 372-392, 479-494 and / or 526-535 of SEQ ID NO:1.

[0185] In certain situations, the compound can block the interaction between YTHDF1 and its fragments or derivatives with m 6 The combination of A, wherein the YTHDF1 and its fragments or derivatives may contain the amino acid sequence shown in any of SEQ ID NO: 1-3, 9-13 and 16-18.

[0186] In some cases, the compound does not significantly or substantially block the interaction between YTHDF1 and its fragments or derivatives with m 6 The combination of A, wherein the YTHDF1 and its fragments or derivatives may contain the amino acid sequence shown in any of SEQ ID NO:4-8.

[0187] In certain circumstances, the compounds of this application can block the interaction between YTHDF1 and its fragments or derivatives with m 6 The combination of A, its IC 50 Values ​​higher than about 7.5 μM (e.g., higher than about 8 μM, higher than about 8.5 μM, higher than about 9 μM, higher than about 9.5 μM, higher than about 10 μM, higher than about 10.5 μM, higher than about 11 μM, higher than about 11.5 μM, higher than about 12 μM, or higher), wherein the YTHDF1 and its fragments or derivatives may comprise the amino acid sequence shown in any of SEQ ID NO:4-8. IC50 values ​​can be determined using any method commonly used in the art, such as fluorescence polarization (FP) analysis, and / or AlphaScreen-based analysis.

[0188] In certain circumstances, the compounds of this application can block the interaction between YTHDF1 and its fragments or derivatives with m 6 The combination of A, its IC 50Values ​​below about 7.5 μM (e.g., below about 6.5 μM, below about 6 μM, below about 5.5 μM, below about 5 μM, below about 4.5 μM, below about 4 μM, below about 3.5 μM, below about 3 μM, below about 2.5 μM, below about 2 μM, below about 1.5 μM, below about 1 μM, below about 0.9 μM, below about 0.8 μM, below about 0.7 μM, below about 0.6 μM, below about 0.5 μM, below about 0.4 μM, below about 0.3 μM, below about 0.2 μM, below about 0.1 μM or lower), wherein the YTHDF1 and its fragments or derivatives may comprise the amino acid sequence shown in any of SEQ ID NO: 1-3, 9-13 and 16-18. IC50 values ​​can be determined using any method commonly used in the art, such as fluorescence polarization (FP) analysis and / or AlphaScreen-based analysis.

[0189] In some cases, compounds containing YTHDF1 depressant are essentially not affected by m 6 A competitive binding to YTHDF1. For example, as determined in an analysis, this analysis is typically used to determine such binding (e.g., as shown in an AlphaScreen-based analysis), by adding (less than about 50%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less) m 6 A, affecting (e.g., reducing) the binding of the compound to YTHDF1 and its fragments or derivatives. For example, as determined in an analysis, which is typically used to determine such binding (e.g., as shown in an AlphaScreen-based analysis), by adding (less than about 50%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5% or less) of the compound of this application, affecting (e.g., reducing) m 6 A binds to YTHDF1 and its fragments or derivatives.

[0190] In some cases, the compound contained in the YTHDF1 depressant may be salvianolic acid, such as salvianolic acid A (SAA), salvianolic acid C (SAC), its prodrug, metabolite, derivative, or a pharmaceutically acceptable salt, ester or amide of any of the above, or any combination thereof.

[0191] In certain cases, YTHDF1 depressants may comprise compounds of Formula I, prodrugs, metabolites, derivatives of compounds of Formula I, or pharmaceutically acceptable salts, esters, or amides of any of the above:

[0192]

[0193] R1 can be selected from C. 1-50 hydrocarbon group, C 1-50 Substituted hydrocarbon group, C 1-50 heterohydrocarbons and C 1-50 Substituted heterohydrocarbon groups.

[0194] In some cases, R1 in Formula I can be (CO)-R2, and R2 can be an optionally substituted alkenyl group. In some cases, R2 can be CH=CH-R3, and R3 can be an optionally substituted aryl group. In some cases, R3 can be a group of Formula II. Where A can be any substituted furan or R6 can be a hydroxyl group, and R5 can be an optional substituted alkenyl group.

[0195] In some cases, A in Equation II can be... And R4 can be

[0196] In some cases, A in Equation II can be... R6 can be a hydroxyl group, R5 is CH=CH-R7, and R7 can be...

[0197] In some cases, the compound contained in the YTHDF1 depressant of this application may contain at least two dihydroxyphenyl moieties.

[0198] In some cases, the compound contained in YTHDF1 depressant may contain at least three dihydroxyphenyl moieties.

[0199] In certain cases, YTHDF1 depressants may comprise compounds of formula III, prodrugs, metabolites, derivatives of compounds of formula III, or pharmaceutically acceptable salts, esters, or amides of any of the above:

[0200] (Formula III), where A can be an optional substituted furan or Furthermore, R6 can be a hydroxyl group, and R5 can be an optional substituted alkenyl group.

[0201] In some cases, A in Equation III can be... And R4 can be

[0202] In some cases, A in Equation III can be... R6 can be a hydroxyl group, R5 can be CH=CH-R7, and R7 can be...

[0203] In certain cases, YTHDF1 depressants may comprise any of the following compounds, prodrugs, metabolites, derivatives of any of the following compounds, or pharmaceutically acceptable salts, esters, or amides of any of the foregoing:

[0204]

[0205] In certain cases, the YTHDF1 depressant may comprise any of the following compounds, a prodrug, metabolite, derivative of any of the following compounds, or a pharmaceutically acceptable salt, ester, or amide of any of the foregoing: (E)-3-(3,4-dihydroxyphenyl)-2-((3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)acryloyl)oxy)propionic acid, and 3-(3,4-dihydroxyphenyl)-2-((E)-3-(2-(((E)-3,4-dihydroxystyryl)-3,4-dihydroxyphenyl)acryloyl)oxy)propionic acid.

[0206] In certain cases, YTHDF1 depressants may comprise any of the following compounds, prodrugs, metabolites, derivatives of any of the following compounds, or pharmaceutically acceptable salts, esters, or amides of any of the foregoing:

[0207]

[0208] In certain cases, the YTHDF1 depressant may comprise any of the following compounds, a prodrug, metabolite, derivative of any of the following compounds, or a pharmaceutically acceptable salt, ester, or amide of any of the foregoing: (R,E)-3-(3,4-dihydroxyphenyl)-2-((3-(2-(3,4-dihydroxyphenyl))-7-hydroxybenzofuran-4-yl)acryloyl)oxy)propionic acid, and (S)-3-(3,4-dihydroxyphenyl)-2-(((E)-3-(2-(((E)-3,4-dihydroxystyryl)-3,4-dihydroxyphenyl)acryloyl)oxy)propionic acid.

[0209] In certain cases, YTHDF1 depressants may comprise any of the following compounds, prodrugs, metabolites, derivatives of any of the following compounds, or pharmaceutically acceptable salts, esters, or amides of any of the foregoing:

[0210]

[0211] In certain cases, the YTHDF1 depressant may comprise any of the following compounds, a prodrug, metabolite, derivative of any of the following compounds, or a pharmaceutically acceptable salt, ester, or amide of any of the foregoing: (S,E)-3-(3,4-dihydroxyphenyl)-2-((3-(2-(3,4-dihydroxyphenyl))-7-hydroxybenzofuran-4-yl)acryloyl)oxy)propionic acid, and (R)-3-(3,4-dihydroxyphenyl)-2-(((E)-3-(2-(((E)-3,4-dihydroxystyryl)-3,4-dihydroxyphenyl)acryloyl)oxy)propionic acid.

[0212] In some cases, YTHDF1 depressants may be achiral or chiral. If a YTHDF1 depressant is chiral, it may have one or more chiral centers and may be a single (R) or (S) enantiomer or a mixture of (R) and (S) enantiomers.

[0213] In some cases, the compounds contained in YTHDF1 depressants may be derived from plants. For example, the compound may be available in plant extracts, such as as part of a plant extract. For example, it may be derived from plants of the genus *Sage* and its active ingredients.

[0214] The compound may be chemically prepared (e.g., from oleochemicals), biochemically generated (e.g., during fermentation), or obtained from plant material, followed optionally by chemical modification. For example, the compound may be (bio)chemically prepared by esterification of 3-(3,4-dihydroxyphenyl)lactic acid with a carboxylic acid.

[0215] In some cases, compounds can be isolated from plant material, such as plant roots. For example, plants may belong to the genus *Salvia*, such as *Salvia miltiorrhiza*, *Salvia cavaleriei*, *Salvia fluva*, *Salvia chinensis*, *Salvia bowleyana*, *Salvia prionitis*, *Salvia officialis*, *Salvia deserta*, and / or *Salvia yunnanensis*. In some cases, compounds are obtained from *Salvia tanshinone*.

[0216] Modified immune cells

[0217] This application provides modified immune cells (e.g., APCs, such as DCs). This application also provides methods for modifying immune cells (e.g., APCs, such as DCs).

[0218] Immune cells may be APCs, such as DCs. APCs (e.g., DCs) may be derived from the subject's bone marrow and / or lymph nodes. DCs may contain one or more of the following: resident CD11b. + Cells (e.g., CD11b) + DC), resident CD8α + Cells (e.g., CD8α) + DC), migratory CD11b + Cells (e.g., CD11b) + DCs, CD11c+ cells (e.g., CD11c+ DCs), and migrating CD103 + Cells (e.g., CD103) + DC).

[0219] Modified APCs (e.g., modified DCs) can exhibit superior performance in cross-sensitized T cells compared to their corresponding unmodified control APCs (e.g., corresponding unmodified control DCs).

[0220] An APC (e.g., a DC) may contain or express one or more tumor-specific antigens (e.g., tumor / cancer-associated antigens provided in this application). In some cases, an APC (e.g., a DC) may be co-cultured with or treated with a mimicry agent such as FLT3L.

[0221] In some cases, immune cells may be APCs (e.g., DCs) obtained from the subject (such as a cancer patient), and in other cases, immune cells (e.g., APCs, such as DCs) may be isolated from tumor tissue.

[0222] Immune cells (e.g., APCs, such as DCs) can be modified with the compounds of this application or YTHDF1 attenuators.

[0223] For example, in an immune cell population (e.g., APCs, such as DCs), one or more cells may have been modified with the compounds of this application or YTHDF1 attenuators. In some cases, the modified immune cells of this application (e.g., mAPCs, such as mDCs) may contain the compounds of this application or YTHDF1 attenuators.

[0224] In certain circumstances, the compound or YTHDF1 attenuator of this application may be permitted to be exposed to immune cells (e.g., mAPCs, such as mDCs) for a period of time sufficient to cause a reduction in the expression and / or activity of YTHDF1 (e.g., at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours or longer). For example, the compound or attenuator may be applied to the culture medium in which immune cells (e.g., mAPCs, such as mDCs) are cultured.

[0225] The compound or YTHDF1 depressant of this application may be applied at concentrations of, for example, at least 1 μM, at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 6 μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM, at least 11 μM, at least 12 μM or higher.

[0226] In some cases, the compound or YTHDF1 attenuator of this application is not applied directly to the immune cells (e.g., APCs or DCs) themselves. Instead, the immune cells (e.g., APCs, such as DCs) may be derived from (e.g., differentiated from, as progeny of, etc.) cells (e.g., ancestors of immune cells) or organisms that have received the compound or YTHDF1 attenuator of this application.

[0227] Immune cells can be human cells, such as human APCs (e.g., DCs).

[0228] In some cases, cellular sources, such as immune cells (e.g., APCs, DCs) or their progenitor cells, may be obtained from the subject before amplification or other modifications. The term "subject" as used herein is intended to include living organisms (e.g., mammals) capable of evoking an immune response. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. Immune cells or their progenitors can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and / or tumors.

[0229] Composition

[0230] The compositions of this application may contain the YTHDF1 depressant of this application and / or the mAPC of this application. In some cases, the compositions may further contain additional / secondary active ingredients of this application.

[0231] In some cases, the composition may be a vaccine composition.

[0232] The compositions of this application may comprise one or more pharmaceutically acceptable excipients. Such pharmaceutically acceptable excipients may include any inactive material that binds to one or more active ingredients of this application (e.g., modified cells or attenuators).

[0233] For example, pharmaceutically acceptable excipients may include one or more of the following: solvents, penetration enhancers, antioxidants, thickeners, ointment bases, protectants, adsorbents, detergents, emollients, preservatives, humectants, buffers, adjuvants, bioavailability enhancers, carriers, flow aids, sweeteners, diluents, dyes / colorants, flavor enhancers, solubilizers (including surfactants), wetting agents, dispersants, suspending agents, stabilizers, and / or isotonic agents.

[0234] In some cases, the composition may contain one or more adjuvants to enhance or increase the immune response associated with the administration of the composition.

[0235] Additional / Second Active Ingredient

[0236] The compounds, YTHDF1 attenuators, cells (e.g., mAPC, mDC) and / or compositions of this application may further contain additional / secondary active ingredients, and / or may be used in combination with additional / secondary active ingredients.

[0237] In certain circumstances, the compounds of this application, YTHDF1 attenuators, cells (e.g., mAPC, mDC) and / or compositions may be administered to subjects who have received, are receiving and / or will receive additional / second active ingredients.

[0238] Additional active ingredients or therapies may be applied before, simultaneously with and / or after the application of the YTHDF1 attenuator, cells (e.g., mAPC, mDC) and / or composition of this application.

[0239] Additional active ingredients may be anticancer agents. For example, additional active ingredients may include cancer immunotherapy. In some cases, additional active ingredients may include immune checkpoint attenuators (e.g., immune checkpoint inhibitors). In some cases, additional active ingredients may include agents selected from: anti-PD-L1 antibodies or their antigen-binding portions, anti-PD-1 antibodies or their antigen-binding portions, anti-CTLA-4 antibodies or their antigen-binding portions, and IDO attenuators.

[0240] For example, additional active ingredients may include antigen-binding fragments or derivatives of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, ipilimumab, and / or any of the above. For example, additional active ingredients may include antibodies (including their antigen-binding portions) capable of competitively binding to the corresponding antigens (PD-1, PD-L1, or CTLA-4, respectively) of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include HCDR3 of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include LCDR3 of any one of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include HCDR2 of any one of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include LCDR2 of any one of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include HCDR1 of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include LCDR1 of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include HCDR3, HCDR2, and HCDR1 of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include LCDR1, LCDR2, and LCDR3 of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include the heavy chain variable region of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab.In some cases, additional active ingredients may include the light chain variable region of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, additional active ingredients may include both the heavy chain and light chain variable regions of any of pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab.

[0241] Suppression methods

[0242] This application provides a method for inhibiting and / or attenuating YTHDF1 activity.

[0243] This application further provides methods for activating immune cells (e.g., APCs, such as DCs), generating immune cells with enhanced anti-tumor activity, preventing and / or reversing the depletion of immune cells (such as immune effector cells, such as T cells), increasing and / or improving the proliferation and / or activity of immune cells (e.g., immune effector cells, such as T cells, e.g., tumor-infiltrating T cells), increasing and / or improving the proliferation and / or activity of tumor-specific immune cells (e.g., immune effector cells, such as T cells), enhancing the cytokine production of immune cells (such as T cells), and / or inhibiting tumor growth, inhibiting the proliferation of tumor cells, and / or killing tumor cells.

[0244] This method may include the steps of applying the YTHDF1 attenuator of this application, cells (e.g., mAPC, mDC), and / or compositions.

[0245] For example, the method may include contacting YTHDF1 or target cells containing YTHDF1 (e.g., immune cells, such as APCs and / or T cells) with the YTHDF1 attenuator of this application, cells (e.g., mAPCs, mDCs), and / or compositions. Contact may be performed ex vivo. In some cases, contact may be performed in vivo.

[0246] In some cases, the method may include introducing the YTHDF1 attenuator and / or composition of this application into the cells (e.g., immune cells, such as APCs and / or T cells). Introduction may be performed ex vivo. In some cases, introduction may be performed in vivo. In some cases, introduction may be performed ex vivo.

[0247] Inhibitor screening methods and kits

[0248] This application provides a method for determining whether a candidate drug is a YTHDF1 depressant.

[0249] This method may include contacting the candidate drug with the YTHDF1 mutant.

[0250] The YTHDF1 mutant may include one or more amino acid substitutions, deletions, and / or additions at one or more residues, corresponding to residues selected from residues 372-392, 479-494, and 526-535 of SEQ ID NO:1. In some cases, the YTHDF1 mutant may include one or more amino acid substitutions, deletions, and / or additions at one or more residues, corresponding to residues selected from residues N378, F382, W384, F480, and H528 of SEQ ID NO:1. In some cases, according to the amino acid sequence shown in SEQ ID NO:1, the YTHDF1 mutant may include one or more amino acid substitutions corresponding to the following amino acid substitutions: N378A, F382A, W384A, F480A, and H528A. In some cases, the YTHDF1 mutant may include amino acid sequences as shown in any of SEQ ID NO:4-8.

[0251] The method may further include determining whether the candidate drug specifically binds to the YTHDF1 mutant of this application.

[0252] If a candidate drug specifically binds to the YTHDF1 mutant of this application, then the candidate drug cannot be a YTHDF1 attenuator.

[0253] The method may further include contacting a candidate agent with a control YTHDF1, its fragments, or derivatives, and determining whether the candidate agent specifically binds to the control YTHDF1, its fragments, or derivatives. In some cases, the control YTHDF1, its fragments, or derivatives may contain at least amino acid residues corresponding to residues N378, F382, W384, F480, and / or H528 of SEQ ID NO:1. In some cases, the control YTHDF1, its fragments, or derivatives may contain at least amino acid residues corresponding to residues 372-392, 479-494, and / or 526-535 of SEQ ID NO:1. In some cases, the control YTHDF1, its fragments, or derivatives may contain amino acid sequences corresponding to any of SEQ ID NO:1-3, 9-13, and 16-18.

[0254] In some cases, the method may further include determining whether the candidate drug specifically binds to control YTHDF1, its fragments, or derivatives.

[0255] If a candidate drug does not specifically bind to the control YTHDF1, its fragments, or derivatives of this application, then the candidate drug cannot be a YTHDF1 attenuator.

[0256] If a candidate drug specifically binds to the control YTHDF1 of this application, its fragments or derivatives, but not to the YTHDF1 mutant of this application, then the candidate drug may be considered a potential YTHDF1 attenuator.

[0257] On the one hand, this application provides YTHDF1 mutants (such as the YTHDF1 mutants described in this application) for example, to screen and / or identify candidate agents that attenuate the activity of YTHDF1.

[0258] On one hand, this application provides a kit containing the YTHDF1 mutant of this application. The kit can be used, for example, to screen and / or identify candidate agents that attenuate the activity of YTHDF1.

[0259] The kit may further contain additional pharmaceutical agents. For example, the kit may contain the control YTHDF1 of this application, its fragments, or derivatives.

[0260] In some cases, the kit may further contain buffers or reagents that can be used in analyses to determine the binding affinity of candidate reagents (e.g., isothermal titration calorimetry (ITC) analysis, surface plasmon resonance (SPR) analysis, and / or micro-thermophoresis (MST) analysis).

[0261] antigen

[0262] The compounds, YTHDF1 attenuators, cells (e.g., mAPC, mDC) and / or compositions of this application may be used to treat diseases, symptoms or conditions associated with antigen expression in subjects in need, and / or may be used to stimulate T cell-mediated immune responses to antigens (e.g., tumor antigens) in subjects in need.

[0263] Furthermore, the compounds, YTHDF1 attenuators, cells (e.g., mAPC, mDC) and / or compositions of this application may be used in combination with an additional / second active ingredient that may induce an increase in one or more antigens (e.g., tumor antigens) in a subject receiving it.

[0264] An antigen can be any molecule capable of eliciting an immune response in a human subject. The immune response may involve the production of antibodies, or the activation of specific immune-active cells, or both. Any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen can be derived from and / or present in a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids containing other biological components.

[0265] In this application, cancer-associated antigens or tumor antigens may be expressed on the surface of cancer cells. In some cases, the cancer-associated antigen itself may be intracellular; however, fragments of such antigens (peptides) may be presented on the surface of cancer cells via the MHC (major histocompatibility complex).

[0266] Examples of cancer / tumor-associated antigens may include, for example, EGFR, HER2 / neu, HER3, HER4, Ep-CAM, CEA, TrAIL, TRAIL receptor 1, TRAIL receptor 2, lymphotoxin-β receptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast activator protein (FAP), hepsin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), prostate-specific membrane antigen (PSMA), VEGF receptor 1, VEGF receptor 2, IGF-1R, TSLP-R, TIE-1, TIE-2, TNF-α and similar weak apoptosis inducers of TNF (TWEAK), and IL-1R.

[0267] In some cases, examples of cancer / tumor-associated antigens may include, for example, CEA, gp100, MAGE family proteins, DAGE, GAGE, RAGE, NY-ESO 1, Melan-A / MART 1, TRP-1, TRP-2, tyrosinase, HER-2 / neu, MUC-1, p53, KSA, PSA, PSMA and / or fragments and modified forms thereof.

[0268] Enhance anti-tumor immunity

[0269] The compounds, YTHDF1 attenuators, cells (e.g., mAPC, mDC), methods, and / or compositions of this application may be used to activate immune cells and / or enhance immune responses, such as antitumor immune responses.

[0270] For example, activated immune cells may have an enhanced (e.g., an enhanced capacity of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more) ability to kill tumor cells or control tumor growth in the body.

[0271] In some cases, CD4 can be observed in immune cell populations. + Increased T-cell proliferation (e.g., an increase of at least approximately 1%, at least approximately 2%, at least approximately 3%, at least approximately 4%, at least approximately 5%, at least approximately 8%, at least approximately 10%, at least approximately 15%, at least approximately 16%, at least approximately 17%, at least approximately 18%, at least approximately 19%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 100%, at least approximately 1.5-fold, at least approximately 2-fold, at least approximately 2.5-fold, at least approximately 3-fold, at least approximately 3.5-fold, at least approximately 4-fold, at least approximately 4.5-fold, or more). In some cases, CD8+ can be observed in the immune cell population. + Increased T-cell proliferation (e.g., an increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more).

[0272] In some cases, CD8+ can be transmitted through the tumor site or surrounding the tumor site. + An increase in the number of cytotoxic T cells (e.g., an increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more) indicates an enhanced antitumor immune response.

[0273] In some cases, it can be transmitted through tumor-invasive CD8 cells. +An increase in cell number (e.g., an increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more) indicates an enhanced antitumor immune response.

[0274] In certain circumstances, increased immune cell activity can be observed through an increase in cytokines (e.g., IFN-γ and / or IL-2) and / or granzyme B produced by immune cells (e.g., an increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more).

[0275] In some cases, the depletion of immune cells can be delayed and / or reversed (e.g., delayed and / or reversed by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more), such as CD8. + Delay and / or reversal of T cell depletion (e.g., delay and / or reversal of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more) indicates increased immune cell activity or enhanced immune response.

[0276] In certain cases, increased CXCR5 expression (e.g., an increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more) can indicate increased immune cell activity or enhanced immune response. Increased expression can be characterized by an increase in the amount / level of CXCR5 intracellularly / on the cell (e.g., an increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more), or by an increase in the amount / level of CXCR5 in the immune cell population. An increase in the number / percentage of cells expressing CXCR5 in an organism (e.g., an immune effector cell population, such as a T cell population) (e.g., an increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more).

[0277] In certain cases, reduced expression of PD-1 (e.g., at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more) can demonstrate increased immune cell activity or enhanced immune response. The reduced expression can be characterized by a decrease in the amount / level of PD-1 intracellularly / onboard (e.g., a decrease of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more), or manifested in immune cell populations. (e.g., a reduction in the number / percentage of cells expressing PD-1 in an immune effector cell population, such as a T cell population) (e.g., a reduction of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more).

[0278] In certain cases, reduced expression of Tim3 (e.g., at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more) can demonstrate increased immune cell activity or enhanced immune response. The reduced expression can be characterized by a decrease in the amount / level of Tim3 intracellularly / onboard (e.g., a decrease of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more), or manifested in immune cell populations. The number / percentage of cells expressing Tim3 in the body (e.g., immune effector cell populations, such as T cell populations) is reduced (e.g., reduced by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more).

[0279] In some cases, PD-1 can be detected in immune cell populations (e.g., immune effector cell populations, such as T cell populations). + Tim3 +A reduction in the number / percentage of cells (e.g., a reduction of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times or more) indicates increased immune cell activity or an enhanced immune response.

[0280] Disease, symptoms or condition

[0281] The compounds, YTHDF1 attenuators, cells (e.g., mAPC, mDC), methods, and / or compositions of this application may be used to treat diseases, symptoms, or conditions, such as treating diseases, symptoms, or conditions associated with the expression of antigens (e.g., cancer / tumor-associated antigens described herein) in subjects of need.

[0282] For example, the disease, symptom, or condition could be cancer.

[0283] In some cases, cancer can be selected from blood cancers, lymphomas, and solid tumors.

[0284] In some implementation schemes, the cancer is selected from melanoma, breast cancer, lung cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, colorectal cancer, kidney cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphoma, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, and leukemia.

[0285] Subjects

[0286] The compounds, YTHDF1 attenuators, cells (e.g., mAPC, mDC), methods, and / or compositions of this application may be administered to subjects (e.g., humans) in need.

[0287] In some cases, participants may be cancer patients. For example, participants may be patients with cancers selected from hematologic malignancies, lymphomas, and solid tumors. In other cases, participants may be patients with cancers selected from melanoma, colon cancer, pancreatic cancer, breast cancer, and lung cancer.

[0288] In some cases, subjects may have received, are receiving, and / or will receive additional treatments. Additional treatments may include anticancer therapies.

[0289] In some cases, anticancer treatment may include cancer immunotherapy. For example, anticancer treatment may include or be an immune checkpoint weakener. In some cases, anticancer treatment may include agents selected from: antiPD-L1 antibodies or their antigen-binding portions, antiPD-1 antibodies or their antigen-binding portions, antiCTLA-4 antibodies or their antigen-binding portions, and IDO weakeners. In some cases, anticancer treatment may include pembrolizumab, nivolumab, cimipril, atezolizumab, avelumab, durvalumab, ipilimumab, and / or antigen-binding fragments or derivatives of any of the above.

[0290] Example

[0291] The following embodiments are provided to provide a complete disclosure and description of how to prepare and use the invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; nt, nucleotide; im, intramuscular injection; ip, intraperitoneal injection; sc, subcutaneous injection; etc. Regarding experimental results (e.g., in a two-sided unpaired Student's t-test), * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, *** indicates p < 0.0001, and ns means not significant.

[0292] Materials and methods

[0293] The following materials and methods are used in the embodiments of this application.

[0294] Protein expression and purification

[0295] The protein YTHDF1 (361-559) (SEQ ID NO:2) and its mutant (SEQ ID NO:4-13) were cloned into the pGEx-6P-1 vector (obtained from YouBio Co, Ltd, catalog number: VT1258), and the His-YTHDF1 (361-559) (SEQ ID NO:3) used in the AlphaScreen analysis was cloned into the modified pET28a vector (obtained from YouBio Co, Ltd, catalog number: VT1207). These vectors were transformed into E. coli BL21(DE3) cells and cultured at 37°C. When the OD value reached 0.6–0.8, 1 mM isopropyl-β-D-thiogalactoside was added to overexpress the protein overnight at 16°C. YTHDF1 (361-559) and its mutants were first purified by glutathione affinity chromatography (GSTrap FF, GE Healthcare), followed by overnight incubation with PPase at 4°C to remove the GST tag. The proteins were then further purified by cation exchange (HiTrap SP, GE Healthcare) and finally by a Superdex 75 10 / 300 column (GE Healthcare). The purified YTHDF1 (361-559) and mutants were then stored in a buffer containing 20 mM Hepes (pH 7.4) and 200 mM NaCl. For His-YTHDF1 (361-559), the proteins were purified sequentially by Ni-NTA chromatography (HisTrap FF, GE Healthcare), followed by cation exchange and a Superdex 75 10 / 300 column. The obtained proteins were then stored in a buffer with the same composition.

[0296] Fluorescence polarization (FP) analysis

[0297] All YTHDF1 (361-559), 5'-FAM labeled containing m 6 A's mRNA (5'-FAM-UUCUUCUGUGG(m 6 A) CUGUG-3', SEQ ID NO: 14) and the candidate compound were diluted with analytical buffer (20 mM Hepes (pH 7.4), 50 mM NaCl, 0.01% (v / v) between 20, 5% (v / v) glycerol). For high-throughput screening (HTS), 1.25 μM MYTHDF1 (361-559) was incubated with 80 μM of the candidate compound on a black 384 plate (Corning, 3575) at room temperature for 30 min. Then 40 nM of 5'-FAM-labeled MYTHDF1 containing m 6Add A's mRNA to the mixture and incubate at 4°C for 1 hour. Use unlabeled mRNA containing m... 6 A mRNA was used as a positive control, and 40 nM of 5'-FAM-labeled mRNA containing m was used alone. 6 A's mRNA regulates the gain factor. Finally, the mixture was measured using Envision Readers (PerkinElmer).

[0298] For the activity assay, 1.25 μM YTHDF1 (361-559) was incubated with the candidate compound diluted to the specified concentration for 30 minutes. Subsequent steps were similar to those for HTS. An equal volume of DMSO was used as a negative control.

[0299] AlphaScreen analysis

[0300] The compound (e.g., SAA or SAC) was diluted from 200 μM in a two-fold gradient with analytical buffer (20 mM Hepes (pH 7.4), 150 mM NaCl, 1 mg / ml BSA, 0.01% (v / v) Triton X-100). Then, 100 nM of Mbis-YTHDF1 (361-559) was incubated with SAA or SAC in analytical buffer at room temperature for 30 min. Then, 10 nM of biotinylated Mbis-YTHDF1 (361-559) was added. 6 A's mRNA (5'-Biotin-UUCUUCUGUGG(m 6 A)CUGUG-3')(SEQ ID NO:15) to bind to YTHDF1(361-559), and using unbiotinylated m 6 A mRNA was used as a positive control. Prior to alpha signal detection, the mRNA was analyzed in a white analytical plate (OptiPlate) under low light. TM Biotin-labeled beads (-384, PerkinElmer) were added with streptavidin donor beads and anti-His receptor beads and incubated at 4°C for 1 hour to ensure adequate binding between the biotin-labeled beads and the streptavidin donor beads, as well as between the His-labeled beads and the anti-His receptor beads. Alpha signals were then detected on Envision Readers (PerkinElmer).

[0301] Regarding the competitive analysis, the compounds (e.g., SAA or SAC) were diluted in the same manner, and the non-biotinylated compounds containing m... 6 A's mRNA was diluted to 400 nM, 200 nM, 50 nM, and 25 nM with analysis buffer. 200 nM His-YTHDF1 (361-559) was then mixed with non-biotinylated mRNA containing... 6The mRNA of A was incubated at 4°C for 10 minutes. Then, a compound (e.g., SAA or SAC) was added and incubated at room temperature for another 30 minutes to react with the mRNA containing A. 6 A's mRNA competes with protein binding. Next, under low light, 20 nM of biotinylated protein containing m... 6 A's mRNA and two kinds of beads were incubated at 4°C for 1 hour before detection.

[0302] NMR analysis

[0303] NMR CPMG experiments were performed at 25 °C using a Bruker Avance III spectrometer (600 MHz proton frequency) with a cryogenic probe (Brukerbiospin, Germany). YTHDF1 (361-559) was diluted to 20 μM, 10 μM, and 5 μM in phosphate buffer (20 mM NaH2PO4, 20 mM Na2HPO4, 150 mM NaCl, pH 7.4, D2O). Compounds (e.g., SAA or SAC) were dissolved in 5% deuterated DMSO to a concentration of 200 μM. Solvent-suppressed 1D sequences were obtained using pulsed sequencing (RD-90°-(τ-180°-τ)n-ACQ). 1 H CPMG. In the presaturation procedure, a 54.78 dB pulse was applied over a 4 s duration of cyclic delay (RD) to eliminate water resonance. Then, the 90° pulse length was modulated to approximately 11.82 μs. Finally, a total of 4 virtual scans and 64 free induction attenuations (FIDs) were collected into 64,000 acquisition points covering a spectral width of 12 kHz (20 ppm) at an acquisition time (ACQ) of 2.73 s.

[0304] Isothermal titration calorimetry

[0305] The purified YTHDF1 (361-559) was dialyzed overnight at 4°C in dialysis buffer (20 mM Hepes (pH 7.4) and 200 mM NaCl). The dialyzed protein was then diluted to 50 μM with dialysis buffer. The compound (e.g., SAA or SAC) was dissolved and diluted to 1 mM with dialysis buffer. Isothermal titration calorimetry (ITC) was performed at 25°C on a Microcal ITC 200 isothermal titration calorimeter (GE Healthcare). 200 μL of 50 μM YTHDF1 (361-559) was loaded into sample cells and stirred continuously at 750 rpm; 40 μL of 1 mM SAA was loaded into a syringe. After a single 0.4 μL injection, the compound (e.g., SAA or SAC) was titrated into YTHDF1 (361-559) with 19 2 μL injections at 180 s intervals. To further enhance the titration effect, the 19 2 μL injections were changed to 5 2.5 μL injections, followed by 14 1.9 μL injections. A 1 mM solution of the compound (e.g., SAA or SAC) was titrated into the dialysis buffer as a control to eliminate the thermal effect of background dilution. The experimental data were analyzed using Microcal ORIGIN (v7.0) software.

[0306] SPR combined analysis

[0307] SPR binding analysis was performed at 25°C on a Biacore T200 instrument (GE Healthcare). YTHDF1 (361-559) was covalently immobilized on a CM5 chip using a standard amine coupling procedure in 10 mM sodium acetate (pH 5.5). Compounds (e.g., SAA or SAC) were then serially diluted with HBS buffer (20 mM Hepes (pH 7.4), 200 mM NaCl, and 0.4% (v / v) DMSO). The diluted compounds (e.g., SAA or SAC) were then injected at a flow rate of 30 μL / min for 60 seconds to bind the immobilized YTHDF (361-559), followed by dissociation at the same flow rate for 600 seconds using HBS buffer. Data analysis using Biacore T200 evaluation software (GE Healthcare) yielded the equilibrium dissociation constant (K0.05) of the compounds (e.g., SAA or SAC). d )value.

[0308] MST Analysis

[0309] Micro-thermophoresis (MST) was performed at room temperature using a non-standard quantitative method on a NanoTemper Technologies micro-thermophoresis system. MST buffer (20 mM Hepes (pH 7.4), 200 mM NaCl, and 0.1 mM...) was used. F-127) was used to dilute the compound (e.g., SAA or SAC) in a two-fold gradient from 1 mM. YTHDF1 (361-559) was diluted to 4 μM with MST buffer. Then, 10 μL of the compound (e.g., SAA or SAC) and 10 μL of YTHDF1 (361-559) were mixed together and incubated at room temperature for 20 min. Before measurement, the mixture was centrifuged at 13000 rpm for 10 min at 4 °C. Finally, the solution was measured using a Monolith NT analyzer. TM The automated non-standard quantitative capillary (NanoTemper Technologies) collects the sample and begins the measurement. The K-values ​​of the compound (e.g., SAA or SAC) are obtained from data analysis using MO affinity analysis software V2.3 (NanoTemper Technologies). d value.

[0310] Hydrogen-deuterium exchange mass spectrometry

[0311] YTHDF1(361-559) was incubated overnight at 4°C with a compound (e.g., SAA) before measurement. In HDX mass spectrometry, hydrogen atoms of YTHDF1(361-559)-apo and YTHDF1(361-559)-SAA were exchanged with deuterium at 10°C for 0 s, 10 s, 30 s, 60 s, 1200 s, 3600 s, and 14400 s, respectively, in a buffer containing 20 mM Hepes (pH 7.4), 200 mM NaCl, and D₂O. The reaction was terminated at 0.5°C using a buffer containing 4 M guanidine hydrochloride, 0.5 M TCEP, and 100 mM citric acid (pH 2.3). After the deuterium labeling reaction, the sample was digested with pepsin immobilized on a column at 4°C to obtain the peptides. These peptides were then separated by HPLC and analyzed separately by mass spectrometry. HDX mass spectrometry data were analyzed in HDExaminer software (v2.4.1), and a threshold of ±5% was set to select peptides with significant changes.

[0312] Cell thermal translocation analysis

[0313] Cell thermal transfer assay (CETSA) was performed according to a previously reported protocol. The 293T cell line (ATCC) used in the analysis was cultured in DMEM medium (Life Technologies) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin / streptomycin (Life Technologies) at 37°C and 5% CO2. Cells were incubated with PBS buffer as a control or with 100 μM SAA for 4 hours, after which they were collected and divided into 12 equal aliquots. The aliquots were heated for 3 minutes at a series of temperatures ranging from 39°C to 59°C, followed immediately by cooling at 4°C for 3 minutes. Cells were then lysed by freeze-thaw in liquid nitrogen, and protein samples were collected by centrifugation. Samples were prepared for Western blot analysis by adding SDS and boiling at 99°C for 5 minutes. GAPDH was used as an internal control in the Western blot analysis. The results were quantified using ImageJ software.

[0314] cell lines and mice

[0315] B16-OVA is an OVA transfection clone derived from the mouse melanoma cell line B16, provided by Y.-X.Fu (UT Southwestern).

[0316] E.G7-OVA is an OVA transfection clone derived from the mouse thymoma cell line E.G7, provided by Chen Dong (Tsinghua University).

[0317] Ythdf1 - / - The mice were produced by the inventors in the laboratory, as described in previous studies (see, for example, Shi, H. et al., Nature 563, 249-253 (2018)).

[0318] Ythdf1 F / F Mice were provided by Bin Shen (Nanjing Medical University), and CD11c... cre The mice were purchased from the Jackson Laboratory.

[0319] OT-I mice are ovalbumin-specific CD8 mice. + TCR transgenic mice were provided by Xiaohuan Guo (Tsinghua University).

[0320] Tumor vaccination and treatment

[0321] To inhibit tumor growth in B6 mice, 5×10 5 One B16-OVA or 1×10 6E.G7-OVA tumor cells were subcutaneously (sc) injected into the flanks of mice. Tumor length (a) and width (b) were measured every two days and analyzed using formula ab. 2 / 2 Calculate tumor volume. For inhibitor treatment, 10 μM SAA or DMSO was administered intraperitoneally (ip) on days 9 and 11 post-tumor inoculation. For other mouse models (rag1) - / - For conditional knockout of Ythdf1, the number of inhibitor treatments and the dosage are the same. For treatment targeting the binding of α-PD-L1 to SAA, 5×10 5 B16-OVA tumor cells were subcutaneously inoculated into the flanks of mice. On day 9 post-inoculation, 100 μg of α-PD-L1 antibody (clone 10F.9G2) or rat immunoglobulin was administered. On days 9 and 11 post-inoculation, 10 μM SAA or DMSO was administered in the same manner.

[0322] FLT3L-DC culture and inhibitor treatment

[0323] From wild type and Ythdf1 - / - Bone marrow was isolated from mice and treated with erythrocyte lysis buffer to remove erythrocytes. Bone marrow cells were suspended in IMDM medium containing 10% fetal bovine serum. To culture FLT3L-DC, the cell concentration was adjusted to 1 × 10⁶ cells / mL. 6 / mL. Cells were cultured with 100 ng / mL FLT3L for 9 days to obtain mature FLT3L-DC. Mature FLT3L-DC was purified using the Easysep Mouse CD11c Positive Selection Kit II and then treated with 10 μM SAA or DMSO in IMDM medium (containing 10% bovine serum and 100 ng / mL FLT3L) for 10 hours.

[0324] DC antigen presentation function analysis

[0325] For in vitro cross-presentation studies, mature FLT3L-DCs were harvested on day 9 and purified using the Easysep Mouse CD11c Positive Selection Kit II. They were then treated with 10 μM SAA or DMSO in IMDM medium (containing 10% bovine serum and 100 ng / mL FLT3L) for 10 hours. After treatment with inhibitors (e.g., SAA or SAC), FLT3L-DCs were co-cultured with necrotic B16-OVA cells for 6 hours. The antigen-containing DCs were then purified and co-cultured with naïve T cells from OT-1 mice at a 1:10 ratio for 96 hours. The co-culture medium was 1640 RPMI containing 10% fetal bovine serum, with or without 1 μg / mL OT-1 (OVA 257-264) peptide. For in vitro DC cross-presentation analysis, four types of DCs (migratory CD11b) were sorted from the draining lymph nodes of SAA-treated B16-OVA tumor-bearing mice on day 12. + DC, migratory CD103 + DC, resident CD11b + DC and resident CD8 + DCs were co-cultured with OT-1 naïve T cells at a ratio of 1:10 for 96 hours with or without OT-1 peptide. IFN-γ production in the supernatant was detected by CBA analysis.

[0326] T cell function analysis

[0327] Tumor-infiltrating leukocytes were cultured in RPMI 1640 medium at a concentration of 5 × 10⁶ cells / mL. 6 T cells were suspended in 96-well plates. T cells were stimulated with phorbol-12-myristate-13-acetate (PMA) (2.5 μg / mL) and iomycin (0.5 μg / mL), while brefeldin A was added to the culture medium. The plates were incubated at 37°C for 2 hours. The total lymphocyte concentration from draining lymph nodes was adjusted to 5 × 10⁶ cells / mL in the 96-well plates. 6 Add 1 μg / mL OT-1 (OVA257-264) peptide to each well and stimulate the peptide for 96 hours. Stain the samples with CD45 and CD8 on ice for 30 minutes. Perform intracellular staining to quantify the production of IFN-γ and granzyme B.

[0328] Flow cytometry

[0329] For flow cytometry analysis and DC sorting, tumors and draining lymph nodes were collected from mice and digested with 100 U / mL collagenase IV and 20 μg / mL deoxyribonuclease I at 37°C for 40 min. Digestion was stopped by FACS buffer (PBS containing 2% FBS and 1 mM EDTA), and samples were filtered through a 70 μm cell sieve. Samples were stained with specific antibodies in FACS buffer on ice for 30 min. Antibody information is described in Table 1 below. All samples were washed with FACS buffer after staining, cells were analyzed on BDFortessa, and sorted using Aria III.

[0330] Table 1

[0331]

[0332]

[0333] CFSE mark

[0334] 1×10 7 Lymphocytes from the initial OT-I mouse lymph nodes were washed twice with PBS and then resuspended in 1 mL PBS. 1 μL of CFSE Tracker was added to the suspension and incubated at 37°C for 5 min. Then, 5 mL of RPMI-1640 medium containing 10% FBS was added to terminate CFSE labeling, and the cells were incubated at room temperature for 5 min. After centrifugation, the CFSE-labeled T cells were resuspended in another 5 mL of RPMI-1640 medium at room temperature for at least 10 min.

[0335] Example 1: Inhibitory activity against YTHDF1

[0336] To discover novel inhibitors of YTHDF1, a high-throughput screening (HTS) method based on fluorescence polarization (FP) analysis was developed, and salvianolic acid A (SAA) was found to be an effective inhibitor. The inhibitory effects of SAA on m... were evaluated in FP analysis. 6 The activity of the interaction between A's mRNA and YTHDF1, such as Figure 1 As shown in a, the obtained IC 50 The value was 2.30 ± 0.11 μM. To further verify the inhibitory activity of SAA, an AlphaScreen-based analysis was performed, and the results were as follows: Figure 1 As shown in b, the obtained IC 50 The value was 0.86 ± 0.06 M, confirming that SAA can effectively block m 6 The combination between A and YTHDF1.

[0337] Another type of salvianolic acid, namely salvianolic acid C (SAC), was also evaluated for its inhibitory effect on YTHDF1 activity using FP analysis, and as... Figure 1 As shown in c, the obtained IC 50 The value is 3.95 μM.

[0338] Then, qualitative and quantitative experiments were performed to explore the binding between SAA and YTHDF1. First, Carr–Purcell–Meiboom–Gill (CPMG) nuclear magnetic resonance (NMR) experiments were conducted. The results are as follows: Figure 1 As shown in d, the compound signal was detected in the CPMG spectrum after the addition of 200 μM SAA, and the signal weakened upon the addition of 5 μM, 10 μM and 20 μM YTHDF1, respectively, indicating direct binding between SAA and YTHDF1.

[0339] Next, the binding affinity between SAA and YTHDF1 was assessed. Isothermal titration calorimetry (ITC) analysis was performed to accurately determine the equilibrium dissociation constant (K₂) between YTHDF1 and SAA. d Three independent ITC analyses were performed on a Microcal iTC200 isothermal titration calorimeter (GE Healthcare). In short, freshly purified YTHDF1 (50 μM) was titrated with 1 mM SAA at 25°C in a buffer containing 20 mM Hepes (pH 7.4) and 200 mM NaCl. Figure 2 As shown in a-2c, SAA at 5.71 μM K d The binding of SAA to YTHDF1 at a certain value confirmed the interaction between SAA and YTHDF1. Furthermore, the enthalpy change (ΔH = -3099 ± 144.1 cal / mol) was less than zero, indicating that SAA can form hydrogen bonds with YTHDF1. Additionally, the entropy change (ΔS = 13.6 cal / mol / deg) was greater than zero, suggesting that the binding of SAA may induce a conformational change in YTHDF1.

[0340] In addition, surface plasmon resonance (SPR) analysis and micro-thermophoretic kinetic (MST) analysis were performed to confirm the binding strength between SAA and YTHDF1. Figure 3 As shown in a-3c, K obtained from SPR analysis d The value was 2.52 μM, and K was obtained from MST analysis. d The value was 4.70 μM, which is consistent with the results from the ITC experiment.

[0341] In conclusion, it can be shown that the compounds of this application (e.g., SAA, SAC, and other compounds of this application) can directly bind to YTHDF1 and block its m in vitro. 6 A binding activity.

[0342] Example 2: Non-competitive inhibition of YTHDF1 activity

[0343] Based on AlphaScreen analysis, 200 nM YTHDF1 and 20 nM biotinylated m 6 A's mRNA, for a fixed concentration of non-biotinylated m... 6 A competitive binding assay was performed on A's mRNA. The results are as follows: Figure 4 As shown in a-4b, in the absence of non-biotinylated m 6 In the case of A mRNA, SAA showed inhibitory activity against YTHDF1 of 0.80 ± 0.08 μM, and this activity was further enhanced when 50 nM, 100 nM, 200 nM, or 400 nM was used with non-biotinylated mRNA containing A. 6 When SAA mRNA was pre-incubated with YTHDF1, its inhibitory activity remained unchanged. These results show that SAA inhibits the function of YTHDF1, but it does not interact with m... 6 A competitive combination YTHDF1.

[0344] Then, hydrogen-deuterium exchange mass spectrometry (HDX MS) experiments were performed to determine the binding site of SAA on YTHDF1. In HDX MS analysis, the HDX behavior of YTHDF1-APO and YTHDF1-SAA at 10 s, 30 s, 60 s, 1200 s, 3600 s, and 14400 s was examined, and the residual plots were analyzed. Figure 5 a) Butterfly picture Figure 5 b) and heatmap ( Figure 6 The analysis revealed changes in deuterium uptake among them. Many peptides showed HDX percentage changes exceeding 5%, indicating that the binding of SAA to YTHDF1 can induce significant conformational changes in the intact protein structure.

[0345] like Figure 7 and Figure 8 As shown in a-8i, YTHDF1 and related peptides exhibit significant structural changes, with the conformational changes mainly occurring in the following three regions: 1) m 6 1) a binding pocket, 2) a long, shallow pocket with certain positively charged amino acids, and 3) the C-terminal α-helix of YTHDF1 (which underwent the most significant changes). These results indicate that SAA binds to one of these three regions of YTHDF1, thereby inducing a conformational change.

[0346] Then, YTHDF1 mutants and C-terminal truncated variants (with amino acid sequences as shown in SEQ ID NO:4-13) were designed, and these mutants and truncated variants were used to test the inhibitory activity of SAA.

[0347] like Figure 9 and Figure 10As shown in f-10j, when using the truncated body (SEQ ID NO:13) or m 6 When A binds to mutants with mutations in the pocket (e.g., mutants with the following mutations K395A (SEQ ID NO:9), Y397A (SEQ ID NO:10), C412A (SEQ ID NO:11), or R506A (SEQ ID NO:12)), SAA reacts with them and m 6 The inhibitory activity of A-binding was similar to that observed for wild-type YTHDF1, as measured in FP experiments. These results indicate that the C-terminal α-helix region and m 6 A-binding pockets are not necessary for SAA binding. Instead, as... Figure 9 and Figure 10 As shown in a-10e, when using mutants (SEQ ID NO:4-8) with one or more mutations in residues 372-392, 479-494, or 526-535 (e.g., mutations W384A, H528A, N378A, F480A, or F382A), SAA is effective against them and m 6 The inhibitory activity of A binding is significantly weaker than that of SAA against wild-type YTHDF1 and m 6 Inhibitory activity of A binding. Further studies using FP analysis showed that these mutations at residues 372-392, 479-494, or 526-535, as well as C-terminal truncations, did not affect YTHDF1's binding to m... 6 Binding affinity of A ( Figure 11 a-11g).

[0348] Based on this, it was found that SAA exerts non-competitive inhibitory activity against YTHDF1 through an allosteric mechanism, at least partially through hydrogen bond interactions between SAA and one or more residues of residues 372-392, 479-494, or 526-535.

[0349] Example 3: Combination of SAA and YTHDF1 in 293T

[0350] In addition, the binding of SAA to YTHDF1 in cells was examined. Cell thermal shift assays (CETSA) were performed using the 293T cell line, which was then collected for heating at a specified temperature. After incubation with 100 μM SAA for 4 h, the presence of YTHDF1 protein in the incubated 293T cells was examined by Western blotting. Figure 12 As shown in a-12b, incubation with SAA improved the stability of YTHDF1, and the curve shifted to the right by approximately 2 degrees, confirming that SAA can directly bind to YTHDF1 in the cytoplasm.

[0351] Example 4: In vivo tumor growth inhibition

[0352] 5×10 5 B16 melanoma cells expressing ovalbumin (OVA) were subcutaneously inoculated into wild-type mice. Subsequently, the tumor-bearing mice were treated with 10 μM SAA on days 9 and 11 post-inoculation, and tumor growth was monitored. Figure 13 As shown in figure a, compared with the control group, tumor growth was observed to be much slower in mice that received SAA.

[0353] In another experiment, wild-type mice were subcutaneously injected with 1×10 6 E.G7-OVA cells were injected into each mouse. Then, on days 9 and 11, each mouse was injected with 10 μM SAA. Tumor growth was monitored. A similar inhibitory trend was observed in the E.G7-OVA lymphoma model. Figure 13 b).

[0354] like Figure 19 As shown, 5×10 5 17 B16-OVA cells were subcutaneously inoculated into C57BL / 6 mice. On day 9 post-implantation, mice were divided into three groups based on tumor size. The mice were intravenously injected with DMSO (n=6), 10 μM SAA (n=6), or 10 μM SAC (n=5), respectively. Tumor growth was monitored. Data are presented as mean ± sem, and "ns" indicates no significance. Unpaired one-tailed t-tests were performed, with * p < 0.05 and ** p < 0.01. Tumor growth was observed to be significantly slower in mice receiving SAA or SAC.

[0355] Example 5 Effect on in vitro proliferation of tumor cells

[0356] In another experiment, tumor cells were treated with SAA in vitro. In short, 5 × 10⁵ cells were treated with different doses of SAA in 96-well plates. 4 B16-OVA tumor cells were counted 12 hours after SAA treatment. It was found that tumor cell proliferation was not affected even with increasing SAA dose. Figure 14 a). These results indicate that SAA does not exert its anti-tumor effect by directly killing tumor cells.

[0357] Example 6: Adaptive immunity is required for the antitumor activity of SAA.

[0358] In another experiment, 5 × 10⁵ SAA were treated with different doses in 96-well plates. 5 CFSE-labeled OT-IT cells were then stimulated with 1 μg / mL OT-I peptide for 24 hours. Dividing T cells were analyzed by FACS. Figure 14As shown in b, SAA does not affect the in vitro proliferation of T cells.

[0359] 1×10 5 One B16-OVA cell was seeded into T / B cell-deficient Rag1 cells. - / - Mice were then injected with 10 μM SAA from day 7 to day 9, and tumor growth was monitored. Wild-type mice were used as a control group. Figure 15 As shown in a, tumor growth is arrested in wild-type mice, but in T / B cell-deficient Rag1 mice... - / - Tumor growth did not stop in mice, indicating that adaptive immunity is required for the maximum anti-tumor therapeutic effect of SAA.

[0360] Example 7: SAA enhances the cross-presentation function of APC.

[0361] Bone marrow-derived cells (wild-type or Ythdf1 gene-deficient) were cultured with FLT3L for 9 days to obtain FLT3L-DCs. These DCs were then treated with 10 μM SAA for 12 hours, followed by co-culturing with necrotic B16-OVA tumor cells for 6 hours. CD11c+ cells were purified and co-cultured with OT-I T cells for 72 hours. IFN-γ production was assessed using an IFN-γ flow cytometry microsphere array.

[0362] like Figure 15 As shown in b, SAA-treated FLT3L-DCs exhibited better performance in cross-sensitized T cells compared to the control group. To compare the efficacy of SAA treatment with Ythdf1 gene knockout, Ythdf1 gene-deficient DCs (derived from Ythdf1) were used. - / - (Obtained from bone marrow) as a positive control. Interestingly, SAA also promotes Ythdf1. - / - Cross-presentation function of FLT3L-DC. These results indicate that the compounds of this application (e.g., SAA) can promote cross-sensitization function of antigen-presenting cells (e.g., DCs) in vitro.

[0363] In addition, on day 12, four types of typical DCs (resident CD11b) were isolated from the draining lymph nodes of SAA-treated B16-OVA tumor-bearing wild-type mice. + Resident CD8α + Migratory CD11b + and migratory CD103 + These DCs were co-cultured with OT-I T cells for 72 hours. Then, IFN-γ production was assessed using an IFN-γ flow cytometry microsphere array. Figure 15As shown in c, all these DC subtypes from SAA-treated mice showed better T-cell cross-sensitization compared to the DMSO-treated group, especially the resident CD8α subtype. + DC and migratory CD103 + DC. These results indicate that the compounds of this application (e.g., SAA) can also promote the cross-sensitization function of antigen-presenting cells (e.g., DCs) in vivo.

[0364] Example 8: The role of SAA in APC to enhance the immune response

[0365] Dendritic cells (DCs) can activate T cells through cross-sensitization and / or direct sensitization. During direct sensitization, DCs can stimulate T cells via surface co-stimulatory molecules such as CD80 / CD86 or cytokines associated with T cell activation. To assess whether direct sensitization was also affected after SAA administration, bone marrow-derived cells (wild-type or Ythdf1-deficient) were co-cultured with FLT3L for 9 days to obtain FLT3L-DCs. These DCs were then treated with 10 μM SAA for 12 hours, followed by co-culturing with necrotic B16-OVA tumor cells for 6 hours. CD11c+ cells were purified and co-cultured with OT-I T cells supplemented with 1 μg / mL OT-I peptide for 72 hours. IFN-γ production was assessed using an IFN-γ flow cytometry microsphere array. Furthermore, on day 12, four types of typical DCs (resident CD11b) were sorted from the draining lymph nodes of SAA-treated B16-OVA tumor-bearing wild-type mice. + Resident CD8α + Migratory CD11b + and migratory CD103 + These DCs were co-cultured with OT-I T cells supplemented with 1 μg / mL OT-I peptide for 72 hours. IFN-γ production was then assessed using an IFN-γ flow cytometry microsphere array.

[0366] like Figure 16 As shown in a-16b, although SAA did not enhance the direct sensitization function of FLT3L-DCs in vitro, three of the four DC subtypes showed improved direct sensitization ability after SAA treatment, indicating that SAA can enhance the direct sensitization ability of DCs in vivo. These results suggest that the compounds of this application (e.g., SAA) can function in APCs (e.g., dendritic cells) to enhance the immune response.

[0367] Example 9: APC is the main target of SAA

[0368] To determine whether DC is the primary target of SAA, Ythdf1 was used. F / F and CD11ccre Ythdf1 F / F mice subcutaneously injected with 2×10 6 Each mouse was injected with 10 μM SAA on days 9 and 11, and tumor growth was monitored. Figure 17 As shown in figure a, Ythdf1 processed by SAA F / F Tumor growth in mice showed similarities to that in CD11c cre Ythdf1 F / F Similar findings were observed in mice; however, in CD11c... cre Ythdf1 F / F No further significant tumor control was observed in mice, indicating that DC is the main target of SAA.

[0369] Example 10: SAA activates tumor-specific T cells

[0370] To investigate the effects of SAA treatment on T cell function, SAA-treated B16-OVA tumor-bearing mice were sacrificed to study tumor-infiltrating T cell (TIL) function. Tumor-infiltrating T cells were first nonspecifically stimulated with phorbol-12-myristate-13-acetate (PMA) and iomycin, followed by intracellular staining to quantify cytokine production (e.g., IFN-γ, granzyme B) via FACS. Figure 17 As shown in b, the levels of cytokines secreted by tumor-infiltrating T cells from mice treated with SAA were higher than those in the DMSO group, indicating that SAA can enhance the effector function of tumor-infiltrating T cells.

[0371] Lymphocytes were then isolated from the draining lymph nodes and stimulated with 1 μg / mL OT1 peptide. The IFN-γ producing cells were then analyzed by FACS. Figure 17 As shown in Figure c, DLN T cells from SAA-treated mice produced significantly more IFN-γ than those in the DMSO group. The results indicate that more tumor-specific T cells were activated in the draining lymph nodes after SAA administration.

[0372] In addition, PD-1 from the SAA treatment group was found. low The expression of CXCR5 in the group was higher than that in the DMSO group. Figure 18 a) Many PD-1 were detected 14 days after tumor inoculation in the case of SAA treatment. low CXCR5 highT cells (exhausted T cell precursors). Furthermore, terminally exhausted T cells (PD-1 and Tim-3 double-positive cells) were assessed 14 days post-tumor inoculation under SAA treatment, and these tumor-infiltrating terminally exhausted T cells (PD-1 and Tim-3 double-positive cells) were observed in SAA-treated mice. + Tim-3 + The frequency of ) decreased ( Figure 18 b) confirmed the enhanced antitumor activity.

[0373] These results show that after SAA treatment, T cells were better sensitized in draining lymph nodes, and the effector function of TILs was significantly improved.

[0374] Example 11: Synergistic effect of binding with immune checkpoint inhibitors

[0375] Subcutaneous injection of 5×10 5 B16-OVA cells were administered to each mouse. 10 μM SAA was injected into each mouse on days 9 and 11. Mice were also treated with 100 μg anti-PD-L1 antibody on day 9. Tumor growth was monitored over time. Figure 18 As shown in c, treatment with SAA or anti-PD-L1 antibodies alone can partially inhibit B16-OVA tumor growth, while combination therapy can significantly inhibit tumor growth and even achieve complete tumor regression. These results further support the idea that SAA can induce improved T cell anti-tumor capabilities through enhanced DC function, and that the combination of immune checkpoint inhibitors (such as anti-PD-L1 antibodies) with compounds of this application (e.g., SAA) can elicit a more durable T cell response.

[0376] like Figure 20 As shown, 5×10 523 B16-OVA cells were subcutaneously inoculated into C57BL / 6 mice. On day 9 post-implantation, mice were divided into two groups based on tumor size. On days 9 and 11 post-implantation, one group was treated intravenously with 10 μM SAA (n=6), and the other group was treated with DMSO (n=6). On day 12 post-implantation, each group was further divided into two subgroups, and each subgroup received an intraperitoneal injection of 200 μg of PD-1 blocking antibody (Bio-X-cell, BE0146, clone: ​​RMP1-14). The control group received PBS and DMSO (n=6). The treatment groups received either an isotype control with SAA or α-PD-1 with DMSO (n=6). The combination group received both SAA and PD-1 blocking antibody (n=5). Tumor growth was monitored. Data are presented as mean ± sem, and “ns” indicates no significance, as determined by an unpaired one-tailed t-test, with “**” p < 0.01 and “****” p < 0.00001. These results further support the idea that binding immune checkpoint inhibitors (such as anti-PD-L1 antibodies) to compounds of this application (e.g., SAA) can elicit a more durable T-cell response.

[0377] Example 12: SAA plays a role in APC to exhibit durable antitumor function.

[0378] like Figure 21 As shown, mature FLT3L DCs were pretreated with DMSO or 10 μM SAA for 10 hours, and then these DCs were co-cultured with necrotic B16-OVA for 6 hours. CD11c was purified. + Cells were used for adoptive transfer. 5 × 10⁶ cells were used. 5 17 B16-OVA cells were subcutaneously inoculated into C57BL / 6 mice (n=17). On day 9 post-implantation, the mice were divided into three groups according to tumor size. 1×10⁶ B16-OVA cells were then subcutaneously inoculated into each group. 5 Six DMSO-treated FLT3L DCs (n=6) or five SAA-treated FLT3L DCs (n=5) were intravenously transferred to tumor-bearing mice. A second batch of adoptive transfers was performed 7 days after the first transfer. Tumor growth was monitored. Data are presented as mean ± sem, and “ns” indicates no significance, assessed by unpaired one-tailed t-tests, with “**” p < 0.01 and “****” p < 0.00001. These results support the durable antitumor function of adoptive FLT3L DCs treated with compounds of this application (e.g., SAA).

[0379] While preferred embodiments of this application have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. This application is not intended to be limited to the specific embodiments provided in the specification. Although this application has been described with reference to the foregoing specification, the description and illustration of embodiments herein are not intended to be construed as limiting. Various changes, modifications, and substitutions will now occur to those skilled in the art without departing from this application. Furthermore, it should be understood that all aspects of this application are not limited to the specific descriptions, configurations, or relative proportions described herein, as this depends on various conditions and variables. It should be understood that various alternatives to the embodiments of this application described herein may be employed in the practice of this application. Therefore, this application is intended to also cover any such substitutions, modifications, variations, or equivalents. The following claims are intended to define the scope of this application, and the methods and structures within the scope of these claims and their equivalents will thereby be covered. SEQUENCE LISTING <110> Shanghai Institute of Materia Medica, Chinese Academy of Sciences; Hangzhou Lingzhi Pharmaceutical Technology Co., Ltd. Shanghai Kangqian Biotechnology Co., Ltd. <120> Compositions and methods for inhibiting YTHDF1 <130> 0128-PA-012CN <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 559 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 1 Met Ser Ala Thr Ser Val Asp Thr Gln Arg Thr Lys Gly Gln Asp Asn 1 5 10 15 Lys Val Gln Asn Gly Ser Leu His Gln Lys Asp Thr Val His Asp Asn 20 25 30 Asp Phe Glu Pro Tyr Leu Thr Gly Gln Ser Asn Gln Ser Asn Ser Tyr 35 40 45 Pro Ser Met Ser Asp Pro Tyr Leu Ser Ser Tyr Tyr Pro Pro Ser Ile 50 55 60 Gly Phe Pro Tyr Ser Leu Asn Glu Ala Pro Trp Ser Thr Ala Gly Asp 65 70 75 80 Pro Pro Ile Pro Tyr Leu Thr Thr Tyr Gly Gln Leu Ser Asn Gly Asp 85 90 95 His His Phe Met His Asp Ala Val Phe Gly Gln Pro Gly Gly Leu Gly 100 105 110 Asn Asn Ile Tyr Gln His Arg Phe Asn Phe Phe Pro Glu Asn Pro Ala 115 120 125 Phe Ser Ala Trp Gly Thr Ser Gly Ser Gln Gly Gln Gln Thr Gln Ser 130 135 140 Ser Ala Tyr Gly Ser Ser Tyr Thr Tyr Pro Pro Ser Ser Leu Gly Gly 145 150 155 160 Thr Val Val Asp Gly Gln Pro Gly Phe His Ser Asp Thr Leu Ser Lys 165 170 175 Ala Pro Gly Met Asn Ser Leu Glu Gln Gly Met Val Gly Leu Lys Ile 180 185 190 Gly Asp Val Ser Ser Ser Ala Val Lys Thr Val Gly Ser Val Val Ser 195 200 205 Ser Val Ala Leu Thr Gly Val Leu Ser Gly Asn Gly Gly Thr Asn Val 210 215 220 Asn Met Pro Val Ser Lys Pro Thr Ser Trp Ala Ala Ile Ala Ser Lys 225 230 235 240 Pro Ala Lys Pro Gln Pro Lys Met Lys Thr Lys Ser Gly Pro Val Met 245 250 255 Gly Gly Gly Leu Pro Pro Pro Pro Ile Lys His Asn Met Asp Ile Gly 260 265 270 Thr Trp Asp Asn Lys Gly Pro Val Pro Lys Ala Pro Val Pro Gln Gln 275 280 285 Ala Pro Ser Pro Gln Ala Ala Pro Gln Pro Gln Gln Val Ala Gln Pro 290 295 300 Leu Pro Ala Gln Pro Pro Ala Leu Ala Gln Pro Gln Tyr Gln Ser Pro 305 310 315 320 Gln Gln Pro Pro Gln Thr Arg Trp Val Ala Pro Arg Asn Arg Asn Ala 325 330 335 Ala Phe Gly Gln Ser Gly Gly Ala Gly Ser Asp Ser Asn Ser Pro Gly 340 345 350 Asn Val Gln Pro Asn Ser Ala Pro Ser Val Glu Ser His Pro Val Leu 355 360 365 Glu Lys Leu Lys Ala Ala His Ser Tyr Asn Pro Lys Glu Phe Glu Trp 370 375 380 Asn Leu Lys Ser Gly Arg Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp 385 390 395 400 Asp Ile His Arg Ser Ile Lys Tyr Ser Ile Trp Cys Ser Thr Glu His 405 410 415 Gly Asn Lys Arg Leu Asp Ser Ala Phe Arg Cys Met Ser Ser Lys Gly 420 425 430 Pro Val Tyr Leu Leu Phe Ser Val Asn Gly Ser Gly His Phe Cys Gly 435 440 445 Val Ala Glu Met Lys Ser Pro Val Asp Tyr Gly Thr Ser Ala Gly Val 450 455 460 Trp Ser Gln Asp Lys Trp Lys Gly Lys Phe Asp Val Gln Trp Ile Phe 465 470 475 480 Val Lys Asp Val Pro Asn Asn Gln Leu Arg His Ile Arg Leu Glu Asn 485 490 495 Asn Asp Asn Lys Pro Val Thr Asn Ser Arg Asp Thr Gln Glu Val Pro 500 505 510 Leu Glu Lys Ala Lys Gln Val Leu Lys Ile Ile Ser Ser Tyr Lys His 515 520 525 Thr Thr Ser Ile Phe Asp Asp Phe Ala His Tyr Glu Lys Arg Gln Glu 530 535 540 Glu Glu Glu Val Val Arg Lys Glu Arg Gln Ser Arg Asn Lys Gln 545 550 555 <210> 2 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 2 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 3 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 3 His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His 1 5 10 15 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Glu Phe 20 25 30 Glu Asn Leu Tyr Phe Gln Gly Ser Val Glu Ser His Pro Val Leu Glu 35 40 45 Lys Leu Lys Ala Ala His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn 50 55 60 Leu Lys Ser Gly Arg Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp 65 70 75 80 Ile His Arg Ser Ile Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly 85 90 95 Asn Lys Arg Leu Asp Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro 100 105 110 Val Tyr Leu Leu Phe Ser Val Asn Gly Ser Gly His Phe Cys Gly Val 115 120 125 Ala Glu Met Lys Ser Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp 130 135 140 Ser Gln Asp Lys Trp Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val 145 150 155 160 Lys Asp Val Pro Asn Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn 165 170 175 Asp Asn Lys Pro Val Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu 180 185 190 Glu Lys Ala Lys Gln Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr 195 200 205 Thr Ser Ile Phe Asp Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu 210 215 220 Glu Glu Val Val Arg Lys Glu Arg Gln Ser Arg Asn Lys Gln 225 230 235 <210> 4 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 4 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Ala Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 5 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 5 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ser Tyr Lys Ala Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195,200 <210> 6 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> THE <400> 6 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Ala Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 7 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 7 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Ala Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 8 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 8 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Ala Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 9 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 9 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Ala Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 10 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 10 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Ala Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 11 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 11 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Ala Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 12 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 12 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Ala Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile Phe Asp 165 170 175 Asp Phe Ala His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Val Val Arg 180 185 190 Lys Glu Arg Gln Ser Arg Asn Lys Gln 195 200 <210> 13 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> Y <400> 13 Gly Pro Ser Val Glu Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala 1 5 10 15 His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg 20 25 30 Val Phe Ile Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile 35 40 45 Lys Tyr Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp 50 55 60 Ser Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 65 70 75 80 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys Ser 85 90 95 Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp Lys Trp 100 105 110 Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp Val Pro Asn 115 120 125 Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp Asn Lys Pro Val 130 135 140 Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln 145 150 155 160 Val Leu Lys Ile Ile Ser Ser Tyr Lys His Thr Thr Ser Ile 165 170 <210> 14 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> mRNA <220> <221> modified_base <222> (1)..(1) <223> FAM labeled u <220> <221> modified_base <222> (12)..(12) <223> m6a <400> 14 uucuucugug gacugug 17 <210> 15 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> mRNA <220> <221> modified_base <222> (1)..(1) <223> biotinylated u <220> <221> modified_base <222> (12)..(12) <223> m6a <400> 15 uucuucugug gacugug 17 <210> 16 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Y1 <400> 16 Lys Ala Ala His Ser Tyr Asn Pro Lys Glu Phe Glu Trp Asn Leu Lys 1 5 10 15 Ser Gly Arg Val Phe 20 <210> 17 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Y2 <400> 17 Ile Phe Val Lys Asp Val Pro Asn Asn Gln Leu Arg His Ile Arg Leu 1 5 10 15 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Y3 <400> 18 Tyr Lys His Thr Thr Ser Ile Phe Asp Asp 1 5 10

Claims

1. Use of modified antigen-presenting cells (mAPCs) in the preparation of a drug, wherein the mAPCs have been treated with a YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) attenuator. in, The YTHDF1 depressant is the following compound or a pharmaceutically acceptable salt thereof: , The drug is intended to treat cancer in subjects who require it. The mAPC is a modified dendritic cell (mDC).

2. The use according to claim 1, wherein the cancer is a hematologic malignancy.

3. The use according to claim 1, wherein the cancer is lymphoma.

4. The use according to claim 1, wherein the cancer is a solid tumor.

5. The use according to claim 1, wherein the cancer is selected from the group consisting of: melanoma, breast cancer, lung cancer, ovarian cancer, liver cancer, cervical cancer, colon cancer, kidney cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, stomach cancer, pancreatic cancer, and testicular cancer.

6. The use according to claim 1, wherein the cancer is brain cancer.

7. The use according to claim 1, wherein the cancer is colorectal cancer.

8. The use according to claim 1, wherein the cancer is skin cancer.

9. The use according to claim 1, wherein the cancer is uterine cancer.

10. The use according to claim 1, wherein the cancer is leukemia.

11. The use according to claim 1, wherein the medicament further comprises a second active ingredient selected from the group consisting of: anti-PD-L1 antibody or its antigen-binding portion, anti-PD-1 antibody or its antigen-binding portion, anti-CTLA-4 antibody or its antigen-binding portion, and IDO inhibitors.

12. The use according to claim 11, wherein the second active ingredient is selected from the group consisting of pembrolizumab, nivolumab, cimipril, atezolizumab, avelumab, durvalumab, ipilimumab, and / or an antigen-binding fragment of any of the above.

13. The use according to claim 11, wherein the second active ingredient is contained in a separate container and is not mixed with the YTHDF1 depressant.

14. Use of the composition in the preparation of a medicament, said composition comprising a YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) attenuator and a second active ingredient, wherein, The YTHDF1 depressant is the following compound or a pharmaceutically acceptable salt thereof: , The second active ingredient is an anti-PD-L1 antibody or its antigen-binding portion. The drug is used to treat melanoma in subjects who require it.

15. The use according to claim 14, wherein it comprises a pharmaceutically acceptable carrier.

16. The use according to claim 14, wherein the second active ingredient is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

17. The use according to claim 14, wherein the second active ingredient is contained in a separate container and is not mixed with the YTHDF1 depressant.

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