Compositions and methods for inhibiting YTHDF1

Abstract

The present disclosure provides compositions and methods for attenuating YTHDF1 activity, and compositions and methods for promoting immune responses. For example, the present disclosure provides YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) attenuators, including compounds that bind to at least one residue of YTHDF1 when bound to YTHDF1.

Description

[Background technology]

[0001] Spontaneous T-cell priming against tumor neoantigens is essential for the clinical efficacy of immunotherapy. However, in many patients, neoantigen recognition is insufficient to induce the sustained T-cell response necessary for complete tumor rejection. Identifying molecular pathways that influence immunoreactivity to tumor neoantigens may provide targets for improving the response to immunotherapy. For example, the most abundant internal mRNA modification is m 6 A is involved in the post-transcriptional regulation of mRNA in various cell types. Furthermore, m 6 A is m 6 The translation efficiency of mRNA can be influenced via the A-binding protein YTH domain-containing family protein 1 (YTHDF1). Previous studies have shown that attenuating YTHDF1 activity in various cells of the immune system (e.g., antigen-presenting cells) may help induce a sufficient and sustained antitumor immune response. However, effective compositions and methods for attenuating YTHDF1 activity are still strongly desired. [Disclosure of the Invention]

[0002] This application provides compositions and methods for attenuating the activity of YTHDF1. This application further provides modified antigen-presenting cells (mAPCs), such as modified dendritic cells, having enhanced activity. The compositions and mAPCs of this application may be used for one or more of the following purposes: 1) activating APCs (e.g., DCs); 2) generating immune cells having enhanced antitumor activity; 3) preventing and / or reversing exhaustion of immune cells (such as T cells); 4) treating diseases, disorders or conditions related to antigen expression in candidates in need; 5) treating cancer in candidates in need; 6) stimulating T cell-mediated immune responses to cancer cells and / or tumor antigens in candidates in need; 7) providing antitumor immunity in candidates in need; 8) increasing and / or improving the proliferation and / or activity of tumor-infiltrating T cells; 9) increasing and / or improving the proliferation and / or activity of tumor-specific T cells; 10) enhancing cytokine production by T cells; 11) enhancing the antitumor response of cancer immunotherapy; 12) inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells. This application further provides a method and composition for enhancing the immune response by combining the YTHDF1 attenuator of this application with a second activator such as an immune checkpoint inhibitor.

[0003] YTHDF1, a member of the YTH domain family, is m 6 It is a "leader" of A modification. For example, by interacting with translation initiation factors, YTHDF1 helps to promote mRNA translation efficiency. Furthermore, dysregulation of YTHDF1 may disrupt the expression balance between oncogenes and tumor suppressors, suggesting a link between YTHDF1 and tumorigenesis. Overexpression of YTHDF1 has been reported to be associated with malignancies such as colorectal cancer (CRC) and hepatocellular carcinoma (HCC). Furthermore, Ythdf1 deficiency (Ythdf1 - / -We found that mice exhibited an enhanced antitumor immune response, suggesting that YTHDF1 is a novel potential therapeutic target. We also found that YTHDF1 is associated with the expression of T cell exhaustion sign genes. Mice lacking YTHDF1 in their T cells showed superior antitumor immunity against lymphoma, solid tumors (such as melanoma and colon cancer), and other types of cancer. In YTHDF1-deficient mice, tumor-infiltrating T cell function was enhanced. Furthermore, the branching of T cell exhaustion was linked to memory-like or stem-like CD8 + The T cells were saved from their fate.

[0004] On the other hand, the present invention provides a YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) weakening agent comprising a compound that, when binding to YTHDF1, binds to at least one residue corresponding to a residue selected from amino acid residues 372-392, 479-494, and 526-535 of SEQ ID NO: 1.

[0005] In some embodiments, when binding to YTHDF1, the compounds contained in the YTHDF1 weakening agent bind to at least one residue corresponding to residues N378, F382, W384, F480, and H528 of SEQ ID NO: 1.

[0006] In some embodiments, the compounds included in the YTHDF1 weakening agent are m 6 This can block the binding to A.

[0007] In some embodiments, the compounds included in the YTHDF1 weakening agent have a relationship to binding to YTHDF1, m 6 It does not compete with A in any meaningful way.

[0008] In some embodiments, the YTHDF1 weakening agent comprises the compound of formula I, a prodrug of the compound of formula I, a metabolite, a derivative, or a pharmaceutically acceptable salt, ester, or amide of any of the above. [ka] , where R1 is selected from the group consisting of C 1-50 hydrocarbyl, C 1-50 substituted hydrocarbyl, C 1-50 heterohydrocarbyl and C 1-50 substituted heterohydrocarbyl.

[0009] In some embodiments, R1 in formula I is (CO)-R2, and R2 is optionally substituted alkenyl. In some embodiments, R2 is CH=CH-R3, and R3 is optionally substituted aryl. In some embodiments, R3 is formula II

Chemical formula

Chemical formula

[0010] In some embodiments, in formula II, A is

Chemical formula

Chemical formula

[0011] ​​​​​​​​​​​​​​​​​​​In some embodiments, the compounds contained in the YTHDF1 weakening agent of the present application include at least two dihydroxyphenyl moieties.

[0013] In some embodiments, the compounds contained in the YTHDF1 weakening agent of the present application include at least three dihydroxyphenyl moieties.

[0014] In some embodiments, the YTHDF1 weakening agent includes the compound of formula III, a prodrug of the compound of formula III, a metabolite, a derivative, or a pharmaceutically acceptable salt, ester, or amide of any of the above. [ka] Here, A is a franc or which can be optionally substituted. [ka] Thus, R6 is a hydroxyl group, and R5 is an optionally substituted alkenyl group.

[0015] In some embodiments, in formula III, A is [ka] And R4 is [ka] That is the case.

[0016] In some embodiments, in formula III, A is [ka] R6 is hydroxyl, R5 is CH=CH-R7, and R7 is [ka] That is the case.

[0017] 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: [ka]

[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. [ka]

[0019] 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. [ka]

[0020] In some embodiments, the compounds included in the YTHDF1 attenuator are derived from plants.

[0021] In some embodiments, the compounds contained in the YTHDF1 attenuator are provided in plant extracts. In some embodiments, the plant is of the genus Salvia. In some embodiments, the plant is Salvia myrthiolyza (Danshen).

[0022] On the other hand, the present application provides modified antigen-presenting cells (mAPCs) that are treated with and / or contain the YTHDF1 attenuator of the present application. In some embodiments, the mAPCs are modified dendritic cells (mDCs).

[0023] On the other hand, the present application provides a composition comprising the YTHDF1 attenuator and / or the mAPC of the present application. In some embodiments, the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is a vaccine composition.

[0024] In some embodiments, the above composition further comprises a second active ingredient. In some embodiments, the second active ingredient is an anticancer agent.

[0025] 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 a drug selected from the group consisting of an anti-PD-L1 antibody or its antigen-binding moiety, an anti-PD-1 antibody or its antigen-binding moiety, an anti-CTLA-4 antibody or its antigen-binding moiety, and an IDO inhibitor.

[0026] In some embodiments, the second active ingredient described above includes pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, and / or antigen-binding fragments or derivatives of any of the above.

[0027] In some embodiments, the second active ingredient described above can cause an increase in one or more tumor antigens in a test subject who receives it.

[0028] In some embodiments, the tumor antigen is selected from the group consisting of 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 versions thereof.

[0029] In some embodiments, the second active ingredient is contained in a separate container and is not mixed with the mAPC or the YTHDF1 attenuator.

[0030] On the other hand, the present invention provides a method for reducing the activity of YTHDF1, which includes administering an effective amount of the present YTHDF1 attenuator.

[0031] In some embodiments, the above method is an in vivo method. In some embodiments, the above method is an in vitro method. In some embodiments, the above method is an ex vivo method.

[0032] On the other hand, the present invention provides a method for determining whether a candidate drug is a YTHDF1 attenuator, comprising contacting the above-mentioned candidate drug with a YTHDF1 mutant having 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.

[0033] In some embodiments, the above YTHDF1 variants include one or more amino acid substitutions, deletions, and / or additions in one or more residues corresponding to residues selected from residues N378, F382, W384, F480, and H528 of SEQ ID NO: 1.

[0034] In some embodiments, the method further includes determining whether the candidate drug specifically binds to the YTHDF1 variant.

[0035] On the other hand, the present application provides a kit containing the YTHDF1 variant of the present application.

[0036] On the other hand, the present application provides the use of a compound in the production of a YTHDF1 weakening agent, which, when binding to YTHDF1, binds to at least one residue corresponding to a residue selected from amino acid residues 372-392, 479-494, and 526-535 of SEQ ID NO: 1.

[0037] In some embodiments, when the compound binds to YTHDF1, it binds to at least one residue corresponding to residues N378, F382, W384, F480, and H528 of SEQ ID NO: 1.

[0038] In some embodiments, the compound is m 6 This can block the binding to A.

[0039] In some embodiments, the compound has a bond to YTHDF1, m 6 It does not compete with A in any meaningful way.

[0040] In some embodiments, the compound comprises the compound of formula I, a prodrug of the compound of formula I, a metabolite, a derivative, or a pharmaceutically acceptable salt, ester, or amide of any of the above. [ka] Here, R1 is C 1-50 Hydrocarbyl, C 1-50 Substituted hydrocarbyl, C 1-50 Heterohydrocarbyl, and C 1-50 The group is selected from those consisting of substituted heterohydrocarbyl compounds.

[0041] In some embodiments, in the compound of formula I, R1 is (CO)-R2, and R2 is an optionally substituted alkenyl. In some embodiments, R2 is CH=CH-R3, and R3 is an optionally substituted aryl.

[0042] In some embodiments, R3 is given by equation II [ka] Here, A is a franc or which can be optionally substituted. [ka] Thus, R6 is a hydroxyl group, and R5 is an optionally substituted alkenyl group.

[0043] In some embodiments, in formula II, A is [ka] And R4 is [ka] That is the case.

[0044] In some embodiments, in formula II, A is [ka] R6 is hydroxyl, R5 is CH=CH-R7, and R7 is [ka] That is the case.

[0045] In some embodiments, the compound comprises at least two dihydroxyphenyl moieties.

[0046] In some embodiments, the compound comprises at least three dihydroxyphenyl moieties.

[0047] In some embodiments, the compound comprises the compound of formula III, a prodrug of the compound of formula III, a metabolite, a derivative, or a pharmaceutically acceptable salt, ester, or amide of any of the above. [ka] Here, A is a franc or which can be optionally substituted. [ka] Thus, R6 is a hydroxyl group, and R5 is an optionally substituted alkenyl group.

[0048] In some embodiments, in formula III, A is [ka] And R4 is [ka] That is the case.

[0049] In some embodiments, A is [ka] R6 is hydroxyl, R5 is CH=CH-R7, and R7 is [ka] That is the case.

[0050] 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. [ka]

[0051] In some embodiments, the compound is derived from a plant. In some embodiments, the compound is provided as a plant extract. In some embodiments, the plant is a plant of the genus Salvia. In some embodiments, the plant is Salvia myrthiolyza (Danshen).

[0052] On the other hand, the present application provides a method for activating APC, which includes administering the YTHDF1 attenuator of the present application to said APC.

[0053] On the other hand, the present invention provides a method for activating a DC, which includes administering the YTHDF1 attenuator of the present invention to the DC.

[0054] On the other hand, the present application provides a method for treating a disease, disorder or condition related to the expression of an antigen in a test subject in need, the method comprising administering to the test subject the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application.

[0055] In some embodiments of the method, the antigen is a tumor antigen.

[0056] In some embodiments of the method, the antigen is a tumor antigen selected from the group consisting of 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 versions.

[0057] In some embodiments of the method, the disease, disorder, or condition is cancer.

[0058] In some embodiments, the cancer is selected from the group consisting of hematological malignancies, lymphomas, and solid tumors.

[0059] In some embodiments, the cancer is selected from the group consisting of 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.

[0060] On the other hand, the present application provides a method for treating cancer in a candidate who requires it, comprising administering to the candidate the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application. In some embodiments, the cancer is selected from the group consisting of hematological cancers, lymphomas, and solid tumors. In some embodiments, the cancer is selected from the group consisting of 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.

[0061] On the other hand, the present application provides a method for stimulating a T cell-mediated immune response to cancer cells and / or tumor antigens in a test subject who requires it, the method comprising administering to the test subject the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application. In some embodiments, the tumor antigen is selected from the group consisting of 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 versions thereof.

[0062] On the other hand, the present application provides a method for providing antitumor immunity to a test subject who requires it, comprising administering to the test subject the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application.

[0063] On the other hand, the present application provides a method for preventing and / or reversing T cell exhaustion in a test subject who requires it, the method comprising administering to the test subject the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application.

[0064] On the other hand, the present application provides a method for enhancing T cell activity in a test subject who requires it, comprising administering to the test subject the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application. In some embodiments, the T cells include tumor-infiltrating T cells. In some embodiments, the T cells include tumor-specific T cells.

[0065] In some embodiments of the method of the present application, the test subject is a cancer patient. In some embodiments, the cancer is selected from the group consisting of hematological cancers, lymphomas, and solid tumors. In some embodiments, the cancer is selected from the group consisting of 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] In some embodiments, the test 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 an immune checkpoint inhibitor. In some embodiments, the anticancer treatment includes a drug selected from the group consisting of an anti-PD-L1 antibody or its antigen-binding moiety, an anti-PD-1 antibody or its antigen-binding moiety, an anti-CTLA-4 antibody or its antigen-binding moiety, and an IDO inhibitor. In some embodiments, the anticancer treatment includes pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, ipilimumab, and / or an antigen-binding fragment or derivative of any of the above. In some embodiments, the anticancer treatment can cause an increase in one or more tumor antigens in the test subject. In some embodiments, the tumor antigen is selected from the group consisting of 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 versions thereof.

[0067] In some embodiments, the method further comprises administering one or more additional anticancer therapies to the test subject. In some embodiments, the additional anticancer therapy includes cancer immunotherapy. In some embodiments, the additional anticancer therapy includes an immune checkpoint inhibitor. In some embodiments, the additional anticancer therapy includes a drug selected from the group consisting of an anti-PD-L1 antibody or its antigen-binding moiety, an anti-PD-1 antibody or its antigen-binding moiety, an anti-CTLA-4 antibody or its antigen-binding moiety, and an IDO inhibitor. In some embodiments, the additional anticancer therapy includes pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, ipilimumab, and / or antigen-binding fragments or derivatives of any of the above. In some embodiments, the additional anticancer therapy can cause an increase in one or more tumor antigens in the test subject. In some embodiments, the tumor antigen is selected from the group consisting of 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 versions thereof.

[0068] On the other hand, this application aims to: 1) activate APCs, 2) activate DCs, 3) generate immune cells with enhanced antitumor activity, 4) prevent and / or reverse the exhaustion of immune cells (such as T cells), 5) treat diseases, disorders or conditions related to antigen expression in candidates in need, 6) treat cancer in candidates in need, 7) stimulate T cell-mediated immune responses against cancer cells and / or tumor antigens in candidates in need, 8) provide antitumor immunity to candidates in need, and 9) treat tumor-infiltrating T cells. The present invention provides the use of the YTHDF1 attenuator, the mAPC, and / or compositions of the present invention in the manufacture of compositions and / or pharmaceuticals used for one or more purposes of increasing and / or improving cell proliferation and / or activity, 10) increasing and / or improving tumor-specific T cell proliferation and / or activity, 11) enhancing T cell cytokine production, 12) enhancing the antitumor response of cancer immunotherapy, and 13) inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells.

[0069] In some embodiments, the cancer or tumor is selected from the group consisting of hematological cancers, lymphomas, and solid tumors. In some embodiments, the cancer or tumor is selected from the group consisting of 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, lymphomas, and leukemia.

[0070] On the other hand, this application aims to: 1) activate APCs; 2) activate DCs; 3) generate immune cells with enhanced antitumor activity; 4) prevent and / or reverse the exhaustion of immune cells (such as T cells); 5) treat diseases, disorders, or conditions related to antigen expression in candidates in need; 6) treat cancer in candidates in need; 7) stimulate T cell-mediated immune responses against cancer cells and / or tumor antigens in candidates in need; 8) provide antitumor immunity to candidates in need; and 9) promote the proliferation of tumor-infiltrating T cells. The present invention provides the use of the YTHDF1 attenuator, the mAPC, and / or compositions of the present invention in combination with additional active ingredients in the manufacture of a pharmaceutical product used for one or more of the purposes of increasing and / or improving the activity of tumor-specific 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 of cancer immunotherapy, and 13) inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells.

[0071] In some embodiments, the additional active ingredient comprises cancer immunotherapy. In some embodiments, the additional active ingredient comprises an immune checkpoint inhibitor. In some embodiments, the additional active ingredient comprises a drug selected from the group consisting of an anti-PD-L1 antibody or its antigen-binding moiety, an anti-PD-1 antibody or its antigen-binding moiety, an anti-CTLA-4 antibody or its antigen-binding moiety, and an IDO inhibitor. In some embodiments, the additional active ingredient comprises pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, ipilimumab, and / or an antigen-binding fragment or derivative of any of the above. In some embodiments, the additional active ingredient can cause an increase in one or more tumor antigens in a test subject who receives it. In some embodiments, the tumor antigen is selected from the group consisting of 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 versions thereof.

[0072] Other aspects and advantages of this disclosure will be readily apparent to those skilled in the art through the following detailed description. This disclosure provides only exemplary embodiments, as described in the following detailed description. As will be understood, this disclosure can be implemented in other different embodiments, and some of its details can be modified in various obvious ways without departing from the present application. Accordingly, the accompanying drawings and specification should be considered illustrative and not limiting. Incorporation by Reference

[0073] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent that each individual publication, patent, and patent application is specifically and individually incorporated by reference. [Brief explanation of the drawing]

[0074] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description and accompanying drawings (also "Figures" and "FIGs" herein) illustrating exemplary embodiments in which the principles of the present invention are employed.

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[0075] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are merely examples. Those skilled in the art will conceive of various modifications, alterations, and substitutions, provided they do not depart from the present invention. It should be understood that various substitutional forms may be applied to the embodiments of the present invention described herein.

[0076] On the other hand, the present application provides a YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) weakening agent. The YTHDF1 weakening agent may include a compound that, when binding to YTHDF1, can bind to at least one residue corresponding to a residue selected from amino acid residues 372-392, 479-494, and 526-535 of SEQ ID NO: 1.

[0077] On the other hand, the present application provides modified antigen-presenting cells (mAPCs), which may be treated with the YTHDF1 attenuator of the present application. In some embodiments, the mAPCs may contain the YTHDF1 attenuator of the present application. The mAPCs may be modified dendritic cells (mDCs).

[0078] On the other hand, the present application provides a composition (such as a pharmaceutical composition). The composition may contain the YTHDF1 attenuator of the present application. Alternatively, the composition may further contain the mAPC of the present application. The composition may contain a pharmaceutically acceptable carrier. In some cases, the composition may be a vaccine composition.

[0079] In some cases, the composition may contain additional or second active ingredients. In this application, the terms “additional active ingredients” and “second active ingredients” may be used interchangeably.

[0080] The additional or second active ingredient may be contained in a separate container and not mixed with the mAPC or YTHDF1 attenuator of the present application.

[0081] In some cases, the additional active ingredient may be contained in the same package or container as the mAPC and / or YTHDF1 attenuator of the present application. In some cases, the additional active ingredient may be contained in a separate container, for example, in a container different from the container containing the mAPC and / or YTHDF1 attenuator of the present application. In some cases, the additional active ingredient may be present in the same container or package as the mAPC and / or YTHDF1 attenuator of the present application but not in direct contact with them (e.g., not mixed).

[0082] On the other hand, the present application provides a method for attenuating the activity of YTHDF1, which may include administering an effective amount of the YTHDF1 attenuator of the present application.

[0083] On the other hand, the present application provides a method for determining whether a candidate drug is a YTHDF1 attenuator. This method may include contacting the candidate drug with a YTHDF1 variant. The YTHDF1 variant may contain one or more amino acid substitutions, deletions, and / or additions in one or more residues selected from residues 372-392, 479-494, and 526-535 of SEQ ID NO: 1.

[0084] On the other hand, the present application provides a kit, which may contain the YTHDF1 variant of the present application.

[0085] On the other hand, this application provides the use of a compound in the production of a YTHDF1 weakening agent. When the compound binds to YTHDF1, it can bind to at least one residue selected from amino acid residues 372-392, 479-494, and 526-535 of SEQ ID NO: 1.

[0086] On the other hand, the present invention provides a method for activating APC. This method may include administering the YTHDF1 attenuator of the present invention to the APC.

[0087] On the other hand, the present application provides a method for activating DCs. This method may include administering the YTHDF1 attenuator of the present application to the DCs.

[0088] On the other hand, the present application provides a method for treating a disease, disorder, or condition related to the expression of an antigen in a test subject who requires it. The method may include administering to the test subject the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application.

[0089] On the other hand, the present application provides a method for inhibiting tumor growth, inhibiting the proliferation of tumor cells, and / or killing tumor cells. The method may include administering the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the compositions of the present application to the tumor and / or tumor cells.

[0090] On the other hand, the present application provides a method for treating cancer in a test subject who requires it. The method may include administering to the test subject the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application.

[0091] On the other hand, the present application provides a method for stimulating a T cell-mediated immune response against cancer cells and / or tumor antigens (for example, in a test subject who requires it). The method may include administering the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application to the test subject.

[0092] On the other hand, the present application provides a method for providing antitumor immunity to a test subject who requires it. The method may include administering to the test subject the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the composition of the present application.

[0093] On the other hand, the present application provides a method for preventing and / or reversing the exhaustion of immune cells, such as immune effector cells (e.g., T cells), in examinees in need. The method may include administering the examinee the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the compositions of the present application.

[0094] On the other hand, the present application provides a method for enhancing the activity of immune cells, such as immune effector cells (e.g., T cells), in examinees who require it. The method may include administering the YTHDF1 attenuator of the present application, the mAPC of the present application, and / or the compositions of the present application to the examinee. In some embodiments, the immune cells include tumor-infiltrating T cells. In some embodiments, the immune cells include tumor-specific T cells.

[0095] On the other hand, the present invention aims to: 1) activate APCs, 2) activate DCs, 3) generate immune cells with enhanced antitumor activity, 4) prevent and / or reverse the exhaustion of immune cells (immune effector cells, e.g., T cells), 5) treat diseases, disorders or conditions related to antigen expression in candidates in need, 6) treat cancer in candidates in need, 7) stimulate immune cell (e.g., immune effector cells such as T cells)-mediated immune responses against cancer cells and / or tumor antigens in candidates in need, 8) provide antitumor immunity to candidates in need, and 9) stimulate immune cells (e.g., T cells, e.g. The present invention provides the use of the YTHDF1 attenuator, mAPC and / or compositions of the present invention in the manufacture of compositions and / or pharmaceuticals used for one or more purposes of increasing and / or improving the proliferation and / or activity of immune effector cells (such as tumor-infiltrating T cells), increasing and / or improving the proliferation and / or activity of tumor-specific immune cells (such as immune effector cells such as T cells), enhancing cytokine production by T cells, enhancing the antitumor response of cancer immunotherapy, and inhibiting tumor growth, inhibiting tumor cell proliferation and / or killing tumor cells.

[0096] On the other hand, the present invention aims to: 1) activate APCs, 2) activate DCs, 3) generate immune cells with enhanced antitumor activity, 4) prevent and / or reverse the exhaustion of immune cells (e.g., immune effector cells such as T cells), 5) treat diseases, disorders or conditions related to antigen expression in candidates in need, 6) treat cancer in candidates in need, 7) stimulate immune cell (e.g., immune effector cells such as T cells)-mediated immune responses against cancer cells and / or tumor antigens in candidates in need, 8) provide antitumor immunity to candidates in need, and 9) stimulate immune cells (e.g., T cells, e.g., tumor cells) The present invention provides the use of the YTHDF1 attenuator, mAPC and / or compositions of the present invention in combination with additional active ingredients in the manufacture of a pharmaceutical product used for one or more of the purposes of: 10) increasing and / or improving the proliferation and / or activity of immune effector cells (such as infiltrating T cells); 11) enhancing cytokine production by T cells; 12) enhancing the antitumor response of cancer immunotherapy; and 13) inhibiting tumor growth, inhibiting tumor cell proliferation and / or killing tumor cells.

[0097] As used herein, the terms “include,” “have,” “can,” and “contain,” and their variations thereto, generally mean open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include multiple references.

[0098] In this specification, the notation of numerical ranges explicitly assumes that each number within that range is represented with a similar degree of precision. For example, the range 6 to 9 assumes the numbers 6, 9, 7, and 8, and the range 6.0 to 7.0 explicitly assumes 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. Therefore, the descriptions of the range forms are merely for convenience and simplification and should not be interpreted as strictly limiting the scope of application of the present invention. The range descriptions should be understood as specifically disclosing not only the individual numbers within that range, but also all possible sub-ranges. For example, a range description like 1-6 should be understood as specifically disclosing subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual values ​​within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Similarly, a range like 95-100% (e.g., 95-96%, 95-97%, 95-98%, 95-99%, 95-99.5%, or above) includes identical values ​​such as 95%, 96%, 97%, 98%, or 99%, with subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99%. This applies regardless of the size of the range.

[0099] When used in relation to quantities, the modifier "approximately" includes the stated value and has contextual meaning (for example, at least the degree of error associated with the measurement of a particular quantity). The modifier "approximately" should be understood as disclosing a range defined by the absolute values ​​of two endpoints. For example, the expression "approximately 2 to approximately 4" also discloses a range of "2 to 4". When referring to measurable values ​​such as quantities or temporal durations, the term "approximately" encompasses and is appropriate for variations of ±20%, possibly ±10%, possibly ±5%, possibly ±1%, and possibly ±0.1% from the specified value.

[0100] As used herein, the term “subject” usually means human or animal. For example, it may refer to any vertebrate, including (but not limited to) mammals (e.g., cattle, pigs, camels, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as crab-eating macaques and chimpanzees) and humans). In some embodiments, the subject is human.

[0101] The term “treat / treated / treating” may be used interchangeably herein and typically refers to a method of treatment that alleviates (reduces) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desirable clinical outcome. In some aspects of this disclosure, beneficial or desirable clinical outcomes include, but are not limited to, relief of symptoms, reduction of the severity of a condition, disorder or disease, stabilization (without exacerbation) of a condition, disorder or disease, delay of the onset of a condition, disorder or disease, or mitigation of the progression of a condition, disorder or disease, improvement and remission (whether partial or complete remission, detectable or undetectable), or enhancement or improvement of a condition, disorder or disease. Treatment also includes extending survival compared to the survival expected without treatment.

[0102] The term "modified / modify / modification" is interchangeable herein and typically means the introduction or generation of alteration or change. When used in the context of genes, the above modifications may include any conventional method that causes an alteration in the activity and / or function of a cell. For example, this could be exposing a cell (e.g., an antigen-presenting cell) to a drug that can modulate the activity and / or function of the cell.

[0103] The terms “attenuating / attenuation / attenuated” are used interchangeably and, as used herein, may refer to the inhibition or reduction of the amount of a target gene or target protein (such as YTHDF1 or a target of YTHDF1), or the inhibition or reduction of the activity of a target gene or target protein (such as YTHDF1 or a target of YTHDF1). Such attenuation can be achieved, for example, by using antibodies or their derivatives, antibody-drug conjugates, fusion proteins, small molecules, antisense molecules, dsRNA, siRNA, shRNA, aptamers and / or gRNA (in combination with, for example, gene editing systems (such as CRIPSR / Cas9)). Alternatively, YTHDF1 can be attenuated, for example, by contacting antigen-presenting cells (e.g., dendritic cells) with a YTHDF1 inhibitor (such as the compounds of this application) to inhibit / block the binding and / or recognition of m6A-modified mRNA by YTHDF1.

[0104] As used herein, the term “small molecule” typically refers to any chemical substance or other component other than polypeptides and nucleic acids that can be used to influence biological processes, particularly the regulation of m6A mRNA modification (e.g., YTHDF1 activity). Small molecules may include any number of therapeutics currently known and used, or any number of therapeutics synthesized in libraries of such molecules for the purpose of screening biological functions. Small molecules are distinguished from macromolecules by size. Small molecules may have molecular weights 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 may include (but are not limited to) organic compounds and their peptide mimes, as well as complexes.

[0105] In its broadest sense as used herein, the term “amino acid” means any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid. In some embodiments, an amino acid is a d-amino acid. In some embodiments, an amino acid is a 1-amino acid. “Standard amino acid” means any of the 20 standard 1-amino acids commonly found in naturally occurring peptides. “Non-standard amino acid” means any amino acid other than a standard amino acid, whether synthetically prepared or obtained from natural sources. In this specification, “synthetic amino acid” includes, but is not limited to, salts, amino acid derivatives (such as amides), and / or substitutions of chemically modified amino acids. Amino acids (including carboxy-terminal and / or amino-terminal amino acids of peptides) can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the cyclic half-life of the peptide without adversely affecting it. Amino acids can be involved in disulfide bonds. An amino acid may contain one or more post-translational modifications, such as the binding of one or more chemical entities (e.g., a methyl group, acetate group, acetyl group, phosphate group, formyl moiety, isoprenoid group, sulfate group, polyethylene glycol moiety, lipid moiety, carbohydrate moiety, biotin moiety, 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.

[0106] As used herein, the term "YTHDF1" generally refers to N6-methyladenosine (m 6A) This refers to YTH N6-methyladenosine RNA-binding protein 1 or a functional fragment thereof, which specifically recognizes and binds to contained RNA and regulates mRNA stability. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human YTHDF1 can be found under accession number NP_060268.2, and the encoding mRNA sequence can be found under accession number NM_017798.4.

[0107] As used herein, the term “YTHDF1 variant” typically refers to a YTHDF1-encoding nucleic acid molecule or YTHDF1 protein that has one or more mutations compared to the corresponding parental or reference (e.g., wild-type) nucleic acid molecule or corresponding parental or reference (e.g., wild-type) YTHDF1 protein that encodes YTHDF1.

[0108] Regarding YTHDF1 mutant proteins, 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 mutant proteins may include deletions, substitutions, and / or additions of one or more amino acids. Mutations can range in size from a single amino acid to a large fragment of a polypeptide. In some embodiments, insertions change the number of amino acids in a polypeptide by adding a fragment of the polypeptide. In some embodiments, deletions change 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 a polypeptide. In some embodiments, substitutions replace one amino acid in a polypeptide with a different amino acid. Substitutions may be conserved amino acid substitutions or non-conserved amino acid substitutions. A "conserved amino acid substitution" generally means replacing an amino acid present in a sequence with a different amino acid that is similar in size, charge, polarity, and / or chemical properties. Examples of conserved substitutions may include the substitution of a nonpolar (hydrophobic) residue, such as isoleucine, valine, and leucine, with another nonpolar residue. Similarly, examples of conserved substitutions include the substitution of a polar (hydrophilic) residue with another polar residue, such as arginine and lysine, glutamine and asparagine, and serine of glycine. Furthermore, substitutions of a basic residue with another basic residue, such as lysine, arginine, or histidine, or substitutions of an acidic residue with another acidic residue, such as aspartic acid or glutamic acid, are conserved substitutions. Examples of "non-conservative substitutions" may include the substitution of a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, or methionine, with a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue with a non-polar residue.

[0109] "Cancer" and "tumor" are interchangeable herein and generally refer to diseases characterized by the uncontrolled proliferation of abnormal cells. Both terms include solid tumors and humoral tumors, such as diffuse and circulating tumors. They include not only malignant but also premalignant cancers and tumors.

[0110] The “diseases, disorders, or conditions related to antigen expression” as described herein typically include, but are not limited to, diseases related to antigen expression or conditions related to cells that express antigens, such as proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndromes, or preleukemia, or non-cancer-related indications related to cells that express or overexpress antigens, such as bacteria, viruses, or cells, such as antigens present on non-cancer cells. Non-cancer-related indications related to antigen expression as described herein include, but are not limited to, autoimmune diseases, inflammatory diseases, and transplantation.

[0111] As used herein, the phrase “disease, disorder or condition related to the expression of a tumor antigen” typically includes, but is not limited to, diseases or conditions related to the expression of a tumor antigen or cells related to the expression of a tumor antigen, such as, for example, proliferative disorders of cancer or malignant tumors, or precancerous conditions of spinal dysplasia, myelodysplastic syndromes or preleukemia, or non-cancer-related indications related to cells expressing a tumor antigen. In one embodiment, the cancer related to the expression of a tumor antigen as described herein is a hematological cancer. In one embodiment, the cancer related to the expression of a tumor antigen as described herein is a solid tumor. Further diseases related to the expression of a tumor antigen as described herein include, but are not limited to, atypical and / or nonclassical cancers, malignant tumors, precancerous conditions or proliferative disorders related to the expression of a tumor antigen as described herein. Non-cancer-related indications related to the expression of a tumor antigen as described herein include, but are not limited to, autoimmune diseases, inflammatory diseases and transplantation. In some embodiments, tumor antigen-expressing cells express or express mRNA encoding a tumor antigen at any given time. In one embodiment, tumor antigen-expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), and the amount of tumor antigen protein can be normal, increased, or decreased.

[0112] As used herein, the terms “activity” and “activation” usually refer to a specific function of a cell. For example, T cell activity may include cytolytic activity, such as cytokine secretion, or helper activity.

[0113] As used herein, the term “immune effector cell” typically refers to cells involved in promoting immune responses, such as immune effector responses. 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 phagocytic cells. As used herein, “immune effector function or immune effector response” typically refers to the function or response of immune effector cells that enhance or promote an immune attack by target cells, for example. For example, immune effector function or response is the property of T cells or NK cells to promote the killing of target cells or to suppress the growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response.

[0114] As used herein, the terms “antigen-presenting cell” or “APC” typically mean a cell (e.g., immune cell) or group of cells (e.g., immune cell group) capable of displaying an antigen on or on its surface. The displayed antigen may be complexed with major histocompatibility complex (MHC), and the antigen may have been processed before display. Examples of APCs include, but are not limited to, macrophages, B cells, and dendritic cells (e.g., Langerhans cells). A cellular immune response may be initiated or enhanced after lymphocytes (e.g., T cells) recognize the antigen presented by the APC. APCs may break down high molecular weight antigens into 10-30 amino acid fragments for loading into HLA class I and II molecules.

[0115] As used herein, the terms “dendritic cell” or “DC” typically refer to antigen-presenting cells. DCs can act as messengers between the innate and adaptive immune systems. For example, DCs may be present in tissues in contact with the external environment, such as the skin, lining of the nose, lungs, stomach, and intestines. They may also be found in the blood in immature disease states. When activated, they migrate to lymph nodes, where they interact with other immune cells, such as T cells and B cells, to initiate and form an adaptive immune response. Immature dendritic cells are also called veil cells. DCs can be leukocytes derived from the bone marrow (BM). They can also be proliferated in vitro from BM and blood using various combinations of growth factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and Flt3 ligand. DCs convert proteins into peptides that are presented on major histocompatibility complex (MHC) molecules, which are recognized by other immune cells, such as T cells, by being specialized to capture and process antigens. DCs may be heterogeneous, such as bone marrow-like and plasmacyto-like DCs. While all dendritic cells (DCs) are capable of taking up, processing, and presenting antigens to naive T cells, DC subtypes possess different markers and, across their generations, differ in their location, migration pathways, detailed immune function, and dependence on infection or inflammatory stimuli. During the development of adaptive immune responses, the phenotype and function of DCs may play a crucial role in tolerance, memory, and / or the initiation of polar T helper 1 (Th1), Th2, and Th17 differentiation.

[0116] In the context of this application, the following abbreviations for commonly existing nucleic acid bases are used: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0117] As used herein, the terms “nucleic acid” or “polynucleotide” typically mean deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations thereof, and polymers of these in single-stranded or double-stranded forms. The term “nucleic acid” includes genes, cDNA, or mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemosynthesized) or recombinant. Unless otherwise specified, the term encompasses nucleic acids including analogs or derivatives of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly includes its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences, as well as sequences explicitly shown. Specifically, degenerate codon substitution is performed by creating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or a 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)).

[0118] The terms “cancer-associated antigen” and “tumor antigen” are interchangeable herein and typically refer to molecules (usually proteins, carbohydrates, or lipids) that are preferentially expressed on the surface of cancer cells in an intact or fragmented form (e.g., MHC / peptides) compared to normal cells and are useful for preferential targeting of drugs to cancer cells. In some embodiments, tumor antigens are markers expressed by both normal and cancer cells. In some embodiments, cancer-associated antigens are cell surface molecules that are overexpressed on cancer cells compared to normal cells, for example, by 1-fold, 2-fold, 3-fold, or more than 3-fold overexpression compared to normal cells. In some embodiments, cancer-associated antigens are cell surface molecules that are improperly synthesized on cancer cells, such as molecules containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, cancer-associated antigens are expressed only on the cell surface of cancer cells in an intact or fragmented form (e.g., MHC / peptides) and are not synthesized or expressed on the surface of normal cells.

[0119] As used herein, the term “specifically binds” typically means a molecule (e.g., small molecule, antibody, or ligand) that recognizes and binds to a homologous ligand protein present in the sample, but does not inherently recognize or bind to other molecules in the sample. In some embodiments, the molecules of this disclosure bind to a target molecule at a rate of approximately 10° -5 Less than M (for example, about 9 × 10) -6 Less than M, approximately 8 x 10 -6 Less than M, approximately 7 x 10 -6 Less than M, approximately 6 x 10 -6 Less than M, approximately 5 x 10 -6 Less than M, approximately 4 x 10 -6 Less than M, approximately 3.5 x 10 -6 Less than M, approximately 3 x 10 -6 Less than M, approximately 2.5 x 10 -6 Less than M, approximately 2 x 10 -6 Less than M, approximately 1 x 10 -6 Less than M, approximately 5 x 10 -7 Less than M, approximately 2 x 10 -7 Less than M, approximately 10 -7 Less than M, approximately 5 x 10 -8 Less than M, approximately 2 x 10-8 Less than M, approximately 10 -8 Less than M, approximately 5 x 10 -9 Less than M, approximately 4 x 10 -9 Less than M, approximately 3 x 10 -9 Less than M, approximately 2 x 10 -9 Less than M, or about 10 -9 It binds specifically with a binding affinity (KD) of less than M.

[0120] K D The ratio of the dissociation rate to the binding rate (k) is usually the ratio of the dissociation rate to the binding rate. off / k on ) may refer to and can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS methods and flow cytometry (FACS, etc.). In some embodiments, K D The value can be appropriately determined using flow cytometry.

[0121] As used herein, the term “anticancer agent” usually means a drug that can inhibit and / or prevent the growth of tumors or cancer cells.

[0122] As used herein, the term “CTLA-4” typically refers to cytotoxic T lymphocyte-associated protein 4, as well as functional fragments thereof, derived from any vertebrate, 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 AAL07473.1). Exemplary sequences of CTLA-4 include the cynomolgus monkey (monkey) CTLA-4 protein (NCBI reference sequence XP_005574071.1). As used herein, the term “CTLA-4” is generally intended to encompass any form of CTLA-4, e.g., 1) naturally occurring raw CTLA-4 molecules, naturally occurring CTLA-4 variants including “full-length” CTLA-4 chains or splice variants or allele variants, 2) any form of CTLA-4 produced by intracellular processing, or 3) full-length, fragment (e.g., truncated, extracellular / transmembrane domain) or modified (e.g., mutant, glycosylated / polyethylene glycolated, His-tagged / immunofluorescence fusion) CTLA-4 subunits produced by recombinant methods.

[0123] 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).

[0124] As used herein, the term "PD-1" typically refers to a programmed cell death protein belonging to the immunoglobulin superfamily and functioning 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 ligands, including PD-L1 and PD-L2. A representative amino acid sequence of human PD-1 is disclosed in NCBI accession number NP_005009.2, and a representative nucleic acid sequence encoding human PD-1 is shown in NCBI accession number NM_005018.2.

[0125] As used herein, the term “PD-L1” typically refers to programmed cell death ligand 1 (PD-L1, see, e.g., Freeman et al., (2000) J. Exp. Med. 192: 1027). A representative amino acid sequence of human PD-L1 is disclosed under NCBI accession number NP_054862.1, and a representative nucleic acid sequence encoding human PD-L1 is shown under NCBI accession number NM_014143.3. PD-L1 binds to receptors PD-1 or B7-1, which are expressed on activated T cells, B cells, and myeloid cells. PD-L1 binding to its receptor induces signaling that suppresses TCR-mediated cytokine production and T cell proliferation. Therefore, PD-L1 is thought to play a crucial role in suppressing the immune system during certain events such as pregnancy, autoimmune diseases, and tissue allogeneic transplantation, enabling tumors and cancer cells to evade immune checkpoints and avoid immune responses.

[0126] As used herein, the terms “anti-PD-1 antibody,” “anti-PD-1 binding domain,” or “PD-1 binding domain” typically refer to an antibody or antigen-binding domain capable of specifically binding to PD-1 (e.g., human or monkey PD-1) with an affinity sufficient to provide use for diagnosis and / or therapy.

[0127] As used herein, the term “antitumor immunity” typically refers to the immune response induced when cancer antigens are recognized by immune cells.

[0128] As used herein, the term “cancer immunotherapy” typically means any therapy designed to induce or enhance a patient’s immune response to cancer cells. For example, cancer immunotherapy includes, but is not limited to, cancer antigen-specific active immunotherapy, therapy with immunomodulators (e.g., activators or inhibitors of immunomodulators, or inhibitors of checkpoint inhibitors), or therapy with cancer cells or mixtures of cancer cell-derived antigens (therapy with cancer cell line-derived antigens). Cancer immunotherapy includes therapies that stimulate or restore the immune system’s ability to fight cancer by inducing, enhancing, or suppressing the immune response. Cancer immunotherapy targets immune activity against disease-specific antigens by increasing immune cell recognition of targets or by reducing disease-related immunosuppression.

[0129] As used herein, the term “tumor-infiltrating T cells” typically refers to T cells that infiltrate a tumor. Tumor-infiltrating T cells may exhibit spontaneous reactivity to autologous tumor antigens. These cells may be found within the tumor stroma and / or within the tumor itself.

[0130] As used herein, the term “IDO inhibitor” typically refers to a drug that inhibits the activity of indoleamine 2,3-dioxygenase (IDO), thereby reversing IDO-mediated immunosuppression. IDO inhibitors can inhibit IDO1 and / or IDO2 (INDOL1). IDO inhibitors can be reversible or irreversible. A “reversible IDO inhibitor” is a compound that reversibly inhibits IDO enzyme activity at either a catalytic or non-catalytic site, while an “irreversible IDO inhibitor” is a compound that irreversibly destroys IDO enzyme activity by forming a covalent bond with the enzyme.

[0131] As used herein, the term “immune checkpoint inhibitor” generally refers to any molecule that directly or indirectly, partially or completely inhibits an immune checkpoint pathway. The function of immune checkpoint pathways is typically thought to be to switch on and off various aspects of the immune system, particularly T cells, as well as other cells such as 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 the appropriate time. 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-exclusive examples of immune checkpoint inhibitors or modulators include fully human noclonal antibodies such as BMS-936558 / MDX-1106, BMS-s936559 / MDX-1105, ipilimumab / 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, TTI-621, and others. Other non-exclusive examples of immune checkpoint modulators (agonists) include antibodies against CD40, OX40, GITR, CD137 (4-1 BB), CD27, ICOS, and TRAIL.According to this disclosure, one or more immune checkpoint modulators may independently be polypeptides or nucleic acid molecules encoding polypeptides, wherein the polypeptides include a domain that binds to a target immune checkpoint and / or inhibits the binding of a ligand to the target immune checkpoint, so as to exert antagonistic function (i.e., can antagonize immune checkpoint-mediated inhibitory signals) or agonist function (i.e., can enhance stimulatory signals via an immune checkpoint). One or more of the above immune checkpoint modulators may be independently selected from peptides (e.g., peptide ligands), soluble domains of innate receptors, RNAi, antisense molecules, antibodies, and protein frameworks. For example, an immune checkpoint modulator may be an antibody. In the context of this disclosure, the term immune checkpoint modulator antibody is used in its broadest sense and includes, for example, natural or artificially engineered full-length antibodies or functional fragments or analogs thereof that can bind to a target immune checkpoint or epitope (and thus retain a target-binding portion). The above antibodies may be of any origin, such as, for example, human antibodies, humanized antibodies, animal antibodies (e.g., rodent or camel antibodies), or chimeric antibodies. The antibodies described above may be of any isotype, and are particularly preferably IgG1 or IgG4 isotypes. Furthermore, the antibodies may be glycosylated or unglycosylated. Standard analytical methods known in the art for evaluating the ability of antibodies to bind to immune checkpoints include, for example, ELISA, Western blot, RIA, and flow cytometry. Antibody binding dynamics (e.g., binding affinity) can also be evaluated by standard analytical methods known in the art, such as Biacore analysis. Where referring to immune checkpoint inhibitors in this application, immune checkpoint modulators may also be used unless it is clear from the wording of the context that otherwise.

[0132] As used herein, the term “exhaustion” typically refers to T-cell exhaustion, a state of T-cell dysfunction that occurs during many chronic infections and cancers. T-cell exhaustion is characterized by reduced T-cell effector function, persistent expression of inhibitory receptors, and / or a transcriptional state different from that of functional effector or memory T cells. T-cell exhaustion impairs the 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” typically refers to the activity or ability to restore at least some of the weakened or reduced antitumor activity of exhausted T cells. Reversing exhaustion may also include preventing T-cell depletion.

[0133] As used herein, the term “T cell-mediated immune response” typically refers to an immune response influenced by the modulation of T cell co-stimulation. Exemplary immune responses include T cell responses such as cytokine production and cytotoxicity. Furthermore, T cell-mediated immune responses also include immune responses indirectly influenced by T cell activation, such as antibody production (humoral responses) and activation of cytokine-responsive cells (such as macrophages).

[0134] As used herein, the term “tumor-specific T cells” typically refers to T lymphocytes that specifically attack and / or destroy tumor cells. For example, they may possess specific receptors (e.g., T cell receptors) that can 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 possess diverse receptors that target a variety of tumor antigens.

[0135] As used herein, the term “substantially non-competitive” typically means that the binding of one molecule or drug to a target does not significantly affect the binding of another molecule or drug to the same target (e.g., 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). For example, such binding is determined by analyses commonly used to determine such binding (e.g., analyses described in the examples herein).

[0136] As used herein, the terms “antigen” or “Ag” typically refer to molecules that trigger an immune response. This immune response may involve antibody production, activation of specific immunological cells, or both. It will be understood by those skilled in the art that virtually any macromolecule, including any protein or peptide, can act as an antigen. Furthermore, antigens may originate from recombinant DNA or genomic DNA. Thus, those skilled in the art will understand that any DNA consisting of a nucleotide sequence or partial nucleotide sequence encoding a protein that triggers an immune response encodes an “antigen” as used herein. Furthermore, it will be understood by those skilled in the art that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. Antigens do not need to be encoded by a “gene.” Antigens may be synthetic, derived from biological samples, or be macromolecules other than polypeptides. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or fluids containing other biological components.

[0137] As used herein, the terms “anti-cancer” or “anti-tumor” typically refer to biological effects that can manifest in various forms, including, but not limited to, reduction in tumor size, decrease in the number of cancer cells, decrease in the number of metastases, extension of life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or improvement of various physiological symptoms associated with cancer. Furthermore, “anti-cancer” or “anti-tumor” effects may also be expressed as the ability to prevent the development of cancer.

[0138] As used herein, the term "hydrocarbyl" usually refers to a moiety consisting only of hydrogen and carbon atoms. Such moieties may include aliphatic and / or aromatic moieties. Such moieties 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. An example of a hydrocarbyl group is C 1-6 C substituted with alkyl (e.g., C1, C2, C3 or C4 alkyl, e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl), aryl (e.g., benzyl), or cycloalkyl (e.g., cyclopropylmethyl) 1-6 This includes, but is not limited to, alkyl groups such as alkyl, cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), and aryl (e.g., phenyl, naphthyl, or fluorenyl).

[0139] As used herein, the term “heterohydrocarbyl” usually refers to any hydrocarbyl group containing one or more heteroatoms. The heteroatoms can be any non-C atoms, such as O, S, or N.

[0140] As used herein, the term “alkenyl” typically refers to a linear or branched alkyl moiety having two, three, four, five, six or more carbon atoms, and, where applicable, at least one double bond in E or Z stereochemistry. The term includes groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl, and 3-hexenyl.

[0141] As used herein, the term “aryl” typically refers to an aromatic ring system containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more ring carbon atoms. Aryls are often phenyl, but may also be polycyclic systems having two or more rings, at least one of which is aromatic. The term includes references to groups such as phenyl, naphthyl, fluorenyl, azlenyl, indenyl, and anthryl.

[0142] As used herein, the term “prodrug” typically refers to a compound that is rapidly converted in vivo to its parent compound, for example, by hydrolysis in the blood.

[0143] As used herein, the term “vaccine” typically means a preparation that provides active acquired immunity to a specific antigen (such as a tumor antigen or a microbial antigen) or to a tissue, cell, or organism of the aforementioned antigen. Vaccines may be prophylactic (to prevent or mitigate the effects of a future disease or disorder) or therapeutic (to treat a disease or disorder that has already occurred, such as cancer).

[0144] YTHDF1 inhibitor

[0145] The YTHDF1 weakening agent of this application may include compounds.

[0146] Such compounds may be polymers. Polymers may be naturally occurring or chemically synthesized organic or inorganic molecules with a molecular weight of about 1,000 daltons or more, or about 1, 2, 3, 5, 7, or 10 trillion daltons or more. Polymers may comprise two or more monomeric subunits or derivatives thereof, linked by covalent bonds, ionic bonds, or other chemical interactions such as hydrogen bonds, ion pairing, base pairing, or charge pairing formed by charge polarization. The monomeric subunits may be different from or identical to each other and, in some embodiments, can form polymers. Polymers may be molecules capable of forming tertiary and / or quaternary structures, whether they have multiple subunits and / or are polymers. Examples of polymers include polynucleotides, DNA, siRNA, snRNA, tRNA, antisense RNA, and RNA including ribozymes, nucleic acid molecules including peptide nucleic acids (PNA), polypeptides, glycopeptides, proteins, carbohydrates, or lipids, or derivatives or combinations thereof, such as nucleic acid molecules or glycoproteins, each containing a peptide nucleic acid moiety. Further examples of polymers include polymer assemblies, such as viruses, viral particles, phages, viroids, prions, and combinations and conjugates thereof.

[0147] Such compounds may be small molecules. Small molecules may be naturally occurring or chemically synthesized organic or inorganic molecules with a dalton content of less than about 1000 daltons, 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 less. Small molecules may be any molecule that is not macromolecule, such as proteins or nucleic acids. "Small molecules" may include molecules containing two or more monomeric subunits, such as dipeptides or dinucleotides.

[0148] Such compounds may include polypeptides or be peptides. In some cases, such compounds may include nucleic acid molecules or be nucleic acid molecules. For example, such compounds may include antibodies or derivatives thereof, antibody-drug conjugates and / or fusion proteins.

[0149] For example, this compound may be able to attenuate the activity of the YTHDF1 protein. For instance, this compound may bind directly or indirectly (e.g., via other molecules) to one or more residues of the YTHDF1 protein. Such binding may cause conformational changes to the structure and / or function of the YTHDF1 protein.

[0150] This compound can specifically bind to YTHDF1 (e.g., human YTHDF1), its fragments, or derivatives. This YTHDF1 protein may have an amino acid sequence represented by SEQ ID NO: 1. In some cases, this 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, this 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, this compound can bind (e.g., specifically) to YTHDF1, its fragments, or derivatives, and this YTHDF1, its fragments, or derivatives may have an amino acid sequence represented by any of SEQ ID NO: 1-3, 9-13, and 16-18. In some cases, this compound does not specifically bind (or substantially does not bind) to YTHDF1 or its fragments or derivatives having an amino acid sequence represented by SEQ ID NO: 4-8.

[0151] In some cases, the compound of the present invention contains approximately 10 amino acids in YTHDF1 and its fragments or derivatives having an amino acid sequence represented by SEQ ID NO: 4-8.-6 M exceeds (for example, approximately 5 x 10) -6 Over M, approximately 10 -5 Over M, approximately 5x10 -5 Over M, approximately 10 -4 Over M, approximately 5x10 -4 Over M, approximately 10 -3 Over M, approximately 5x10 -3 (M or more) K d It can be joined by value. This K d The value can be determined using any method commonly used in this field, such as isothermal titration calorimetry (ITC) analysis, surface plasmon resonance (SPR) analysis, and / or microscale thermophoresis (MST) analysis.

[0152] In some cases, the compounds of this application contain / have about 10 amino acids in YTHDF1 and its fragments or derivatives (for example, those containing / having amino acid sequences represented by SEQ ID NO: 1-3, 9-13, and 16-18 as described in this application). -5 Less than M (for example, about 9x10) -6 Less than M, approximately 8x10 -6 Less than M, approximately 7x10 -6 Less than M, approximately 6x10 -6 Less than M, approximately 5x10 -6 Less than M, approximately 4x10 -6 Less than M, approximately 3.5 x 10 -6 Less than M, approximately 3x10 -6 Less than M, approximately 2.5 x 10 -6 Less than M, approximately 2x10 -6 Less than M, approximately 1x10 -6 Less than M, approximately 5x10 -7 Less than M, approximately 2x10 -7 Less than M, approximately 10 -7 Less than M, approximately 5x10 -8 Less than M, approximately 2x10 -8 Less than M, approximately 10 -8 Less than M, approximately 5x10 -9 Less than M, approximately 4x10 -9 Less than M, approximately 3x10 -9 Less than M, approximately 2x10 -9 Less than M, or about 10 -9 K (less than M) dIt can be joined by value. This K d The values ​​can be determined using any method commonly used in this field, such as isothermal titration calorimetry (ITC) analysis, surface plasmon resonance (SPR) analysis, and / or microscale thermophoresis (MST) analysis.

[0153] In some cases, when this compound binds to YTHDF1, it may bind (e.g., specifically) to at least one residue (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, at least 30, at least 35, at least 40, or more residues) selected from amino acid residues 372-392, 479-494, and 526-535 of SEQ ID NO:1. In some cases, when a compound binds to YTHDF1, it may bind to multiple residues, and at least one of the bound residues (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, at least 30, at least 35, at least 40, or more) may correspond to residues selected from amino acid residues 372-392, 479-494, and 526-535 of SEQ ID NO: 1.

[0154] In some cases, when the compound binds to YTHDF1, it may bind to at least one residue (e.g., at least two, at least three, at least four, or at least five) selected from residues N378, F382, W384, F480, and H528 of SEQ ID NO: 1.

[0155] The compounds contained in the YTHDF1 weakening agent are m 6It may be possible to block binding to A. This YTHDF1 protein may have an amino acid sequence represented by SEQ ID NO: 1. In some cases, YTHDF1 and 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, YTHDF1 and 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.

[0156] In some cases, this compound may have an amino acid sequence represented by SEQ ID NO: 1-3, 9-13, or 16-18, as well as fragments or derivatives of YTHDF1 and its derivatives. 6 This can block the binding to A.

[0157] In some cases, this compound may have an amino acid sequence represented by SEQ ID NO: 4-8 of YTHDF1 and its fragments or derivatives. 6 It does not significantly or substantially block binding to A.

[0158] In some cases, the compound of the present application may have an amino acid sequence represented by SEQ ID NO: 4-8 of YTHDF1 and its fragments or derivatives m 6 Bonding to A is greater than approximately 7.5 μM (for example, greater than approximately 8 μM, greater than approximately 8.5 μM, greater than approximately 9 μM, greater than approximately 9.5 μM, greater than approximately 10 μM, greater than approximately 10.5 μM, greater than approximately 11 μM, greater than approximately 11.5 μM, greater than approximately 12 μM, or more) IC 50 The IC50 value can be blocked. The IC50 value can be determined using any method commonly used in this field, such as fluorescence polarization (FP) analysis and / or AlphaScreen-based analysis.

[0159] In some cases, the compound of the present application may have an amino acid sequence represented by any of SEQ ID NOs: 1 to 3, 9 to 13, 16 to 18, a fragment or a derivative of YTHDF1, and m 6 The binding to A can be blocked with an IC 50 value of less than about 7.5 μM (for example, less than about 6.5 μM, less than about 6 μM, less than about 5.5 μM, less than about 5 μM, less than about 4.5 μM, less than about 4 μM, less than about 3.5 μM, less than about 3 μM, less than about 2.5 μM, less than about 2 μM, less than about 1.5 μM, less than about 1 μM, less than about 0.9 μM, less than about 0.8 μM, less than about 0.7 μM, less than about 0.6 μM, less than about 0.5 μM, less than about 0.4 μM, less than about 0.3 μM, less than about 0.2 μM, less than about 0.1 μM or less). The IC50 value can be determined using any method commonly used in the art, such as fluorescence polarization (FP) analysis and / or AlphaScreen-based analysis.

[0160] In some cases, the compound contained in the YTHDF1 attenuator does not substantially compete with m 6 A for binding to YTHDF1. For example, the binding of the compound to YTHDF1 and its fragments or derivatives is affected (e.g., decreased) by the addition of m 6 A (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), as determined by an assay commonly used to determine such binding (e.g., as shown by an AlphaScreen-based assay). For example, m 6Binding to YTHDF1 of A and its fragments or derivatives is affected (e.g., reduced) by the addition of the compounds of the present application (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), as determined by an assay generally used to determine such binding (e.g., as shown by AlphaScreen-based assays).

[0161] In some cases, the compounds included in the YTHDF1 attenuator may be salvianolic acids such as salvianolic acid A (SAA), salvianolic acid C (SAC), their prodrugs, metabolites, derivatives, or pharmaceutically acceptable salts, esters or amides of any of the above, or any combination thereof.

[0162] In some cases, the YTHDF1 attenuator may comprise 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,

Chemical formula

[0163] In some cases, R1 in formula I is (CO)-R2, and R2 may be optionally substituted alkenyl. In some cases, R2 is CH=CH-R3, and R3 may be optionally substituted aryl. In some cases, R3 is of formula II

Chemical formula

Chem.

[0164] In some cases, in formula II, A is

Chem.

Chem.

[0165] In some cases, in formula II, A is

Chem.

Chem.

[0166] In some cases, the compound contained in the YTHDF1 attenuator of the present application can contain at least two dihydroxyphenyl moieties.

[0167] In some cases, the compound contained in the YTHDF1 attenuator can contain at least three dihydroxyphenyl moieties.

[0168] In some cases, the YTHDF1 attenuator can contain 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

Chem.

Chemical formula

[0169] In some cases, in formula III, A is

Chemical formula

Chemical formula

[0170] In some cases, in formula III, A is

Chemical formula

Chemical formula

[0171] In some cases, the YTHDF1 attenuator may include 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:

Chemical formula

[0172] [[ID=�6]] In some cases, the YTHDF1 weakening agent may include 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: (E)-3-(3,4-dihydroxyphenyl)-2-((3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)acryloyl)oxy)propanoic acid, and 3-(3,4-dihydroxyphenyl)-2-((E)-3-(2-(((E)-3,4-dihydroxystyryl)-3,4-dihydroxyphenyl)acryloyl)oxy)propanoic acid.

[0173] In some cases, the YTHDF1 weakening agent may include 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: [ka]

[0174] In some cases, the YTHDF1 weakening agent may include 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: (R,E)-3-(3,4-dihydroxyphenyl)-2-((3-(2-(3,4-dihydroxyphenyl))-7-hydroxybenzofuran-4-yl)acryloyl)oxy)propanoic acid, and (S)-3-(3,4-dihydroxyphenyl)-2-(((E)-3-(2-((E)-3,4-dihydroxystyryl)-3,4-dihydroxyphenyl)acryloyl)oxy)propanoic acid.

[0175] In some cases, the YTHDF1 weakening agent may include 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: [ka]

[0176] In some cases, the YTHDF1 weakening agent may include 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: (S,E)-3-(3,4-dihydroxyphenyl)-2-((3-(2-(3,4-dihydroxyphenyl))-7-hydroxybenzofuran-4-yl)acryloyl)oxy)propanoic acid, and (R)-3-(3,4-dihydroxyphenyl)-2-(((E)-3-(2-((E)-3,4-dihydroxystyryl)-3,4-dihydroxyphenyl)acryloyl)oxy)propanoic acid.

[0177] In some cases, the YTHDF1 weakening agent may be achiral or chiral, and if the YTHDF1 weakening agent 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.

[0178] In some cases, the compounds contained in the YTHDF1 attenuator may be derived from plants. For example, the compound may be provided in a plant extract, for instance, as part of a plant extract. For example, it may be derived from Salvia plants and their active ingredients.

[0179] The compound may be chemically produced (e.g., from oleochemicals), biochemically generated (e.g., by a fermentation process), or obtained from plant extracts, which may subsequently undergo chemical modification. For example, the compound may be (bio)chemically produced by esterifying 3-(3,4-dihydroxyphenyl)lactic acid with a carboxylic acid.

[0180] In some cases, the compound can be isolated from plant material such as plant roots. For example, the plant 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, the compound can be obtained from Salvia miltiorrhiza (Danshen).

[0181] Modified immune cells

[0182] This invention provides modified immune cells (e.g., APCs such as DCs). This invention also provides a method for modifying immune cells (e.g., APCs such as DCs).

[0183] These immune cells may be APCs such as DCs. These APCs (e.g., DCs) may originate from the examinee's bone marrow and / or lymph nodes. These DCs may include one or more of the following: resident CD11b + Cells (e.g., CD11b) + DC), residing in 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).

[0184] Modified APCs (e.g., modified DCs) may exhibit superior performance in cross-priming T cells compared to their corresponding unmodified control APCs (e.g., their corresponding unmodified control DCs).

[0185] APC (e.g., DC) may contain or express one or more tumor-specific antigens (e.g., tumor / cancer-associated antigens provided herein). In some cases, APC (e.g., DC) may be co-cultured or treated with an irritant (e.g., FLT3L).

[0186] In some cases, these immune cells may be APCs (e.g., DCs) obtained from the test subject (e.g., cancer patients), and in some cases, these immune cells (e.g., APCs such as DCs) may be isolated from tumor tissue.

[0187] These immune cells (e.g., APCs such as DCs) may be modified with the compound of the present invention or a YTHDF1 attenuator.

[0188] For example, in a population of immune cells (e.g., APCs such as DCs), one or more cells may be modified with the compound of the present invention or a YTHDF1 attenuator. In some cases, the modified immune cells of the present invention (e.g., mAPCs such as mDCs) may contain the compound of the present invention or a YTHDF1 attenuator.

[0189] In some cases, the compound of the present invention or the YTHDF1 attenuator may be exposed to immune cells (mAPCs such as mDCs) for a period of time sufficient to cause a decrease 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 more), for example, the compound or attenuator may be administered in a culture medium for immune cells (mAPCs such as mDCs).

[0190] The compounds of this application or YTHDF1 attenuators may be administered 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.

[0191] In some cases, the compounds of the present application or YTHDF1 attenuators may not be applied directly to immune cells (e.g., APCs or DCs) themselves, but rather these immune cells (e.g., APCs such as DCs) may originate from cells (e.g., precursor cells of immune cells) or organisms that have received the compounds of the present application or YTHDF1 attenuators (e.g., differentiated from or descended from them).

[0192] These immune cells could be human cells, such as human APCs (e.g., DCs).

[0193] In some cases, a source of cells, such as immune cells (APCs, e.g., DCs) or their precursor cells, may be obtained from the examinee before expansion or other modifications. The term “examinee” as used herein is intended to encompass organisms (e.g., mammals) capable of eliciting an immune response. Examples of examinees include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. Immune cells or their precursor cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, infection site tissue, ascites, pleural fluid, spleen tissue, and / or tumors.

[0194] Composition

[0195] The composition of the present application may comprise the YTHDF1 attenuator and / or the mAPC of the present application. In some cases, the composition may further comprise an additional / second active ingredient of the present application.

[0196] In some cases, this composition may be a vaccine composition.

[0197] The compositions of the present application may comprise one or more pharmaceutically acceptable excipients. Such pharmaceutically acceptable excipients may comprise any inert substances to be combined with one or more active ingredients of the present application (e.g., modified cells or attenuators).

[0198] For example, pharmaceutically acceptable excipients may include one or more of the following: solvents, penetration enhancers, antioxidants, thickeners, ointment bases, protective agents, adsorbents, lubricants, emollients, preservatives, humectants, buffers, adjuvants, bioavailability enhancers, carriers, flow enhancers, sweeteners, diluents, dyes / colorants, flavor enhancers, solubilizers (including surfactants), wetting agents, dispersants, suspending agents, stabilizers, and / or isotonic agents.

[0199] In some cases, this composition may contain one or more adjuvants to enhance or increase the immune response associated with the administration of the composition.

[0200] Additional / second active ingredient

[0201] The compounds, YTHDF1 attenuators, cells (e.g., mAPCs, mDCs) and / or compositions of the present invention may further contain an additional / secondary active ingredient and / or be used in combination thereof.

[0202] In some cases, the compounds, YTHDF1 attenuators, cells (e.g., mAPCs, mDCs), and / or compositions of the present application may be administered to examinees who have received, are receiving, and / or will receive additional / secondary active ingredients.

[0203] Additional active ingredients or therapies may be administered before, concurrently with, and / or after the administration of the YTHDF1 attenuator, cells (e.g., mAPCs, mDCs), and / or compositions of the present invention.

[0204] This additional active ingredient may be an anticancer agent. For example, this additional active ingredient may include cancer immunotherapy. In some cases, this additional active ingredient may include an immune checkpoint attenuator (e.g., an immune checkpoint inhibitor). In some cases, this additional active ingredient may include a drug selected from the group consisting of an anti-PD-L1 antibody or its antigen-binding moiety, an anti-PD-1 antibody or its antigen-binding moiety, an anti-CTLA-4 antibody or its antigen-binding moiety, and an IDO attenuator.

[0205] For example, the additional active ingredient may include an antigen-binding fragment or derivative of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, ipilimumab, and / or any of the above. For example, this additional active ingredient may include an antibody (including its antigen-binding moiety) that can compete with pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab for binding to the corresponding antigen (which is PD-1, PD-L1, or CTLA-4, respectively). In some cases, this additional active ingredient may include the HCDR3 of any of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include any of the LCDR3 of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include any of the HCDR2 of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include any of the LCDR2 of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include HCDR1 of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include HCDR1 of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include HCDR3, HCDR2, and HCDR1 of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab.In some cases, this additional active ingredient may include LCDR1, LCDR2, and LCDR3 of any of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of any of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include the heavy chain variable region of any of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include the light chain variable region of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab. In some cases, this additional active ingredient may include the heavy chain variable region and light chain variable region of pembrolizumab, nivolumab, semiprimab, atezolizumab, avelumab, durvalumab, and / or ipilimumab.

[0206] Inhibition method

[0207] This invention provides a method for inhibiting and / or attenuating the activity of YTHDF1.

[0208] The present invention further provides a method for activating immune cells (e.g., APCs such as DCs) to generate immune cells with enhanced antitumor activity, preventing and / or reversing exhaustion of immune cells (e.g., immune effector cells such as T cells), increasing and / or improving the proliferation and / or activity of immune cells (e.g., T cells, immune effector cells such as tumor-infiltrating T cells), and / or increasing and / or improving the proliferation and / or activity of tumor-specific immune cells (e.g., immune effector cells such as T cells), enhancing cytokine production by immune cells (e.g., T cells), and / or inhibiting tumor growth, inhibiting tumor cell proliferation, and / or killing tumor cells.

[0209] Such a method may include the step of administering the YTHDF1 attenuator, cells (e.g., mAPC, mDC), and / or composition of the present invention.

[0210] For example, this method may involve contacting YTHDF1, or target cells containing YTHDF1 (e.g., immune cells such as APCs and / or T cells), with the YTHDF1 attenuator, cells (e.g., mAPCs, mDCs), and / or compositions of the present invention. This contact can be carried out ex vivo. In some cases, this contact can be carried out in vivo.

[0211] In some cases, this method may involve introducing the YTHDF1 attenuator and / or composition of the present application into the cells (e.g., immune cells such as APCs and / or T cells). This introduction can be performed ex vivo. In some cases, this introduction can be performed in vivo. In some cases, this introduction can be performed ex vivo.

[0212] Inhibitor screening method and kit

[0213] This invention provides a method for determining whether a candidate drug is a YTHDF1 attenuator.

[0214] This method may involve contacting the candidate drug with the YTHDF1 variant.

[0215] This YTHDF1 variant may contain one or more amino acid substitutions, deletions, and / or additions in one or more residues selected from residues 372-392, 479-494, and 526-535 of SEQ ID NO: 1. In some cases, this YTHDF1 variant may contain one or more amino acid substitutions, deletions, and / or additions in one or more residues selected from residues N378, F382, W384, F480, and H528 of SEQ ID NO: 1. In some cases, this YTHDF1 variant may contain one or more amino acid substitutions corresponding to the following amino acid substitutions based on the amino acid sequence represented by SEQ ID NO: 1: N378A, F382A, W384A, F480A, and H528A. In some cases, this YTHDF1 variant may have an amino acid sequence represented by any of SEQ ID NOs: 4-8.

[0216] This method may further include determining whether a candidate drug specifically binds to the YTHDF1 variant of the present invention.

[0217] If this candidate drug specifically binds to the YTHDF1 variant of the present invention, it may not be a YTHDF1 attenuator.

[0218] This method may further include contacting the candidate drug with control YTHDF1, its fragment, or derivative, and determining whether the candidate drug specifically binds to the control YTHDF1, its fragment, or derivative. In some cases, the control YTHDF1, its fragment, or derivative 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, this control YTHDF1, its fragment, or derivative 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 fragment, or derivative may have an amino acid sequence represented by any of SEQ ID NOs: 1-3, 9-13, and 16-18.

[0219] In some cases, this method may further include determining whether a candidate drug specifically binds to control YTHDF1, a fragment thereof, or a derivative thereof.

[0220] If this candidate drug does not specifically bind to the control YTHDF1, its fragments, or derivatives of the present invention, then this candidate drug may not be a YTHDF1 attenuator.

[0221] If this candidate drug specifically binds to the control YTHDF1, its fragments, or derivatives, but does not specifically bind to the YTHDF1 variants of the present invention, then this candidate drug may be considered a potential YTHDF1 attenuator.

[0222] On the other hand, the present application provides, for example, YTHDF1 variants (such as the YTHDF1 variants described herein) for screening and / or determining the activity of candidate drugs that attenuate YTHDF1.

[0223] On the other hand, the present application provides a kit containing the YTHDF1 variant of the present application. This kit can be used, for example, to screen and / or determine the activity of candidate drugs that attenuate YTHDF1.

[0224] This kit may further contain additional agents. For example, this kit may contain the control YTHDF1 of the present application, a fragment thereof, or a derivative thereof.

[0225] In some cases, this kit may further contain buffers or drugs used for analysis to determine the binding affinity of candidate drugs (e.g., isothermal titration calorimetry (ITC) analysis, surface plasmon resonance (SPR) analysis, and / or microscale thermophoresis (MST) analysis).

[0226] Antigen

[0227] The compounds, YTHDF1 attenuators, cells (e.g., mAPCs, mDCs) and / or compositions of the present application may be used to treat diseases, disorders or conditions related to antigen expression in examinees in need, and / or to stimulate a T cell-mediated immune response to an antigen (e.g., a tumor antigen) in examinees in need.

[0228] Furthermore, the compounds, YTHDF1 attenuators, cells (e.g., mAPCs, mDCs) and / or compositions of the present application may be used in combination with an additional / secondary active ingredient which may cause an increase in one or more antigens (e.g., tumor antigens) in a test subject receiving it.

[0229] This antigen can be any molecule that can induce an immune response in, for example, a human test subject. This immune response may involve antibody production, activation of specific immune cells, or both. Virtually any macromolecule, including proteins or peptides, can function as an antigen. This antigen may originate from and / or be present in a biological sample. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or fluids containing other biological components.

[0230] In this application, cancer-associated antigens or tumor antigens may be expressed on the surface of cancer cells. In some cases, cancer-associated antigens themselves may be intracellular, but fragments of such antigens (peptides) may also be presented on the surface of cancer cells by MHC (major histocompatibility complex).

[0231] Examples of cancer / tumor-associated antigens include, for example, EGFR, HER2 / neu, HER3, HER4, Ep-CAM, CEA, TrAIL, TRAIL receptor 1, TRAIL receptor 2, lymphotoxin-beta receptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast-activating 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 (TWEAKs) of TNF, IL-1R.

[0232] In some cases, examples of these cancer / tumor-associated antigens 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 versions thereof.

[0233] Enhancement of antitumor immunity

[0234] The compounds, YTHDF1 attenuators, cells (e.g., mAPCs, mDCs), methods, and / or compositions of this application may be used to activate immune cells and / or to enhance immune responses, such as antitumor immune responses.

[0235] For example, activated immune cells may have an improved ability to kill tumor cells or control tumor growth in vivo (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).

[0236] In some cases, in a population of immune cells, CD4 + Increased T cell proliferation (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) may be observed. In some cases, CD8 +An increase in T cell proliferation (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) may be observed.

[0237] In some cases, an enhanced antitumor immune response may affect CD8 within or around the tumor site. + This may be indicated by an increase in the number of cytotoxic T cells (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).

[0238] In some cases, an enhanced antitumor immune response may lead to tumor infiltration of CD8 +This may be revealed by an increase in the number of T cells (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).

[0239] In some cases, increased activity of immune cells (e.g., T cells) may be manifested by increased production of cytokines (e.g., IFN-γ and / or IL-2) and / or granzyme B by immune cells (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).

[0240] In some cases, increased activity of immune cells or enhancement of the immune response may be due to CD8 +Delayed and / or reversed T cell exhaustion (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), and delayed and / or reversed immune cell exhaustion (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%). This may be revealed by (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).

[0241] In some cases, increased immune cell activity or enhanced immune response may be manifested by increased CXCR5 expression (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).Increased expression is characterized by an increase in the amount / level of CXCR5 inside / on cells (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) or by an increase in the number / percentage of cells expressing CXCR5 within a population of immune cells (e.g., a population of immune effector cells such as a population of T cells) (e.g., at least about 1%, at least about 2 It may be characterized by (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).

[0242] In some cases, increased immune cell activity or enhanced immune response may be manifested by a decrease in PD-1 expression (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).The decrease in expression is characterized by a decrease in the amount / level of PD-1 inside / on cells (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) or by a decrease in the number / percentage of cells expressing PD-1 within an immune cell population (e.g., an immune effector cell population such as a T cell population) (e.g., at least about 1%, at least about 2 It may be characterized by (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).

[0243] In some cases, increased immune cell activity or enhanced immune response may be manifested by a decrease in Tim3 expression (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).The decrease in expression is characterized by a decrease in the amount / level of Tim3 inside / on cells (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) or by a decrease in the number / percentage of cells expressing Tim3 within a population of immune cells (e.g., a population of immune effector cells such as a population of T cells) (e.g., at least about 1%, at least about 2 It may be characterized by (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).

[0244] In some cases, increased activity of immune cells or enhancement of the immune response is due to PD-1 in a population of immune cells (e.g., a population of immune effector cells such as a population of T cells). + Tim3 +This may be evident from a decrease in the number and / or percentage of cells (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).

[0245] Disease, disorder or medical condition

[0246] The compounds, YTHDF1 attenuators, cells (e.g., mAPCs, mDCs), methods, and / or compositions of this application may be used to treat diseases, disorders, or conditions, such as treating diseases, disorders, or conditions related to the expression of antigens (e.g., cancer / tumor-related antigens as described herein) in examinees who require treatment.

[0247] For example, this disease, disorder, or condition could be cancer.

[0248] In some cases, this cancer may be selected from a group consisting of hematological malignancies, lymphomas, and solid tumors.

[0249] In some embodiments, the cancer is selected from the group consisting of 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.

[0250] Subject

[0251] The compounds, YTHDF1 attenuators, cells (e.g., mAPCs, mDCs), methods, and / or compositions of this application may be administered to examinees (e.g., humans) who require them.

[0252] In some cases, this candidate may be a cancer patient. For example, this candidate may be a cancer patient selected from the group consisting of hematological cancers, lymphomas, and solid tumors. In some cases, this candidate may be a cancer patient selected from the group consisting of melanomas, colon cancers, pancreatic cancers, breast cancers, and lung cancers.

[0253] In some cases, this candidate may have received, are receiving, and / or will receive additional treatment. This additional treatment may be cancer treatment.

[0254] In some cases, this anticancer therapy may include cancer immunotherapy. For example, this anticancer therapy may include, or may be, an immune checkpoint attenuator. In some cases, this anticancer therapy may include a drug selected from the group consisting of an anti-PD-L1 antibody or its antigen-binding moiety, an anti-PD-1 antibody or its antigen-binding moiety, an anti-CTLA-4 antibody or its antigen-binding moiety, and an IDO attenuator. In some cases, this anticancer therapy may include pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, and / or antigen-binding fragments or derivatives of any of the above.

[0255] Example

[0256] The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of how the present invention can be carried out and used, and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent all or only experiments performed. Although efforts have been made to ensure the accuracy of the numerical values ​​used (quantity, temperature, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise specified, parts refer to parts by weight, molecular weight to weight-average molecular weight, temperature to degrees Celsius, and pressure to atmospheric pressure or near atmospheric pressure. Standard abbreviations are used, for example, bp to base pair, kb to kilobase, pl to picoliters, s or sec to seconds, min to minutes, h or hr to hours, aa to amino acid, nt to nucleotide, im to intramuscular injection, ip to intraperitoneal injection, and sc to subcutaneous injection. For the experimental results (e.g., two-tailed unpaired Student's t-test), * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and *** indicates p<0.0001, while ns means that the result is not statistically significant.

[0257] Materials and methods

[0258] In the embodiments of this application, the following materials and methods were used.

[0259] Protein expression and purification

[0260] The protein YTHDF1 (361-559) (SEQ ID NO: 2) and its variants (SEQ ID NOs: 4-13) were cloned into the pGEx-6P-1 carrier (obtained from YouBio, catalog number: VT1258). Additionally, His-YTHDF1 (361-559) (SEQ ID NO: 3), used in AlphaScreen analysis, was cloned into the modified pET28a carrier (obtained from YouBio, catalog number: VT1207). These carriers were transformed into E. coli BL21 (DE3) cells and cultured at 37°C. Once the OD value reached 0.6-0.8, 1 mM isopropyl-β-D-thiogalactopyranoside was added, and the protein was overexpressed overnight at 16°C. YTHDF1(361-559) and its variants were first purified by glutathione affinity chromatography (GSTrap FF, GE Healthcare) and incubated overnight at 4°C with PPase to remove the GST tag. The proteins were then further purified via cation exchange (HiTrap SP, GE Healthcare) and finally via a Superdex 75 10 / 300 column (GE Healthcare). Finally, the purified YTHDF1(361-559) and variants were retained in a buffer containing 20 mM Hepes (pH 7.4) and 200 mM NaCl. For His-YTHDF1(361-559), the proteins were sequentially purified by Ni-NTA chromatography (HisTrap FF, GE Healthcare), followed by cation exchange and a Superdex 75 10 / 300 column. The resulting proteins were then retained in a buffer containing the same components.

[0261] Fluorescence polarization (FP) analysis

[0262] YTHDF1 (361~559), 5'-FAM labeled m 6 A-containing mRNA (5'-FAM-UUCUUCUGUGG (m 6A) CUGUG-3', SEQ ID NO: 14) and all candidate compounds were diluted in analytical buffer (20 mM Hepes (pH 7.4), 50 mM NaCl, 0.01% (v / v) tween 20, 5% (v / v) glycerol). For high-throughput screening (HTS), 1.25 μM YTHDF1 (361-559) was incubated with 80 μM of the candidate compound in a black 384 plate (Corning, 3575) at room temperature for 30 minutes. Next, 40 nM 5'-FAM-labeled m 6 A-containing mRNA was added to the mixture and incubated at 4°C for 1 hour. Unlabeled mRNA was added. 6 A-containing mRNA was used as a positive control, and 40 nM 5'-FAM-labeled mRNA was used. 6 The gain factor was adjusted using only A-containing mRNA. Finally, the mixture was measured using Envision Readers (PerkinElmer).

[0263] For the activity test, 1.25 μM YTHDF1 (361-559) was incubated with candidate compounds diluted to the specified concentrations for 30 minutes. Subsequent steps were the same as in the HTS test. An equal volume of DMSO was then used as a negative control.

[0264] AlphaScreen analysis

[0265] The compound (e.g., SAA or SAC) was diluted from 200 μM using a 2x gradient method with analytical buffer (20 mM Hepes (pH 7.4), 150 mM NaCl, 1 mg / ml BSA, 0.01% (v / v) TritonX-100). Next, 100 nM His-YTHDF1 (361-559) was incubated with SAA or SAC in analytical buffer at room temperature for 30 minutes. Then, 10 nM biotinylated m 6 A-containing mRNA (5'-biotin-UUCUUCUGUGG (m 6A) Add CUGUG-3') (SEQ ID NO: 15) and bind to YTHDF1 (361~559), then debiotinylated m 6 A-containing mRNA was used as a positive control. Before detecting the Alpha signal, streptavidin donor beads and anti-His acceptor beads were added to a white analysis plate (OptiPlate®-384, PerkinElmer) in the dark and incubated at 4°C for 1 hour to ensure sufficient binding between the biotin tag and streptavidin donor beads, and between the His tag and anti-His acceptor beads. The Alpha signal was then detected using Envision Readers (PerkinElmer).

[0266] For competitive analysis, dilute the compound (e.g., SAA or SAC) in the same manner and debiotinize it. 6 A-containing mRNA was diluted to 400 nM, 200 nM, 50 nM, and 25 nM with analytical buffer. 200 nM of His-YTHDF1 (361-559) was analyzed for unbiotinylated mRNA. 6 It was incubated with A-containing mRNA at 4°C for 10 minutes. Then, m 6 To compete with A-containing mRNA for protein binding, a compound (e.g., SAA or SAC) was added and incubated at room temperature for a further 30 minutes. Next, 20 nM biotinylated m 6 A-containing mRNA and two types of beads were added in the dark and incubated at 4°C for 1 hour before detection.

[0267] NMR analysis

[0268] NMR CPMG experiments were performed at 25°C using a Bruker Avance III spectrometer (proton frequency 600 MHz) equipped with a cryogenic probe (Bruker biospin, Germany). YTHDF1 (361-559) was diluted to 20 μM, 10 μM, and 5 μM with phosphate buffer (20 mM NaH2PO4, 20 mM Na2HPO4, 150 mM NaCl, pH 7.4, D2O). The compound (e.g., SAA or SAC) was dissolved in 5% deuterated DMSO to a concentration of 200 μM. Solvent suppression 1D 1 The H CPMG was obtained via a pulse sequence (RD-90°-(τ-180°-τ) n-ACQ). In the pre-saturation procedure, a 54.78 dB pulse was applied with a recycle delay (RD) duration of 4 s to eliminate water resonance. Next, the 90° pulse length was modulated to approximately 11.82 μs. Finally, a total of four dummy scans and 64 free induction decays (FIDs) were collected at 64,000 acquisition points covering a spectral width of 12 kHz (20 ppm) with an acquisition time (ACQ) of 2.73 s.

[0269] Isothermal titration calorimetry

[0270] Purified YTHDF1 (361-559) was dialyzed overnight at 4°C with dialysis buffer (20 mM Hepes (pH 7.4) and 200 mM NaCl). The dialyzed protein was then diluted to 50 μM with dialysis buffer. Compounds (e.g., SAA or SAC) were dissolved and diluted to 1 mM with dialysis buffer. Isothermal titration calorimetry (ITC) was performed at 25°C using a Microcal ITC 200 isothermal titration calorimetry (GE Healthcare). 200 μL of 50 μM YTHDF1 (361-559) was packed into a sample cell and continuously stirred at 750 rpm. 40 μL of 1 mM SAA was packed into a syringe. After a single injection of 0.4 μL, the compound (e.g., SAA or SAC) was first titrated to YTHDF1 (361-559) by 19 injections of 2 μL at 180 s intervals. Next, to more clearly demonstrate the titration effect, the 19 injections of 2 μL were calculated to be changed to 5 injections of 2.5 μL, and then to 14 injections of 1.9 μL. Additionally, to eliminate the thermal effect of background dilution, a titration of 1 mM of the compound (e.g., SAA or SAC) into dialysis buffer was performed as a control. Experimental data were analyzed using Microcal ORIGIN (v7.0) software (Microcal Software).

[0271] SPR binding analysis

[0272] SPR binding analysis was performed using a Biacore T200 instrument (GE Healthcare) at 25°C. YTHDF1(361-559) was covalently immobilized on a CM5 tip by a standard amine binding procedure under conditions of 10 mM sodium acetate (pH 5.5). The compound (e.g., SAA or SAC) was then gradient diluted with HBS buffer (20 mM Hepes (pH 7.4), 200 mM NaCl, and 0.4% (v / v) DMSO). Next, the diluted compound (e.g., SAA or SAC) was injected at a flow rate of 30 μL / min for 60 seconds to bind to the immobilized YTHDF1(361-559), followed by dissociation by injection of HBS buffer at the same flow rate for 600 seconds. The equilibrium dissociation constant (K) of the compound (e.g., SAA or SAC) was determined. d The values ​​were generated from data analysis using Biacore T200 evaluation software (GE Healthcare).

[0273] MST analysis

[0274] Microscale thermophoresis (MST) analysis was performed at room temperature using a label-free microscale thermophoresis apparatus (NanoTemper Technologies). The compound (e.g., SAA or SAC) was diluted from 1 mM using a 2x gradient method with MST buffer (20 mM Hepes (pH 7.4), 200 mM NaCl, and 0.1 mM Pluronic® F-127). YTHDF1(361-559) was then diluted to 4 μM with MST buffer. Next, 10 μL of the compound (e.g., SAA or SAC) was mixed with 10 μL of YTHDF1(361-559) and incubated at room temperature for 20 minutes. Prior to measurement, the mixture was centrifuged at 13000 rpm for 10 minutes at 4°C. Finally, the sample was collected using a Monolith NT® automated label-free capillary (NanoTemper Technologies) and measurement was initiated. K of a compound (e.g., SAA or SAC) dThe values ​​were obtained from data analysis using MO affinity analysis software V2.3 (NanoTemper Technologies).

[0275] Hydrogen-deuterium exchange mass spectrometry

[0276] Prior to measurement, YTHDF1(361-559) was incubated overnight at 4°C with a compound (e.g., SAA). Hydrogen-deuterium exchange mass spectrometry (HDX MS) was performed by exchanging hydrogen atoms of YTHDF1(361-559)-apo and YTHDF1(361-559)-SAA with deuterium at 10°C for 0, 10, 30, 60, 1200, 3600, and 14400 seconds, respectively, using buffers containing 20 mM Hepes (pH 7.4), 200 mM NaCl, and D2O. The reaction was then stopped at 0.5°C using a buffer containing 4 M guanidine hydrochloride, 0.5 M TCEP, and 100 mM citrate (pH 2.3). After the deuterium labeling reaction, the sample was digested at 4°C with pepsin immobilized on a column to obtain the peptide. Next, these peptides were separated by HPLC and analyzed by mass spectrometry. The HDX MS data was analyzed using HDExaminer software (v2.4.1), and peptides showing significant changes were selected with a threshold of ±5%.

[0277] Cell thermal shift assay

[0278] Cellular thermal shift analysis (CETSA) was performed according to a previously reported protocol. The 293T cell line (ATCC) used in the analysis was cultured in DMEM medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin / streptomycin (Life Technologies) at 37°C and 5% CO2. As a control, the 293T cells were incubated with PBS buffer or with 100 μM SAA, harvested after 4 hours, and divided into 12 equal portions. The divided portions were heated for 3 minutes over a range of temperatures from 39°C to 59°C, and immediately cooled to 4°C for 3 minutes. The cells were then lysed by freeze-thaw with liquid nitrogen, and protein samples were collected by centrifugation. Samples for Western blot detection were prepared by adding SDS and boiling at 99°C for 5 minutes. GAPDH was used as an internal reference for Western blot analysis. Quantitative analysis of the results was performed using ImageJ software.

[0279] Cell lines and mice

[0280] B16-OVA is a transfection clone derived from the mouse melanoma cell line B16, provided by Y.-X. Fu (UT Southwestern).

[0281] E.G7-OVA is a transfection clone derived from the mouse thymic lymphoma cell line E.G7, provided by Chen Dong (Tsinghua University).

[0282] Ythdf1 - / - The mice were generated in the laboratory by the inventors, as described in previous studies (see, for example, Shi, H. et al., Nature 563, 249-253 (2018)).

[0283] Ythdf1 F / F The mice were provided by Bin Shen (Nanjing Medical University) and were CD11c cre The mice were purchased from Jackson Laboratory.

[0284] OT-I mice are ovalbumin-specific CD8 + These are TCR gene-modified mice, provided by Xiaohuan Guo (Tsinghua University).

[0285] Tumor inoculation and treatment

[0286] Regarding tumor growth in B6 mice, 5 × 10 5 Individual B16-OVA or 1×10 6 Individual E.G7-OVA tumor cells were subcutaneously (sc) inoculated into the flanks of mice. The length (a) and width (b) of the tumors were measured every two days, and the tumor volume was calculated using formula ab. 2 The calculation was performed using a factor of 2. For inhibitor therapy, 10 μM SAA or DMSO was injected intraperitoneally (ip) on days 9 and 11 after tumor inoculation. Other mouse models (rag1 - / - For conditional disruption of the Ythdf1 gene, the number and dose of inhibitor treatments were the same. For combination therapy with α-PD-L1 and SAA, 5 × 10 5 100 B16-OVA tumor cells were subcutaneously inoculated into the flanks of mice. α-PD-L1 antibody (cloning 10F.9G2) or 100 μg of rat immunoglobulin was administered 9 days after tumor inoculation. 10 μM of SAA or DMSO was similarly administered on 9 and 11 days after tumor injection.

[0287] FLT3L-DC culture and inhibitor treatment

[0288] Bone marrow wild type and Ythdf1 - / - The cells were isolated from mice and treated with erythrocyte lysis buffer to remove erythrocytes. IMDM medium containing 10% fetal bovine serum was used to suspend the bone marrow cells. The cell concentration was set to 1 × 10⁶ to culture FLT3L-DCs. 6The concentration was adjusted to / mL. Cells were cultured in 100 ng / mL FLT3L for 9 days to obtain mature FLT3L-DCs. Mature FLT3L-DCs were purified using the EasySep Mouse CD11c Positive Selection Kit II and treated with 10 μM SAA or DMSO in IMDM medium (containing 10% bovine serum and 100 ng / mL FLT3L) for 10 hours.

[0289] DC antigen-presentation function analysis

[0290] For the in vitro cross-presentation study, mature FLT3L-DCs were collected on day 9, purified using the EasySep Mouse CD11c Positive Selection Kit II, and treated with 10 μM SAA or DMSO in IMDM medium (containing 10% bovine serum and 100 ng / mL FLT3L) for 10 hours. After inhibitor treatment (e.g., SAA or SAC), FLT3L-DCs were co-cultured with necrotic B16-OVA cells for 6 hours. Next, the DCs from which the antigen was obtained were purified and co-cultured with naive OT-1 mouse T cells in 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 the ex vivo DC cross-presentation analysis, four types of DCs (migrating CD11b) were identified. + DC, migration CD103 + DC, resident CD11b + DC and resident CD8 + DCs were selected from the lymph nodes in the inflow region of SAA-treated B16-OVA-carrying mice on day 12. These DCs were co-cultured with OT-1 naive T cells at a ratio of 1:10 for 96 hours, with or without the presence of OT-1 peptide. IFN-γ production in the supernatant was detected by CBA analysis.

[0291] T cell function analysis

[0292] Tumor-infiltrating leukocytes were present in a 96-well plate at a concentration of 5 × 10⁴ cells per mL.6 The cells were then resuspended in RPMI 1640 medium. T cells were stimulated with phorbol-12-myristate-13-acetate (PMA) (2.5 μg / mL) and ionomycin (0.5 μg / mL), and brefeldin A was added to the medium, followed by incubation at 37°C for 2 hours. The total lymphocyte cell concentration from the influx region lymph nodes was 5 × 10⁴ cells per mL in a 96-well plate. 6 The cells were prepared accordingly. 1 μg / mL of OT-1 (OVA 257-264) peptide was added to the wells and stimulated for 96 hours. The samples were stained with CD45 and CD8 on ice for 30 minutes. Intracellular staining was performed to quantify the production of IFN-γ and granzyme B.

[0293] Flow cytometry

[0294] For flow cytometry analysis and DC sorting, tumors and inflow region lymph nodes were collected from mice and digested with 100 U / mL collagenase IV and 20 μg / mL DNase I at 37°C for 40 minutes. Digestion was stopped with FACS buffer (PBS containing 2% FBS and 1 mM EDTA), and the samples were filtered through a 70 μm cell strainer. The samples were stained with specific antibodies in FACS buffer on ice for 30 minutes. Antibody information is listed in Table 1 below. After staining, all samples were washed with FACS buffer, and cells were analyzed in BD Fortessa and sorted in Aria III. [Table 1]

[0295] CFSE labeling

[0296] 1 x 10⁶ lymph nodes from naive OT-I mice 7Each lymphocyte was washed twice with PBS and resuspended in 1 mL of PBS. 1 μL of CFSE Tracker was added to the suspension and incubated at 37°C for 5 minutes. Next, 5 mL of RPMI-1640 medium containing 10% FBS was added to stop the CFSE labeling process, and the cells were incubated at room temperature for 5 minutes. After centrifugation, the CFSE-labeled T cells were suspended in another 5 mL of RPMI-1640 medium at room temperature for at least 10 minutes.

[0297] Example 1 Inhibitory activity against YTHDF1

[0298] To discover novel inhibitors of YTHDF1, a high-throughput screening (HTS) approach based on fluorescence polarization (FP) analysis was developed, and salvianolic acid A (SAA) was found to be an effective inhibitor. 6 The activity of SAA, which inhibits the interaction between A-containing mRNA and YTHDF1, was evaluated by FP analysis. As shown in Figure 1a, 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 obtained IC50 was shown in Figure 1b. 50 The value is 0.86 ± 0.06 M, and SAA is m 6 It was confirmed that the binding between A and YTHDF1 can be effectively blocked.

[0299] Inhibition of YTHDF1 activity by another salvianolic acid, salvianolic acid C (SAC), was also evaluated by FP analysis, and the obtained IC was shown in Figure 1c. 50 The value was 3.95 μM.

[0300] Next, qualitative and quantitative experiments were performed to investigate the binding between SAA and YTHDF1. First, nuclear magnetic resonance (NMR) Carr-Purcell-Meiboom-Gill (CPMG) experiments were performed. As shown in Figure 1d, after the addition of 200 μM SAA, a signal of the compound was detected in the CPMG spectrum, and the signal decreased with the addition of 5 μM, 10 μM, and 20 μM YTHDF1, respectively, indicating a direct binding between SAA and YTHDF1.

[0301] Next, the binding affinity between SAA and YTHDF1 was evaluated. Isothermal titration calorimetry (ITC) analysis was performed to determine the equilibrium dissociation constant (K) between YTHDF1 and SAA. d The ITC analysis was precisely tested. This ITC analysis was performed three times independently using a Microcal iTC200 isothermal titration calorimeter (GE Healthcare). Briefly, 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. As shown in Figures 2a-2c, SAA was 5.71 μM K d The bond between SAA and YTHDF1 was confirmed. Furthermore, the enthalpy change (δH = -3099 ± 144.1 cal / mol) was less than zero, indicating that SAA can form hydrogen bonding interactions with YTHDF1. In addition, the entropy change (δS = 13.6 cal / mol / deg) was greater than zero, indicating that SAA binding may induce a conformational change in YTHDF1.

[0302] Furthermore, surface plasmon resonance (SPR) analysis and microscale thermophoresis (MST) analysis were performed to confirm the binding strength between SAA and YTHDF1. As shown in Figures 3a-3c, a binding strength of 2.52 μM was obtained from the SPR analysis. d A value was obtained, and from MST analysis, 4.70 μM K was found. d Values ​​were obtained, and these results are consistent with the results of the ITC experiment.

[0303] In summary, the compounds of this application (e.g., SAA, SAC, and other compounds of this application) directly bind to YTHDF1 and their m 6 We can conclude that it is possible to block A-binding activity.

[0304] Example 2 Non-competitive inhibition of YTHDF1 activity

[0305] 200 nM YTHDF1 and 20 nM biotinylated m 6 Using A-containing mRNA, AlphaScreen analysis was performed to determine a predetermined concentration of non-biotinized mRNA. 6 Competitive binding experiments were performed on A-containing mRNA. As shown in Figures 4a-4b, the inhibitory activity of SAA against YTHDF1 was observed in non-biotinylated mRNA. 6 In the absence of A-containing mRNA, the concentration is 0.80 ± 0.08 μM, and non-biotinylated mRNA concentrations of 50 nM, 100 nM, 200 nM, or 400 nM are also present. 6 When A-containing mRNA was pre-incubated with YTHDF1, this inhibitory activity remained unchanged. These results indicate that SAA inhibits the function of YTHDF1, but its binding to YTHDF1 is m 6 This indicates that it does not compete with A.

[0306] Next, 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 was examined at 10s, 30s, 60s, 1200s, 3600s, and 14400s, and the changes in deuterium uptake between these times are shown in residual plots (Figure 5a), butterfly plots (Figure 5b), and heatmaps (Figure 6). Many peptides showed HDX percentage changes of more than 5%, indicating that the binding of SAA to YTHDF1 can induce significant conformational changes in the intact protein structure.

[0307] As shown in Figures 7 and 8a-8i, YTHDF1 and related peptides undergo significant structural changes, and these conformational changes are mainly due to the following 1)m 6The binding occurred in three regions: 1) a long, shallow pocket containing a specific positively charged amino acid, and 2) the C-terminal α-helix of YTHDF1 (which underwent the most significant change). These results suggest that SAA bound to YTHDF1 in one of these three regions, inducing a conformational change.

[0308] Next, YTHDF1 mutants and C-terminal cleavages (having amino acid sequences represented by SEQ ID NOs: 4-13) were designed, and their inhibitory activity against SAA was tested.

[0309] As shown in Figures 9 and 10f-10j, the section (SEQ ID NO: 13) or m 6 When mutants with mutations in the A-binding pocket (for example, 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)) are used, as measured in FP experiments, m 6 The inhibitory activity of SAA against binding to A was similar to that observed for wild-type YTHDF1. These results suggest that the C-terminal α-helix region and m 6 This indicates that the A-binding pocket is not essential for SAA binding. Conversely, as shown in Figures 9 and 10a-10e, when there are mutants (SEQ ID NOs: 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), m 6 The inhibitory activity of SAA against binding to A was significantly weaker than that against wild-type YTHDF1. Further studies using FP analysis showed that these mutations at residues 372-392, 479-494, or 526-535, as well as the C-terminal cleavage, were more effective against YTHDF1 and m 6 This demonstrated that it did not affect the binding affinity with A (Figures 11a-11g).

[0310] Therefore, it was found that SAA exerts non-competitive inhibitory activity against YTHDF1 through an allosteric mechanism and at least partially through hydrogen bonding interactions formed between SAA and one or more residues within residues 372-392, 479-494, or 526-535.

[0311] Example 3 Binding of SAA and YTHDF1 in 293T

[0312] Furthermore, the binding of SAA to YTHDF1 in cells was also investigated. Cellular thermal shift analysis (CETSA) was performed using 293T cell lines collected for heating at a predetermined temperature. After incubating these cells with 100 μM SAA for 4 hours, YTHDF1 protein in the incubated 293T cells was examined by Western blotting. As shown in Figures 12a–12b, the stability of YTHDF1 improved after incubation with SAA, with the curve shifting approximately 2 degrees to the right, confirming that SAA can directly bind to YTHDF1 in the cytoplasm.

[0313] Example 4 Inhibition of tumor growth in vivo

[0314] 5 x 10 5 B16 melanoma cells expressing ovalbumin (OVA) were subcutaneously inoculated into wild-type mice. Subsequently, tumor-bearing mice were treated with 10 μM SAA 9 and 11 days after tumor inoculation, and tumor growth was monitored. As shown in Figure 13a, tumor growth was observed to be much slower in mice treated with SAA compared to the control group.

[0315] In another experiment, wild-type mice were given 1 × 10⁶ 6 Each mouse was subcutaneously injected with E.G7-OVA cells. Subsequently, on days 9 and 11, each mouse was injected with 10 μM of SAA. Tumor growth was monitored. A similar inhibitory trend was observed in the E.G7-OVA lymphoma model (Figure 13b).

[0316] As shown in Figure 19, 5 × 10 5B16-OVA cells were subcutaneously inoculated into C57BL / 6 mice (n=17). Nine days after transplantation, the mice were divided into three groups based on tumor size. Each group received intravenous injection of DMSO (n=6), 10 μM SAA (n=6), or 10 μM SAC (n=5), respectively. Tumor growth was monitored. Data are shown as mean ± sem, and "ns" means no significant difference. Unpaired one-sided t-tests showed "*" p<0.05 and "**" p<0.01. Tumor growth was observed to be much slower in mice that received SAA or SAC.

[0317] Example 5 Effect on tumor cell proliferation in vitro

[0318] In another experiment, tumor cells were treated with SAA in vitro. Simply put, 5 × 10 4 Individual B16-OVA tumor cells were treated with varying doses of SAA in 96-well plates. Cell counts were measured 12 hours after SAA treatment. Tumor cell proliferation was found to be unaffected by increasing the SAA dose (Figure 14a). These results suggest that SAA does not exert its antitumor effect by directly killing tumor cells.

[0319] Example 6 Adaptive immunity is required for SAA antitumor activity

[0320] In another experiment, 5 × 10 5 Individual CFSE-labeled OT-I T cells were treated with varying doses of SAA in 96-well plates, and then the cells were stimulated with 1 μg / mL of OT-I peptide for 24 hours. The divided T cells were analyzed by FACS. As shown in Figure 14b, SAA was unable to affect T cell proliferation in vitro.

[0321] T / B cell-deficient Rag1 - / - 1x10 on the mouse 5Each mouse was inoculated with B16-OVA cells, and 10 μM of SAA was injected into each mouse from day 7 to day 9. Tumor growth was monitored, and wild-type mice were used as a control group. As shown in Figure 15a, tumor growth stopped in wild-type mice, but T / B cell-deficient Rag1 - / - In mice, tumor growth was not stopped, suggesting that adaptive immunity is necessary for SAA to exert its maximum antitumor therapeutic effect.

[0322] Example 7 SAA enhanced the cross-presentation function of APC

[0323] Bone marrow-derived cells (wild-type or Ythdf1 gene-deficient) were co-cultured with FLT3L for 9 days to obtain FLT3L-DCs. These DCs were treated with 10 μM SAA for 12 hours, and then co-cultured 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 evaluated by IFN-γ cytometry bead array.

[0324] As shown in Figure 15b, FLT3L-DCs treated with SAA showed superior ability in T cell crosspriming compared to the control group. To compare the effectiveness of SAA treatment with the effectiveness of Ythdf1 gene knockout, Ythdf1 gene-deficient DCs (Ythdf1 - / - SAA (obtained from bone marrow) was used as a positive control. Interestingly, SAA was Ythdf1 - / - The cross-presentation function of FLT3L-DCs may also be enhanced. These results indicate that the compounds of this application (e.g., SAA) can enhance the cross-priming function of antigen-presenting cells (e.g., DCs) in vitro.

[0325] Furthermore, on day 12, four types of classical DCs (resident CD11b) were extracted from the inflow area lymph nodes of SAA-treated B16-OVA-carrying wild-type mice. + , resident CD8α + , migration CD11b + and wandering CD103 +) were selected. These DCs were co-cultured with OT-I T cells for 72 hours. Next, IFN-γ production was evaluated by IFN-γ cytometry bead array. As shown in Figure 15c, all of these DC subtypes from SAA-treated mice, particularly resident CD8α, were selected from the DMSO-treated group. + DC and Play CD103 + DCs showed superior T cell cross-priming. These results indicate that the compound of this invention (e.g., SAA) can promote the cross-priming function of antigen-presenting cells (e.g., DCs) even in vivo.

[0326] Example 8 SAA functions in APCs to enhance the immune response

[0327] Dendritic cells can activate T cells through cross-priming and / or direct priming. In the direct priming process, DCs can stimulate T cells via surface costimulatory molecules such as CD80 / CD86 or cytokines associated with T cell activation. To assess whether direct priming function is also affected after SAA administration, bone marrow-derived cells (wild-type or Ythdf1 gene-deficient) were cultured with FLT3L for 9 days to obtain FLT3L-DCs. These DCs were treated with 10 μM SAA for 12 hours, and then the FLT3L-DCs were co-cultured 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 by IFN-γ cytometry bead array. Furthermore, on day 12, four types of classical DCs (resident CD11b) were collected from lymph nodes in the influx region of SAA-treated B16-OVA-carrying wild-type mice. + , resident CD8α + , migration CD11b + and wandering CD103 + These DCs were selected. These DCs were co-cultured with OT-I T cells supplemented with 1 μg / mL of OT-I peptide for 72 hours. Next, IFN-γ production was evaluated by IFN-γ cytometry bead array.

[0328] As shown in Figures 16a-16b, SAA was unable to enhance the direct priming function of FLT3L-DCs in vitro, but three of the four DC subtypes showed improved direct priming ability after SAA treatment, demonstrating that SAA can enhance the in vivo direct priming ability of DCs. These results indicate that the compound of the present invention (e.g., SAA) can function in APCs (e.g., dendritic cells) to enhance the immune response.

[0329] Example 9 APCs are the main targets of SAA

[0330] To determine whether DC is a primary target of SAA, Ythdf1 F / F and CD11c cre Ythdf1 F / F 2x10 on the mouse 6 Each mouse was subcutaneously injected with B16-OVA cells. Then, on days 9 and 11, 10 μM of SAA was injected into each mouse, and tumor growth was monitored. As shown in Figure 17a, SAA-treated Ythdf1 F / F Tumor growth in mice is associated with CD11c cre Ythdf1 F / F The situation was similar to what was observed in mice, but CD11c cre Ythdf1 F / F In mice, no further significant tumor control effects were observed, indicating that DCs are the primary target of SAA.

[0331] Example 10 SAA activates tumor-specific T cells

[0332] To investigate the effect of SAA treatment on T cell function, we examined the function of tumor-infiltrating T cells (TILs) using SAA-treated B16-OVA-carrying mice. First, tumor-infiltrating T cells were nonspecifically stimulated using phorbol-12-myristate-13-acetate (PMA) and ionomycin, and intracellular staining was performed to quantify cytokine (e.g., IFN-γ, granzyme B) production by FACS. As shown in Figure 17b, tumor-infiltrating T cells from SAA-treated mice secreted higher levels of cytokines than those from the DMSO group, indicating that SAA can enhance the effector function of tumor-infiltrating T cells.

[0333] Next, lymphocytes were isolated from lymph nodes in the inflow region, stimulated with 1 μg / mL of OT1 peptide, and IFN-γ-producing cells were analyzed by FACS. As shown in Figure 17c, DLN T cells from SAA-treated mice produced significantly more IFN-γ than those from the DMSO group. The results indicated that more tumor-specific T cells were activated in the lymph nodes in the inflow region after SAA administration.

[0334] Furthermore, PD-1 from the SAA treatment group low The population was found to express far more CXCR5 than the DMSO group (Figure 18a), and PD-1 low CXCR5 high T cells (precursor exhausted T cells) were detected 14 days after tumor inoculation when treated with SAA. Furthermore, terminal exhausted T cells (PD-1 and Tim-3 double-positive cells) were evaluated 14 days after tumor inoculation when treated with SAA, and these tumor-infiltrating terminal exhausted T cells (PD-1) were identified. + Tim-3 + The frequency of ) was observed to decrease in SAA-treated mice (Figure 18b), confirming enhanced antitumor activity.

[0335] These results indicate that after SAA treatment, T cells were better primed in the lymph nodes in the inflow region, and the effector function of TILs was significantly improved.

[0336] Example 11 Synergistic effect by combination with immune checkpoint inhibitors

[0337] In wild-type mice, 5 x 10 5 B16-OVA cells were subcutaneously injected into mice. On days 9 and 11, 10 μM of SAA was injected into each mouse. On day 9, mice were also treated with 100 μg of anti-PD-L1 antibody. Tumor growth was monitored over time. As shown in Figure 18c, SAA or anti-PD-L1 antibody monotherapy could partially inhibit B16-OVA tumor growth, while combination therapy could dramatically suppress tumors and even achieve complete tumor regression. These results further support the idea that SAA can induce improved T-cell antitumor capacity through enhancement of DC function, and that immune checkpoint inhibitors (such as anti-PD-L1 antibodies), when combined with the compound of the present invention (e.g., SAA), can induce a more sustained T-cell response.

[0338] As shown in Figure 20, 5 × 10 510 B16-OVA cells were subcutaneously inoculated into C57BL / 6 mice (n=23). Nine days after transplantation, the mice were divided into two groups based on tumor size. On days 9 and 11 post-transplant, 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-inoculation, each of the two groups was further divided into other groups and administered 200 μg of PD-1 blocking antibody (Bio-X-cell, BE0146, cloning: RMP1-14) via ip injection. The control group was treated with PBS and DMSO (n=6). The single-treatment group was injected with either an isotype control containing SAA or α-PD-1 containing DMSO (n=6). The combination group was treated with SAA and PD-1 blocking antibody (n=5). Tumor growth was monitored. The data are presented as mean ± sem, and "ns" indicates no significant difference. Unpaired one-sided t-tests showed "**" p<0.01 and "****" p<0.00001. These results further support the idea that immune checkpoint inhibitors (such as anti-PD-L1 antibodies) can induce a more sustained T-cell response when combined with the compound of the present invention (e.g., SAA).

[0339] Example 12 SAA functions in APCs to exert a sustained antitumor function

[0340] As shown in Figure 21, mature FLT3L DCs were pre-treated 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 used for adoptive transfer. + The cells were purified. 5 × 10 5 1 x 10¹ B16-OVA cells were subcutaneously inoculated into C57BL / 6 mice (n=17). Nine days after transplantation, the mice were divided into three groups based on tumor size. 5Individual DMSO-treated FLT3L DCs (n=6) or SAA-treated FLT3L DCs (n=5) were intravenously transplanted into tumor-bearing mice. Seven days after the first transplant, a second batch of adoptive transplants was performed. Tumor growth was monitored. Data are shown as mean ± sem, where "ns" means no significant difference, and by unpaired one-sided t-tests, "**" p<0.01 and "****" p<0.00001. These results support the demonstration of sustained antitumor activity by adoptive-transplanted FLT3L DCs treated with the present compound (e.g., SAA).

[0341] Even though preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that these embodiments are merely given as examples. The present invention is not limited to the embodiments described herein. Although the present invention has been described herein above, the description and explanation of embodiments herein should not be constrained. Those skilled in the art will conceive of various changes, modifications and substitutions as long as they do not depart from the present invention. It should be understood that the present invention is not in any respect limited to the specific description, arrangement or relative ratio of the various conditions and variables described herein. It should be understood that various alternative forms of the embodiments described herein may be used when the present invention is implemented. Accordingly, it is expected that the present invention will cover all such alternatives, modifications, variations or equivalents. The following claims limit the scope of the present invention and include methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for determining whether a candidate drug is a YTHDF1 attenuator, The candidate drug is brought into contact with a YTHDF1 mutant that contains one or more amino acid substitutions, deletions, and / or additions at one or more residues selected from residues 372-392, 479-494, and 526-535 of SEQ ID NO:

1. To determine whether the candidate drug specifically binds to the YTHDF1 variant, and If the candidate drug specifically binds to the YTHDF1 variant, it is determined that the candidate drug is not a YTHDF1 attenuator. Methods that include...

2. The YTHDF1 variant includes one or more amino acid substitutions, deletions, and / or additions in one or more residues selected from residues N378, F382, W384, F480, and H528 of SEQ ID NO:

1. The method according to claim 1.

3. A method for determining whether a candidate drug is a YTHDF1 attenuator, The candidate drug is brought into contact with a control YTHDF1 containing one or more amino acid residues corresponding to residues selected from residues 372-392, 479-494, and 526-535 of SEQ ID NO:

1. To determine whether the candidate drug specifically binds to the control YTHDF1, and If the candidate drug does not specifically bind to the control YTHDF1, it is determined that the candidate drug is not a YTHDF1 attenuator. Methods that include...

4. A method for determining whether a candidate drug is a YTHDF1 attenuator, The candidate drug is brought into contact with a YTHDF1 mutant that contains one or more amino acid substitutions, deletions, and / or additions at one or more residues selected from residues 372-392, 479-494, and 526-535 of SEQ ID NO:

1. The candidate drug is brought into contact with a control YTHDF1 containing one or more amino acid residues corresponding to residues selected from residues 372-392, 479-494, and 526-535 of SEQ ID NO:

1. To determine whether the candidate drug specifically binds to the YTHDF1 variant and / or the control YTHDF1, and If the candidate drug does not specifically bind to the YTHDF1 variant but specifically binds to control YTHDF1, then the candidate drug is determined to be a potential YTHDF1 attenuator. Methods that include...

5. The use of compounds in the manufacture of YTHDF1 weakening agents, When the compound binds to YTHDF1, it binds to at least one residue corresponding to a residue selected from amino acid residues 372-392, 479-494, and 526-535 of SEQ ID NO: 1, The aforementioned compound comprises any of the following compounds, or a pharmaceutically acceptable salt thereof. use: 【Chemistry 1】

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