Engineered immune effector cell and application of engineered immune effector cell in combination with CBL-b inhibitor

Abstract

The invention relates to engineered immune effector cells and combined application of the engineered immune effector cells and other active substances (such as PDL1-IL15 fusion protein). The combined application relates to preparation of drugs for treating cancers or tumors, a method for treating cancers or tumors, a method for amplifying immune cells in vitro and the like.

Description

Engineered immune effector cells and their application in combination with CBL-b inhibitors

[0001] Citation of Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202210499532.9 filed on May 9, 2022 and Chinese Patent Application No. 202310305627.7 filed on March 24, 2023, and the contents of these two applications are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present application relates to the fields of bioengineering and cell therapy. Specifically, the present application provides engineered immune effector cells and their combined use with CBL-b inhibitors and / or other active substances (such as PDL1-IL15 fusion protein). Background Art

[0004] Existing cell therapy technologies include chimeric antigen receptor (CAR)-modified T cells or NK cells (CAR-T or CAR-NK) therapy targeting tumor antigens.

[0005] CAR-T therapy has been shown to be effective in treating B-cell malignancies and multiple myeloma, but it still has some shortcomings, including: patients may not be able to provide enough T cells for engineering, the preparation cycle of autologous CAR-T cells is long, CAR-T therapy may induce cell-release factor syndrome (CRS), neurotoxicity and autoimmune toxicity, and it is not ideal in treating solid tumors.

[0006] Unlike T cells, natural killer (NK) cells do not require antigen presentation via MHC molecules to recognize and kill target cells. This makes them suitable for direct preparation using an "off-the-shelf" model, using NK cells from the peripheral blood or umbilical cord blood of healthy donors. Furthermore, NK cells do not cause graft-versus-host disease (GVHD) in an allogeneic setting, making them suitable as universal cell product candidates. They also do not induce CRS and have a better safety profile. Furthermore, NK cells can be activated by accessory cells such as monocytes, macrophages, and dendritic cells, secreting active cytotoxic factors. CAR-NK cells have demonstrated rapid and potent tumor cell cytotoxicity in in vitro and in vivo studies, but their poor durability often results in a short-lived effect, failing to completely eliminate solid tumors or control tumor recurrence.

[0007] Casitas B-lineage lymphoma proto-oncogene-b (CBL-b) is an E3 ligase that negatively regulates T cell and NK cell activation and is a negative regulatory immune checkpoint. Studies have shown that CBL-b is upregulated in exhausted T cells, and knockout of CBL-b can restore the effector function of exhausted T cells or CAR-T cells. IL2 and IL15 activate NK cells and significantly upregulate CBL-b expression on the NK cell surface. This upregulation in CBL-b expression, in turn, inhibits the cytotoxicity of activated NK cells against tumor cells, acting as a negative feedback regulator during NK cell activation. Therefore, CBL-b is an important immune regulatory target.

[0008] There is an urgent need in the art to provide solutions to one or more of the shortcomings of existing cell therapy methods.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect, the present application provides use of a combination of a CBL-b inhibitor and immune effector cells in the preparation of a medicament for treating cancer or tumors, wherein the immune effector cells express or do not express a chimeric antigen receptor.

[0011] In a second aspect, the present application provides a pharmaceutical combination product for treating cancer or tumors, comprising a CBL-b inhibitor and immune effector cells, wherein the immune effector cells express or do not express a chimeric antigen receptor.

[0012] In a third aspect, the present application provides a method for treating cancer or tumors, wherein the method comprises administering to a subject in need thereof an effective amount of a CBL-b inhibitor and immune effector cells, wherein the immune effector cells express or do not express a chimeric antigen receptor.

[0013] In a fourth aspect, the present application provides a method for expanding immune effector cells in vitro, the method comprising: expanding and culturing the immune effector cells in the presence of a CBL-b inhibitor.

[0014] In a fifth aspect, the present application provides engineered NK cells, wherein the NK cells express a chimeric antigen receptor that specifically binds to Claudin18.2, and the chimeric antigen receptor includes a CD8α signal peptide, an antigen binding region that specifically binds to Claudin18.2, a CD8α hinge region, a CD8α transmembrane region, a CD28 co-stimulatory domain and CD3ζ.

[0015] In a sixth aspect, the present application provides use of a combination of the NK cells described in the fifth aspect and T cells expressing a chimeric antigen receptor that specifically binds to Claudin18.2 in the preparation of a drug for treating cancer or tumors.

[0016] In a seventh aspect, the present application provides a cellular medicine for treating cancer or tumors, which comprises the NK cells described in the fifth aspect and T cells expressing a chimeric antigen receptor that specifically binds to Claudin18.2.

[0017] In an eighth aspect, the present application provides a method for treating cancer or tumors, comprising administering to a subject in need thereof an effective amount of the NK cells described in the fifth aspect, and optionally, administering T cells expressing a chimeric antigen receptor that specifically binds to Claudin18.2.

[0018] In a ninth aspect, the present application provides use of any one of the following combinations (a)-(c) in the preparation of a medicament for treating tumors or cancer:

[0019] (a) immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,

[0020] (b) immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,

[0021] (c) Immune effector cells expressing chimeric antigen receptors, CBL-b inhibitors, and PDL1-IL15 fusion proteins.

[0022] In a tenth aspect, the present application provides a pharmaceutical combination product for treating cancer or tumors, comprising any one of the following (a)-(c):

[0023] (a) immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,

[0024] (b) immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,

[0025] (c) Immune effector cells expressing chimeric antigen receptors, CBL-b inhibitors, and PDL1-IL15 fusion proteins.

[0026] In an eleventh aspect, the present application provides a method for treating cancer or tumors, comprising administering to a subject in need thereof an effective amount of immune effector cells expressing a chimeric antigen receptor, the method further comprising: (1) stimulating the immune effector cells with a CBL-b inhibitor prior to administration, and / or (2) administering to the subject an effective amount of a PDL1-IL15 fusion protein.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 shows schematic diagrams of different exemplary CAR-NK and CAR-T plasmid structures of the present application.

[0029] FIG2A shows the in vitro expansion results of NK cells transfected with 18.2-CAR1 (NK) or untransfected NK cells (UTNK).

[0030] FIG2B shows the FACS detection results of CAR transfection efficiency of 18.2-CAR1(NK) and UTNK cells.

[0031] FIG2C shows the killing effect of 18.2-CAR1(NK) and UTNK cells on NUGC4-luc cells for 4 hours, with effector-target ratios (E:T) of 10:1, 2:1, 1:1, and 1:2, respectively.

[0032] FIG2D shows the sequential killing effects of 18.2-CAR1(NK) and UTNK cells on the CHOK1-18.2 cell line over time at different effector-target ratios as shown.

[0033] FIG2E shows the cytokine IFN-γ secretion levels when 18.2-CAR1(NK) and UTNK cells kill CHOK1-18.2 cells at different effector-target ratios as shown.

[0034] FIG3A shows the tumor growth curves of each group of mice in the NUGC4-luc mouse subcutaneous tumor model within 21 days.

[0035] FIG3B shows the body weight change rate of each group of mice in the NUGC4-luc mouse subcutaneous tumor model within 21 days.

[0036] FIG3C shows the tumor inhibition rate (TGI%) of each group of mice in the NUGC4-luc mouse subcutaneous tumor model within 21 days.

[0037] Figure 3D shows the FACS test results of the number of NK cells in the peripheral blood of each group of mice in the NUGC4-luc mouse subcutaneous tumor model on days 5, 9, 13, and 18.

[0038] Figure 3E shows the results of human IL-15 cytokine secretion detection in the peripheral blood serum of each group of mice in the NUGC4-luc mouse subcutaneous tumor model on days 5, 9, 13, and 18.

[0039] Figure 3F shows the FACS test results of the proportion of NK cells in tumor tissue lymphocytes in each group of mice in the NUGC4-luc mouse subcutaneous tumor model after dissection on the 22nd day.

[0040] Figure 3G shows the PCR detection results of the CAR copy number in the spleen and tumor tissues of each group of mice in the NUGC4-luc mouse subcutaneous tumor model after autopsy on day 22.

[0041] Figure 4A shows the fluorescence intensity curves of tumor cell growth in the peritoneal area of ​​each group of mice in the NUGC4-luc mouse peritoneal tumor model over 20 days.

[0042] FIG4B shows the weight change rate of mice in each group in the NUGC4-luc mouse peritoneal tumor model within 20 days.

[0043] Figure 4C shows the FACS test results of the proportion of NK cells in lymphocytes in the peripheral blood of each group of mice in the NUGC4-luc mouse peritoneal tumor model on the 5th day, the 12th day and the 19th day.

[0044] Figure 4D shows the results of human IL-15 cytokine secretion detection in the peripheral blood serum of each group of mice in the NUGC4-luc mouse peritoneal tumor model on the 5th day, the 12th day and the 19th day.

[0045] Figure 5A shows the killing effect of 18.2-CAR1(NK), 18.2-CAR3(T), and the combination of 18.2-CAR1(NK) and 18.2-CAR3(T) on NUGC4-luc cells for 24 hours and 48 hours, where E:T is the ratio of NK cells: target cells = 1:2, and T cells: target cells = 1:4.

[0046] Figure 5B shows the killing effect of 18.2-CAR1 (NK) and CBL-bi-1 on NUGC4-luc cells for 24 hours; E:T is the ratio of NK cells to target cells = 1:6.

[0047] Figure 5C shows the multiple rounds of continuous killing effects of 18.2-CAR1 (NK) in combination with CBL-bi-3, and 18.2-CAR4 (T) in combination with CBL-bi-3 on NUGC4-luc cells, where E:T is the NK cell: target cell ratio = 1:24, and the T cell: target cell ratio = 1:16.

[0048] FIG6 shows the multiple rounds of continuous real-time killing effects of 18.2-CAR1(NK), 18.2-CAR3(T), CBL-bi-1, and the combination of the two or the combination of the three on the CHOK1-18.2 cell line.

[0049] Figure 7A shows the CAR transfection efficiency of 18.2-CAR2(T) cells detected by FACS after 9 days of culture and expansion with the addition of CBL-bi-1 and CBL-bi-2.

[0050] FIG7B shows the expression level of the CD4 cell memory phenotype of 18.2-CAR2(T) cells detected by FACS after 9 days of culture and expansion with the addition of CBL-bi-1 and CBL-bi-2.

[0051] FIG7C shows the expression level of the CD8 cell memory phenotype of 18.2-CAR2(T) cells detected by FACS after 9 days of culture and expansion with the addition of CBL-bi-1 and CBL-bi-2.

[0052] Figure 7D shows the CD69 expression levels of CD4 and CD8 cells of 18.2-CAR2 (T) cells detected by FACS after 9 days of culture and expansion with the addition of CBL-bi-1 and CBL-bi-2.

[0053] Figure 7E shows the killing effect of 18.2-CAR2 (T) cells on NUGC4-luc cells for 24 hours after 9 days of culture and expansion with the addition of CBL-bi-1 and CBL-bi-2, where the E:T ratio is 5:1, 2:1, 1:1, and 1:2.

[0054] Figure 7F shows the level of CD107a expression on the T cell surface and the level of IFN-γ released during the 24-hour killing of NUGC4-luc cells by 18.2-CAR2 (T) cells after 9 days of culture and expansion with the addition of CBL-bi-1 and CBL-bi-2, where the E:T ratio is 1:1.

[0055] Figure 7G shows the level of CD107a expression on the T cell surface and the level of IFN-γ released after 18.2-CAR2 (T) cells were cultured and expanded for 9 days with the addition of CBL-bi-1 and CBL-bi-2, and then cultured for 24 hours without the addition of target cells.

[0056] FIG8 shows the CAR transfection efficiency of BCMA-CAR1(NK) and BCMA-CAR2(NK) after 15 days of expansion in culture medium supplemented with CBL-bi-4 as determined by FACS.

[0057] Figure 9 shows a schematic diagram of the dual-target chimeric antigen receptor structure targeting BCMA and GPRC5D.

[0058] FIG10 shows the positive rate detection results of BCMA-GPRC5D CAR-NK with or without the addition of CBL-bi.

[0059] FIG11 shows the results of multiple rounds of killing experiments of NCI H929+10% NCI H929-hBCMA-KO cells using BCMA-GPRC5D CAR-NK in combination with CBL-b inhibitor and / or PDL1-IL15.

[0060] FIG12A shows the anti-tumor efficacy test scheme of BCMA-GPRC5D CAR-NK in combination with CBL-b inhibitor in Molp8-luc mouse tumor model.

[0061] Figures 12B-12E respectively show the tumor growth fluorescence signal ROI values ​​(Figures 12B and 12C), body weight changes (Figure 12D), and mouse survival rate (Figure 12E) of the Molp8-luc mouse tumor model using BCMA-GPRC5D CAR-NK in combination with a CBL-b inhibitor, wherein the BI-CAR21-CBL-b inhibitor in Figure 12B represents the BI-CAR21 treatment group in Table 9, i.e., the treatment group with only BI-CAR21 without the addition of a CBL-b inhibitor.

[0062] Detailed Description of the Invention

[0063] Unless otherwise defined herein, scientific and technical terms related to the present application shall have the meanings understood by those of ordinary skill in the art.

[0064] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0065] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0066] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0067] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0068] Chimeric antigen receptor (CAR)

[0069] Chimeric antigen receptor refers to an artificial immune effector cell surface receptor that is modified to express on immune effector cells and specifically binds to an antigen, and it at least includes: (1) an extracellular antigen binding domain, such as a variable heavy chain or light chain of an antibody; (2) a transmembrane domain that anchors CAR into immune effector cells; and (3) an intracellular signaling domain. CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, using the extracellular antigen binding domain in a non-MHC restricted manner. In some specific embodiments, optionally, the extracellular domain of the chimeric antigen receptor also includes a signal peptide and / or a hinge region, and / or the intracellular domain of the chimeric antigen receptor also includes a costimulatory domain.

[0070] As used herein, the term "signal peptide" in the context of chimeric antigen receptors refers to a segment of a protein or polypeptide that is used to guide the protein or polypeptide into the secretory pathway and translocate to the cell membrane and / or cell surface. An example of a signal peptide is the CD8α signal peptide.

[0071] As used herein, the terms "antigen binding region" or "target binding sequence" in the context of a chimeric antigen receptor are used interchangeably and refer to the portion responsible for binding to the target of the CAR (e.g., a tumor antigen on a tumor cell). The form of the antigen binding region can be similar to the structure of an antibody, such as scFv.

[0072] As used herein, "specific binding" refers to the ability of an antigen-binding molecule (e.g., antibodies and fragments thereof, variants of the antigen's natural ligand, and natural variants thereof) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically reflected in terms of the equilibrium dissociation constant (KD), with a lower KD indicating higher affinity. For antibodies, for example, high affinity typically refers to a KD of about 10E-6 M or less, about 10E-7 M or less, about 10E-8 M or less, about 10E-9 M or less, about 10E-10 M or less, 10E-11 M or less, or 10E-12 M or less. KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0073] The term "antibody" is used in the broadest sense herein to refer to a polypeptide or combination of polypeptides that comprises sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0074] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0075] The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lama guanicoe) and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0076] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0077] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, scFv, and VHH.

[0078] Papain digestion of intact antibodies generates two identical antigen-binding fragments, called "Fab" fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains carry free thiol groups. Pepsin treatment produces a F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0079] The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).

[0080] The term "nanoantibody" in this article refers to heavy chain antibodies that are naturally missing light chains and exist in camels and other organisms. Cloning their variable regions can produce single-domain antibodies consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0081] The terms "VHH domain" and "single domain antibody" (sdAb) are used interchangeably and have the same meaning. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy chain variable region by cloning the variable region of a heavy chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy chain variable region.

[0082] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0083] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0084] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0085] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0086] The term "IMGT numbering system" herein generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.

[0087] As used herein, the term "hinge region" in the context of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane region and the antigen binding region. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antigen binding region. The hinge region may be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. The hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.

[0088] As used herein, the term "transmembrane (TM) region" in the context of a chimeric antigen receptor refers to a polypeptide structure that enables a chimeric antigen receptor to be expressed on the surface of an immune effector cell (e.g., a lymphocyte, NK cell, or NKT cell) and guides the immune effector cell to respond to a target cell. The transmembrane domain can be natural or synthetic, or it can be derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor binds to the target antigen, the transmembrane domain can carry out signal transduction.

[0089] As used herein, the term "intracellular signaling domain" in the context of a chimeric antigen receptor refers to a portion of a protein that transduces effector function signals and directs cells to perform designated functions. The intracellular signaling domain is responsible for primary signaling within the cell after the antigen binding domain binds to the antigen, thereby leading to activation of immune effector cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune effector cells in which the CAR is expressed. Exemplary intracellular signaling domains include CD3ζ.

[0090] As used herein, the term "costimulatory domain" in the context of a chimeric antigen receptor refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes after binding to an antigen. The costimulatory domain can be from CD28, 4-1BB or 2B4, but is not limited thereto.

[0091] As used herein, the term "immune effector cell" or "effector cell" refers to a cell that participates in an immune response, such as a cell that promotes immune effector responses. Examples of immune effector cells include T cells, such as α / βT cells and γ / δT cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes, and TIL (tumor infiltrating lymphocytes). Immune effector cells or effector cells include nucleic acids encoding chimeric antigen receptors and / or express chimeric antigen receptors, which are different according to the type of immune effector cells or effector cells, such as CAR-T, CAR-NK, CAR-M, etc.

[0092] As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced into one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored). For the purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires achieving maximum alignment over the full length of the compared sequences. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0093] As used herein, "vector" is a material composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmids and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0094] The term "drug" herein refers to a substance used to prevent, treat and diagnose a disease, including a "drug composition" or a "drug combination".

[0095] The term "pharmaceutical composition" herein refers to a preparation that exists in a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain additional ingredients that have unacceptable toxicity to the subject to whom the pharmaceutical composition is administered. Pharmaceutical compositions include combinations separated in time and / or space, as long as they can work together to achieve the purpose of this application. For example, the ingredients contained in the pharmaceutical composition (such as CAR- immune cells according to the present application) can be applied to an individual as a whole, or separately. When the ingredients contained in the pharmaceutical composition are applied separately to an individual, the ingredients can be applied to the individual simultaneously or sequentially. The pharmaceutical composition according to the present application may include conventional components of cell culture to maintain the activity of CAR- immune cells. Pharmaceutically acceptable carriers may also include water, buffered aqueous solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The pharmaceutical composition or pharmaceutical preparation according to the present application can be administered by any appropriate route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc. The composition according to the present application may contain a wetting agent, an emulsifier or a buffer substance as an additive.

[0096] As used herein, the term "combination" or "pharmaceutical combination" or "pharmaceutical combination product" refers to a non-fixed combination, wherein the active agent and at least one additional active agent can be administered separately, simultaneously or within time intervals, particularly where these time intervals allow the combination partners to exhibit a cooperative (e.g., synergistic) effect. The term "non-fixed combination" means that the active ingredients (e.g., one active agent and at least one additional active agent) are both administered to a patient as separate entities, simultaneously or sequentially without specific time limits, wherein such administration provides therapeutically effective levels of both compounds in the patient's body.

[0097] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0098] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or organic acid, and a salt formed between a compound and an inorganic base or an organic base.

[0099] The term "pharmaceutically acceptable excipients or excipients" refers to those excipients that are non-irritating to organisms and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0100] As used herein, the terms "subject," "object," "individual," and "patient" refer to an organism that is being treated for a particular disease or condition, such as cancer or an infectious disease, as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as cattle, bison, buffalo, elk, and yaks, etc.), cattle, sheep, horses, and bison, etc., that are being treated for a disease or condition, such as a cell proliferative disorder, such as cancer or an infectious disease.

[0101] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of a cell proliferative disorder (such as cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a disease or disease, as well as subjects susceptible to a disease or disease, or subjects intending to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0102] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0103] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the terms "tumor" or "neoplasm" refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0104] In this article Indicates the junction site.

[0105] In this paper, the bonds depicted by solid and dashed lines Indicates a single bond or a double bond.

[0106] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge and dotted wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0107] The term "tautomer" refers to functional group isomers resulting from the rapid movement of an atom in a molecule between two positions. The compounds of the present application may exhibit tautomerism. Tautomeric compounds may exist as two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to separate a single tautomer usually produce a mixture whose physical and chemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical characteristics within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; whereas in phenols, the enol form predominates. The present application encompasses all tautomeric forms of the compounds.

[0108] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0109] The compounds of the present application may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, and therefore the compounds of the present application may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E-type and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the definition of the compounds of the present application and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of these isomers and mixtures thereof involved in the substituents are also within the definition of the compounds of the present application. The compounds of the present invention containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0110] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0111] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes that the event or situation occurs and that the event or situation does not occur. For example, an ethyl group is "optionally" substituted with halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any sterically impossible and / or incomposable replacement or substitution pattern will not be introduced.

[0112] When any variable (such as R 1a 、R 2a ) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is represented by two R 1a is replaced, then each R 1a There are independent options.

[0113] When the number of a linking group is 0, such as -(CR13 R 14 )0-, indicating that the linking group is a bond.

[0114] When one of the variables is selected from a chemical bond or is absent, it means that the two groups it connects are directly connected, such as When L represents a bond, it means that the structure is actually

[0115] When the linking group mentioned in this article does not specify its connection direction, its connection direction is arbitrary. L in the formula is selected from "-NR 7 CH2-", at this time L can be connected with the ring D from left to right to form "ring D-NR 7 CH2-", or you can connect ring D from right to left to form "ring D-CH2NR 7 -".

[0116] When a substituent's bond crosses two atoms in a ring, the substituent may be bonded to any atom in the ring. Represents R 10 Substitution can occur at any of positions 1, 2, and 3 on the benzene ring.

[0117] In this article, C m -C n For example, "C1-C6" means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0118] The term "alkyl" refers to a group of the formula C n H 2n+1 The term "C1-C6 alkyl" is understood to mean a straight or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. Specific examples of the alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl. The term "C1-C3 alkyl" is understood to mean a linear or branched saturated monovalent hydrocarbon group having 1 to 3 carbon atoms. The "C1-C6 alkyl" may include a "C1-C3 alkyl".

[0119] The term "alkoxy" refers to a monovalent group derived from a straight-chain or branched alcohol by the loss of a hydrogen atom from a hydroxyl group, and can be understood as "alkyloxy" or "alkyl-O-." The term "C1-C6 alkoxy" can be understood as "C1-C6 alkyloxy" or "C1-C6 alkyl-O-," and the term "C1-C3 alkoxy" can be understood as "C1-C3 alkyloxy" or "C1-C3 alkyl-O-." The "C1-C6 alkoxy" group may further include a "C1-C3 alkoxy group."

[0120] The term "alkenyl" refers to a linear or branched, monovalent, unsaturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms and having at least one double bond. The term "C2-C4 alkenyl" is understood to mean a linear or branched, unsaturated, monovalent hydrocarbon group containing one or more double bonds and having 2, 3, or 4 carbon atoms. "C2-C4 alkenyl" is preferably a C2 or C3 alkenyl group. It should be understood that when the alkenyl group contains more than one double bond, the double bonds may be separated or conjugated. Specific examples of the alkenyl group include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl or (Z)-1-methylprop-1-enyl, etc.

[0121] The term "alkynyl" refers to a straight or branched monovalent unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, having at least one triple bond. The term "C2-C4 alkynyl" may be understood to mean a straight or branched unsaturated monovalent hydrocarbon group containing one or more triple bonds and having 2, 3 or 4 carbon atoms. Examples of "C2-C4 alkynyl" include, but are not limited to, ethynyl (-C≡CH), propynyl (-C≡CCH3, -CH2C≡CH), but-1-ynyl, but-2-ynyl or but-3-ynyl. "C2-C4 alkynyl" may include "C2-C3 alkynyl," and examples of "C2-C3 alkynyl" include ethynyl (-C≡CH), prop-1-ynyl (-C≡CCH3), prop-2-ynyl (propargyl).

[0122] The term "cycloalkyl" refers to a fully saturated carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. Unless otherwise indicated, the carbon ring is typically a 3- to 10-membered ring. The term "C3-C 10"Cycloalkyl" is understood to mean a saturated monovalent monocyclic, bicyclic, spirocyclic or bridged ring having 3 to 10 carbon atoms. Specific examples of the cycloalkyl group include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, and the like. The term "C3-C 10 "Cycloalkyl" may include "C3-C8 cycloalkyl", "C3-C8 cycloalkyl" may include "C3-C6 cycloalkyl", and "C3-C6 cycloalkyl" may include "C3-C4 cycloalkyl". The term "C3-C6 cycloalkyl" may be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms, and specific examples include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0123] The term "cycloalkyloxy" may be understood as "cycloalkyl-O-".

[0124] The term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon group containing at least one carbon-carbon double bond. "C3-C6 cycloalkenyl" refers to a non-aromatic cyclic hydrocarbon group having 3-6 carbon atoms as ring atoms and containing at least one carbon-carbon double bond. Specific examples of C3-C6 cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, and cyclohexadienyl.

[0125] The term "heterocyclyl" refers to a fully saturated or partially saturated (heteroaromatic group that is not aromatic as a whole) monovalent monocyclic, fused, spiro or bridged ring group containing 1 to 5 heteroatoms or heteroatomic groups (i.e., heteroatom-containing groups) in the ring atoms, wherein the "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)2NH-, S(=O)NH- or -NHC(=O)NH-. The term "4-10 membered heterocyclyl" refers to a heterocyclyl group having 4, 5, 6, 7, 8, 9 or 10 ring atoms, and containing 1 to 5 heteroatoms or heteroatomic groups independently selected from the above-mentioned heteroatoms or heteroatomic groups in the ring atoms. The term "4-10 membered nitrogen-containing heterocyclic group" refers to a 4-10 membered heterocyclic group containing at least one nitrogen atom among its ring atoms, and "4-10 membered nitrogen-containing heterocyclic group" includes "6-10 membered nitrogen-containing heterocyclic group". The term "4-7 membered monocyclic nitrogen-containing heterocyclic group" refers to a 4-7 membered heterocyclic group in the form of a monocyclic ring containing at least one nitrogen atom among its ring atoms. “4-10 membered heterocyclyl” includes “6-10 membered heterocyclyl”, and “6-10 membered heterocyclyl” further includes “4-7 membered heterocyclyl”, wherein specific examples of 4-membered heterocyclyl include but are not limited to azetidinyl or oxetanyl; specific examples of 5-membered heterocyclyl include but are not limited to tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazolyl or 2,5-dihydro-1H-pyrrolyl; specific examples of 6-membered heterocyclyl include but are not limited to tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl or 4H-[1,3,4]thiadiazinyl; specific examples of 7-membered heterocyclyl include but are not limited to diazepanyl; specific examples of 8-membered heterocyclyl include but are not limited to 6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazolyl. The heterocyclic group may also be a bicyclic group, wherein specific examples of 5,5-membered bicyclic groups include but are not limited to hexahydrocyclopenta[c]pyrrol-2(1H)-yl; specific examples of 5,6-membered bicyclic groups include but are not limited to hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl or 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl. Optionally, the heterocyclic group may be a benzo-fused ring group of the above-mentioned 4-7-membered heterocyclic groups, specific examples of which include but are not limited to dihydroisoquinolinyl and the like."4-10 membered heterocyclyl" may include "5-10 membered heterocyclyl", "4-7 membered heterocyclyl", "5-7 membered heterocyclyl", "5-6 membered heterocyclyl", "6-8 membered heterocyclyl", "4-10 membered heterocycloalkyl", "5-10 membered heterocycloalkyl", "4-7 membered heterocycloalkyl", "5-6 membered heterocycloalkyl", "6-8 membered heterocycloalkyl", etc. "4-7 membered heterocyclyl" may further include "4-6 membered heterocyclyl", "5-7 membered heterocyclyl", "5-6 membered heterocyclyl", "4-7 membered heterocycloalkyl", "4-6 membered heterocycloalkyl", "5-6 membered heterocycloalkyl", etc. In the present application, although some bicyclic heterocyclyl groups partially contain a benzene ring or a heteroaromatic ring, the heterocyclyl group as a whole is still non-aromatic.

[0126] The term "heterocyclyloxy" may be understood as "heterocyclyl-O-".

[0127] The term "heterocycloalkyl" refers to a fully saturated monovalent cyclic group in the form of a monocyclic, fused, bridged or spirocyclic ring, wherein the ring atoms of the ring contain 1-5 heteroatoms or heteroatomic groups (i.e., heteroatomic groups containing heteroatoms), wherein the "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)2NH-, S(=O)NH- or -NHC(=O)NH-. The term "4-10 membered heterocycloalkyl" refers to a heterocycloalkyl group having 4, 5, 6, 7, 8, 9 or 10 ring atoms, wherein the ring atoms of the heterocycloalkyl group contain 1-5 heteroatoms or heteroatomic groups independently selected from the above-mentioned heteroatoms or heteroatomic groups. “4-10 membered heterocycloalkyl” includes “4-7 membered heterocycloalkyl”, wherein specific examples of 4 membered heterocycloalkyl include but are not limited to azetidinyl, oxetanyl or thietanyl; specific examples of 5 membered heterocycloalkyl include but are not limited to tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl or tetrahydropyrazolyl; specific examples of 6 membered heterocycloalkyl include but are not limited to piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1,4-thioxanyl, 1,4-dioxanyl, thiomorpholinyl, 1,3-dithianyl or 1,4-dithianyl; specific examples of 7 membered heterocycloalkyl include but are not limited to azepanyl, oxetanyl or thiepanyl.

[0128] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. The aryl group may have 6-14 carbon atoms or 6-10 carbon atoms. The term "C6-C 10"Aryl" is understood to be a monovalent aromatic monocyclic or bicyclic group having 6 to 10 carbon atoms. In particular, a ring having 6 carbon atoms ("C6 aryl"), for example phenyl; or a ring having 10 carbon atoms ("C 10 "aryl"), for example naphthyl.

[0129] The term "aryloxy" may be understood as "aryl-O-".

[0130] The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system having aromatic character, which contains at least one ring atom selected from N, O, S, and the remaining ring atoms are C. The term "5-10 membered heteroaryl" is understood to include monovalent monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 ring atoms, and containing 1, 2, 3, 4 or 5, preferably 1, 2 or 3 heteroatoms independently selected from N, O and S. In particular, the heteroaryl group is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl or thiadiazolyl, and the like, and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl or isoindolyl, and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl or isoquinolyl, and the like; or acinyl, indolizinyl, purinyl, and the like, and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl or phenoxazinyl, and the like. The term "5-10 membered nitrogen-containing heteroaryl" refers to a 5-10 membered heteroaryl group containing at least one nitrogen atom among its ring atoms, and "5-10 membered nitrogen-containing heteroaryl" includes "5-6 membered nitrogen-containing heteroaryl". The term "5-6 membered heteroaryl" refers to an aromatic ring system having 5 or 6 ring atoms, and containing 1, 2 or 3, preferably 1 or 2 heteroatoms independently selected from N, O and S. The term "6 membered heteroaryl" refers to an aromatic ring system having 6 ring atoms, and containing 1, 2 or 3, preferably 1 or 2 N as a heteroatom. The term "5-10 membered heteroaryl" includes "5-6 membered heteroaryl".

[0131] The term "heteroaryloxy" may be understood as "heteroaryl-O-".

[0132] The term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0133] The term "hydroxy" refers to an -OH group.

[0134] The term "cyano" refers to a -CN group.

[0135] The term "mercapto" refers to a -SH group.

[0136] The term "amino" refers to a -NH2 group.

[0137] The term "nitro" refers to a -NO2 group.

[0138] The present application also includes isotopically labeled compounds of the present application that are identical to those described herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0139] Certain isotope-labeled compounds of the present application (e.g. 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0140] After extensive research and testing, the inventors of this application have developed engineered immune effector cells and their combined use with and / or other active substances (such as PDL1-IL15 fusion protein). The combined application involves the preparation of drugs for treating cancer or tumors, methods for treating cancer or tumors, methods for expanding immune cells in vitro, etc.

[0141] In a first aspect, the present application provides a use of a combination of a CBL-b inhibitor and immune effector cells in the preparation of a medicament for treating cancer or tumors, wherein the immune effector cells express or do not express a chimeric antigen receptor.

[0142] In a second aspect, the present application provides a pharmaceutical combination product for treating cancer or tumors, comprising a CBL-b inhibitor and immune effector cells, wherein the immune effector cells express or do not express a chimeric antigen receptor.

[0143] In a third aspect, the present application provides a method for treating cancer or tumors, wherein the method comprises administering to a subject in need thereof an effective amount of a CBL-b inhibitor and immune effector cells, wherein the immune effector cells express or do not express a chimeric antigen receptor.

[0144] The following specific embodiments described for CBL-b inhibitors, immune effector cells, chimeric antigen receptors, cancer or tumors are applicable to the first to third aspects, and, unless there is a contradiction, also apply to the various embodiments of the invention described in any aspect of this application.

[0145] In some specific embodiments, the CBL-b inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0146] in,

[0147] Selected from any one of the following: i) C=C-A3, wherein A3 is selected from CR 11a R 11b NR 12 , O or S; ii) A1-C=A3, wherein A1 is selected from C or N, A3 is selected from CR 11c or N; iii) A1-A2-A3, wherein A1 and A2 are independently selected from C(R 11 ) n or N, A3 selected from CR 11a R 11b NR 12 , O or S;

[0148] n is selected from 0 or 1;

[0149] R 11a 、R 11b 、R 11c 、R 11 、R 12are independently selected from H, halogen, OH, CN, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl or C3-C6 cycloalkyloxy, wherein said C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl or C3-C6 cycloalkyloxy is optionally replaced by R 11d replace;

[0150] The Q ring is selected from phenyl, 5-6 membered heteroaryl or 5-7 membered heterocyclic group, wherein the phenyl, 5-6 membered heteroaryl or 5-7 membered heterocyclic group is optionally replaced by R 10 replace;

[0151] R 10 is selected from halogen, =O, OH, NH2, NO2, CN, C1-C6 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 cycloalkyloxy, C3-C6 cycloalkyl-NH-, 4-7 membered heterocyclyl, 4-7 membered heterocyclyloxy or 4-7 membered heterocyclyl-NH-, wherein NH2, C1-C6 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 cycloalkyloxy, C3-C6 cycloalkyl-NH-, 4-7 membered heterocyclyl, 4-7 membered heterocyclyloxy or 4-7 membered heterocyclyl-NH- is optionally replaced by R 10a replace;

[0152] Y 1 、Y 2 、Y 3 and Y 4 Independently selected from CR b or N;

[0153] X is selected from halogen, CN, OH, COOH, CONH2, C1-C6 alkyl, C1-C6 alkoxy, wherein C1-C6 alkyl or C1-C6 alkoxy is optionally replaced by R e substituted, ring B is selected from optionally R 3 The following groups substituted: 4-10 membered nitrogen-containing heterocyclic group or 5-10 membered nitrogen-containing heteroaryl group, ring D is selected from optionally replaced by R 6 Substituted with the following groups: C3-C 10 Cycloalkyl, 4-10 membered heterocyclyl, phenyl or 5-10 membered heteroaryl, and ring D is connected to L via a non-N atom, and L is selected from a bond, -NR 7 -、-NR 7 CH2-, -O-, -C(=O)-, -C(=O)NH- or -CR 8 R 9 -;

[0154] R b Selected from H, halogen, OH, CN, C1-C6 alkyl, C1-C6 alkoxy, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, NHC(O)(C1-C6 alkyl), NHC(O)-O(C1-C6 alkyl), N(C1-C6 alkyl)C(O)-O(C1-C6 alkyl), NHS(O)2(C1-C6 alkyl), C3-C6 cycloalkyl, C3 -C6 cycloalkyl-O-, C3-C6 cycloalkyl-NH-, N(C3-C6 cycloalkyl)2, NHC(O)-C3-C6 cycloalkyl, NHS(O)2-C3-C6 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclyloxy, 4-7 membered heterocyclyl-NH-, N(4-7 membered heterocyclyl)2, NHC(O)-4-7 membered heterocyclyl, NHS(O)2-4-7 membered heterocyclyl, C6-C 10 Aryl, C6-C 10 Aryloxy, C6-C 10 Aryl-NH-, N(C6-C 10 Aryl)2, NHC(O)-C6-C 10 Aryl, NHS(O)2-C6-C 10 Aryl, 5-10 membered heteroaryl, 5-10 membered heteroaryloxy or 5-10 membered heteroaryl-NH-, N(5-10 membered heteroaryl)2, NHC(O)-5-10 membered heteroaryl, NHS(O)2-5-10 membered heteroaryl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, C6-C 10 Aryl or 5-10 membered heteroaryl is optionally replaced by R 2a replace;

[0155] Or two R b The C atoms connected thereto together form a C3-C6 cycloalkenyl, phenyl, 4-7 membered heterocyclic group or 5-6 membered heteroaryl, wherein the C3-C6 cycloalkenyl, phenyl, 4-7 membered heterocyclic group or 5-6 membered heteroaryl is optionally replaced by R 2a replace;

[0156] R 4 、R 5 、R 7 、R 8 and R 9 Each of them is independently selected from H, halogen, OH, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl or C1-C6 alkoxy is optionally replaced by R 4a replace;

[0157] or R 8 、R 9and the atoms to which they are attached together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally further replaced by R 8a replace;

[0158] or R 4 、R 5 and the atoms to which they are attached together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally further replaced by R 8a Replacement; or when p is taken from 2, two R 4 and the atoms to which they are attached together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally further replaced by R 8a replace;

[0159] or R 4 、R 5 Together they form =O;

[0160] R 3 and R 6 Independently selected from halogen, CN, =O, OH, NO2, C1-C6 alkyl, OR 6a SR 6a 、N(R 6a )2、S(O)2R 6a 、S(O)2N(R 6a )2、S(O)R 6a 、S(O)N(R 6a )2、C(O)R 6a 、C(O)OR 6a 、C(O)N(R 6a )2、C(O)N(R 6a )OR 6a 、OC(O)R 6a 、OC(O)N(R 6a )2、N(R 6a )C(O)OR 6a 、N(R 6a )C(O)R 6a 、N(R 6a )C(O)N(R 6a )2、N(R 6a )C(NR 6a )N(R 6a )2、N(R 6a )S(O)2N(R 6a )2、N(R 6a )S(O)2R 6a 、C3-C 10Cycloalkyl, 4-7 membered heterocyclic group, 6-10 membered aryl or 5-10 membered heteroaryl, wherein C1-C6 alkyl, C3-C 10 Cycloalkyl, 4-7 membered heterocyclic group, C6-C 10 The 5- to 10-membered aryl or heteroaryl group is optionally further substituted by R 3a replace;

[0161] Every R 6a independently selected from H, C1-C6 alkyl, phenyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, wherein the C1-C6 alkyl, phenyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl is optionally further replaced by R 6b Substitution, or two R on a N atom 6a Together with the N to which it is attached, it forms a 4-7 membered heterocyclic group or a 5-6 membered heteroaryl group, wherein the 4-7 membered heterocyclic group or the 5-6 membered heteroaryl group is optionally further replaced by R 6b replace;

[0162] R 3a 、R 4a 、R 6b and R e independently selected from halogen, OH, CN, =O, C1-C6 alkyl, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, COOH or C1-C6 alkoxy, wherein said C1-C6 alkyl or C1-C6 alkoxy is further optionally replaced by R f replace;

[0163] R f is selected from halogen, OH, =O, NH2, NH(C1-C6 alkyl) or N(C1-C6 alkyl)2;

[0164] R 11d 、R 2a and R 10a Independently selected from halogen, OH, CN, =O, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy or halo-C1-C6 alkoxy;

[0165] R 1 and R 2 independently selected from H, halogen, CN, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, C1-C6 alkoxy, C3-C 10 Cycloalkyl or 4-10 membered heterocyclic group, wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C 10 Cycloalkyl or 4-10 membered heterocyclic group is optionally replaced by R 1a replace,

[0166] or R 1 、R 2 The atoms connected to it together form C3-C 10 Cycloalkyl or 4-10 membered heterocyclic group, the C3-C 10 Cycloalkyl or 4-10 membered heterocyclic group is optionally replaced by R 1b replace;

[0167] or R 1 、R b and their respective atoms and bonds together form a C3-C6 cycloalkenyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkenyl or the 4-7 membered heterocyclic group is optionally replaced by R 1d replace;

[0168] R 1a and R 1b independently selected from halogen, OH, CN, =O, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl or C1-C6 alkoxy is optionally further substituted by R 1c replace;

[0169] R 1c is selected from halogen, OH, CN, =O, NH2 or COOH;

[0170] W is selected from (CR 13 R 14 ) k W1; said W1 is selected from 5-10 membered heteroaryl or 4-10 membered heterocyclic group, said 5-10 membered heteroaryl, 4-10 membered heterocyclic group is optionally replaced by R 15 Replacement; R 13 、R 14 R is independently selected from H, halogen, OH, C1-C6 alkyl or C1-C6 alkoxy; 15 Selected from halogen, OH, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, C3-C 10 Cycloalkyl or 4-7 membered heterocyclic group, wherein the C1-C6 alkyl, C3-C 10 Cycloalkyl or 4-7 membered heterocyclic group is optionally replaced by R 15a replace;

[0171] or R 1 With R 13 and their respective atoms and bonds together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally replaced by R 13a replace;

[0172] R 8a 、R13a and R 1d R is independently selected from halogen, OH, CN, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl or C1-C6 alkoxy is optionally further substituted with halogen; 15a Selected from halogen, =O, OH, CN or C1-C6 alkyl;

[0173] k is selected from 0 or 1;

[0174] p is selected from 0, 1 or 2.

[0175] In some embodiments, Selected from: i) C=C-A3, wherein A3 is selected from CR 11a R 11b NR 12 , O or S; or ii) A1-C=A3, wherein A1 is selected from C or N, A3 is selected from CR 11c or N.

[0176] In some embodiments, Selected from A1-C=A3, wherein A1 is selected from C or N, and A3 is selected from CR 11c or N.

[0177] In some embodiments, Selected from A1-C=A3, wherein A1 is selected from N, and A3 is selected from CR 11c or N.

[0178] In some embodiments, Selected from C=C-A3, wherein A3 is selected from CR 11a R 11b NR 12 , O or S.

[0179] In some embodiments, Selected from C=C-A3, wherein A3 is selected from NR 12 , O or S.

[0180] In some embodiments, Selected from A1-A2-A3, wherein A1 and A2 are independently selected from C(R 11 ) n or N, A3 is selected from NR 12 , O or S.

[0181] In some embodiments, is selected from A1-A2-A3, wherein A1 is selected from N, A2 is selected from C(R 11 ) n , A3 is selected from NR 12 .

[0182] In some embodiments, R 11a 、R 11b 、R 11c 、R 11 、R 12 are independently selected from H, halogen, OH, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl or C3-C6 cycloalkyloxy, wherein said C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl or C3-C6 cycloalkyloxy is optionally replaced by R 11d replace.

[0183] In some embodiments, R 11a 、R 11b 、R 11c 、R 11 、R 12 Each of them is independently selected from H, halogen, OH, NH2, C1-C6 alkyl or C3-C6 cycloalkyl, wherein the C1-C6 alkyl or C3-C6 cycloalkyl is optionally replaced by R 11d replace.

[0184] In some embodiments, R 11d Selected from halogen, OH, NH2, C1-C6 alkyl or halogenated C1-C6 alkyl.

[0185] In some embodiments, n is selected from 0.

[0186] In some embodiments, R 11c is selected from H, halogen or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally replaced by R 11d replace.

[0187] In some embodiments, R 11c is selected from H or halogen.

[0188] In some embodiments, R 11c Selected from F.

[0189] In some embodiments, R 12 is selected from H, halogen, OH, C1-C6 alkyl or C3-C6 cycloalkyl, wherein the C1-C6 alkyl or C3-C6 cycloalkyl is optionally replaced by R 11d replace.

[0190] In some embodiments, R 12 Selected from H or C1-C3 alkyl.

[0191] In some embodiments, R 12 Selected from H or methyl.

[0192] In some embodiments, R 12 Selected from methyl.

[0193] In some embodiments, Selected from one of the following situations: i) C=C-A3, wherein A3 is selected from N-CH3, O or S; ii) A1-C=A3, wherein A1 is selected from N, and A3 is selected from N or CF; iii) A1-A2-A3, wherein A1 is selected from N, A2 is selected from C, and A3 is selected from N-CH3.

[0194] In some embodiments, Selected from A1-C=A3, wherein A1 is selected from N, and A3 is selected from N.

[0195] In some embodiments, the Q ring is selected from the group consisting of 10 Substituted phenyl, pyridyl, pyrimidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl or 5-7 membered heterocyclyl containing 1 or 2 N as heteroatoms.

[0196] In some embodiments, the Q ring is selected from the group consisting of 10 Substituted with the following groups: phenyl,

[0197] In some embodiments, the Q ring is selected from the group consisting of 10 Replaced

[0198] In some embodiments, R 10 is selected from halogen, =O, OH, NH2, CN, C1-C6 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 cycloalkyl-O-, C3-C6 cycloalkyl-NH-, 4-7 membered heterocyclyl, 4-7 membered heterocyclyloxy or 4-7 membered heterocyclyl-NH-, wherein the NH2, C1-C6 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 alkoxy or C3-C6 cycloalkyl is optionally replaced by R 10a replace.

[0199] In some embodiments, R 10 is selected from halogen, =O, NH2, C1-C6 alkyl, C2-C4 alkynyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 cycloalkyl-O- or C3-C6 cycloalkyl-NH-, wherein the NH2, C1-C6 alkyl, C2-C4 alkynyl, C1-C6 alkoxy or C3-C6 cycloalkyl is optionally replaced by R 10a replace.

[0200] In some embodiments, R10 is selected from halogen, =O, NH2, C1-C6 alkyl, C2-C4 alkynyl, C1-C6 alkoxy, C3-C6 cycloalkyl, wherein the NH2, C1-C6 alkyl, C2-C4 alkynyl, C1-C6 alkoxy or C3-C6 cycloalkyl is optionally replaced by R 10a replace.

[0201] In some embodiments, R 10a is selected from halogen, OH, NH2, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy or halogenated C1-C6 alkoxy.

[0202] In some embodiments, R 10a Selected from halogen, OH, C1-C6 alkyl or halogenated C1-C6 alkyl.

[0203] In some embodiments, R 10a Selected from F or CH3.

[0204] In some embodiments, R 10 is selected from ═O, F, Cl, Br, CN, methyl, ethyl, ethynyl, CF 3 , CHF 2 , methoxy, N(CH 3 ) 2 , cyclopropyl, cyclopropyl-O— or cyclopropyl-NH—.

[0205] In some embodiments, R 10 is selected from ═O, F, Cl, ethynyl, CF 3 , CHF 2 , methoxy, N(CH 3 ) 2 , cyclopropyl, cyclopropyl-O- or cyclopropyl-NH-.

[0206] In some embodiments, R 10 Selected from =O, F, Cl, Br, CN, methyl, ethyl, ethynyl, CF3, methoxy, N(CH3)2 or cyclopropyl.

[0207] In some embodiments, R 10 Selected from =O, F, Cl, ethynyl, CF3, methoxy, N(CH3)2 or cyclopropyl.

[0208] In some embodiments, Selected from

[0209] In some embodiments, Selected from

[0210] In some embodiments, Selected from

[0211] In some embodiments, Selected from

[0212] In some embodiments, Selected from

[0213] In some embodiments, Selected from

[0214] In some embodiments, Selected from

[0215] In some embodiments, Y 1 、Y 2 Independently selected from CR b or N, Y 3 and Y 4 Independently selected from CR b .

[0216] In some embodiments, Y 1 、Y 2 、Y 3 and Y 4 All CR b .

[0217] In some embodiments, Y 1 and Y 2 Both N, Y 3 and Y 4 Independently selected from CR b .

[0218] In some embodiments, Y 1 N, Y 2 、Y 3 and Y 4 Independently selected from CR b .

[0219] In some embodiments, R b is selected from H, halogen, OH, C1-C6 alkyl, C1-C6 alkoxy, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl-O-, C3-C6 cycloalkyl-NH-, 4-7 membered heterocyclyl-O-, 4-7 membered heterocyclyl-NH- or 5-10 membered heteroaryl, wherein said C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or 5-10 membered heteroaryl is optionally replaced by R 2a replace.

[0220] In some embodiments, R b Selected from H, halogen or optionally R 2a Substituted from the following groups: methyl, ethoxy, NHCH3, NHEt, NH(i-Pr), pyrazolyl, cyclopropyl-O-, cyclobutyl-NH- or oxetanyl-O-.

[0221] In some embodiments, R 2a is selected from halogen, OH, =O, C1-C3 alkyl or C1-C3 alkoxy.

[0222] In some embodiments, R 2a Selected from halogen, OH or C1-C3 alkyl.

[0223] In some embodiments, R 2a is selected from F, OH or methyl.

[0224] In some embodiments, R b Selected from H, F, CF3, OEt, NHCH3, NHEt, NH(i-Pr),

[0225] In some embodiments, R b 、R 1 and their respective atoms and bonds together form a C3-C6 cycloalkenyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally replaced by R 1d replace.

[0226] In some embodiments, R b 、R 1 and their respective atoms and bonds together form an optionally R 1d Substituted C3-C6 cycloalkenyl.

[0227] In some embodiments, R 1d Selected from halogen, OH, C1-C3 alkyl or halogenated C1-C3 alkyl.

[0228] In some embodiments, R b 、R 1 and the atoms and bonds to which they are attached together form a cyclopentenyl group.

[0229] In some embodiments, Selected from

[0230] In some embodiments, Selected from

[0231] In some embodiments, Selected from

[0232] In some embodiments, X is selected from or optionally R e Substituted C1-C6 alkyl.

[0233] In some embodiments, X is selected from

[0234] In some embodiments, X is selected from

[0235] In some embodiments, Ring B is selected from the group consisting of 3 substituted 5-10 membered nitrogen-containing heteroaryl, 4-7 membered monocyclic nitrogen-containing heterocyclic group or 6-10 membered nitrogen-containing heterocyclic group.

[0236] In some embodiments, Ring B is selected from the group consisting of 3 Substituted groups: tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl,

[0237] In some embodiments, Ring B is selected from the group consisting of 3 Substituted groups: tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl,

[0238] In some embodiments, R 3 Selected from halogen, OH, =O, CN, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C6-C 10 Aryl or 5-10 membered heteroaryl, the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C6-C 10 Aryl or 5-10 membered heteroaryl is optionally further substituted by R 3a replace.

[0239] In some embodiments, R 3 is selected from halogen, OH, =O, C1-C3 alkyl, C3-C6 cycloalkyl or phenyl, wherein the C1-C3 alkyl, C3-C6 cycloalkyl or phenyl is optionally further replaced by R 3a replace.

[0240] In some embodiments, R 3a is selected from halogen, OH, =O or C1-C3 alkoxy.

[0241] In some embodiments, R 3aSelected from F.

[0242] In some embodiments, R 3 Selected from =O, OH, F, methyl, CF3, cyclopropyl or phenyl.

[0243] In some embodiments, R 4 、R 5 independently selected from H, halogen, OH or optionally replaced by R 4a Substituted C1-C3 alkyl; or R 4 、R 5 and its connected atoms together form an optionally R 8a Substituted C3-C6 cycloalkyl; or R 4 、R 5 Together form =O; or when p is taken from 2, the two R 4 and its connected atoms together form an optionally R 8a Substituted C3-C6 cycloalkyl.

[0244] In some embodiments, R 4 、R 5 Independently selected from H, halogen, OH or optionally replaced by R 4a Substituted C1-C3 alkyl; or R 4 、R 5 and its connected atoms together form an optionally R 8a Substituted C3-C6 cycloalkyl; or R 4 、R 5 Together they form =O.

[0245] In some embodiments, R 4a is selected from halogen, OH or C1-C3 alkoxy.

[0246] In some embodiments, R 4a Selected from F or OH.

[0247] In some embodiments, R 8a Selected from halogen, OH or C1-C3 alkyl.

[0248] In some embodiments, R 4 、R 5 are independently selected from H, methyl, hydroxymethyl or CF3, or R 4 、R 5 and the atoms to which it is attached together form a cyclopropyl group, or R 4 、R 5 Together they form =O.

[0249] In some embodiments, p is selected from 1.

[0250] In some embodiments, p is selected from 0.

[0251] In some embodiments, p is selected from 2, and both R 4 and its connected atoms together form an optionally R 8a Substituted C3-C6 cycloalkyl.

[0252] In some embodiments, p is selected from 2, and both R 4 and the atoms to which it is attached together form a cyclopropyl or cyclobutyl group.

[0253] In some embodiments, Selected from the following groups:

[0254] In some embodiments, Selected from the following groups:

[0255] In some embodiments, Ring D is selected from the group consisting of 6 The following groups are substituted: C3-C6 cycloalkyl, 5-6 membered heteroaryl, 4-7 membered monocyclic nitrogen-containing heterocyclic group or 6-10 membered nitrogen-containing heterocyclic group, and ring D is connected to L through a non-N atom.

[0256] In some embodiments, Ring D is selected from the group consisting of 6 Substituted groups: cyclopropyl, cyclobutyl, cyclopentyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl, isothiazolyl or pyridinyl.

[0257] In some embodiments, Ring D is selected from the group consisting of 6 Substituted groups: cyclopentyl, piperidinyl, pyridinyl,

[0258] In some embodiments, R 6 Selected from halogen, OH, CN, =O or optionally replaced by R 3a Substituted C1-C3 alkyl.

[0259] In some embodiments, R 6 Selected from halogen, =O, OH or C1-C3 alkyl.

[0260] In some embodiments, R 6 is selected from F or methyl.

[0261] In some embodiments, L is selected from a bond, -NR 7 -、-NR 7 CH2-, -O- or -CR8 R 9 -.

[0262] In some embodiments, R 7 、R 8 and R 9 independently selected from H, halogen, C1-C3 alkyl or OH.

[0263] In some embodiments, R 7 、R 8 and R 9 are independently selected from H or halogen.

[0264] In some embodiments, R 7 Selected from H.

[0265] In some embodiments, R 8 、R 9 Selected from H or F.

[0266] In some embodiments, L is selected from a bond, -NH-, -NHCH2-, -O-, -C(F)2-, or -CH2-.

[0267] In some embodiments, Selected from the following groups:

[0268] In some embodiments, X is selected from the group consisting of e Substituted C1-C6 alkyl.

[0269] In some embodiments, R e is selected from halogen, OH, C1-C3 alkyl, NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2 or C1-C3 alkoxy, wherein the C1-C3 alkyl or C1-C3 alkoxy is further optionally replaced by R f replace.

[0270] In some embodiments, R e is selected from C1-C3 alkyl, N(C1-C3 alkyl)2 or C1-C3 alkoxy, wherein the C1-C3 alkyl or C1-C3 alkoxy is further optionally replaced by R f replace.

[0271] In some embodiments, R f Selected from halogen, OH or N(C1-C6 alkyl)2.

[0272] In some embodiments, R f Selected from N(CH3)2.

[0273] In some embodiments, Re Selected from N(CH3)2, CH2N(CH3)2 or

[0274] In some embodiments, X is selected from

[0275] In some embodiments, X is selected from the following groups:

[0276] In some embodiments, X is selected from the following groups:

[0277] In some embodiments, X is selected from

[0278] In some embodiments, X is selected from

[0279] In some embodiments, R 1 、R 2 The atoms to which it is attached together form a C3-C8 cycloalkyl or a 4-10 membered heterocyclic group, wherein the C3-C8 cycloalkyl or the 4-10 membered heterocyclic group is optionally replaced by R 1b replace.

[0280] In some embodiments, R 1 、R 2 The atoms to which it is connected together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally replaced by R 1b replace.

[0281] In some embodiments, R 1 、R 2 The atoms to which it is attached together form an optionally R 1b Substituted: cyclobutyl, spiro[2,3]hexyl or oxetanyl.

[0282] In some embodiments, R 1b is selected from halogen, OH, CN, =O, NH2, C1-C3 alkyl or C1-C3 alkoxy, wherein the C1-C3 alkyl or C1-C3 alkoxy is optionally replaced by R 1c replace.

[0283] In some embodiments, R 1b is selected from halogen, CN, C1-C3 alkyl or C1-C3 alkoxy, wherein the C1-C3 alkyl or C1-C3 alkoxy is optionally replaced by R 1c replace.

[0284] In some embodiments, R1c is selected from halogen, OH or CN.

[0285] In some embodiments, R 1c Selected from CN.

[0286] In some embodiments, R 1b is selected from F, CN, methyl, methoxy or CH2CN.

[0287] In some embodiments, R 1 and R 2 independently selected from H, halogen, CN, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl or 4-7 membered heterocyclyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl or 4-7 membered heterocyclyl is optionally replaced by R 1a The C3-C8 cycloalkyl group or the 4-7 membered heterocyclic group may be spiro, bridged or fused.

[0288] In some embodiments, R 1 and R 2 independently selected from H, halogen, CN, C1-C3 alkyl, C1-C3 alkoxy or C3-C6 cycloalkyl, wherein the C1-C3 alkyl, C1-C3 alkoxy or C3-C6 cycloalkyl is optionally replaced by R 1a replace.

[0289] In some embodiments, R 1 and R 2 independently selected from H or C1-C3 alkyl, said C1-C3 alkyl being optionally replaced by R 1a replace.

[0290] In some embodiments, R 1a is selected from halogen, OH, CN, =O, NH2, C1-C3 alkyl or C1-C3 alkoxy.

[0291] In some embodiments, R 1 and R 2 are independently selected from H or methyl, or R 1 、R 2 The atoms to which it is attached together form the following group:

[0292] In some embodiments, R 1 、R 2 The atoms to which it is attached together form the following group:

[0293] In some embodiments, R 1、R 2 The atoms to which it is attached together form the following group:

[0294] In some embodiments, R 1 and R 2 are independently selected from H or methyl.

[0295] In some embodiments, W is selected from -(CR 13 R 14 )W1.

[0296] In some embodiments, R 13 、R 14 are independently selected from H, halogen, OH or methyl.

[0297] In some embodiments, R 13 、R 14 Both are H.

[0298] In some embodiments, R 1 With R 13 and their respective atoms and bonds together form an optionally R 13a Substituted C3-C6 cycloalkyl.

[0299] In some embodiments, R 13a Selected from C1-C3 alkyl.

[0300] In some embodiments, R 13a Selected from methyl.

[0301] In some embodiments, R 1 With R 13 and their respective atoms and bonds together form

[0302] In some embodiments, W is selected from W1.

[0303] In some embodiments, W1 is selected from the group consisting of 15 substituted 5- to 10-membered heteroaryl or 6- to 10-membered heterocyclic group.

[0304] In some embodiments, W1 is selected from the group consisting of 15 Substituted 5-10 membered heteroaryl.

[0305] In some embodiments, W1 is selected from the group consisting of 15 substituted 5-membered heteroaryl or 8-membered heterocyclic group.

[0306] In some embodiments, W1 is selected from the group consisting of 15 Substituted 5-membered heteroaryl.

[0307] In some embodiments, W1 is selected from the group consisting of 15 Substituted groups include pyrrolyl, thienyl, furyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, triazolyl, oxazolyl, isoxazolyl, oxadiazolyl, or 6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazolyl.

[0308] In some embodiments, W1 is selected from the group consisting of 15 Substituted pyrrolyl, thienyl, furyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, triazolyl, oxazolyl, isoxazolyl or oxadiazolyl.

[0309] In some embodiments, W1 is selected from the group consisting of 15 Substituted groups include triazolyl, oxazolyl, isoxazolyl, oxadiazolyl, or 6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazolyl.

[0310] In some embodiments, W1 is selected from the group consisting of 15 Substituted: triazolyl, oxazolyl, isoxazolyl or oxadiazolyl.

[0311] In some embodiments, R 15 is selected from halogen, OH, NH2, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally replaced by R 15a replace.

[0312] In some embodiments, R 15 is selected from OH, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally replaced by R 15a replace.

[0313] In some embodiments, R 15 is selected from methyl or cyclopropyl, wherein the methyl or cyclopropyl is optionally replaced by R 15a replace.

[0314] In some embodiments, R 15a is selected from halogen, OH or CN.

[0315] In some embodiments, R 15a Selected from F.

[0316] In some embodiments, R 15 is selected from methyl, CHF2 or cyclopropyl.

[0317] In some embodiments, W1 is selected from the following groups:

[0318] In some embodiments, W1 is selected from the following groups:

[0319] In some embodiments, W is selected from

[0320] In some embodiments, the compound of formula (I) of the present application is selected from the compound of formula (II):

[0321] Among them, Y 1 、Y 2 、Y 3 、Y 4 , X, Q, W, R 1 and R 2 As defined above.

[0322] In some embodiments, the compound of formula (I) of the present application is selected from the compound of formula (III):

[0323] Among them, Z 1 , Z 2 , Z 3 Independently selected from CH, CR 10 or N; R 10 、Y 1 、Y 2 、Y 3 、Y 4 , X, W, R 1 and R 2 As defined above.

[0324] In some embodiments, the compound of formula (I) of the present application is selected from the compound of formula (IV):

[0325] where R 10 、Y 1 、Y 2 、Y 3 、Y 4 , X, W, R 1 and R 2 As defined above. In some embodiments, the compound of formula (I) of the present application is selected from the following compounds:

[0326] The present application also provides a CBL-b inhibitor and the use of the CBL-b inhibitor in the inventions of various aspects of the present application. The CBL-b inhibitor is a compound of formula (V) or a pharmaceutically acceptable salt thereof.

[0327] in,

[0328] Z1 and Z2 are independently selected from CR a or N;

[0329] Z3 is selected from C, CH or N;

[0330] Y1, Y2, Y3 and Y4 are independently selected from CR b or N;

[0331] X is selected from halogen, CN, OH, COOH, CONH2, C1-C6 alkyl, C1-C6 alkoxy, wherein C1-C6 alkyl or C1-C6 alkoxy is optionally replaced by R e substituted, ring B is selected from optionally R 3 The following groups substituted: 4-10 membered nitrogen-containing heterocyclic group or 5-10 membered nitrogen-containing heteroaryl group, ring D is selected from optionally replaced by R 6 Substituted with the following groups: C3-C 10 Cycloalkyl, 4-10 membered heterocyclyl, phenyl or 5-10 membered heteroaryl, and ring D is connected to L by a non-N atom, and L is selected from a bond, -NR 7 -、-NR 7 CH2-, -O-, -C(=O)-, -C(=O)NH- or -CR 8 R 9 -;

[0332] R b selected from H, halogen, OH, CN, C1-C6 alkyl, C1-C6 alkoxy, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, NHC(O)(C1-C6 alkyl), NHS(O)2(C1-C6 alkyl), C3-C6 cycloalkyl, C3-C6 cycloalkyl-O-, C3-C6 cycloalkyl-NH-, N(C3-C6 cycloalkyl)2, NHC(O)-C3-C6 cycloalkyl, NHS(O)2-C3-C6 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclyloxy, 4-7 membered heterocyclyl-NH-, N(4-7 membered heterocyclyl)2, NHC(O)-4-7 membered heterocyclyl, NHS(O)2-4-7 membered heterocyclyl, C6-C6 10 Aryl, C6-C 10 Aryloxy, C6-C 10 Aryl-NH-, N(C6-C 10Aryl)2, NHC(O)-C6-C 10 Aryl, NHS(O)2-C6-C 10 Aryl, 5-10 membered heteroaryl, 5-10 membered heteroaryloxy or 5-10 membered heteroaryl-NH-, N(5-10 membered heteroaryl)2, NHC(O)-5-10 membered heteroaryl, NHS(O)2-5-10 membered heteroaryl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, C6-C 10 Aryl or 5-10 membered heteroaryl is optionally replaced by R 2a replace;

[0333] Or two R b The C atoms connected thereto together form a C3-C6 cycloalkenyl, phenyl, 4-7 membered heterocyclic group or 5-6 membered heteroaryl, wherein the C3-C6 cycloalkenyl, phenyl, 4-7 membered heterocyclic group or 5-6 membered heteroaryl is optionally replaced by R 2a replace;

[0334] R a 、R 4 、R 5 、R 7 、R 8 and R 9 Each of them is independently selected from H, halogen, OH, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl or C1-C6 alkoxy is optionally replaced by R 4a replace;

[0335] or R 8 、R 9 and the atoms to which they are connected together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, or R 4 、R 5 and the atoms to which they are attached together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally further replaced by R 8a Replace, or R 4 、R 5 Together they form =O;

[0336] R 3 and R 6 Independently selected from halogen, CN, =O, NO2, C1-C6 alkyl, OR 6a SR 6a 、N(R 6a )2、S(O)2R 6a 、S(O)2N(R 6a )2、S(O)R 6a 、S(O)N(R 6a )2、C(O)R 6a、C(O)OR 6a 、C(O)N(R 6a )2、C(O)N(R 6a )OR 6a 、OC(O)R 6a 、OC(O)N(R 6a )2、N(R 6a )C(O)OR 6a 、N(R 6a )C(O)R 6a 、N(R 6a )C(O)N(R 6a )2、N(R 6a )C(NR 6a )N(R 6a )2、N(R 6a )S(O)2N(R 6a )2、N(R 6a )S(O)2R 6a 、C3-C 10 Cycloalkyl, 4-7 membered heterocyclic group, 6-10 membered aryl or 5-10 membered heteroaryl, wherein C1-C6 alkyl, C3-C 10 Cycloalkyl, 4-7 membered heterocyclyl, 6-10 membered aryl or 5-10 membered heteroaryl are optionally further substituted by R 3a replace;

[0337] R 6a is selected from H, C1-C6 alkyl, phenyl, 4-7 membered heterocyclic group or 5-6 membered heteroaryl, wherein the C1-C6 alkyl, phenyl, 4-7 membered heterocyclic group or 5-6 membered heteroaryl is optionally further replaced by R 6b Substitution, or two R on a N atom 6a Together with the N to which it is attached, it forms a 4-7 membered heterocyclic group or a 5-6 membered heteroaryl group, wherein the 4-7 membered heterocyclic group or the 5-6 membered heteroaryl group is optionally further replaced by R 6b replace;

[0338] R 3a 、R 4a 、R 6b and R e Independently selected from halogen, OH, CN, =O, NH2, COOH or C1-C6 alkoxy;

[0339] Q is phenyl or 6-membered heteroaryl, said phenyl or 6-membered heteroaryl being optionally replaced by R 10 replace;

[0340] Or Q is a group as shown below: Where Z4 and Z5 are independently selected from CR c R d NR 11, O, S, or S(=O)2, represents a single bond or a double bond, and when For double bonds, m is 1. When it is a single bond, m is 1 or 2;

[0341] R 10 Selected from halogen, OH, NH2, CN, C1-C6 alkyl, C1-C6 alkoxy, C3-C 10 Cycloalkyl or 4-7 membered heterocyclic group, the C1-C6 alkyl, C1-C6 alkoxy, C3-C 10 Cycloalkyl or 4-7 membered heterocyclic group is optionally replaced by R 10a replace;

[0342] R 2a and R 10a Independently selected from halogen, OH, CN, =O, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy or halo-C1-C6 alkoxy;

[0343] R c 、R d and R 11 independently selected from H, halogen, OH, CN, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl or C1-C6 alkoxy, wherein said C1-C6 alkyl or C1-C6 alkoxy is optionally replaced by R 11a Replace, or R c 、R d Together form =O, or R c 、R d The atoms connected to it together form C3-C 10 Cycloalkyl or 4-7 membered heterocyclic group, the C3-C 10 Cycloalkyl or 4-7 membered heterocyclic group is optionally replaced by R 11b replace;

[0344] or R c With R 11 and their respective atoms together form a 4-7 membered heterocyclic group, wherein the 4-7 membered heterocyclic group is optionally replaced by R 11c replace;

[0345] R 11a 、R 11b and R 11c Independently selected from halogen, OH, =O, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2 or C1-C6 alkyl;

[0346] R 1 and R 2independently selected from H, halogen, CN, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, C1-C6 alkoxy, C3-C 10 Cycloalkyl or 4-10 membered heterocyclic group, wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C 10 Cycloalkyl or 4-10 membered heterocyclic group is optionally replaced by R 1a replace,

[0347] or R 1 、R 2 The atoms connected to it together form C3-C 10 Cycloalkyl or 4-10 membered heterocyclic group, the C3-C 10 Cycloalkyl or 4-10 membered heterocyclic group is optionally replaced by R 1b replace;

[0348] R 1a and R 1b independently selected from halogen, OH, CN, =O, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl or C1-C6 alkoxy is optionally further substituted by R 1c replace;

[0349] R 1c is selected from halogen, OH, CN, =O, NH2 or COOH;

[0350] W is selected from (CR 12 R 13 ) k W1, wherein W1 is selected from 5-10 membered heteroaryl or 4-10 membered heterocyclic group, wherein the 5-10 membered heteroaryl, 4-10 membered heterocyclic group is optionally replaced by R 14 Replacement, R 12 、R 13 R is independently selected from H, halogen, OH, C1-C6 alkyl or C1-C6 alkoxy, 14 Selected from halogen, OH, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, C3-C 10 Cycloalkyl or 4-7 membered heterocyclic group, wherein the C1-C6 alkyl, C3-C 10 Cycloalkyl or 4-7 membered heterocyclic group is optionally replaced by R 14a replace;

[0351] or R 1 With R 12 and their respective atoms and bonds together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally replaced by R 12a replace;

[0352] R 8a and R 12a are independently selected from halogen, OH, CN, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl or C1-C6 alkoxy is optionally further substituted with halogen, R 14a Selected from halogen, =O, OH, CN or C1-C6 alkyl;

[0353] p and k are independently selected from 0 or 1.

[0354] In some embodiments, Z1 and Z2 are independently selected from CH or N.

[0355] In some embodiments, Z1 and Z2 are both CH.

[0356] In some embodiments, Z1 is CH and Z2 is N.

[0357] In some embodiments, Z1 is N and Z2 is CH.

[0358] In some embodiments, Y1, Y2, Y3 and Y4 are all CR b .

[0359] In some embodiments, Y1 and Y2 are independently selected from CR b or N, Y3 and Y4 are all CR b .

[0360] In some embodiments, Y1 and Y2 are both N, and Y3 and Y4 are both CR b .

[0361] In some embodiments, Y1 is N, and Y2, Y3, and Y4 are all CR b .

[0362] In some embodiments, R b is selected from H, halogen, OH, C1-C6 alkyl, C1-C6 alkoxy, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl-O-, C3-C6 cycloalkyl-NH-, 4-7 membered heterocyclyl-O-, 4-7 membered heterocyclyl-NH- or 5-10 membered heteroaryl, wherein said C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or 5-10 membered heteroaryl is optionally replaced by R 2a replace.

[0363] In some embodiments, R b Selected from H, halogen or optionally R 2aSubstituted from the following groups: methyl, ethoxy, NHCH3, NHEt, NH(i-Pr), pyrazolyl, cyclopropyl-O-, cyclobutyl-NH- or oxetanyl-O-.

[0364] In some embodiments, R 2a is selected from halogen, OH, =O, C1-C3 alkyl or C1-C3 alkoxy.

[0365] In some embodiments, R 2a Selected from halogen, OH or C1-C3 alkyl.

[0366] In some embodiments, R 2a is selected from F, OH or methyl.

[0367] In some embodiments, R b Selected from H, F, CF3, ethoxy, OCH2CHF2, NHCH3, NHEt, NH(i-Pr), NHCH2CH2OH,

[0368] In some embodiments, Selected from

[0369] In some embodiments, Selected from

[0370] In some embodiments, X is selected from

[0371] In some embodiments, Ring B is selected from the group consisting of 3 substituted 5-10 membered nitrogen-containing heteroaryl, 4-7 membered monocyclic nitrogen-containing heterocyclic group or 6-10 membered nitrogen-containing heterocyclic group.

[0372] In some embodiments, Ring B is selected from the group consisting of 3 Substituted from the following groups: pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, azetidinyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, azepanyl,

[0373] In some embodiments, Ring B is selected from the group consisting of 3 Substituted groups: pyrazolyl, azetidinyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, azepanyl,

[0374] In some embodiments, R 3is selected from halogen, OH, =O, CN, C1-C6 alkyl, C1-C6 alkoxy, 6-10 membered aryl or 5-10 membered heteroaryl, wherein the C1-C6 alkyl, C1-C6 alkoxy, 6-10 membered aryl or 5-10 membered heteroaryl is optionally further replaced by R 3a replace.

[0375] In some embodiments, R 3 is selected from halogen, OH, =O, CN, C1-C3 alkyl, C1-C3 alkoxy or phenyl, wherein the C1-C3 alkyl, C1-C3 alkoxy or phenyl is optionally further replaced by R 3a replace.

[0376] In some embodiments, R 3a is selected from halogen, OH, =O or C1-C3 alkoxy.

[0377] In some embodiments, R 3a is selected from F, OH or methoxy.

[0378] In some embodiments, R 3 Selected from =O, OH, F, CN, methyl, CF3, hydroxymethyl, methoxy, or phenyl.

[0379] In some embodiments, R 4 、R 5 Independently selected from H, halogen, OH or optionally replaced by R 4a Substituted C1-C3 alkyl, or R 4 、R 5 Together they form =O.

[0380] In some embodiments, R 4a is selected from halogen, OH or C1-C3 alkoxy.

[0381] In some embodiments, R 4a Selected from F.

[0382] In some embodiments, R 4 、R 5 are independently selected from H, methyl, CF3 or ethyl, or R 4 、R 5 Together they form =O.

[0383] In some embodiments, p is selected from 1.

[0384] In some embodiments, p is selected from 0.

[0385] In some embodiments, Selected from the following groups:

[0386] In some embodiments, Ring D is selected from the group consisting of 6 The following groups are substituted: C3-C6 cycloalkyl, 5-6 membered heteroaryl, 4-7 membered monocyclic heterocyclyl or 6-10 membered heterocyclyl, and ring D is connected to L through a non-N atom.

[0387] In some embodiments, Ring D is selected from the group consisting of 6 Substituted groups: cyclopropyl, cyclobutyl, cyclopentyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl or isothiazolyl.

[0388] In some embodiments, R 6 Selected from halogen, OH, CN, =O or optionally replaced by R 3a Substituted C1-C3 alkyl.

[0389] In some embodiments, R 6 Selected from halogen, =O, OH or C1-C3 alkyl.

[0390] In some embodiments, R 6 is selected from F, =O or methyl.

[0391] In some embodiments, L is selected from a bond, -NR 7 -、-NR 7 CH2-, -O- or -CR 8 R 9 -.

[0392] In some embodiments, R 7 、R 8 and R 9 Independently selected from H, C1-C3 alkyl or OH.

[0393] In some embodiments, R 7 Selected from H or methyl.

[0394] In some embodiments, R 8 、R 9 Selected from H.

[0395] In some embodiments, L is selected from a bond, -NCH3-, -NHCH2-, -O-, or -CH2-.

[0396] In some embodiments, Selected from the following groups:

[0397] In some embodiments, X is selected from the following groups:

[0398] In some embodiments, X is selected from

[0399] In some embodiments, Q is optionally replaced by R 10 substituted phenyl or 6-membered heteroaryl.

[0400] In some embodiments, Q is optionally replaced by R 10 Substituted: phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl.

[0401] In some embodiments, Q is optionally replaced by R 10 Substituted: phenyl, pyridyl, pyridazinyl or pyrimidinyl.

[0402] In some embodiments, Q is optionally replaced by R 10 Substituted phenyl.

[0403] In some embodiments, R 10 is selected from halogen, OH, CN, C1-C6 alkyl or C1-C6 alkoxy, wherein the C1-C6 alkyl or C1-C6 alkoxy is optionally replaced by R 10a replace.

[0404] In some embodiments, R 10 is selected from halogen, C1-C3 alkyl or C1-C3 alkoxy, wherein the C1-C3 alkyl or C1-C3 alkoxy is optionally replaced by R 10a replace.

[0405] In some embodiments, R 10a Selected from halogen, OH or C1-C3 alkyl.

[0406] In some embodiments, R 10a Selected from F or OH.

[0407] In some embodiments, R 10 is selected from F, Cl, methyl, methoxy, CH2OH or CF3.

[0408] In some embodiments, Q is selected from Where Z4 and Z5 are independently selected from CR c R d NR 11 , O, S or SO2, represents a single bond or a double bond, and when For double bonds, m is 1. When it is a single bond, m is 1 or 2.

[0409] In some embodiments, R c 、R d and R 11 independently selected from H, halogen, OH, NH2 or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally replaced by R 11a Replace, or R c 、R d Together form =O, or R c 、R d The atoms to which it is attached together form a C3-C6 cycloalkyl group, wherein the C3-C6 cycloalkyl group is optionally replaced by R 11b replace.

[0410] In some embodiments, R c 、R d and R 11 Independently selected from H, halogen, OH or optionally replaced by R 11a Substituted C1-C3 alkyl, or R c 、R d Together form =O, or R c 、R d The atoms to which it is attached together form an optionally R 11b Substituted C3-C4 cycloalkyl.

[0411] In some embodiments, R c With R 11 and their respective atoms together form a 4-7 membered heterocyclic group, wherein the 4-7 membered heterocyclic group is optionally replaced by R 11c replace.

[0412] In some embodiments, R 11a 、R 11b and R 11c Independently selected from halogen, OH, =O, NH2 or C1-C3 alkyl.

[0413] In some embodiments, R 11a 、R 11b and R 11c are independently selected from halogen, OH or ═O.

[0414] In some embodiments, R c 、R d and R 11 are independently selected from H, halogen or methyl, or R c 、R d Together form =O, or R c 、R d The atoms to which it is attached together form a cyclopropyl group.

[0415] In some embodiments, R c and R d The two and the atoms to which they are connected together form a cyclopropyl group.

[0416] In some embodiments, Z4 and Z5 are independently selected from CH2, CF2, CHCH3, C(CH3)2, NCH3, C=O, O, S, S(=O)2 or Or Z4-Z5 together form

[0417] In some embodiments, Q is selected from m is 1 or 2.

[0418] In some embodiments, Q is selected from m is 1.

[0419] In some embodiments, Q is selected from

[0420] In some embodiments, Q is selected from phenyl, Wherein a represents the bond shared by Q and the 6-membered ring in the parent core, and b represents the bond shared by Q and the 5-membered ring in the parent core.

[0421] In some embodiments, Q is selected from phenyl, Wherein a represents the bond shared by Q and the 6-membered ring in the parent core, and b represents the bond shared by Q and the 5-membered ring in the parent core.

[0422] In some embodiments, Q is selected from phenyl, Wherein a represents the bond shared by Q and the 6-membered ring in the parent core, and b represents the bond shared by Q and the 5-membered ring in the parent core.

[0423] In some embodiments, Q is selected from Wherein a represents the bond shared by Q and the 6-membered ring in the parent core, and b represents the bond shared by Q and the 5-membered ring in the parent core.

[0424] In some embodiments, Q is selected from Wherein a represents the bond shared by Q and the 6-membered ring in the parent core, and b represents the bond shared by Q and the 5-membered ring in the parent core.

[0425] In some embodiments, R 1 、R 2The atoms to which it is attached together form a C3-C8 cycloalkyl or a 4-10 membered heterocyclic group, wherein the C3-C8 cycloalkyl or the 4-10 membered heterocyclic group is optionally replaced by R 1b replace.

[0426] In some embodiments, R 1 、R 2 The atoms to which it is connected together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or the 4-7 membered heterocyclic group is optionally replaced by R 1b replace.

[0427] In some embodiments, R 1 、R 2 The atoms to which it is attached together form an optionally R 1b Substituted: cyclobutyl, spiro[2,3]hexyl or oxetanyl.

[0428] In some embodiments, R 1b is selected from halogen, OH, CN, =O, NH2, C1-C3 alkyl or C1-C3 alkoxy, wherein the C1-C3 alkyl or C1-C3 alkoxy is optionally replaced by R 1c replace.

[0429] In some embodiments, R 1b is selected from halogen, CN, C1-C3 alkyl or C1-C3 alkoxy, wherein the C1-C3 alkyl or C1-C3 alkoxy is optionally replaced by R 1c replace.

[0430] In some embodiments, R 1c is selected from halogen, OH or CN.

[0431] In some embodiments, R 1c Selected from CN.

[0432] In some embodiments, R 1b is selected from F, CN, methyl, methoxy or CH2CN.

[0433] In some embodiments, R 1 and R 2 independently selected from H, halogen, CN, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl or 4-7 membered heterocyclyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl or 4-7 membered heterocyclyl is optionally replaced by R 1a The C3-C8 cycloalkyl group or the 4-7 membered heterocyclic group may be spiro, bridged or fused.

[0434] In some embodiments, R1 and R 2 independently selected from H, halogen, CN, C1-C3 alkyl, C1-C3 alkoxy or C3-C6 cycloalkyl, wherein the C1-C3 alkyl, C1-C3 alkoxy or C3-C6 cycloalkyl is optionally replaced by R 1a replace.

[0435] In some embodiments, R 1 and R 2 independently selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally replaced by R 1a replace.

[0436] In some embodiments, R 1a is selected from halogen, OH, CN, =O, NH2, C1-C3 alkyl or C1-C3 alkoxy.

[0437] In some embodiments, R 1 and R 2 are independently selected from H, methyl or cyclobutyl, or R 1 、R 2 The atoms to which it is attached together form the following group:

[0438] In some embodiments, R 1 、R 2 The atoms to which it is attached together form the following group:

[0439] In some embodiments, R 1 、R 2 The atoms to which it is attached together form the following group:

[0440] In some embodiments, R 1 and R 2 are independently selected from H, methyl or cyclobutyl.

[0441] In some embodiments, W is selected from -(CR 12 R 13 )W1.

[0442] In some embodiments, R 12 、R 13 are independently selected from H, halogen, OH or methyl.

[0443] In some embodiments, R 12 、R 13 Both are H.

[0444] In some embodiments, R1 With R 12 and their respective atoms and bonds together form an optionally R 12a Substituted C3-C6 cycloalkyl.

[0445] In some embodiments, R 12a Selected from C1-C3 alkyl.

[0446] In some embodiments, R 12a Selected from methyl.

[0447] In some embodiments, R 1 With R 12 and their respective atoms and bonds together form

[0448] In some embodiments, W is selected from W1.

[0449] In some embodiments, W1 is selected from the group consisting of 14 substituted 4-10 membered heterocyclic group.

[0450] In some embodiments, W1 is selected from the group consisting of 14 Substituted 5-10 membered heteroaryl.

[0451] In some embodiments, W1 is selected from the group consisting of 14 Substituted 5-membered heteroaryl.

[0452] In some embodiments, W1 is selected from the group consisting of 14 Substituted groups such as pyrrole, thiophene, furan, pyrazole, imidazole, thiazole, isothiazole, thiadiazole, triazole, oxazole, isoxazole, oxadiazole,

[0453] In some embodiments, W1 is selected from the group consisting of 14 Substituted groups such as triazole, oxazole, isoxazole or oxadiazole.

[0454] In some embodiments, W1 is selected from the group consisting of 14 Substituted groups:

[0455] In some embodiments, R 14 is selected from halogen, OH, NH2, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally replaced by R 14a replace.

[0456] In some embodiments, R 14is selected from OH, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally replaced by R 14a replace.

[0457] In some embodiments, R 14 is selected from methyl or cyclopropyl, wherein the methyl or cyclopropyl is optionally replaced by R 14a replace.

[0458] In some embodiments, R 14a is selected from halogen, OH or CN.

[0459] In some embodiments, R 14a Selected from F.

[0460] In some embodiments, R 14 is selected from methyl, CHF2 or cyclopropyl.

[0461] In some embodiments, W1 is selected from the following groups:

[0462] In some embodiments, W is selected from

[0463] In some embodiments, the compound of formula (V) of the present application is selected from the compound of formula (VI):

[0464] wherein j is selected from 0, 1, 2 or 3, Z1, Z2, Y1, Y2, Y3, Y4, X, W, R 1 、R 2 and R 10 As defined in formula (V).

[0465] In some embodiments, the compound of formula (V) of the present application is selected from the compound of formula (VII):

[0466] Among them, Z1, Z2, Z3, Z4, Z5, Y1, Y2, Y3, Y4, X, W, R 1 、R 2 and m are as defined in formula (V).

[0467] In some embodiments, the compound of formula (V) of the present application is selected from the following compounds:

[0468] In some specific embodiments, the CBL-b inhibitor has the structure of the CBL-b inhibitor described in WO 2020 / 264398 A1, the entire text of WO 2020 / 264398 A1 is incorporated into this application by reference.

[0469] In some specific embodiments, the CBL-b inhibitor has the structure of the CBL-b inhibitor described in WO 2020 / 210508 A1, the entire text of WO 2020 / 210508 A1 is incorporated into this application by reference.

[0470] In some specific embodiments, the CBL-b inhibitor is a compound having the following chemical formula or a pharmaceutically acceptable salt thereof:

[0471] In some specific embodiments, the CBL-b inhibitor is formulated as an oral or injectable formulation.

[0472] The pharmaceutical composition containing the CBL-b inhibitor of the present application can be prepared by combining the CBL-b inhibitor of the present application with suitable pharmaceutically acceptable excipients. For example, it can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0473] Typical routes of administration of the CBL-b inhibitor salts or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0474] The pharmaceutical composition containing the CBL-b inhibitor of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, and the like.

[0475] In some embodiments, the pharmaceutical compositions of the CBL-b inhibitors of the present application are in oral form. For oral administration, the pharmaceutical compositions can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present application to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, and the like for oral administration to patients. Solid oral compositions can be prepared by conventional mixing, filling, or tableting methods. For example, they can be prepared by mixing the active compound with a solid excipient, optionally grinding the resulting mixture, adding other suitable excipients as needed, and then granulating the mixture to form a tablet or dragee core. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, or flavoring agents. The pharmaceutical compositions can also be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms.

[0476] In some specific embodiments, the cancer or tumor is selected from a hematological tumor or a solid tumor. In some specific embodiments, the cancer or tumor is a solid tumor. In some specific embodiments, the solid tumor is selected from gastric cancer, liver cancer, colorectal cancer, renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer, and sarcoma.

[0477] In some embodiments in which immune effector cells express chimeric antigen receptors, the tumor or cancer refers to a tumor or cancer in which the surface of the tumor cell or cancer cell expresses an antigen molecule targeted by the chimeric antigen receptor. Taking Claudin18.2 involved in the present application as an example, in some embodiments, the tumor is a tumor that highly expresses Claudin18.2 (Claudin18.2+). In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) refers to a tumor cell population in which at least 60% of the tumor cells express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) refers to a tumor cell population in which at least 70% of the tumor cells express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) refers to a tumor cell population in which at least 80% of the tumor cells express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) refers to a tumor cell population in which at least 90% of the tumor cells express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) refers to a tumor cell population in which at least 95% of the tumor cells express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) refers to a tumor cell population in which at least 98% of the tumor cells express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) refers to a tumor cell population in which at least 99% of the tumor cells express Claudin18.2. The same applies to the antigen molecules BCMA, GPRC5D and other tumor antigen molecules involved in this application.

[0478] In some specific embodiments, the immune effector cells are selected from the group consisting of: (1) T cells expressing chimeric antigen receptors; (2) NK cells expressing chimeric antigen receptors; (3) T cells expressing chimeric antigen receptors and NK cells expressing chimeric antigen receptors.

[0479] In some specific embodiments, the immune effector cells comprise T cells expressing chimeric antigen receptors and / or NK cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the T cells and the chimeric antigen receptors expressed by the NK cells bind to the same or different antigens or antigenic epitopes.

[0480] In some specific embodiments, the immune effector cells express a chimeric antigen receptor, and the antigen bound by the chimeric antigen receptor is independently selected from Claudin18.2, BCMA, GPRC5D, or any combination thereof. For example, the antigen can be Claudin18.2, BCMA, or GPRC5D alone, a pairwise combination of Claudin18.2, BCMA, and GPRC5D, or a combination of Claudin18.2, BCMA, or GPRC5D.

[0481] In some specific embodiments, the chimeric antigen receptor of the present application comprises an antibody or antibody fragment targeting CLDN18.2 as an antigen-binding region, and the present application also separately provides such an antibody or antibody fragment targeting CLDN18.2.

[0482] In some specific embodiments, the VH or VL sequence of the antibody or antibody fragment targeting CLDN18.2 is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9:

[0483] VH sequence (SEQ ID NO: 8):

[0484] VL sequence (SEQ ID NO:9):

[0485] According to the Kabat numbering system, the VH / VL represented by SEQ ID NOs: 8-9 have the following CDRs:

[0486] In some specific embodiments, the antibody or antibody fragment is a scFv, and the scFv is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the following sequence:

[0487] In some specific embodiments, the chimeric antigen receptor of the present application comprises an antibody or antibody fragment targeting BCMA as an antigen binding region, and the present application also separately provides such an antibody or antibody fragment targeting BCMA.

[0488] In some specific embodiments, the antibody or antibody fragment targeting BCMA is a nanobody or VHH. In some specific embodiments, the antibody or antibody fragment targeting BCMA has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identity to the following SEQ ID NO: 18 or SEQ ID NO: 19:

[0489] In some specific embodiments, the BCMA-targeting VHHs are connected in tandem via a linker.

[0490] According to the IMGT numbering system, the VHHs represented by SEQ ID NOs: 18-19 have the following CDRs:

[0491] In some specific embodiments, the present application further provides antibodies or antibody fragments with different sequence compositions targeting BCMA.

[0492] In some specific embodiments, the BCMA antibody or antibody fragment is a nanobody or VHH. In some specific embodiments, the BCMA antibody or antibody fragment has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identity compared to the following SEQ ID NO: 38:

[0493] According to the Kabat numbering system, the VHH shown in SEQ ID NO: 38 has the following CDRs:

[0494] In some specific embodiments, the chimeric antigen receptor of the present application comprises an antibody or antibody fragment targeting GPRC5D as an antigen binding region, and the present application also separately provides such an antibody or antibody fragment targeting GPRC5D.

[0495] In some specific embodiments, the antibody or antibody fragment targeting GPRC5D is a nanobody or VHH. In some specific embodiments, the antibody or antibody fragment targeting GPRC5D has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identity with the following sequence of SEQ ID NO: 42:

[0496] According to the Kabat numbering system, the VHH shown in SEQ ID NO: 42 has the following CDRs:

[0497] In some specific embodiments, the immune effector cells comprise T cells expressing a chimeric antigen receptor, and the chimeric antigen receptor expressed by the T cells comprises a signal peptide, an antigen binding region, a hinge region, a transmembrane region, and an intracellular signaling domain.

[0498] In some specific embodiments, the chimeric antigen receptor comprises a CD8α signal peptide, an antigen-specific binding sequence, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 costimulatory domain, and CD3ζ.

[0499] In some specific embodiments, the CD8α signal peptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the following SEQ ID NO:7.

[0500] In some specific embodiments, the hinge region is a CD8α hinge region. In some specific embodiments, the CD8α hinge region has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the following SEQ ID NO: 26 or SEQ ID NO: 37:

[0501] In some specific embodiments, the hinge region is a CD28 hinge region. In some specific embodiments, the CD28 hinge region has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the sequence of SEQ ID NO: 27:

[0502] In some specific embodiments, the transmembrane region can be selected from the group consisting of the CD8α transmembrane region, the CD28 transmembrane region, and the NKG2D transmembrane region.

[0503] In some specific embodiments, the CD8α transmembrane region has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the following sequence:

[0504] In some specific embodiments, the CD28 transmembrane region has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the following sequence:

[0505] In some specific embodiments, the NKG2D transmembrane region has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the following sequence:

[0506] In some specific embodiments, the CD3ζ has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the sequence of SEQ ID NO: 31:

[0507] In some specific embodiments, the costimulatory domain is from CD28, 4-1BB or 2B4.

[0508] In some specific embodiments, the CD28 costimulatory domain has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the sequence of SEQ ID NO:32:

[0509] In some specific embodiments, the 4-1BB costimulatory domain has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the sequence of SEQ ID NO: 33:

[0510] In some specific embodiments, the 2B4 costimulatory domain has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the sequence of SEQ ID NO:34:

[0511] In some specific embodiments, the chimeric antigen receptor is further linked to a cytokine or chemokine via a self-cleaving peptide. In some specific embodiments, the self-cleaving peptide has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the sequence of SEQ ID NO: 35:

[0512] In some specific embodiments, the cytokine or chemokine is IL15. In some specific embodiments, the IL15 has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the following sequence:

[0513] In some specific embodiments, the antigen binding region or target binding sequence is in the form of a scFv having a VH-linker-VL or VL-linker-VH structure.

[0514] In some specific embodiments, the chimeric antigen receptor comprises the sequence of any one of SEQ ID NOs: 2-4, or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to any one of SEQ ID NOs: 2-4.

[0515] In some specific embodiments, the immune effector cells comprise NK cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the NK cells include a signal peptide, an antigen binding region or a target binding sequence, a hinge region, a transmembrane region, a co-stimulatory domain and CD3ζ.

[0516] In some specific embodiments, the chimeric antigen receptor further comprises a cytokine or chemokine linked to the CD3ζ via a self-cleaving peptide. In some specific embodiments, the cytokine is IL15.

[0517] In some specific embodiments, the chimeric antigen receptor expressed by the NK cell includes a CD8α signal peptide, an antigen binding region or a target binding sequence, a CD8α hinge region, a CD8α or CD28 or NKG2D transmembrane region, a CD28 or 2B4 or 4-1BB costimulatory domain and CD3ζ, and a cytokine or chemokine preferably connected to the CD3ζ by a self-cleaving peptide. In some specific embodiments, the cytokine is IL15.

[0518] In some specific embodiments, the chimeric antigen receptor expressed by the NK cell comprises the sequence shown in any one of SEQ ID NO:1, 5-6 and 46, or comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to any one of SEQ ID NO:1, 5-6.

[0519] In a fourth aspect, the present application further provides a method for expanding immune effector cells in vitro, the method comprising: expanding and culturing the immune effector cells in the presence of a CBL-b inhibitor.

[0520] In some specific embodiments, the immune effector cells are T cells or NK cells. In some specific embodiments, the T cells or NK cells are T cells expressing chimeric antigen receptors or NK cells expressing chimeric antigen receptors.

[0521] In some specific embodiments, the concentration of the CBL-b inhibitor during the culture process is 0.1 nM to 10 μM, for example, 1 μM.

[0522] In some specific embodiments, the T cells or NK cells are transfected with a chimeric antigen receptor, and the CBL-b inhibitor is added before or after transfection with a nucleic acid molecule expressing the chimeric antigen receptor.

[0523] In some specific embodiments, the CBL-b inhibitor is added when the T cells or NK cells begin to be activated, or the CBL-b inhibitor is added 4-7 days after the T cells or NK cells are activated and cultured.

[0524] In a fifth aspect, the present application also provides an engineered NK cell, wherein the NK cell expresses a chimeric antigen receptor that specifically binds to Claudin18.2, and the chimeric antigen receptor includes a CD8α signal peptide, an antigen binding region that specifically binds to Claudin18.2, a CD8α hinge region, a CD8α transmembrane region, a CD28 costimulatory domain, and CD3ζ. In some specific embodiments, the chimeric antigen receptor further includes a cytokine or chemokine connected to the CD3ζ via a self-cleaving peptide. In some specific embodiments, the cytokine is IL15.

[0525] In some specific embodiments, the antigen-binding region is in the form of an scFv. In some specific embodiments, the antigen-binding region is an scFv having a VH-connecting peptide-VL structure. In some specific embodiments, the VH comprises HCDR1-HCDR3 as shown in SEQ ID NOs: 10-12, and the VL comprises LCDR1-LCDR3 as shown in SEQ ID NOs: 13-15. In some specific embodiments, the VH has a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the sequence of SEQ ID NO:8, and the VL has a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the sequence of SEQ ID NO:9; most preferably, the scFv has a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the sequence of SEQ ID NO:16-17.

[0526] In some specific embodiments, the chimeric antigen receptor has a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the sequence shown in SEQ ID NO:1.

[0527] In a sixth aspect, the present application also provides the use of the aforementioned NK cells in combination with T cells expressing a chimeric antigen receptor that specifically binds to Claudin18.2 in the preparation of a drug for treating cancer or tumors.

[0528] In a seventh aspect, the present application also provides a cellular medicine for treating cancer or tumors, which comprises the aforementioned NK cells and T cells expressing a chimeric antigen receptor that specifically binds to Claudin18.2.

[0529] In an eighth aspect, the present application further provides a method for treating cancer or tumors, comprising administering to a subject in need thereof an effective amount of the aforementioned NK cells, and optionally, administering T cells expressing a chimeric antigen receptor that specifically binds to Claudin18.2.

[0530] In some specific embodiments, the chimeric antigen receptor expressed by the T cell comprises a CD8α signal peptide, a target binding sequence, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 costimulatory domain, and CD3ζ. In some specific embodiments, the antigen binding region is in the form of a scFv having a VH-linker-VL or VL-linker-VH structure.

[0531] In some specific embodiments, the chimeric antigen receptor expressed by the T cell comprises any one of SEQ ID NOs: 2-4, or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to any one of SEQ ID NOs: 2-4.

[0532] In a ninth aspect, the present application provides use of any one of the following combinations (a)-(c) in the preparation of a medicament for treating a tumor or cancer:

[0533] (a) immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,

[0534] (b) immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,

[0535] (c) Immune effector cells expressing chimeric antigen receptors, CBL-b inhibitors, and PDL1-IL15 fusion proteins.

[0536] In a tenth aspect, the present application provides a pharmaceutical combination product for treating cancer or tumors, comprising any one of the following (a)-(c):

[0537] (a) immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,

[0538] (b) immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,

[0539] (c) Immune effector cells expressing chimeric antigen receptors, CBL-b inhibitors, and PDL1-IL15 fusion proteins.

[0540] In an eleventh aspect, the present application provides a method for treating cancer or tumors, comprising administering to a subject in need thereof an effective amount of immune effector cells expressing a chimeric antigen receptor, the method further comprising: (1) stimulating the immune effector cells with a CBL-b inhibitor prior to administration, and / or (2) administering to the subject an effective amount of a PDL1-IL15 fusion protein.

[0541] In some specific embodiments, the antigen bound by the chimeric antigen receptor is independently selected from Claudin18.2, BCMA, GPRC5D, or any combination thereof. For example, the antigen can be Claudin18.2, BCMA, or GPRC5D alone, a pairwise combination of Claudin18.2, BCMA, and GPRC5D, or Claudin18.2, BCMA, or GPRC5D. In some embodiments, the chimeric antigen receptor comprises a target binding sequence that specifically binds to BCMA and / or GPRC5D.

[0542] In some specific embodiments, the antigen binding region comprises a target binding sequence that specifically binds to BCMA and GPRC5D and is in the form of a VHH.

[0543] In some specific embodiments, the VHH comprises a first set of HCDR1-HCDR3 having sequences shown in SEQ ID NOs: 39-41 and / or a second set of HCDR1-HCDR3 having sequences shown in SEQ ID NOs: 43-45.

[0544] In some specific embodiments, the VHH has a sequence as shown in SEQ ID NO:38 and / or 42, or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to SEQ ID NO:38 and / or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the sequence of SEQ ID NO:42.

[0545] In some specific embodiments, the sequence is SEQ ID NO:46 or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical to the sequence of SEQ ID NO:46.

[0546] In some embodiments, the immune effector cells are selected from T cells (e.g., cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages, and dendritic cells. In some embodiments, the immune effector cells are NK cells.

[0547] In some embodiments, the PDL1-IL15 fusion protein has the sequence shown in SEQ ID NO:47.

[0548] In some embodiments, the tumor or cancer is a B-cell lymphoma. In some embodiments, the tumor or cancer is multiple myeloma (MM). In some embodiments, the multiple myeloma is refractory or relapsed multiple myeloma.

[0549] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application and is not intended to limit the present invention in any respect. Those skilled in the art can make various modifications and variations to the embodiments described. Example

[0550] The present application will be further described below with reference to specific examples, and the advantages and features of the present application will become clearer as the description proceeds. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or those recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0551] The embodiments of the present application are merely exemplary and do not constitute any limitation on the scope of the present application. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present application may be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements shall fall within the scope of protection of the present application.

[0552] Example 1 Construction of Chimeric Antigen Receptor and Viral Packaging

[0553] Referring to Figure 1, chimeric antigen receptors for NK cells or T cells are constructed by cloning the nucleic acid encoding the chimeric antigen receptor for NK cells into a retroviral shuttle plasmid, and the nucleic acid encoding the chimeric antigen receptor for T cells into a lentiviral shuttle plasmid, and then packaging the virus using a packaging plasmid. Detailed information on chimeric antigen receptor and plasmid construction and viral packaging is as follows:

[0554] Table 1. Amino acid sequences of various elements in chimeric antigen receptors

[0555] Plasmid Construction: Synthesize a polynucleotide encoding the chimeric antigen receptor and add restriction sites and shuttle plasmid homology sequences at both ends. Digest the shuttle plasmid with restriction endonucleases, and recover and purify the linearized plasmid by agarose gel electrophoresis. Ligate the polynucleotide synthesized in the above steps with the linearized vector using the recombinase 5× In-Fusion HD enzyme (TaKaRa, Cat# ST0344). The reaction mixture is as follows: 2 μl of polynucleotide fragment (50 ng / μl), 1 μl of linearized plasmid (50 ng / μl), 2 μl of 5× HD In-Fusion enzyme, and 5 μl of ddH2O. Mix thoroughly, centrifuge briefly, and incubate at 50°C for 15 minutes. Add 10 μl of the recombinant reaction product to 100 μl of competent bacterial cells. Incubate on ice for 5 minutes. Spread the transformed bacterial suspension evenly on LB plates containing 100 μg / ml ampicillin and incubate in an incubator for 12-16 hours. Randomly select 3-5 clones from each plate for sequencing verification. Transfer the correctly sequenced bacterial suspension to 100 ml of LB liquid medium containing 100 μg / ml ampicillin and culture overnight at 37°C. Extract the plasmid using the MN Endotoxin-Free Plasmid Extraction Kit (MN, Cat# 740420.50). After quantification, dilute to 1000 ng / μl with endotoxin-free ultrapure water.

[0556] Lentivirus preparation: 293T cells (Cell Bank of the Chinese Academy of Sciences, Cat# GNHu17) were seeded into 100 mm culture dishes and cultured in DMEM (Gibco, Cat# 10566016) supplemented with 10% FBS (Gibco, Cat# 10099141). Plasmid transfection was performed when the cells reached approximately 70% confluency: a lentiviral shuttle plasmid expressing the chimeric antigen receptor was mixed with lentiviral packaging plasmids (pRRE, pRSV-Rev, and pVSV-G), and then added to 1.2 ml of Opti-MEM medium (Thermofisher Scientific, Cat# 31985070). Add 35 μl of Fugene HD (Promega, Cat# 04709691001), mix thoroughly, and incubate at room temperature for 15 minutes. Finally, the mixture was added to 293T cells and cultured at 37° C. under 5% CO 2 for 2 days. The supernatant was collected, filtered through a 0.45 μm filter membrane, and concentrated for later use.

[0557] Preparation of retrovirus: One day before virus packaging, 293T cells (purchased from ATCC) were seeded into culture dishes at 1E7 cells / 10cm. When transfecting cells, the packaging plasmid and retroviral shuttle plasmid were mixed and added to α-MEM medium. In another centrifuge tube containing α-MEM medium, HD transfection reagent (Promega, E2311). Add the diluted transfection reagent dropwise to the diluted plasmid, mix thoroughly, and let it stand at room temperature for 15 minutes. Add the mixture to the culture dish containing 293T cells, shake gently 10 times to mix thoroughly, and place in the incubator for incubation. Three days after cell transfection, harvest the virus. Transfer 10 ml of the virus-containing culture supernatant to a 50 ml centrifuge tube and centrifuge at 1250 rpm at 4°C for 5 minutes to remove dead 293T cells. Filter and concentrate the supernatant for later use.

[0558] Example 2 Preparation and Functional Evaluation of CAR-NK Targeting CLDN18.2

[0559] 1. Preparation of CAR-NK Cells

[0560] Fresh peripheral blood mononuclear cells (PBMCs) from healthy donors were centrifuged at 500 g for 7 minutes at room temperature. The supernatant was discarded and NK cells were isolated using the Human NK Cell Isolation Kit (Stemcell, 17955). The isolated NK cells were activated with K562 cells and transfected with CAR:

[0561] On day 0, cells were counted using AO / PI and mixed at a ratio of NK:K562 = 1:2. The mixed cells were added to a non-treated 6-well plate at 2 ml / well (the culture medium was NK cell culture medium containing 200 IU / ml human IL-2 (Miltenyi Biotec, 130-114-429)) and cultured at 37°C, 5% CO2.

[0562] On day 4, 3 ml of culture medium was added to each well.

[0563] On the fifth day, 24-well plates were coated with 7 μg / ml RetroNectin reagent (Takara, T202), 500 μl per well, at 4°C overnight.

[0564] On day 6, add retrovirus containing 18.2-CAR1(NK) to a 24-well plate and centrifuge at 2000g for 60 minutes at 4-8°C. Discard the supernatant. Add 3E5 activated NK cells to the 24-well plate at a concentration of 1:1.5 cells / well, centrifuge at 400g for 5 minutes at room temperature, and incubate at 37°C with 5% CO2.

[0565] On day 7, the transfected NK cells were transferred to non-treated 6-well plates and cultured for another 6-7 days.

[0566] The proliferation of NK cells during culture was examined, and the results are shown in Figure 2A. Compared with UTNK, the in vitro expansion capacity of NK cells transfected with 18.2-CAR1(NK) was significantly improved, with the expansion fold exceeding 150-fold on day 13 (i.e., 7 days after transfection).

[0567] 2. Detection of CAR transfection efficiency

[0568] On day 7 after transfection, a certain number of NK cells were collected, washed, and diluted to 2E6 cells / mL with PBS. Fc receptor blocker (BioLegend, Cat#422302) was added at a ratio of 50 μl / mL of cell diluent. The cells were incubated at room temperature for 10 minutes, and then 100 μl per well was transferred to a 96-well FACS reaction plate. 100 μl of 2 μg / μl CLDN18.2-His-tagged protein (Acro, Cat#CL2-H5546) was added to each well and incubated on ice for 20 minutes. The cells were washed twice by centrifugation with FACS buffer, and 100 μl of THE™ His-tag antibody (Genscript, Cat#A00612) was added to each well and incubated on ice for 20 minutes. The cells were washed three times by centrifugation with FACS buffer, and the cells were resuspended in 100 μl of FACS buffer. The cells were detected and analyzed using a flow cytometer (BD, CANTO II). The results are shown in FIG2B . The expression rate of 18.2-CAR1(NK) reached 88.5%, indicating a good viral transfection efficiency.

[0569] 3. Detection of the killing effect of CAR-NK cells on gastric cancer cells that highly express CLDN18.2

[0570] The NUGC4 stable transgenic cell line NUGC4-luc that highly expresses human CLDN18.2 and bacterial luciferase (Luciferase) and the CHO-K1 stable transgenic cell line CHOK1-18.2 that highly expresses human CLDN18.2 were constructed as target cells to detect the killing effect of 18.2-CAR1 (NK) and UTNK on target cells.

[0571] 4-hour tumor cell cytotoxicity assay: 20,000 target NUGC4-luc cells were plated onto a 96-well plate. 18.2-CAR1(NK) cells or UTNK cells were co-cultured at effector-to-target (E:T) ratios of 10:1, 2:1, 1:1, and 1:2, respectively. Samples were collected 4 hours later, and the firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added. Bioluminescence values ​​were read using a PE EnSight microplate reader. The results are shown in Figure 2C. Compared to UTNK cells, 18.2-CAR1(NK) cells exhibited strong cytotoxicity against NUGC4-luc cells within a short period of time. At high E:T ratios, 18.2-CAR1(NK) cells achieved 100% cytotoxicity against NUGC4-luc cells.

[0572] Real-time cell killing analysis experiment: Target cells CHOK1-18.2 were added to the E-plate 96-well plate at 20,000 cells / well. The E-Plate 96-well plate was placed in a real-time label-free cell analyzer (Agilent, Cat#RTCA MP) for culture. On the next day, NK cells with corresponding effector-target ratios were added and cultured for another 3 days. During the culture process, the cell index (CI) was detected every 15 minutes, and the growth of target cells was recorded. The results are shown in Figure 2D. 18.2-CAR1 (NK) cells showed 100% killing efficiency against CHOK1-18.2 at E:T=4:1, 2:1, and 1:1, but UTNK had no obvious killing effect on CHOK1-18.2 at high E:T. After NK cells and target cells were co-cultured for 72 hours, ELISA kit (BD OptEIA TM Human interferon-γ (IFN-γ) secretion levels in cell culture medium were measured using a Human IFN-γ ELISA Set (Cat. No. 555142). The results are shown in Figure 2E: Across the four E:T ratio experimental groups, IFN-γ secretion by 18.2-CAR1 (NK) cells was significantly higher than that by the UTNK control group, exhibiting a significant dose-dependent relationship.

[0573] 4. Detection of the anti-tumor efficacy of CLDN18.2-NK cells in the NUGC4-luc mouse subcutaneous tumor model

[0574] The anti-tumor effect of 18.2-CAR1(NK) cells was evaluated using a mouse gastric cancer subcutaneous xenograft model: 5E6 NUGC4-luc cells in the logarithmic growth phase and in good growth condition were subcutaneously inoculated into NPG mice (combined immunodeficient mice). On the 7th day after inoculation, the long and short diameters a and b of the mouse tumors were measured, and the tumor volume V (mm) was calculated. 3 )=1 / 2×(a×b 2), tumor volume was selected according to the random number principle. 3 Mice were randomly divided into groups. On the 8th day after tumor inoculation, NK cells (2E7 / mouse) were injected into the tail vein. The day of NK cell injection was designated as day 0 (D0). The grouping of mice is shown in Table 2. The tumor volume and body weight of the mice were continuously observed and measured. The measurements were recorded twice a week and the tumor inhibition rate (TGI) was calculated as follows: (tumor volume of mice in the PBS group - tumor volume of mice in the experimental group) / tumor volume of mice in the PBS group × 100%).

[0575] Table 2. Grouping of in vivo anti-tumor experiments

[0576] The results of mouse tumor volume measurement are shown in Figure 3A. Group G2 showed no tumor suppression after injection of UTNK cells, and its tumor growth rate was consistent with that of group G1 (PBS control group). Group G3, injected with 18.2-CAR1(NK) cells, showed significant tumor growth inhibition starting on day 14, reaching a 68% tumor inhibition rate by day 21 (Figure 3C). Mice in group G3 died on day 22 after injection, likely due to the higher injection dose, rapid in vivo expansion of 18.2-CAR1(NK), and inflammatory responses induced by IL-15 secretion. Body weight decreased (<10%) on day 18, while no significant changes were observed in the other groups (Figure 3B). To examine the role of secreted IL-15 on the in vivo expansion and tumor homing of 18.2-CAR1(NK) cells, the number of NK cells in peripheral blood and the amount of IL-15 secreted in serum were measured on days 5, 9, 13, and 18 after cell injection. As shown in Figure 3D, human NK cells (CD56 + CD3 -) No expansion was found in the G3 group, while the number of NK cells in the peripheral blood of mice injected with 18.2-CAR1(NK) cells gradually increased, reaching a peak of 1E4 NK cells / 25μl blood sample on the 18th day. Correspondingly, the secretion of human IL-15 in the serum of mice in the G2 group remained at baseline, while the secretion of human IL-15 in the serum of mice in the G3 group gradually increased, reaching a peak of 350pg / ml on the 13th day (Figure 3E). These results show that IL-15 in the CAR structure is effectively expressed and secreted in animals and supports the continuous expansion of 18.2-CAR1(NK) cells in solid tumor models. On the 22nd day after NK cell injection, the mice were dissected and organ samples were collected to quantitatively study the infiltration of NK cells in the spleen and tumor. As shown in Figures 3F and 3G, compared with the UTNK group, a higher proportion of 18.2-CAR1(NK) cells were found in the spleen and tumor tissues of mice in the 18.2-CAR1(NK) group, indicating that the IL-15-secreting 18.2-CAR1(NK) cells successfully infiltrated the immune organs and had a good tumor homing effect, which was consistent with the tumor control rate results.

[0577] 5. Detection of the Antitumor Efficacy of CLDN18.2-NK Cells in the NUGC4-luc Mouse Tumor Peritoneal Model

[0578] The anti-tumor efficacy of 18.2-CAR1(NK) cells was evaluated using a mouse gastric cancer model with intraperitoneal inoculation. NPG mice (combined immunodeficient mice) were intraperitoneally inoculated with 5E5 NUGC4-luc cells in logarithmic growth and in good condition. On day 10 after inoculation, the total fluorescence intensity (total flux) of the intraperitoneal tumor cells was measured to assess tumor growth. Mice with tumor cell fluorescence intensities of approximately 3E8 photons / second (p / s) in a specific abdominal region (ROI) were randomly assigned to groups. On day 10 after tumor inoculation, UTNK and 18.2-CAR1(NK) cells were injected into the tail vein at a volume of 200 μl per mouse. Day 0 (D0) of NK cell injection was designated as day 0. Mouse grouping and NK cell injection status are shown in Table 3. Peritoneal tumor growth and body weight changes were continuously observed and measured.

[0579] Table 3. Grouping of in vivo peritoneal model anti-tumor experiment

[0580] The results of bioluminescence intensity detection of mouse peritoneal tumors are shown in Figure 4A. The peritoneal tumors grew rapidly in the PBS control group G1 and the UTNK injection group G2. The 18.2-CAR1 (NK) cell-injected group G3 effectively delayed tumor growth, but did not completely eliminate the tumor. All mice in the treatment groups and the control group died due to tumor growth on the 24th day after treatment. The weight changes of mice in each group were not significant, showing a slow growth trend (Figure 4B). The proportion of NK cells in the peripheral blood and the secretion of human IL-15 in the serum of mice injected with 18.2-CAR1 (NK) cells in the G3 group gradually increased, reaching a peak on the 19th day, with NK cells accounting for 18% of peripheral blood lymphocytes (Figure 4C) and the average secretion of human IL-15 reaching 500pg / ml (Figure 4D). The results showed that reducing the dose of 18.2-CAR1(NK) effectively controlled the inflammatory response and toxicity caused by IL-15 secretion and rapid expansion of CAR-NK cells. However, due to the low expansion level of 18.2-CAR1(NK), it was unable to effectively control tumor growth and prolong animal survival.

[0581] Example 3 Synthesis of CBL-b Inhibitor

[0582] (1) Synthesis of the compounds shown in the following table

[0583] Table 4 Code names and chemical structures of CBL-b inhibitors

[0584] 1. Synthesis of CBL-bi-1 and its isomers

[0585] Step 1: Preparation of (S)-trifluoro(3-methylpiperidin-1-yl)methylborate potassium (2B)

[0586] To (S)-3-methylpiperidine hydrochloride (2A, 540 mg, 3.98 mmol), potassium (bromomethyl)trifluoroborate (960 mg, 4.78 mmol), potassium carbonate (605 mg, 4.38 mmol), and potassium iodide (67 mg, 0.40 mmol) was added anhydrous tetrahydrofuran (10 mL), and the atmosphere was purged with nitrogen three times. The mixture was heated to reflux under nitrogen and stirred for 12 hours. The reaction mixture was cooled to room temperature, 100 mL of acetone was added, and the mixture was filtered. The solid was washed with acetone, and the filtrate was evaporated to dryness to afford 420 mg of the crude title compound 2B as a gel, which was used directly in the next reaction. MS m / z (ESI): 162.1 [M-KF+H]+.

[0587] Step 2: Preparation of 7-bromo-9-(trifluoromethyl)-4H-pyrido[1,2-a]pyrimidin-4-one (2D)

[0588] To a mixture of 5-bromo-3-(trifluoromethyl)pyridinyl-2-amine (2C, 8.00 g, 33.2 mmol) and 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (7.42 g, 39.8 mmol) was added 1,2-dichlorobenzene (100 mL). The mixture was heated to 110°C and stirred for 4 hours, then raised to 200°C and stirred for another 3 hours. The reaction mixture was cooled to room temperature and directly subjected to normal phase column chromatography (petroleum ether:ethyl acetate = 3:1) to afford the title compound 2D (4.9 g) as a solid. MS m / z (ESI): 293.1 / 295.1 [M+H]+.

[0589] Step 3: Preparation of 7-[[(3S)-3-methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyridin[1,2-a]pyrimidin-4-one (2E)

[0590] To 7-bromo-9-(trifluoromethyl)-4H-pyrido[1,2-a]pyrimidinyl-4-one (2D, 410 mg, 1.40 mmol), (S)-trifluoro(3-methylpiperidin-1-yl)methylborate potassium (2B, 460 mg, 2.10 mmol), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1' To a mixture of [(1,4-dioxane, 2',4',6'-triisopropylbiphenyl)] palladium (II) (110 mg, 0.14 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (110 mg, 0.14 mmol), and potassium carbonate (133 mg, 4.20 mmol) was added 1,4-dioxane (20 mL) and water (5 mL). The nitrogen atmosphere was evacuated three times, and then heated to 100°C under nitrogen protection and stirred for 4 hours. The reaction solution was cooled to room temperature, the solvent was removed by rotary evaporation, and the residue was subjected to reverse phase column chromatography (chromatographic column: Flash silica gel column chromatography (acetonitrile:water=60:40) afforded the title compound 2E (210 mg) as a solid. MS m / z (ESI): 326.1 [M+H]+.

[0591] Step 4: Preparation of 3-iodo-7-[[(3S)-3-methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyridin[1,2-a]pyrimidin-4-one (2F)

[0592] 7-[[(3S)-3-Methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyrido[1,2-a]pyrimidinyl-4-one (2E, 210 mg, 0.66 mmol) was dissolved in acetonitrile (10 mL). Iodine (197 mg, 0.77 mmol) and ceric ammonium nitrate (342 mg, 0.65 mmol) were added, and the mixture was stirred at room temperature for 12 hours. Excess iodine was quenched with saturated aqueous sodium sulfite solution, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness to obtain the title compound 2F as a crude solid (240 mg), which was used directly in the next reaction. MS m / z (ESI): 452.0 [M+H].

[0593] Step 5: 3-[3-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]phenyl]-7-[[(3S)-3-methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyridin[1,2-a]pyrimidin-4-one (Compound 2), 3-(3-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)phenyl)-7-( Preparation of ((S)-3-methylpiperidin-1-yl)methyl)-9-(trifluoromethyl)-4H-pyrido[1,2-a]pyrimidin-4-one and 3-(3-((1R,3S)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)phenyl)-7-(((S)-3-methylpiperidin-1-yl)methyl)-9-(trifluoromethyl)-4H-pyrido[1,2-a]pyrimidin-4-one

[0594] To a solution of 3-iodo-7-[[(3S)-3-methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyrido[1,2-a]pyrimidinyl-4-one (2F, 240 mg, 0.53 mmol), 4-methyl-3-[3-methyl-1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]cyclobutyl]-1,2,4-triazole (188 mg, 0.53 mmol) and potassium carbonate (221 mg, 1.60 mmol) in dioxane / water (25 mL, 4:1) was added [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (39 mg, 0.53 mmol). The resulting mixture was heated to 80°C under nitrogen and stirred for 4 hours. The reaction solution was cooled to room temperature, and the solvent was removed by rotary evaporation. The residue was directly subjected to normal phase column chromatography (methanol: dichloromethane = 1:10), and then to reverse phase column chromatography (chromatographic column: Flash silica gel column chromatography (acetonitrile:water = 75:25) gave solid CBL-bi-5 (130 mg). MS m / z (ESI): 551.0 [M+H] + . Compound 2 was separated by SFC (instrument: CAS-SH-ANA-SFC-G (Agilent 1260 with DAD detector); column: ChiralPak AS-3 column, 150 mm length, 4.6 mm inner diameter, 3 μm particle size; mobile phase A: CO2, mobile phase B: IPA (containing 0.05% DEA); gradient: mobile phase B from 5% to 40% in 4.5 minutes, followed by elution with 5% mobile phase B for 1.5 minutes; flow rate: 2.5 mL / min; column temperature: 40°C; automatic back pressure regulator (ABPR): 100 bar) to obtain isomeric solids CBL-bi-1 (tR = 4.52 min, 70 mg) and CBL-bi-6 (tR = 4.69 min, 15 mg).

[0595] CBL-bi-1:

[0596] MS m / z(ESI):551.2[M+H] + ;

[0597] 1 H NMR(400MHz, CDCl3)δ9.17(s,1H),8.58(s,1H),8.25(br.s.,1H),7.97(s,1H),7. 88(s,1H),7.63(d,J=8.0Hz,1H),7.45(t,J=8.0Hz,1H),7.32(d,J=8.0Hz,1H),3. 56(br.s.,2H),3.25(s,3H),2.99-2.84(m,2H),2.84-2.62(m,5H),2.10-1.94(m, 1H),1.78-1.53(m,6H),1.19-1.10(m,3H),1.02-0.91(m,1H),0.90-0.82(m,3H).

[0598] CBL-bi-6:

[0599] MS m / z(ESI):551.2[M+H] + ;

[0600] 1H NMR(400MHz, CDCl3)δ9.16(s,1H),8.59-8.55(m,1H),8.25(s,1H),8.03(s,1H),7.71(s ,1H),7.62-7.57(m,1H),7.45-7.39(m,1H),7.23-7.16(m,1H),3.56(s,2H),3.31(s,3H ),3.26-3.16(m,2H),2.83-2.69(m,2H),2.65-2.54(m,1H),2.41-2.31(m,2H),2.06-1. 97(m,1H),1.71-1.54(m,5H),1.39-1.23(m,2H),1.18-1.11(m,3H),0.89-0.85(m,3H).

[0601] 2. Synthesis of CBL-bi-2

[0602] Step 1: Preparation of (S)-trifluoro(3-methylpiperidin-1-yl)methylborate potassium (3B)

[0603] To (S)-3-methylpiperidine hydrochloride (3A, 540 mg, 3.98 mmol), potassium (bromomethyl)trifluoroborate (960 mg, 4.78 mmol), potassium carbonate (605 mg, 4.38 mmol), and potassium iodide (67 mg, 0.40 mmol) was added anhydrous tetrahydrofuran (10 mL), and the atmosphere was purged with nitrogen three times. The mixture was heated to reflux under nitrogen and stirred for 12 hours. The reaction mixture was cooled to room temperature, 100 mL of acetone was added, and the mixture was filtered. The solid was washed with acetone, and the filtrate was evaporated to dryness to afford 420 mg of the crude title compound as a light yellow gum, which was used directly in the next reaction.

[0604] MS m / z(ESI):162.1[M-KF+H] + .

[0605] Step 2: Preparation of 4-bromo-6-fluorobenz[cd]indol-2(1H)-one (3C)

[0606] At room temperature, 4-bromobenz[cd]indol-2(1H)-one (1J, 0.2 g, 0.8 mmol) was dissolved in N,N-dimethylformamide (5 mL), and acetic acid (48.4 mg, 0.8 mmol) and 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane di(tetrafluoroborate) salt (Selectflour) (428.4 mg, 1.2 mmol) were added in sequence. The system was heated to 50°C and reacted for 8 hours. After cooling to room temperature, ethyl acetate (50 ml) was added and washed with water (30 ml x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by reverse phase silica gel column (chromatographic column: The residue was purified by flash silica gel column, water:acetonitrile = 95:5-5:95) and freeze-dried to obtain the title compound as a solid (40.0 mg, yield 19%).

[0607] MS m / z(ESI):266[M+H] + .

[0608] Step 3: Preparation of (S)-6-fluoro-4-((3-methylpiperidin-1-yl)methyl)benz[cd]indol-2(1H)-one (3D)

[0609] At room temperature, the compound 4-bromo-6-fluorobenz[cd]indol-2(1H)-one (3C, 40.0 mg, 150.0 μmol), potassium (S)-trifluoro((3-methylpiperidin-1-yl)methyl)borate (98.6 mg, 450 μmol), XphosPdG2 (23.6 mg, 30.1 μmol), Xphos (28.7 mg, 60.1 μmol) and potassium carbonate (83.1 mg, 601.4 μmol) were added to a reaction tube. The argon atmosphere was replaced, and 1,4-dioxane (2 mL) and water (0.5 mL) were added. The system was heated to 100° C. and reacted for 2 hours. The mixture was cooled to room temperature and filtered. The resulting residue was purified by reverse phase silica gel column (chromatographic column: The residue was purified by flash silica gel column, water:acetonitrile = 95:5-5:95) and freeze-dried to give the title compound (20 mg) as a solid.

[0610] MS m / z(ESI):299[M+H] + .

[0611] Step 4: Preparation of (S)-6-fluoro-1-(3-(3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)phenyl)-4-((3-methylpiperidin-1-yl)methyl)benzo[cd]indol-2(1H)-one (Compound 3)

[0612] At room temperature, (S)-6-fluoro-4-((3-methylpiperidin-1-yl)methyl)benzo[cd]indol-2(1H)-one (3D, 20.0 mg, 66.6 μmol), 3-(1-(3-iodophenyl)-3-methylcyclobutyl)-4-methyl-4H-1,2,4-triazole (1G, 23.5 mg, 66.6 μmol), potassium carbonate (18.4 mg, 133.2 μmol) and cuprous iodide (2.5 mg, 13.3 μmol) were added to a reaction tube. The atmosphere was replaced with argon, and anhydrous N,N-dimethylformamide (2 ml) and (1R,2R)-(-)-N,N'-dimethyl-1,2-cyclohexanediamine (4.7 mg, 33.3 μmol) were added. The system was heated to 100°C and reacted for 2 hours. The reaction was cooled to room temperature and filtered. The resulting residue was purified by reverse phase silica gel column (chromatographic column: The residue was purified by flash silica gel column, water:acetonitrile = 95:5-5:95) and freeze-dried to give the title compound (6.9 mg) as a solid.

[0613] MS m / z(ESI):524.0[M+H] + ;

[0614] 1 H-NMR (400MHz, DMSO) δ8.32(s,1H),8.20(d,J=7.2Hz,2H),7.63–7.25(m,5H),6.89(dd,J=7.8,2.9Hz,1H),3.83–3.74(m,2H),3.26(s,3H),2. 89(s,2H),2.77(t,J=11.4Hz,2H),2.57(d,J=6.5Hz,2H),1.96(t,J=10.3Hz,1H),1.72–1.40(m,6H),1.09(d,J=5.4Hz,3H),0.92–0.78(m,4H).

[0615] 3. The synthetic route of CBL-bi-3 is described in patent application WO 2020 / 264398 A1.

[0616] 4. The synthetic route of CBL-bi-4 is described in patent application WO 2020 / 210508 A1.

[0617] (II) Detection of biological activity of the compounds shown in Table 4

[0618] 1. Detection of the inhibitory effects of compounds CBL-bi-1, CBL-bi-5, CBL-bi-6, and CBL-bi-2 on the interaction between CBL-b protein and UbcH5B-Ub

[0619] Experimental Methods: Eu-Ubquitin (Cisbio) was incubated with UbcH5B (ENZO) and E1 (ENZO) at 37°C for 4 hours to prepare Eu-Ubquitin-UbcH5B. Eu-Ubquitin-UbcH5B was aliquoted and stored at -80°C. CBL-b activity assays were performed in 384-well plates (Perkin Elmer). 100 nL of a 3-fold serial dilution of compound (final concentration 10 μM to 0.5 nM, starting at 10 μM, 3-fold dilutions, 10 points, 10th point at 0.5 nM) was incubated with 5 μL of 50 nM Biotin-CBL-b protein (Sigma) at room temperature for 1 hour in a reaction buffer consisting of 50 mM HEPES pH 7.0 (Gibco), 100 mM NaCl (Sigma), 0.01% Triton X-100 (Sigma), 0.01% BSA (Sigma), and 1 mM DTT (Invitrogen). 5 μL of a Src cocktail (40 nM Src (R&D), 2 mM ATP (Sigma), and 10 mM MgCl2 (Sigma)) was added to the reaction plate and incubated at room temperature for 3 hours. 10 μL of detection solution (12.5 nM Strepdividin-XL665 (Cisbio), 500 nM Eu-Ubquitin-UbcH5B, 120 nM EDTA (Invitrogen), 0.004% BSA (Sigma)) was added to the reaction plate and incubated overnight at room temperature. The HTRF signal (665 nm / 615 nm) was read on Envision (Perkin Elmer). IC was calculated using IDBS XLfit. 50 The experimental results are shown in Table 5.

[0620] Table 5 IC values ​​of compounds inhibiting the interaction between CBL-b protein and UbcH5B-Ub 50

[0621] 2. Detection of the effects of compounds CBL-bi-1 and CBL-bi-2 on the activation of IL-2 release in Jurkat T cells

[0622] 96-well cell plates (Corning) were coated with 2 μg / mL Anti-Human CD3 Clone OKT3 (BD) at 37°C for 4 hours. A 3-fold serial dilution of the compound (final concentration 10 μM-4.6 nM, starting concentration 10 μM, 3-fold dilution, 8 points, the 8th point is 4.6 nM) was incubated with 220 μL 1.11E6 / mL Jurkat T cells (ATCC) for 1 hour, 5 μL 45 μg / mL Anti-Human CD28 Clone CD28.2 (BD) was added, mixed evenly, and 100 μL was transferred to the aforementioned CD3-coated cell plate. The cells were cultured in a 37°C cell culture incubator for 48 hours. The supernatant was collected and the IL-2 release was detected using an IL-2 ELISA kit (BD). EC was analyzed using Prism. 50 The experimental results are shown in Table 6.

[0623] Table 6 ECs of compounds activating Jurkat T cells to release IL2 50

[0624] Example 4 CLDN18.2 CAR-T Preparation

[0625] T cells were isolated from fresh PBMC using the Stemcell Easy Sep Kit (Stemcell, Cat#19055). The isolated T cells were transferred to culture dishes pre-coated with 1 μg / ml CD3 / CD28 antibodies (MY-ebioscience-16-0289-85, MY-ebioscience-16-0037-85). The culture medium consisted of X-VIVO15 (Lonza, Cat#BEBP02-054Q), 5% human AB serum (Gemini, Cat#100-512), 100 U / ml penicillin-streptomycin (Gibco, Cat#15140-122), and 200 IU / ml human IL-2 (Beijing Shuanglu, Cat#S19991007). Cell counts were performed twice a week, and passages were expanded when the cell density reached 2.5E6 cells / ml.

[0626] Healthy T cells were plated at 1E6 cells / well onto a 12-well cell culture plate pre-coated with 5 μg / ml recombinant human fibrin (Takara, Cat#T100B). 50 μl of concentrated lentivirus and 10 μg / ml polybrene (Sigma-Aldrich, Cat#TR-1003) were added and centrifuged at 1000 g for 1 hour at 4°C. After centrifugation, the cells were cultured at 37°C in 5% CO2, counted twice weekly, and passaged when the cell density reached 2.5E6 cells / ml.

[0627] Example 5 In vitro killing functional evaluation of CLDN18.2 CAR-T and CLDN18.2 CAR-NK combination, CLDN18.2 CAR-T combined with CBL-b inhibitor, and CLDN18.2 CAR-NK combined with CBL-b inhibitor

[0628] 18.2-CAR1 (NK) cells and 18.2-CAR3 (T) cells were prepared according to Examples 2 and 4, and CBL-bi-1 and CBL-bi-3 were synthesized according to Example 3 for use in the combination regimen of this example.

[0629] 1. Detecting the killing effect of CLDN18.2 CAR-T and NK cells on tumor cells

[0630] Target NUGC4-luc cells were plated on a 96-well plate at 20,000 cells / well. NK cells and T cells were added to the 96-well plate and co-cultured with the target cells at a ratio of 1:2 for NK cells and 1:4 for T cells. Samples were taken at 24 and 48 hours, and the firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added. The bioluminescence value was read using a PE EnSight microplate reader. The results are shown in Figure 5A. Compared with the use of NK cells (UTNK or 18.2-CAR1 (NK) cells) or CAR-T cells alone, the combination group showed a stronger killing effect. In addition, UTNK and 18.2-CAR1 (NK) cells had a strong killing effect on NUGC4-luc cells at 24 hours, but the killing effect was significantly reduced at 48 hours. The combined group still maintained strong killing activity at 48 hours, which was basically equivalent to the 24-hour killing effect of the combined group, and was significantly better than the killing ability of the NK group alone at 48 hours.

[0631] 2. Detect the killing effect of CBL-b inhibitors combined with NK cells on tumor cells

[0632] Target NUGC4-luc cells were plated on a 96-well plate at 20,000 cells / well. NK cells were added at a 1:6 NK cell:target cell ratio, and CBL-bi-1 was added at a final concentration of 1 μM. After 24 hours of co-culture, samples were collected and the firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added. Bioluminescence values ​​were read using a PE EnSight microplate reader. The results are shown in Figure 5B. At a 1:6 NK cell:target cell ratio, the CBL-b inhibitor (CBL-bi-1, at a final concentration of 1 μM in the culture medium) effectively enhanced the cytotoxicity of UTNK and 18.2-CAR1 (NK) cells against NUGC4-luc cells, with the killing efficiency increasing by approximately 16%.

[0633] 3. Detection of the multi-round killing effect of CBL-b inhibitors combined with CLDN18.2 CART or CLDN18.2 CARNK cells

[0634] NUGC4-luc cells were plated at 40,000 cells / well in a 96-well plate. NK cells or T cells were co-cultured at a ratio of 1:24 (NK cell:target cell) or 1:16 (T cell:target cell) with CBL-bi-3 at a final concentration of 1 μM. After 72 hours, samples were collected and the firefly luciferase substrate, D-luciferin (Abcam, Cat#ab143655), was added. Bioluminescence was measured using a PE EnSight microplate reader to calculate the killing rate. After 72 hours of co-culture, the cells were resuspended, and half the volume of the cell suspension was co-cultured with the same number of fresh NUGC4-luc cells for the next round of killing. This process was repeated for a total of three rounds of killing. As shown in Figure 5C, CBL-bi-3 effectively enhanced the killing ability of 18.2-CAR1 (NK) cells and NUGC4-luc cells, and significantly prolonged the duration of killing until the third round. Similarly, the combined use of CBL-bi-3 and 18.2-CAR4(T) cells effectively prolonged the CAR-T killing ability. Under the condition of relatively low target efficiency, the killing efficiency of the combination group reached 100% in the third round.

[0635] The above results show that the combined use of CLDN18.2 CAR-T and CLDN18.2 CAR-NK, as well as the combined use of CLDN18.2 CAR-NK and CBL-b inhibitors can overcome the shortcoming of the short-term efficacy of CAR-NK cells used alone; it also shows that the combined use of CLDN18.2 CAR-T and CBL-b inhibitors can reduce the amount of CAR-T used and improve the safety of cell therapy.

[0636] Example 6 In vitro evaluation of the killing function of the combination of CLDN18.2 CAR-T, CLDN18.2 CAR-NK, and CBL-b inhibitor

[0637] 18.2-CAR1(NK) and 18.2-CAR3(T) were prepared according to Examples 2 and 4, and CBL-bi-1 was synthesized according to Example 3 for use in the combined scheme of this example.

[0638] Real-time cell-based multi-round killing assay: Healthy target CHOK1-18.2 cells were plated onto a 96-well E-plate at 20,000 cells per well. The 96-well E-plate was placed in a real-time, label-free cell analyzer (Agilent, Cat# RTCA MP) for incubation. The next day, CAR-NK cells, CAR-T cells, and CBL-b inhibitors were added at the appropriate effector-target ratios (E:T ratio: NK cell: target cell = 1:6, T cell: target cell = 1:8). Culture was continued for 72 hours. During the incubation period, the cell index (CI) was measured every 15 minutes to record target cell growth. After 72 hours, half the culture volume was removed and the same number of fresh CHOK1-18.2 cells were added to the same well for the next round of killing assay. This cycle was repeated for a total of three rounds of killing. As shown in Figure 6, the 18.2-CAR3 (T) cell group and the 18.2-CAR3 (T) cell + CBL-bi-1 group had relatively weak killing effects on CHOK1-18.2 cells and did not completely control the growth of target cells in the first round of killing. The killing ability of the 18.2-CAR1 (NK) group was only maintained until the second round. However, the killing ability of the 18.2-CAR1 (NK) cell + CBL-bi-1 group, the 18.2-CAR1 (NK) cell + 18.2-CAR3 (T) cell group, and the 18.2-CAR1 (NK) cell + 18.2-CAR3 (T) cell + CBL-bi-1 group was maintained until the third round. In the third round of killing experiments, only the combination of the three still showed a complete inhibitory effect on target cells. This result shows that CBL-b inhibitors can effectively activate NK and T cells, showing significant synergy, further improving the killing function of the combined use of CAR-NK cells + CAR-T cells to resist multiple rounds of tumor cell attacks and effectively reduce tumor recurrence.

[0639] Example 7 Effect of using culture medium supplemented with CBL-b inhibitor on CAR-T cell activity

[0640] 18.2-CAR2(T) cells were prepared with reference to Example 4, and 1 μM CBL-bi-1 or CBL-bi-2 was added to the culture medium to stimulate T cells throughout the process.

[0641] 1. FACS detection of CAR transfection efficiency and T cell phenotype

[0642] Referring to the CAR transfection efficiency detection method described in Example 2, CAR expression in 18.2-CAR2 (T) cells was measured on day 9 (day 7 after transfection) of expansion culture with the addition of CD3 / CD28 antibodies. The results are shown in Figure 7A. The culture conditions containing CBL-bi-1 and CBL-bi-2 effectively improved the CAR transfection efficiency. Compared with the 18.2-CAR2 (T) control group, the T cell CAR expression in the CBL-bi-1 and CBL-bi-2 groups increased by approximately 10%.

[0643] Flow cytometry was also used to detect the phenotype of T cells 9 days after transfection, and the central memory T cells (CM, CD62L) of CD4 T cells and CD8 T cells were detected. + CD45RO + ), Effector Memory T Cell (EM, CD62L - CD45RO + ), stem cell-like memory T cells (SCM, CD62L + CD45RO - ) and effector T cells (Effector T cells, EF, CD62L - CD45RO - ) and the expression of CD69, a T cell activation phenotype. As shown in Figures 7B-7C, compared with the 18.2-CAR2(T) control group, the addition of CBL-bi-1 and CBL-bi-2 had no effect on the memory phenotype of 18.2-CAR2(T) cells. As shown in Figure 7D, compared with the 18.2-CAR2(T) control group, the addition of CBL-bi-1 and CBL-bi-2 to the culture medium significantly increased the expression of CD69, an activation phenotype of CD4 T and CD8 T cells.

[0644] Detection of the cytotoxic effect of 2-CAR2(T) cells on gastric cancer cells that highly express CLDN18.2

[0645] Tumor cell cytotoxicity assay: 20,000 target NUGC4-luc cells were plated onto a 96-well plate and co-cultured with 18.2-CAR2(T) cells or UT cells at E:T ratios of 5:1, 2:1, 1:2, and 1:2. Samples were collected after 24 hours, and the firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added. Bioluminescence values ​​were read using a PE EnSight microplate reader. The results are shown in Figure 7E. Compared to the 18.2-CAR2(T) cell control group, the addition of CBL-bi-1 and CBL-bi-2 to the culture medium significantly enhanced the cytotoxicity of 18.2-CAR2(T) cells against NUGC4-luc cells at low E:T ratios.

[0646] To assess CD107a expression and IFN-γ secretion, a cell degranulation marker, 20,000 target NUGC4-luc cells were plated in a 96-well plate. Different 18.2-CAR2 (T) cells and UT cells were added at an E:T ratio of 1:1. 1 μl of fluorescently labeled CD107a antibody (BioLegend, Cat. 328620) was added to each well. After 1 hour of incubation, 1 μl of Monensin Solution (eBioscience Cat. No. 00-4505) and 1 μl of Brefeldin A Solution (eBioscience Cat. No. 00-4506) were added to each well. Five hours later, T cell surface antigens were labeled using flow cytometry. The cells were washed once with buffer and centrifuged, and the supernatant was discarded. 100 μl of Intracellular Fixation Buffer (eBioscience Cat. No. 88-8824) was added, fixed by vortexing, and incubated at room temperature in the dark for 20-60 minutes. After washing, 100 μl of 1X Permeabilization Buffer (eBioscience Cat.No.88-8824) was added to each well, and IFN-γ antibody (BioLegend, Cat.506506) was added and incubated at room temperature in the dark for 20-60 minutes. Wash twice with 1X Permeabilization Buffer, resuspend in 100 μl FACS buffer, and detect and analyze the results using FACS (CANTOII, BD). As shown in Figure 7F, compared with the control group CAR-T, the addition of CBL-bi-1 and CBL-bi-2 to the culture medium significantly increased the secretion of IFN-γ and the expression of surface CD107a by 18.2-CAR2 (T) cells. In the absence of tumor cell stimulation, there was no significant change in the secretion of IFN-γ and the expression of surface CD107a by UT cells in each group and 18.2-CAR2 (T) cells in each group (Figure 7G).

[0647] Example 8 Effect of CBL-b inhibitor-added culture medium on BCMA CAR-NK cell activity

[0648] NK cells expressing BCMA-CAR1 (NK) and BCMA-CAR2 (NK) were prepared with reference to Example 2, and on the 4th day (Day 4) of K562 cell activation and NK cell expansion, 1 μM CBL-bi-4 was added to the culture medium to stimulate NK cells. The CAR transfection efficiency on the 15th day (Day 15) of the expansion culture was detected by FACS:

[0649] The cells were washed twice with PBS buffer, and 2E5 cells were taken after counting. 50 μl Fc receptor blocker (BioLegend, Cat#422302) was added and incubated at room temperature for 10 minutes. 100 μl FITC-hBCMA-his (ACRO Biosystems, BCA-HF254) with a final concentration of 2 μg / ml was added and incubated at 4°C in the dark for 1 hour. Washed three times with FACS buffer by centrifugation, the cells were suspended with 100 μl FACS buffer, and the results were detected and analyzed using a flow cytometer (BD, CANTO II). As shown in Figure 8, the culture conditions containing CBL-bi-4 can effectively improve the CAR transfection efficiency of CAR-NK cells. Compared with the BCMA-CAR1 (NK) and BCMA-CAR2 (NK) cell control groups and UTNK groups, the NK cell CAR expression in the CBL-bi-4 group was increased by about 15%.

[0650] Example 9 Combination of CAR-NK targeting GPRC5D and BCMA with CBL-b inhibitors and / or PDL1-IL15

[0651] 9.1 Construction of BCMA-GPRC5D CAR-NK

[0652] A dual-target chimeric antigen receptor targeting BCMA and GPRC5D was designed and constructed, including a CD8α signal peptide (SP), anti-GPRC5D antibody and anti-BCMA, a CD8α hinge region, an NKG2D-F transmembrane region, a 2B4 co-stimulatory domain, and CD3ζ, as well as IL15 connected via a self-cleaving peptide P2A. BCMA employed the VHH shown in SEQ ID NO:38, and GPRC5D employed the VHH shown in SEQ ID NO:42. The BCMA-GPRC5D CAR-NK structure is shown in Figure 9 . The sequences are shown in Table 7.

[0653] Table 7 BCMA-GPRC5D CAR amino acid sequence

[0654] 9.2 CBL-b Inhibitors Increase CAR Positive Rate

[0655] By constructing a conventional plasmid in the art, viral packaging, PBMC induction into NK cells, and NK cell activation and viral infection to construct a CAR-NK method, BCMA-GPRC5D CAR-NK (BI-CAR21) was prepared, and CBL-bi-4 (final concentration 1 μM) was added during the activation and transfection of NK cells. Six days after retroviral transfection of NK cells, the CAR positivity of NK cells was detected by a chimeric antigen receptor expression rate detection method. The results showed that after the addition of CBL-b inhibitors, the CAR positivity rate of NK cells increased. Exemplarily, as shown in Figure 10, the expression rate of BI-CAR21 was detected by BCMA, wherein the expression rate of BI-CAR increased from 80% to 92.5% after the addition of CBL-b inhibitors, compared with the control to which the CBL-b inhibitors were not added.

[0656] 9.3 CBL-b inhibitors and / or PDL1-IL15 enhance the killing effect of CAR-NK cells

[0657] The multi-round killing effect of CAR-NK (BI-CAR21) targeting GPRC5D and BCMA in combination with CBL-b inhibitor and / or PDL1-IL15 on NCI H929 + 10% NCI H929-hBCMA-KO cells was tested. The experimental design is shown in Table 8, wherein the CBL-b inhibitor and its addition method are shown in Section 9.2, and the PDL1-IL15 is added during the incubation of target cells and NK cells. Among them, the NCI H929-hBCMA-KO cells are H929 cell lines in which BCMA is knocked out using conventional genetic manipulation methods.

[0658] Furthermore, the target cells NCI H929 and NCI H929-hBCMA-KO were both transfected with the luciferase gene to express luciferase. Fluorescence intensity was measured using a luciferase reporter gene assay to reflect cell viability and NK cell cytotoxicity. The cytotoxicity calculation formula is as follows:

[0659] Killing rate = (target cell well reading - test well reading) / target cell well reading × 100%.

[0660] The results of multiple rounds of killing are shown in Figure 11. Using CBL-b inhibitors to stimulate NK cells during culture or adding PDL1-IL15 fusion protein during CAR-NK cell killing can enhance the killing effect of BCMA-GPRC5D CAR-NK. The combination of CBL-b inhibitors and PDL1-IL15 can further enhance the killing effect of BCMA-GPRC5D CAR-NK. The G5 group still maintained a high target cell killing rate at R5-1d, which was significantly higher than that of the G3 and G4 groups.

[0661] Table 8 Experimental design

[0662] Note: “ / ” means not added.

[0663] The sequence information of PDL1-IL15 is as follows:

[0664] Example 10. Antitumor efficacy test of CAR-NK targeting GPRC5D and BCMA combined with CBL-b inhibitor in Molp8-luc mouse tumor model

[0665] Anti-tumor efficacy tests were conducted using a mouse bone marrow cancer intravenous transplant tumor model. The details are as follows:

[0666] BCMA-GPRC5D CAR-NK cells were prepared according to the method of Example 9.

[0667] Molp8-Luc cells expressing bacterial luciferase were constructed by conventional methods in the art and cultured using conventional methods. Subsequently, Molp8-Luc cells in the logarithmic growth phase and in good growth condition were collected and inoculated into the tail vein of NPG mice (combined immunodeficient mice) with a total of 5×10 5 cells. The weight and tumor fluorescence signal values ​​of mice were measured on the first day after tumor inoculation. Mice weighing approximately 21.81 to 27.25 g were randomly selected according to the random number principle, with an average weight of 23.98 g for random grouping. On the first day after tumor inoculation, frozen-thawed CAR-NK cells (8×10 6 The injection volume was 200 μl / mouse, and the CAR-NK cell injection date was day 0 (D0). The mouse grouping and CAR-NK cell injection status are shown in Table 9. Tumor growth fluorescence signal ROI value and body weight changes were continuously monitored using an IVIS in vivo imager, and measurements were recorded once a week and the survival rate of the mice was observed.

[0668] Table 9. Grouping of in vivo anti-tumor experiments

[0669] The results of fluorescence signal detection of mouse tumor growth are shown in Figure 12B, which show that:

[0670] On the 29th day after the injection of CAR-NK cells, the BI-CAR21+CBL-b inhibitor treatment group was able to significantly inhibit the tumor growth burden of mice, of which two animals were cured of tumors, one animal had less tumor recurrence, and the tumor burden was reduced; the BI-CAR21 treatment group was able to inhibit the growth rate of mouse tumors, but could not control the progression of tumors in the group of animals. All 5 animals in the group had tumors that recurred and grew, and 3 animals died; the photon quantity statistics are shown in Figure 12C. At the same time, the weight of the animals monitored during the experiment in the BI-CAR21+CBL-b inhibitor treatment group showed an overall upward trend, and no adverse reactions after administration were shown; the weight of the animals in the BI-CAR21 treatment group showed a fluctuating trend during the experiment, which was caused by weight loss in a single animal in the group (as shown in Figure 12D);

[0671] During the experiment, survival rates were continuously monitored for 30 days. Due to the highly invasive tumor and rapid tumor progression in this model, one animal in the PBNK treatment group died starting on day 24, and the mortality rate was 100% by day 28. One animal in the BI-CAR21 treatment group died starting on day 25, and the mortality rate was 60% by day 30. During clinical observation, the animals in the BI-CAR21+CBL-b inhibitor treatment group were in better overall condition, with no deaths. This group demonstrated a long-lasting therapeutic effect while ensuring good safety (as shown in Figure 12E).

Claims

1. Use of a combination of a CBL-b inhibitor and immune effector cells expressing or not expressing a chimeric antigen receptor in the preparation of a medicament for treating cancer or tumors.

2. A pharmaceutical combination product for treating cancer or tumors, comprising a CBL-b inhibitor and immune effector cells, wherein the immune effector cells express or do not express a chimeric antigen receptor.

3. The use according to claim 1 or the pharmaceutical combination product according to claim 2, wherein: The immune effector cells are selected from the group consisting of: (1) T cells expressing chimeric antigen receptors; (2) NK cells expressing chimeric antigen receptors; and (3) T cells expressing chimeric antigen receptors and NK cells expressing chimeric antigen receptors.

4. The use or pharmaceutical combination according to any one of claims 1 to 3, wherein The CBL-b inhibitor is a compound having the following chemical formula or a pharmaceutically acceptable salt thereof:

5. The use or pharmaceutical combination product according to any one of claims 1 to 4, wherein The CBL-b inhibitor is formulated as an oral or injectable preparation.

6. The use or pharmaceutical combination product according to any one of claims 1 to 5, wherein The cancer or tumor is selected from a blood tumor or a solid tumor, preferably a solid tumor. Preferably, the solid tumor is selected from gastric cancer, liver cancer, colorectal cancer, renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcoma.

7. The use or pharmaceutical combination product according to any one of claims 1 to 6, wherein The immune effector cells include T cells expressing chimeric antigen receptors and / or NK cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the T cells and the chimeric antigen receptors expressed by the NK cells bind to the same or different antigens or antigenic epitopes.

8. The use or pharmaceutical combination product according to any one of claims 1 to 7, wherein The immune effector cells express chimeric antigen receptors, and the antigens bound by the chimeric antigen receptors are independently selected from Claudin18.2, BCMA, GPRC5D, or any combination thereof.

9. The use or pharmaceutical combination product according to any one of claims 1 to 8, wherein The immune effector cells comprise T cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the T cells comprise a signal peptide, an antigen binding region, a hinge region, a transmembrane region, and an intracellular signaling structural region; Preferably, the chimeric antigen receptor comprises a CD8α signal peptide, an antigen binding region, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 co-stimulatory domain and CD3ζ, preferably, the antigen binding region is in the form of a scFv having a VH-linker-VL or VL-linker-VH structure; Preferably, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NOs: 2-4, or comprises a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NOs: 2-4.

10. The use or pharmaceutical combination product according to any one of claims 1 to 9, wherein: The immune effector cells comprise NK cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the NK cells comprise a signal peptide, an antigen binding region, a hinge region, a transmembrane region, a co-stimulatory domain, and CD3ζ; Preferably, the chimeric antigen receptor further comprises a cytokine or chemokine linked to the CD3ζ via a self-cleaving peptide, the cytokine being for example IL15; More preferably, the chimeric antigen receptor expressed by the NK cell includes a CD8α signal peptide, an antigen binding region, a CD8α hinge region, a CD8α or CD28 or NKG2D transmembrane region, a CD28 or 2B4 or 4-1BB costimulatory domain and CD3ζ, and preferably A cytokine or chemokine linked to the CD3ζ via a self-cleaving peptide, such as IL15; Most preferably, the chimeric antigen receptor expressed by the NK cell comprises the sequence shown in any one of SEQ ID NOs: 1, 5-6 and 46, or comprises a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NOs: 1, 5-6 and 46.

11. A method for treating cancer or tumor, wherein: The method comprises administering to a subject in need thereof an effective amount of a CBL-b inhibitor and immune effector cells that express or do not express a chimeric antigen receptor.

12. The method according to claim 11, wherein The immune effector cells are selected from the group consisting of: (1) T cells expressing chimeric antigen receptors; (2) NK cells expressing chimeric antigen receptors; and (3) T cells expressing chimeric antigen receptors and NK cells expressing chimeric antigen receptors.

13. The method according to any one of claims 11 to 12, wherein: The CBL-b inhibitor is a compound having the following chemical formula or a pharmaceutically acceptable salt thereof:

14. The method according to any one of claims 11 to 13, wherein: The CBL-b inhibitor is formulated as an oral or injectable preparation.

15. The method according to any one of claims 11 to 14, wherein: The cancer or tumor is selected from a blood tumor or a solid tumor, preferably a solid tumor. Preferably, the solid tumor is selected from gastric cancer, liver cancer, colorectal cancer, renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcoma.

16. The method according to any one of claims 11 to 15, wherein: The immune effector cells include T cells expressing chimeric antigen receptors and / or NK cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the T cells and the chimeric antigen receptors expressed by the NK cells bind to the same or different targets.

17. The method according to any one of claims 11 to 16, wherein: The immune effector cells express chimeric antigen receptors, and the antigens bound by the chimeric antigen receptors are independently selected from Claudin18.2, BCMA, GPRC5D, or any combination thereof.

18. The method according to any one of claims 11 to 17, wherein: The immune effector cells comprise T cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the T cells comprise a signal peptide, an antigen binding region, a hinge region, a transmembrane region, and an intracellular signaling structural region; Preferably, the chimeric antigen receptor comprises a CD8α signal peptide, an antigen binding region, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 co-stimulatory domain and CD3ζ, preferably, the antigen binding region is in the form of a scFv having a VH-linker-VL or VL-linker-VH structure; Preferably, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NOs: 2-4, or comprises a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NOs: 2-4.

19. The method according to any one of claims 11 to 18, wherein: The immune effector cells comprise NK cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the NK cells comprise a signal peptide, an antigen binding region, a hinge region, a transmembrane region, a co-stimulatory domain, and CD3ζ; Preferably, the chimeric antigen receptor further comprises a cytokine or chemokine linked to the CD3ζ via a self-cleaving peptide, the cytokine being for example IL15; More preferably, the chimeric antigen receptor expressed by the NK cell comprises a CD8α signal peptide, an antigen binding region, a CD8α hinge region, a CD8α or CD28 or NKG2D transmembrane region, a CD28, 2B4 or 4-1BB costimulatory domain and CD3ζ, and a cytokine or chemokine preferably linked to the CD3ζ via a self-cleaving peptide, such as IL15; Most preferably, the chimeric antigen receptor expressed by the NK cell comprises the sequence shown in any one of SEQ ID NOs: 1, 5-6 and 46, or comprises a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NOs: 1, 5-6 and 46.

20. A method for expanding immune effector cells in vitro, the method comprising: The immune effector cells are expanded and cultured in the presence of a CBL-b inhibitor.

21. The method according to claim 20, wherein The immune effector cells are T cells or NK cells. Preferably, the T cells or NK cells are T cells expressing chimeric antigen receptors or NK cells expressing chimeric antigen receptors.

22. The method according to any one of claims 20-21, wherein: The concentration of the CBL-b inhibitor during the culture process is 0.1 nM to 10 μM, for example, 1 μM.

23. The method according to any one of claims 21 to 22, wherein: The T cells or NK cells are transfected with a chimeric antigen receptor, and the CBL-b inhibitor is added before or after transfection of a nucleic acid molecule expressing the chimeric antigen receptor.

24. The method according to claim 23, wherein The CBL-b inhibitor is added when the T cells or NK cells begin to be activated, or after the T cells or NK cells are activated and cultured for 4-7 days, the CBL-b inhibitor is added.

25. The method according to any one of claims 20 to 24, wherein: The CBL-b inhibitor has the following chemical formula or a pharmaceutically acceptable salt of a compound having the following chemical formula:

26. An engineered NK cell, wherein: The NK cells express a chimeric antigen receptor that specifically binds to Claudin18.2, and the chimeric antigen receptor includes a CD8α signal peptide, an antigen binding region that specifically binds to Claudin18.2, a CD8α hinge region, a CD8α transmembrane region, a CD28 co-stimulatory domain and CD3ζ. Preferably, the chimeric antigen receptor also includes a cytokine or chemokine connected to the CD3ζ via a self-cleaving peptide, the cytokine is, for example, IL15.

27. The NK cell according to claim 26, wherein The antigen-binding region is in the form of an scFv, preferably an scFv having a VH-connecting peptide-VL structure, more preferably, the VH comprises HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NOs: 10-12, and the VL comprises LCDR1, LCDR2 and LCDR3 having the sequences shown in SEQ ID NOs: 13-15; more preferably, the VH has the sequence shown in SEQ ID NO: 8 or a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 8, and the VL has the sequence shown in SEQ ID NO: 9 or a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 9; most preferably, the scFv has the sequence shown in SEQ ID NO: 16 or 17 or a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 16 or 17.

28. The NK cell according to any one of claims 26-27, wherein The chimeric antigen receptor has the sequence shown in SEQ ID NO: 1 or a sequence that is at least 80% identical to the sequence shown in SEQ ID NO:

1.

29. Use of a combination of the NK cells according to any one of claims 26 to 28 and T cells expressing a chimeric antigen receptor that specifically binds to Claudin18.2 in the preparation of a medicament for treating cancer or tumor.

30. A cellular medicine for treating cancer or tumor, comprising the NK cell according to any one of claims 26 to 28 and a T cell expressing a chimeric antigen receptor that specifically binds to Claudin18.

2.

31. The use according to claim 29 or the cell medicine according to claim 30, wherein: The chimeric antigen receptor expressed by the T cell comprises a CD8α signal peptide, an antigen binding region, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 co-stimulatory domain and CD3ζ, preferably, the antigen binding region is in the form of a scFv having a VH-linker-VL or VL-linker-VH structure; Preferably, the chimeric antigen receptor expressed by the T cell comprises a sequence of any one of SEQ ID NOs: 2-4 or a sequence having at least 80% identity with a sequence of any one of SEQ ID NOs: 2-4.

32. The use or cell medicine according to any one of claims 29 to 31, wherein The cancer or tumor is selected from a blood tumor or a solid tumor, preferably a solid tumor. Preferably, the solid tumor is selected from gastric cancer, liver cancer, colorectal cancer, renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcoma.

33. A method for treating cancer or tumor, comprising administering to a subject in need thereof an effective amount of the NK cells of any one of claims 26-28, and optionally administering T cells expressing a chimeric antigen receptor that specifically binds to Claudin18.

2.

34. The method according to claim 33, wherein The chimeric antigen receptor expressed by the T cell comprises a CD8α signal peptide, an antigen binding region, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 co-stimulatory domain and CD3ζ, preferably, the antigen binding region is in the form of a scFv having a VH-linker-VL or VL-linker-VH structure; Preferably, the chimeric antigen receptor expressed by the T cell comprises a sequence of any one of SEQ ID NOs: 2-4 or a sequence having at least 80% identity with a sequence of any one of SEQ ID NOs: 2-4.

35. The method according to any one of claims 33-34, wherein the cancer or tumor is selected from a hematological tumor or a solid tumor, preferably a solid tumor, preferably, the solid tumor is selected from gastric cancer, liver cancer, colorectal cancer, renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcoma.

36. Use of any one of the following combinations (a) to (c) in the preparation of a medicament for treating tumors or cancer: (a) immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors, (b) immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein, (c) Immune effector cells expressing chimeric antigen receptors, CBL-b inhibitors, and PDL1-IL15 fusion proteins.

37. A pharmaceutical combination product for treating cancer or tumors, comprising any one of the following (a) to (c): (a) immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors, (b) immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein, (c) Immune effector cells expressing chimeric antigen receptors, CBL-b inhibitors, and PDL1-IL15 fusion proteins.

38. The use according to claim 36 or the pharmaceutical combination product according to claim 37, wherein The chimeric antigen receptor binds to one or more antigens or antigenic epitopes, preferably, the antigens are independently selected from Claudin18.2, BCMA, GPRC5D or any combination thereof; Preferably, the chimeric antigen receptor comprises a CD8α signal peptide, an antigen binding region that specifically binds to BCMA and / or GPRC5D, a CD8α hinge region, a CD8α transmembrane region, a CD28 co-stimulatory domain, and CD3ζ. Preferably, the chimeric antigen receptor further comprises a cytokine or chemokine connected to the CD3ζ via a self-cleaving peptide, such as IL15. Preferably, the antigen-binding region is in the form of a VHH, more preferably, the VHH comprises a first set of HCDR1-HCDR3 having sequences shown in SEQ ID NOs: 39-41 and / or a second set of HCDR1-HCDR3 having sequences shown in SEQ ID NOs: 43-45, more preferably, the VHH has a sequence shown in SEQ ID NOs: 38 and / or 42, or a sequence that is at least 80% identical to SEQ ID NO: 38 and / or a sequence that is at least 80% identical to a sequence shown in SEQ ID NO: 42; Preferably, the chimeric antigen receptor has the sequence shown in SEQ ID NO: 46 or a sequence that is at least 80% identical to the sequence shown in SEQ ID NO:

46.

39. The use or pharmaceutical combination according to any one of claims 36 to 38, wherein The immune effector cells are selected from T cells (e.g., cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells; preferably, the immune effector cells are NK cells.

40. The use or pharmaceutical combination according to any one of claims 36 to 39, wherein the CBL-b inhibitor is a compound having the following chemical formula or a pharmaceutically acceptable salt thereof:

41. The use or pharmaceutical combination according to any one of claims 36 to 40, wherein The PDL1-IL15 fusion protein has the sequence shown in SEQ ID NO:

47.

42. The use or pharmaceutical combination according to any one of claims 36 to 41, wherein The tumor or cancer is B-cell lymphoma; more preferably, it is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.

43. A method of treating cancer or tumor, the method comprising administering to a subject in need thereof an effective amount of immune effector cells expressing a chimeric antigen receptor, the method further comprising: (1) prior to administration, stimulating the immune effector cells with a CBL-b inhibitor, and / or (2) administering an effective amount of PDL1-IL15 fusion protein to the subject.

44. The method according to claim 43, wherein The chimeric antigen receptor binds to one or more antigens or antigenic epitopes, preferably, the antigens are independently selected from Claudin18.2, BCMA, GPRC5D or any combination thereof; Preferably, the chimeric antigen receptor comprises a CD8α signal peptide, an antigen binding region that specifically binds to BCMA and / or GPRC5D, a CD8α hinge region, a CD8α transmembrane region, a CD28 co-stimulatory domain, and CD3ζ. Preferably, the chimeric antigen receptor further comprises a cytokine or chemokine connected to the CD3ζ via a self-cleaving peptide, such as IL15. Preferably, the antigen-binding region is in the form of a VHH, more preferably, the VHH comprises a first set of HCDR1-HCDR3 having sequences shown in SEQ ID NOs: 39-41 and / or a second set of HCDR1-HCDR3 having sequences shown in SEQ ID NOs: 43-45, more preferably, the VHH has a sequence shown in SEQ ID NOs: 38 and / or 42, or a sequence that is at least 80% identical to SEQ ID NO: 38 and / or a sequence that is at least 80% identical to a sequence shown in SEQ ID NO: 42; Preferably, the chimeric antigen receptor has the sequence shown in SEQ ID NO: 46 or a sequence that is at least 80% identical to the sequence shown in SEQ ID NO:

46.

45. The method according to any one of claims 43-44, wherein: The immune effector cells are selected from T cells (e.g., cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells; preferably, the immune effector cells are NK cells.

46. ​​The method according to any one of claims 43 to 45, wherein: The CBL-b inhibitor is a compound having the following chemical formula or a pharmaceutically acceptable salt thereof:

47. The method according to any one of claims 43 to 46, wherein: The PDL1-IL15 fusion protein has the sequence shown in SEQ ID NO:

47.

48. The method according to any one of claims 43 to 47, wherein: The tumor or cancer is B-cell lymphoma; more preferably, it is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.

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