WO2023217067A1 - Engineered immune effector cell and use thereof in combination with cbl-b inhibitor - Google Patents

Engineered immune effector cell and use thereof in combination with cbl-b inhibitor Download PDF

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WO2023217067A1
WO2023217067A1 PCT/CN2023/092686 CN2023092686W WO2023217067A1 WO 2023217067 A1 WO2023217067 A1 WO 2023217067A1 CN 2023092686 W CN2023092686 W CN 2023092686W WO 2023217067 A1 WO2023217067 A1 WO 2023217067A1
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cells
chimeric antigen
cancer
seq
cbl
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PCT/CN2023/092686
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French (fr)
Chinese (zh)
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王义芳
王超
麦万笋
姜福伟
王庆杨
杨翠青
李正涛
曹卓晓
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上海先博生物科技有限公司
先声再明医药有限公司
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Publication of WO2023217067A1 publication Critical patent/WO2023217067A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present application relates to the fields of bioengineering and cell therapy. Specifically, the present application provides engineered immune effector cells and their combined application with CBL-b inhibitors and/or other active substances (such as PDL1-IL15 fusion protein).
  • CAR Chimeric Antigen Receptor
  • NK cells CAR-T or CAR-NK
  • CAR-T therapy has been proven to be effective in treating B-cell malignancies and multiple myeloma, but there are still shortcomings, including: patients may not be able to provide enough T cells for engineering, and the production cycle of autologous CAR-T cells is long.
  • CAR-T therapy may cause cell-releasing factor syndrome (CRS), neurotoxicity and autoimmune toxicity, as well as unsatisfactory results in the treatment of solid tumors.
  • CRS cell-releasing factor syndrome
  • NK natural killer cells
  • MHC molecules to recognize and kill target cells. They are suitable for the "spot" mode, using NK from peripheral blood or umbilical cord blood of healthy donors. Cells were prepared directly.
  • NK cells do not cause graft versus host disease (GVHD) in an allogeneic environment and are suitable as universal cell product candidates. They do not cause CRS and have better safety.
  • GVHD graft versus host disease
  • NK cells can be activated by auxiliary cells such as monocytes, macrophages, and dendritic cells to secrete active killing factors.
  • CAR-NK cells have shown rapid and potent killing effect on tumor cells in in vitro and in vivo studies, but poor persistence usually results in short-lived drug effects and inability to completely eliminate solid tumors and control tumor recurrence.
  • Casitas B-lineage lymphoma proto-oncogene-b (CBL-b) is an E3 ligase that negatively regulates T cell or NK cell activation and is a negative regulatory immune checkpoint.
  • CBL-b gene is up-regulated in exhausted T cells, and the knockout of CBL-b gene can restore the effector function of exhausted T cells or CAR-T cells.
  • IL2 and IL15 activate NK cells and significantly upregulate the expression of CBL-b on the surface of NK cells.
  • the upregulation of CBL-b expression in turn inhibits the killing effect of activated NK cells on tumor cells and plays a negative feedback regulation in the activation process of NK cells. effect. It can be seen that CBL-b is an important immune regulatory target.
  • the present application provides the use of a combination of a CBL-b inhibitor and an immune effector cell, the immune effector cell expressing or not expressing a chimeric antigen receptor, in the preparation of a medicament for treating cancer or tumors.
  • the present application provides a pharmaceutical combination product for treating cancer or tumors, which includes a CBL-b inhibitor and an immune effector cell that expresses or does not express a chimeric antigen receptor.
  • the present application provides a method of treating cancer or tumors, wherein the method includes administering to a subject in need thereof an effective amount of a CBL-b inhibitor and an immune effector cell that expresses or does not express chimeric antigen receptor.
  • the present application provides a method for amplifying immune effector cells in vitro, which method includes: amplifying and culturing the immune effector cells in the presence of a CBL-b inhibitor.
  • 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 CD8 ⁇ signal peptide and specifically binds to Claudin18.2. 2’s antigen-binding region, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, CD28 costimulatory domain and CD3 ⁇ .
  • the present application provides the use of a combination of the NK cells described in the fifth aspect and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2 in the preparation of drugs for the treatment of cancer or tumors.
  • the present application provides a cellular drug for treating cancer or tumors, which includes the NK cells described in the fifth aspect and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2.
  • the present application provides a method for treating cancer or tumors, the method comprising administering to a subject in need an effective amount of the NK cells described in the fifth aspect, and optionally, administering NK cells that express specific binding to Claudin18.2 of chimeric antigen receptor T cells.
  • this application provides the use of any combination of the following (a)-(c) in the preparation of a drug for treating tumors or cancer:
  • the present application provides a pharmaceutical combination product for treating cancer or tumors, which includes any one of the following (a)-(c):
  • the present application provides a method for treating cancer or tumors, the method comprising administering an effective amount of immune effector cells expressing chimeric antigen receptors to a subject in need thereof, the method further comprising: (1) Before administration, use a CBL-b inhibitor to stimulate the immune effector cells, and/or (2) administer an effective amount of PDL1-IL15 fusion protein to the subject.
  • Figure 1 shows a schematic structural diagram of different exemplary CAR-NK and CAR-T plasmids of the present application.
  • Figure 2A shows the in vitro expansion results of NK cells transfected with 18.2-CAR1 (NK) or untransfected NK cells (UTNK).
  • Figure 2B shows the FACS detection results of CAR transfection efficiency of 18.2-CAR1 (NK) and UTNK cells.
  • Figure 2C shows the killing effect of 18.2-CAR1 (NK) and UTNK cells on NUGC4-luc cells for 4 hours.
  • the target ratios (E:T) are 10:1, 2:1, 1:1 and 1:2 respectively.
  • Figure 2D shows the sequential killing effect of 18.2-CAR1 (NK) and UTNK cells on the CHOK1-18.2 cell line over time at the different efficacy-to-target ratios shown.
  • Figure 2E shows the secretion levels of cytokine IFN- ⁇ when 18.2-CAR1 (NK) and UTNK cells kill CHOK1-18.2 cells at the different efficacy-target ratios shown.
  • Figure 3A shows the tumor growth curves of each group of mice within 21 days in the NUGC4-luc mouse subcutaneous tumor model.
  • Figure 3B shows the body weight change rate of mice in each group within 21 days in the NUGC4-luc mouse subcutaneous tumor model.
  • FIG. 3C shows the tumor inhibition rate (TGI%) of each group of mice within 21 days in the NUGC4-luc mouse subcutaneous tumor model.
  • Figure 3D shows the FACS detection 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.
  • Figure 3E shows the detection results of human IL-15 cytokine secretion in peripheral blood serum of each group of mice in the NUGC4-luc mouse subcutaneous tumor model on days 5, 9, 13 and 18.
  • Figure 3F shows the FACS detection results of the proportion of NK cells in tumor tissue lymphocytes after dissection of mice in each group of mice in the NUGC4-luc mouse subcutaneous tumor model on day 22.
  • Figure 3G shows the PCR detection results of CAR copy number in the spleen and tumor tissue of each group of mice in the NUGC4-luc mouse subcutaneous tumor model after dissection on day 22.
  • Figure 4A shows the fluorescence intensity curve of tumor cell growth in the abdominal area of mice in each group of mice in the NUGC4-luc mouse abdominal tumor model within 20 days.
  • Figure 4B shows the body weight change rate of mice in each group within 20 days in the NUGC4-luc mouse abdominal tumor model.
  • Figure 4C shows the FACS detection results of the proportion of NK cells in lymphocytes in the peripheral blood of each group of mice in the NUGC4-luc mouse abdominal tumor model on days 5, 12 and 19.
  • Figure 4D shows the detection results of human IL-15 cytokine secretion in peripheral blood serum of each group of mice in the NUGC4-luc mouse abdominal tumor model on days 5, 12 and 19.
  • Figure 6 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 three on the CHOK1-18.2 cell line.
  • Figure 7A shows the CAR transfection efficiency of 18.2-CAR2(T) cells detected by FACS after culture and expansion for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
  • Figure 7B shows the expression level of CD4 cell memory phenotype of 18.2-CAR2(T) cells detected by FACS after culture and expansion for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
  • Figure 7C shows the expression level of CD8 cell memory phenotype of 18.2-CAR2(T) cells detected by FACS after culture and expansion for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
  • Figure 7D shows the CD69 expression levels of CD4 and CD8 cells in 18.2-CAR2(T) cells detected by FACS after culture and expansion for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
  • Figure 7E shows the killing effect of 18.2-CAR2(T) cells on NUGC4-luc cells for 24 hours after culture and expansion with the addition of CBL-bi-1 and CBL-bi-2 for 9 days, where E:T is 5:1, 2: 1, 1:1, 1:2.
  • Figure 7F shows that 18.2-CAR2(T) cells killed NUGC4-luc cells for 24 hours after being cultured and expanded for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
  • Figure 7G shows that 18.2-CAR2(T) cells were cultured and expanded with the addition of CBL-bi-1 and CBL-bi-2 for 9 days, and then cultured for 24 hours without the addition of target cells, expressing CD107a levels on the surface of T cells and releasing IFN- ⁇ level.
  • Figure 8 shows the CAR transfection efficiency of BCMA-CAR1(NK) and BCMA-CAR2(NK) detected by FACS after amplification in culture medium supplemented with CBL-bi-4 for 15 days.
  • Figure 9 shows a schematic structural diagram of a dual-target chimeric antigen receptor targeting BCMA and GPRC5D.
  • Figure 10 shows the positive rate detection results of BCMA-GPRC5D CAR-NK with or without the addition of CBL-bi.
  • Figure 11 shows the results of multiple rounds of killing experiments on NCI H929+10% NCI H929-hBCMA-KO cells using BCMA-GPRC5D CAR-NK in combination with CBL-b inhibitors and/or PDL1-IL15.
  • Figure 12A shows the anti-tumor efficacy trial scheme of BCMA-GPRC5D CAR-NK combined with CBL-b inhibitor in Molp8-luc mouse tumor model.
  • Figures 12B-12E respectively show the tumor growth fluorescence signal ROI value (Figure 12B and 12C), body weight changes (Figure 12D), and Mouse survival rate (Figure 12E), where the BI-CAR21-CBL-b inhibitor in Figure 12B represents the BI-CAR21 treatment group in Table 9, that is, the treatment group with only BI-CAR21 and no CBL-b inhibitor.
  • compositions including A and B
  • composition containing other components in addition to A and B All fall within the scope of the aforementioned “a composition”.
  • CAR Chimeric antigen receptor
  • Chimeric antigen receptors refer to artificial immune effector cell surface receptors engineered to be expressed on immune effector cells and specifically bind to antigens, which at least include: (1) an extracellular antigen-binding domain, such as a variable recombination domain of an antibody; chain or light chain; (2) a transmembrane domain that anchors the CAR into immune effector cells; and (3) an intracellular signaling domain.
  • CARs are able to utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • the extracellular domain of the chimeric antigen receptor further includes a signal peptide and/or a hinge region, and/or the intracellular domain of the chimeric antigen receptor further Includes costimulatory domain.
  • signal peptide in the context of a chimeric antigen receptor refers to the fragment of a protein or polypeptide that serves to guide the protein or polypeptide into the secretory pathway, translocation to the cell membrane and/or cell surface.
  • An example of a signal peptide is the CD8 ⁇ signal peptide.
  • antigen-binding region or “target-binding sequence” in the context of chimeric antigen receptors are used interchangeably and refer to the region responsible for binding to the target of the CAR (e.g., a tumor antigen on a tumor cell) part.
  • the antigen-binding region may be in a form similar to the structure of an antibody, such as scFv.
  • an antigen-binding molecule e.g., an antibody and fragments thereof, a natural ligand of an antigen, and a variant of a natural variant
  • an antigen-binding molecule specifically binds an antigen and substantially the same antigen, typically with high affinity, but Does not bind unrelated antigens with high affinity.
  • Affinity is usually reflected by the equilibrium dissociation constant (KD), where a lower KD indicates higher affinity.
  • high affinity usually refers to having about 10E-6M or less, about 10E-7M or less, about 10E-8M or less, about 10E-9M or less, about 10E-10M or less, 10E -11M or lower or 10E-12M or lower KD.
  • KD KD/Ka, where Kd represents the dissociation rate and Ka represents the association rate.
  • the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis determination.
  • antibody is used herein in its broadest sense and refers to a polypeptide that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen. or peptide combinations.
  • Antibody herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives.
  • Such scaffolds include antibody-derived scaffolds, which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds, which contain, for example, biocompatible polymers.
  • Such supports may also include non- Antibody-derived scaffolds, such as scaffold proteins known in the art for grafting CDRs, include but are not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical “quadruple chain antibody”, which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to such a polypeptide chain, which It 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 in the direction from the N end to the C end; and, 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 composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • CH1 domain a heavy chain constant region
  • HR hinge region
  • CH2 domain heavy chain constant region
  • CH3 domain heavy chain constant region in the direction from the N end to the C end
  • the full-length antibody is of the IgE isotype, it
  • Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • IgA can be divided into IgA1 and IgA2.
  • Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.
  • Antibodies herein also include antibodies that do not contain light chains, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Alpaca ( Heavy-chain antibodies (HCAbs) produced by Vicugna pacos and others, as well as immunoglobulin neoantigen receptors (Ig new antigen receptor, IgNAR) discovered in sharks and other cartilaginous fishes.
  • HCAbs Heavy-chain antibodies
  • Ig new antigen receptor, IgNAR immunoglobulin neoantigen receptors
  • the "antibodies” herein can be derived from any animal, including but not limited to humans and non-human animals.
  • the non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camelids and llamas. , ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (such as shark).
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have the ability to bind Antigen capabilities.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, scFv and VHH.
  • Papain digestion of intact antibodies generates two identical antigen-binding fragments, termed "Fab” fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy 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 domain carry free thiol groups. Pepsin treatment produces an F(ab')2 fragment with two antigen binding sites (two Fab fragments) and part of the Fc region.
  • scFv single-chain variable fragment
  • 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.
  • 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 provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
  • a disulfide bond may also exist between the VH and VL of scFv, forming a disulfide-linked Fv (dsFv).
  • anobody in this article refers to the natural heavy chain antibody lacking the light chain that exists in camels and other bodies. Cloning its variable region can obtain a single domain antibody 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.
  • VHH domain and “single domain antibody” (single domain antibody, sdAb) in this article have the same meaning and can be used interchangeably. They refer to the variable region of a cloned heavy chain antibody, which is constructed from only one heavy chain variable region.
  • a single-domain antibody is the smallest fully functional antigen-binding fragment.
  • CH1 light chain and heavy chain constant region 1
  • variable region herein refers to the region of the heavy or light chain of an antibody involved in enabling the antibody to bind to the antigen.
  • Heavy chain variable region is used interchangeably with “VH” and “HCVR”
  • light chain variable region is used interchangeably.
  • VL can be used interchangeably with “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably in this article, and usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region.
  • HVR hypervariable region
  • VH heavy chain variable region
  • VL light chain variable region
  • This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • LCDR light chain variable region
  • frame region or "FR region” is used interchangeably and refers to those amino acid residues other than CDRs in the heavy or light chain variable region of an antibody.
  • FR region usually, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR CDR
  • 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 M.P.
  • CDR herein can be annotated and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but are not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of differently defined amino acid residues.
  • 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).
  • Chothia numbering system 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).
  • IMGT numbering system in this article generally refers to the 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.
  • IMGT ImMunoGeneTics information system
  • the term "hinge region" in the context of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that serves to connect the transmembrane region and the antigen-binding region. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antigen-binding region.
  • the hinge region may be derived in whole or in part from a natural molecule, 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 completely synthetic hinge sequence.
  • transmembrane (TM) region in the context of a chimeric antigen receptor refers to one that enables the chimeric antigen receptor to be expressed on the surface of immune effector cells (e.g., lymphocytes, NK cells, or NKT cells), and a polypeptide structure that guides the cellular response of immune effector cells against target cells.
  • the transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain enables signaling when the chimeric antigen receptor binds to the target antigen.
  • intracellular signaling domain in the context of a chimeric antigen receptor refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specified function.
  • the intracellular signaling domain is responsible for primary intracellular signal transmission after the antigen-binding domain binds the antigen, leading to the activation of immune effector cells and immune responses.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune effector cell in which the CAR is expressed.
  • Exemplary intracellular signaling domains include CD3 ⁇ .
  • 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 effective activation and function of T lymphocytes upon binding to an antigen.
  • the costimulatory domain can be from CD28, 4-1BB or 2B4, but is not limited thereto.
  • immune effector cell refers to cells that participate in an immune response, such as promoting an immune effector response.
  • immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and myeloid-derived phagocytes, and TILs (tumor infiltrating lymphocytes) etc.
  • Immune effector cells or effector cells contain nucleic acids encoding chimeric antigen receptors and/or express chimeric antigen receptors. Depending on the type of immune effector cells or effector cells, they are CAR-T, CAR-NK, CAR-M, etc. .
  • percent (%) sequence identity and “percent (%) sequence identity” are interchangeable and refer to the alignment of sequences and the introduction of gaps, if necessary, to achieve maximum percent sequence identity ( For example, for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and nonhomologous sequences may be ignored for comparison purposes) followed by the amino acid (or nucleotide) of the candidate sequence ) residues are identical to the amino acid (or nucleotide) residues of the reference sequence.
  • alignment can be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% decrease in to 100% sequence identity.
  • the length of the candidate sequences aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. .
  • 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.
  • a "vector” is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell.
  • Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid 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, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • drug herein refers to substances used to prevent, treat and diagnose diseases, including “pharmaceutical compositions” or “drug combinations”.
  • composition refers to a preparation that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain unacceptable toxicity to the subject administered the pharmaceutical composition of additional ingredients.
  • Pharmaceutical compositions include combinations that are separated in time and/or space, so long as they can act together to achieve the purposes of the present application.
  • the components contained in the pharmaceutical composition eg, CAR-immune cells according to the present application
  • the ingredients contained in the pharmaceutical composition can be administered to the individual as a whole, or separately.
  • the ingredients contained in the pharmaceutical composition are administered to the individual separately, the ingredients may be administered to the individual simultaneously or sequentially.
  • Pharmaceutical compositions 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, aqueous buffer solutions, isotonic saline solutions such as PBS (phosphate buffer saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, 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 suitable route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc.
  • the compositions according to the present application may contain wetting agents, emulsifiers or buffer substances as additives.
  • non-fixed combination means that the active ingredients (e.g., one active agent and at least one additional active agent) are administered to a patient simultaneously or sequentially without specific time limits, as separate entities, wherein such Administration provides therapeutically effective levels of both compounds in the patient.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without multiple toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salts refers to salts of pharmaceutically acceptable acids or bases, including salts of compounds with inorganic or organic acids, and salts of compounds with inorganic or organic bases.
  • pharmaceutically acceptable excipients or excipients refers to those excipients that have no obvious irritating effect on the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.
  • the terms "subject,” “subject,” “individual,” and “patient” refer to an organism undergoing treatment for a particular disease or disorder (eg, cancer or infectious disease) as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.
  • treatment refers to surgical or therapeutic treatment with the purpose of preventing, slowing down (reducing) undesirable physiological changes or pathologies in the subject, such as cell proliferative disorders (e.g., cancer). or pass progression of infectious diseases).
  • Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented.
  • slow down, alleviation, weakening, alleviation, alleviation their meanings also include elimination, disappearance, non-occurrence, etc.
  • the term "effective amount” refers to an amount of a therapeutic agent that is effective when administered alone or in combination with another therapeutic agent to a cell, tissue or subject to prevent or alleviate the symptoms of a disease or the progression of the disease. "Effective amount” also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • cancer refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers.
  • tumor or “tumor” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” as used herein are not mutually exclusive.
  • bonds depicted by solid and dashed lines Represents a single or double bond.
  • tautomer refers to a functional group isomer 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 can exist in two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually yield a mixture whose physical and chemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form is dominant; in phenols, the enol form is dominant. This application encompasses all tautomeric forms of the compounds.
  • stereoisomer refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.
  • the compounds of the present application may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so the compounds of the present application may exist in specific geometric or stereoisomer forms.
  • Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S) )-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic or other mixtures thereof, such as enantiomers or diastereomers Enriched mixtures, all of the above isomers and their mixtures are within the scope of the definition of compounds in this application.
  • asymmetric carbon atoms there may be additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms in substituents such as alkyl groups. These isomers and their mixtures involved in all substituents are also included in Within the scope of the definition of compounds in this application.
  • the compounds of the present application containing asymmetric atoms can be isolated in optically active pure form or in the racemic form.
  • the optically active pure form can be resolved from the racemic mixture, or by using chiral starting materials or chiral forms. Reagent synthesis.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence state of the specific atom is normal and the substituted compound is stable.
  • the ethyl group is "optionally" substituted by halogen, which means that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (CH 2 CH 2 F, CH 2 CH 2 Cl, etc.), or polysubstituted. (CHFCH 2 F, CH 2 CHF 2 , CHFCH 2 Cl, CH 2 CHCl 2, etc.) or completely substituted (CF 2 CF 3 , CF 2 CCl 3 , CCl 2 CCl 3, etc.). It will be understood by those skilled in the art that any substitution or substitution pattern that is sterically impossible and/or cannot be synthesized will not be introduced for any group containing one or more substituents.
  • variable e.g., R 1a , R 2a
  • R 1a a variable in the composition or structure of a compound
  • its definition in each instance is independent. For example, if a group is substituted by 2 R 1a , there is a separate option for each R 1a .
  • linking group When the number of a linking group is 0, such as -(CR 13 R 14 ) 0 -, it means that the linking group is a bond.
  • the direction of connection is arbitrary.
  • L in when the structural unit When L in is selected from “-NR 7 CH 2 -", L can be connected to ring D in the direction from left to right to form “ring D-NR 7 CH 2 -", or it can be connected in the direction from right to left The direction connects ring D to form "ring D-CH 2 NR 7 -".
  • substituents bond When a substituent's bond is cross-linked to two atoms on a ring, the substituent can be bonded to any atom on the ring.
  • structural unit It means that R 10 can be substituted at any one of positions 1, 2, and 3 on the benzene ring.
  • Cm - Cn refers to having an integer number of carbon atoms in the range of mn.
  • C 1 -C 6 means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
  • alkyl refers to a hydrocarbon group of the general formula C n H 2n+1 , which alkyl group may be straight or branched.
  • C 1 -C 6 alkyl is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1, 2, 3, 4, 5 or 6 carbon atoms.
  • alkyl group examples 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-di Methylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.
  • C 1 -C 3 alkyl is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1 to 3 carbon atoms.
  • the "C 1 -C 6 alkyl group” may include “C 1 -C 3 alkyl group”.
  • alkoxy refers to a monovalent group produced by losing a hydrogen atom on a hydroxyl group of a straight-chain or branched alcohol, and can be understood as “alkyloxy” or “alkyl-O-”.
  • C 1 -C 6 alkoxy is understood to mean “C 1 -C 6 alkyloxy” or “C 1 -C 6 alkyl-O-”; the term “C 1 -C 3 alkoxy” It can be understood as “C 1 -C 3 alkyloxy” or "C 1 -C 3 alkyl-O-”.
  • the "C 1 -C 6 alkoxy group” may further include "C 1 -C 3 alkoxy group”.
  • alkenyl refers to a linear or branched monovalent unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one double bond.
  • C 2 -C 4 alkenyl is understood to mean a straight-chain or branched unsaturated monovalent hydrocarbon radical containing one or more double bonds and having 2, 3 or 4 carbon atoms, "C 2 -C " Alkenyl” is preferably C 2 or C 3 alkenyl. It will be understood that where the alkenyl group contains more than one double bond, the double bonds may be isolated or conjugated to each other.
  • alkenyl group examples 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 wait.
  • alkynyl refers to a linear or branched monovalent unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one triple bond.
  • C 2 -C 4 alkynyl is understood to mean a straight-chain or branched unsaturated monovalent hydrocarbon radical which contains one or more triple bonds and has 2, 3 or 4 carbon atoms.
  • Examples of “C 2 -C 4 alkynyl” include, but are not limited to, ethynyl (-C ⁇ CH), propynyl (-C ⁇ CCH 3 , -CH 2 C ⁇ CH), but-1-ynyl, butyl -2-alkynyl or but-3-ynyl.
  • C 2 -C 4 alkynyl may include “C 2 -C 3 alkynyl", and examples of “C 2 -C 3 alkynyl” include ethynyl (-C ⁇ CH), prop-1-ynyl (-C ⁇ CCH 3 ), prop-2-ynyl (propargyl).
  • cycloalkyl refers to a fully saturated carbocyclic ring that exists in the form of a single ring, a branched ring, a bridged ring or a spiro ring. Unless otherwise indicated, the carbocyclic ring is generally 3 to 10 membered.
  • C 3 -C 10 cycloalkyl is understood to mean a saturated monovalent monocyclic, paracyclic, spirocyclic or bridged ring having 3 to 10 carbon atoms.
  • cycloalkyl group examples 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, etc.
  • C 3 -C 10 cycloalkyl may include “C 3 -C 8 cycloalkyl", “C 3 -C 8 cycloalkyl” may include “C 3 -C 6 cycloalkyl”, “C 3 -C 6 cycloalkyl” may include “C 3 -C 4 cycloalkyl”.
  • C 3 -C 6 cycloalkyl can be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring with 3 to 6 carbon atoms. Specific examples include but are not limited to cyclopropyl, cyclobutyl, cyclopropyl, cyclobutyl, Pentyl or cyclohexyl, etc.
  • cycloalkyloxy is understood to mean “cycloalkyl-O-”.
  • cycloalkenyl refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical containing at least one carbon-carbon double bond.
  • C 3 -C 6 cycloalkenyl refers to a non-aromatic cyclic hydrocarbon having 3 to 6 carbon atoms as ring atoms and containing at least one carbon-carbon double bond.
  • Specific examples of C 3 -C 6 cycloalkenyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.
  • heterocyclyl refers to a fully saturated or partially saturated (not aromatic heteroaromatic as a whole) monovalent monocyclic, paracyclic, spirocyclic or bridged cyclic group whose ring atoms contain 1- 5 heteroatoms or heteroatom groups (i.e.
  • 4-10 membered heterocyclyl refers to a heterocyclyl with a number of ring atoms of 4, 5, 6, 7, 8, 9 or 10, and its ring atoms contain 1 to 5 independently selected from the above. of heteroatoms or heteroatom groups.
  • the term "4-10-membered nitrogen-containing heterocyclic group” refers to a 4-10-membered heterocyclic group containing at least 1 N atom in its ring atom, and "4-10-membered nitrogen-containing heterocyclic group” includes "6-10-membered nitrogen-containing heterocyclic group”. Nitrogen-containing heterocyclic group".
  • 4-7 membered monocyclic nitrogen-containing heterocyclyl group refers to a 4-7 membered heterocyclyl group in the form of a monocyclic ring and containing at least 1 N atom in the ring atom.
  • 4-10 membered heterocyclyl includes “6-10 membered heterocyclyl”
  • 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; 5-membered heterocyclyl Specific examples include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydroxazolyl or 2,5-dihydroxazolyl.
  • 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianiyl Alkyl, tetrahydropyridyl or 4H-[1,3,4]thiadiazinyl; specific examples of 7-membered heterocyclyl include but are not limited to diazacycloheptyl; specific examples of 8-membered heterocyclyl Including but not limited to 6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazolyl.
  • the heterocyclic group can also be a bicyclic group, wherein, 5,5-membered
  • bicyclic groups include, but are not limited to, hexahydrocyclopenta[c]pyrrole-2(1H)-yl
  • 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.
  • the heterocyclic group can be a benzo-fused cyclic group of the above 4-7 membered heterocyclic group.
  • Specific examples include but are not limited to dihydrogen Isoquinolinyl, etc.
  • "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” and other ranges, "4-7 membered heterocyclyl” may further include "4-6 membered heterocyclyl", "5-7 “Membered heterocyclyl”, “5-6 membered heterocyclyl”, “4
  • heterocyclyloxy is understood to mean “heterocyclyl-O-”.
  • heterocycloalkyl refers to a monovalent cyclic group that is fully saturated and exists in the form of a single ring, a branched ring, a bridged ring or a spiro ring, and the ring atoms of the ring contain 1-5 heteroatoms or Heteroatom groups (i.e.
  • 4-10 membered heterocycloalkyl refers to a heterocycloalkyl group with a number of ring atoms of 4, 5, 6, 7, 8, 9 or 10, and its ring atoms contain 1 to 5 independently selected from the above The heteroatom or heteroatom group.
  • 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 thibutanyl; Specific examples of 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl or tetrahydrofuranyl.
  • 6-membered heterocycloalkyl include but are not limited to piperidyl, 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 aza Cycloheptyl, oxeptanyl or thieptanyl.
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated ⁇ electron system.
  • Aryl groups can have 6-14 carbon atoms or 6-10 carbon atoms.
  • C 6 -C 10 aryl is understood to mean a monovalent aromatic monocyclic or bicyclic radical having 6 to 10 carbon atoms.
  • rings with 6 carbon atoms (“C 6 aryl”), for example phenyl; or rings with 10 carbon atoms (“C 10 aryl”), for example naphthyl.
  • aryloxy is understood to mean “aryl-O-”.
  • heteroaryl refers to an aromatic monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, and S, and the remaining ring atoms are C.
  • heteroaryl refers to an aromatic monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, and S, and the remaining ring atoms are C.
  • 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 it contains 1, 2, 3, 4 or 5, preferably 1, 2 or 3 heteroatoms independently selected from N, O and S.
  • heteroaryl group is selected from the group consisting of thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl base, oxadiazolyl, triazolyl or thiadiazolyl, etc.
  • benzo derivatives such as benzofuryl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisox Azolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl or isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl, etc. and their Benzo derivatives, such as quinolinyl, quinazolinyl or isoquinolinyl, etc.; or azocinyl, indolinyl, purinyl, etc.
  • 5-10-membered nitrogen-containing heteroaryl refers to a 5-10-membered heteroaryl containing at least 1 N atom in its ring atom, and "5-10-membered nitrogen-containing heteroaryl” includes "5-6-membered Nitrogen-containing heteroaryl”.
  • 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 .
  • 6-membered heteroaryl refers to an aromatic ring system having 6 ring atoms and which contains 1, 2 or 3, preferably 1 or 2 N as heteroatoms.
  • 5-10 membered heteroaryl includes “5-6 membered heteroaryl”.
  • heteroaryloxy is understood to mean “heteroaryl-O-”.
  • halo or halogen refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
  • hydroxy refers to the -OH group.
  • cyano refers to the -CN group.
  • mercapto refers to the -SH group.
  • amino refers to the -NH group .
  • nitro refers to the -NO2 group.
  • the present application also includes compounds of the present application that are the same as those described herein, but are isotopically labeled in which one or more atoms are replaced by an atom having an atomic weight or mass number different from that typically found in nature.
  • isotopes that may 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 respectively N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
  • isotopically labeled compounds of the present application can be used in compound and/or substrate tissue distribution analysis. Tritiated (ie 3 H) and carbon-14 (ie 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 substituting an isotopically labeled reagent for a non-isotopically labeled reagent by following procedures similar to those disclosed in the Schemes and/or Examples below.
  • the inventor of the present application has developed engineered immune effector cells and their combined application with and/or other active substances (such as PDL1-IL15 fusion protein).
  • the combined application involves drugs for the treatment of cancer or tumors. Methods for preparing and treating cancer or tumors, methods for amplifying immune cells in vitro, etc.
  • the present application provides the use of a combination of a CBL-b inhibitor and immune effector cells, which express or do not express chimeric antigen receptors, in the preparation of a medicament for the treatment of cancer or tumors.
  • the present application provides a pharmaceutical combination product for treating cancer or tumors, which includes a CBL-b inhibitor and immune effector cells that express or do not express chimeric antigen receptors.
  • the application provides a method of treating cancer or tumors, wherein the method comprises administering to a subject in need thereof an effective amount of a CBL-b inhibitor and an immune effector cell that expresses or does not Expresses chimeric antigen receptor.
  • CBL-b inhibitors immune effector cells, chimeric antigen receptors, cancers or tumors are applicable to the first to third aspects, and, in the absence of contradiction, are also applicable to this application any aspect of various embodiments of the invention.
  • the CBL-b inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,
  • C CA 3
  • a 3 is selected from CR 11a R 11b , NR 12 , O or S
  • a 3 is selected from CR 11c or N
  • a 1 -A 2 -A 3 said A 1 and A 2 are independently selected from C(R 11 ) n or N
  • a 3 is selected from CR 11a R 11b , NR 12 , O or S;
  • n is selected from 0 or 1;
  • R 11a , R 11b , R 11c , R 11 , R 12 are independently selected from H, halogen, OH, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) ) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 cycloalkyloxy, wherein the C 1 -C 6 alkyl Base, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 cycloalkyloxy is optionally substituted by R 11d ;
  • Q ring is selected from phenyl, 5-6-membered heteroaryl or 5-7-membered heterocyclyl, and the phenyl, 5-6-membered heteroaryl or 5-7-membered heterocyclyl is optionally substituted by R 10 ;
  • Y 1 , Y 2 , Y 3 and Y 4 are independently selected from CR b or N;
  • R b is selected from H, halogen, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 Alkyl) 2 , NHC(O)(C 1 -C 6 alkyl), NHC(O)-O(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl)C(O)- O(C 1 -C 6 alkyl), NHS(O) 2 (C 1 -C 6 alkyl), C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl-O-, C 3 - C 6 cycloalkyl-NH-, N(C 3 -C 6 cycloalkyl) 2 , NHC(O)-C 3 -C 6 cycloalkyl, NHS(O) 2 -C 3 -C 6 cycloalkyl , 4
  • R b and the C atom to which they are connected together form a C 3 -C 6 cycloalkenyl group, a phenyl group, a 4-7 membered heterocyclyl group or a 5-6 membered heteroaryl group, and the C 3 -C 6 cycloalkenyl group , phenyl, 4-7-membered heterocyclyl or 5-6-membered heteroaryl optionally substituted by R 2a ;
  • R 4 , R 5 , R 7 , R 8 and R 9 are independently selected from H, halogen, OH, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, the C 1 -C 6 alkyl
  • the base or C 1 -C 6 alkoxy group is optionally substituted by R 4a ;
  • R 8 , R 9 and their connected atoms together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group is optional further replaced by R 8a ;
  • R 4 , R 5 and their connected atoms together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group is optional Further substituted by R 8a ; or when p is taken from 2, two R 4 and its connected atoms together form a C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl group, the C 3 -C 6 ring Alkyl or 4-7 membered heterocyclyl is optionally further substituted by R 8a ;
  • Each R 6a is independently selected from H, C 1 -C 6 alkyl, phenyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, the C 1 -C 6 alkyl, phenyl,
  • the 4-7-membered heterocyclyl or 5-6-membered heteroaryl is optionally further substituted by R 6b , or the two R 6a on one N atom and the N to which it is connected together form a 4-7-membered heterocyclyl or 5-6
  • One-membered heteroaryl, the 4-7-membered heterocyclyl or 5-6-membered heteroaryl is optionally further substituted by R 6b ;
  • R 1 and R 2 are independently selected from H, halogen, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 cycloalkyl or 4-10 membered heterocyclyl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 Cycloalkyl or 4-10 membered heterocyclyl is optionally substituted by R 1a ,
  • R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 10 cycloalkyl group or a 4-10 membered heterocyclyl group, and the C 3 -C 10 cycloalkyl group or 4-10 membered heterocyclyl group is optionally R 1b substitution;
  • the group is optionally substituted by R 1d ;
  • W is selected from (CR 13 R 14 ) k W 1 ; said W 1 is selected from 5-10-membered heteroaryl or 4-10-membered heterocyclyl, and said 5-10-membered heteroaryl, 4-10-membered heterocyclic group
  • the ring group is optionally substituted by R 15 ;
  • R 13 and R 14 are independently selected from H, halogen, OH, C 1 -C 6 alkyl or C 1 -C 6 alkoxy;
  • R 15 is selected from halogen, OH, NH 2 , NH (C 1 -C 6 alkyl), N (C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl, wherein The C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 15a ;
  • the group is optionally substituted by R 13a ;
  • R 8a , R 13a and R 1d are independently selected from halogen, OH, CN, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, said C 1 -C 6 alkyl or C 1 -C 6 The alkoxy group is optionally further substituted by halogen;
  • k is selected from 0 or 1;
  • p is selected from 0, 1 or 2.
  • Selected from A 1 -C A 3 , said A 1 is selected from C or N, and A 3 is selected from CR 11c or N.
  • Selected from A 1 -C A 3 , said A 1 is selected from N, and A 3 is selected from CR 11c or N.
  • Selected from A 1 -A 2 -A 3 , said A 1 and A 2 are independently selected from C(R 11 ) n or N, and A 3 is selected from NR 12 , O or S.
  • a 1 is selected from N
  • a 2 is selected from C(R 11 ) n
  • a 3 is selected from NR 12 .
  • R 11a , R 11b , R 11c , R 11 , R 12 are independently selected from H, halogen, OH, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 - C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 cycloalkyloxy, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 cycloalkyloxy is optionally substituted by R 11d .
  • R 11a , R 11b , R 11c , R 11 , R 12 are independently selected from H, halogen, OH, NH 2 , C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl. , the C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl group is optionally substituted by R 11d .
  • R 11d is selected from halogen, OH, NH 2 , C 1 -C 6 alkyl, or haloC 1 -C 6 alkyl.
  • n is selected from 0.
  • R 11c is selected from H, halogen, or C 1 -C 6 alkyl optionally substituted with R 11d .
  • R 11c is selected from H or halogen.
  • R 11c is selected from F.
  • R 12 is selected from H, halogen, OH, C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl, said C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl The group is optionally substituted by R 11d .
  • R 12 is selected from H or C 1 -C 3 alkyl.
  • R 12 is selected from H or methyl.
  • R 12 is selected from methyl.
  • Selected from A 1 -C A 3 , said A 1 is selected from N, and A 3 is selected from N.
  • the Q ring is selected from the following groups optionally substituted with R 10 : phenyl, pyridyl, pyrimidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, iso Thiazolyl or 5-7 membered heterocyclyl group containing 1 or 2 N as heteroatoms.
  • the Q ring is selected from the following groups optionally substituted with R 10 : phenyl,
  • the Q ring is selected from optionally substituted with R 10
  • R 10a is selected from halogen, OH, NH 2 , C 1 -C 6 alkyl, haloC 1 -C 6 alkyl, C 1 -C 6 alkoxy, or haloC 1 -C 6 alkoxy.
  • R 10a is selected from halogen, OH, C 1 -C 6 alkyl, or haloC 1 -C 6 alkyl.
  • R 10a is selected from F or CH 3 .
  • Y 1 , Y 2 are independently selected from CR b or N, and Y 3 and Y 4 are independently selected from CR b .
  • Y 1 , Y 2 , Y 3 and Y 4 are each CR b .
  • Y 1 and Y 2 are both N and Y 3 and Y 4 are independently selected from CR b .
  • Y 1 is N and Y 2 , Y 3 and Y 4 are independently selected from CR b .
  • R b is selected from H, halogen, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 3 -C 6 cycloalkyl -O-, C 3 -C 6 cycloalkyl -NH-, 4-7 membered heterocyclyl -O-, 4-7 membered heterocycle -NH- or 5-10 membered heteroaryl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, 4-7 membered heterocyclyl Or 5-10 membered heteroaryl is optionally substituted by R 2a .
  • R b is selected from H, halogen, or optionally substituted by R 2a : methyl, ethoxy, NHCH 3 , NHEt, NH(i-Pr), pyrazolyl, cyclopropyl -O-, cyclobutyl-NH- or oxetanyl-O-.
  • R 2a is selected from halogen, OH, or C 1 -C 3 alkyl.
  • R 2a is selected from F, OH, or methyl.
  • R b is selected from H, F, CF 3 , OEt, NHCH 3 , NHEt, NH(i-Pr),
  • R b , R 1 and their respective connected atoms and bonds together form a C 3 -C 6 cycloalkenyl or 4-7 membered heterocyclyl group, the C 3 -C 6 cycloalkyl or 4 -7-membered heterocyclyl optionally substituted by R 1d .
  • R b , R 1 and their respective atoms and bonds to which they are attached together form a C 3 -C 6 cycloalkenyl group optionally substituted by R 1d .
  • R 1d is selected from halogen, OH, C 1 -C 3 alkyl, or haloC 1 -C 3 alkyl.
  • R b , R 1 and their respective atoms and bonds to which they are attached together form cyclopentenyl.
  • X is selected from Or C 1 -C 6 alkyl optionally substituted by Re .
  • X is selected from
  • X is selected from
  • Ring B is selected from a 5-10 membered nitrogen-containing heteroaryl optionally substituted by R3 , a 4-7 membered monocyclic nitrogen-containing heterocyclyl, or a 6-10 membered nitrogen-containing heterocyclyl.
  • Ring B is selected from the following groups optionally substituted by R : tetrahydropyrrolyl, piperidinyl, piperazinyl, Morpholinyl,
  • Ring B is selected from the following groups optionally substituted by R : tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl,
  • the heteroaryl group is optionally further substituted with R 3a .
  • R 3a is selected from F.
  • R 4a is selected from halogen, OH, or C 1 -C 3 alkoxy.
  • R 4a is selected from F or OH.
  • R 8a is selected from halogen, OH, or C 1 -C 3 alkyl.
  • p is selected from 1.
  • p is selected from 0.
  • p is selected from 2, and two R 4 and the atoms to which they are attached together form a C 3 -C 6 cycloalkyl group optionally substituted by R 8a .
  • p is selected from 2, and two R 4 and the atoms to which they are attached together form cyclopropyl or cyclobutyl.
  • Ring D is selected from the following groups optionally substituted by R: C3-C6 cycloalkyl , 5-6 membered heteroaryl, 4-7 membered monocyclic nitrogen-containing heterocyclyl, or 6-10 membered nitrogen-containing heterocyclic group, and ring D is connected to L with a non-N atom.
  • Ring D is selected from the following groups optionally substituted by R: cyclopropyl , cyclobutyl, cyclopentyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, Pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl, isothiazolyl or pyridyl.
  • Ring D is selected from the following groups optionally substituted by R : cyclopentyl, piperidyl, pyridyl,
  • R6 is selected from F or methyl.
  • L is selected from bond , -NR7- , -NR7CH2- , -O-, or -CR8R9- .
  • R 7 , R 8 and R 9 are independently selected from H, halogen, C 1 -C 3 alkyl, or OH.
  • R 7 , R 8 and R 9 are independently selected from H or halogen.
  • R is selected from H.
  • R 8 , R 9 are selected from H or F.
  • L is selected from the group consisting of bond, -NH-, -NHCH2- , -O-, -C(F) 2- , or -CH2- .
  • X is selected from C 1 -C 6 alkyl optionally substituted by Re .
  • Re is selected from halogen, OH, C 1 -C 3 alkyl, NH 2 , NH(C 1 -C 3 alkyl), N(C 1 -C 3 alkyl) 2 or C 1 -C 3 alkoxy group, wherein the C 1 -C 3 alkyl group or C 1 -C 3 alkoxy group is further optionally substituted by R f .
  • Re is selected from C 1 -C 3 alkyl, N(C 1 -C 3 alkyl) 2 or C 1 -C 3 alkoxy, wherein said C 1 -C 3 alkyl Or the C 1 -C 3 alkoxy group is further optionally substituted by R f .
  • R f is selected from halogen, OH, or N(C 1 -C 6 alkyl) 2 .
  • R f is selected from N(CH 3 ) 2 .
  • Re is selected from N(CH 3 ) 2 , CH 2 N(CH 3 ) 2 or
  • X is selected from
  • X is selected from the following groups:
  • X is selected from the following groups:
  • X is selected from
  • X is selected from
  • R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 8 cycloalkyl group or a 4-10 membered heterocyclyl group .
  • the ring group is optionally substituted by R 1b .
  • R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group
  • the ring group is optionally substituted by R 1b .
  • R 1 , R 2 and the atoms to which they are attached together form the following group optionally substituted by R 1b : cyclobutyl, spiro[2,3]hexyl, or oxetanyl.
  • R 1b is selected from halogen, CN, C 1 -C 3 alkyl or C 1 -C 3 alkoxy, any of which C 1 -C 3 alkyl or C 1 -C 3 alkoxy Selected to be replaced by R 1c .
  • R 1c is selected from halogen, OH, or CN.
  • R 1c is selected from CN.
  • R lb is selected from F, CN, methyl, methoxy, or CH 2 CN.
  • R 1 and R 2 are independently selected from H, halogen, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl, the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 1a , and the C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl may be a spiro ring, a bridged ring or a Ring form.
  • R 1 and R 2 are independently selected from H, halogen, CN, C 1 -C 3 alkyl, C 1 -C 3 alkoxy, or C 3 -C 6 cycloalkyl, the C 1 -C 3 alkyl, C 1 -C 3 alkoxy or C 3 -C 6 cycloalkyl is optionally substituted by R 1a .
  • R 1 and R 2 are independently selected from H or C 1 -C 3 alkyl, which C 1 -C 3 alkyl is optionally substituted with R 1a .
  • R 1 and R 2 are independently selected from H or methyl, or R 1 , R 2 and the atoms to which they are connected together form the following group:
  • R 1 , R 2 and the atoms to which they are connected together form the following group:
  • R 1 , R 2 and the atoms to which they are connected together form the following group:
  • R 1 and R 2 are independently selected from H or methyl.
  • W is selected from -(CR 13 R 14 )W 1 .
  • R 13 , R 14 are independently selected from H, halogen, OH, or methyl.
  • R 13 and R 14 are both H.
  • R 1 and R 13 and the atoms and bonds to which each is attached together form a C 3 -C 6 cycloalkyl group optionally substituted by R 13a .
  • R 13a is selected from C 1 -C 3 alkyl.
  • R 13a is selected from methyl.
  • R 1 and R 13 and their respective atoms and bonds to which they are attached together form
  • W is selected from W 1 .
  • W 1 is selected from 5-10 membered heteroaryl or 6-10 membered heterocyclyl optionally substituted by R 15 .
  • W 1 is selected from 5-10 membered heteroaryl optionally substituted with R 15 .
  • W 1 is selected from 5-membered heteroaryl or 8-membered heterocyclyl optionally substituted with R 15 .
  • W 1 is selected from 5-membered heteroaryl optionally substituted with R 15 .
  • W is selected from the following groups optionally substituted with R : pyrrolyl, thienyl, furyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tris Azolyl, oxazolyl, isoxazolyl, oxadiazolyl or 6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazolyl.
  • W is selected from the following groups optionally substituted with R : pyrrolyl, thienyl, furyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tris Azolyl, oxazolyl, isoxazolyl or oxadiazolyl.
  • W is selected from the group consisting of triazolyl, oxazolyl, isoxazolyl, oxadiazolyl, or 6,7-dihydro-5H-pyrrole, optionally substituted with R 15 And [2,1-c][1,2,4]triazolyl.
  • W 1 is selected from the group consisting of triazolyl, oxazolyl, isoxazolyl, or oxadiazolyl, optionally substituted with R 15 .
  • R 15 is selected from halogen, OH, NH 2 , C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl, said C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl Alkyl is optionally substituted with R 15a .
  • R 15 is selected from OH, C 1 -C 3 alkyl, or C 3 -C 6 cycloalkyl, which C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl is optionally replaced by R 15a substituted.
  • R 15 is selected from methyl or cyclopropyl, which is optionally substituted with R 15a .
  • R 15a is selected from halogen, OH, or CN.
  • R 15a is selected from F.
  • R15 is selected from methyl, CHF2 , or cyclopropyl.
  • W is selected from the following groups:
  • W is selected from the following groups:
  • W is selected from
  • compounds of formula (I) of the present application are selected from compounds of formula (II):
  • Y 1 , Y 2 , Y 3 , Y 4 , X, Q, W, R 1 and R 2 are as defined above.
  • compounds of formula (I) of the present application are selected from compounds of formula (III):
  • Z 1 , Z 2 , Z 3 are 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 are as defined above.
  • compounds of Formula (I) of the present application are selected from compounds of Formula (IV):
  • R 10 , Y 1 , Y 2 , Y 3 , Y 4 , X, W, R 1 and R 2 are as defined above.
  • compounds of formula (I) of the present application are selected from the following compounds:
  • the CBL-b inhibitor is a compound of formula (V) or a pharmaceutically acceptable compound thereof. Salt,
  • Z 1 and Z 2 are independently selected from CR a or N;
  • Z 3 is selected from C, CH or N;
  • Y 1 , Y 2 , Y 3 and Y 4 are independently selected from CR b or N;
  • R b is selected from H, halogen, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 Alkyl) 2 , NHC(O)(C 1 -C 6 alkyl), NHS(O) 2 (C 1 -C 6 alkyl), C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl Base -O-, C 3 -C 6 cycloalkyl -NH-, N(C 3 -C 6 cycloalkyl) 2 , NHC(O)-C 3 -C 6 cycloalkyl, NHS(O) 2 - C 3 -C 6 cycloalkyl, 4-7 membered heterocycle base, 4-7-membered heterocyclyloxy group, 4-7-membered heterocyclyl-NH-, N(4-7-membered heterocycl
  • R b and the C atom to which they are connected together form a C 3 -C 6 cycloalkenyl group, a phenyl group, a 4-7 membered heterocyclyl group or a 5-6 membered heteroaryl group, and the C 3 -C 6 cycloalkenyl group , phenyl, 4-7-membered heterocyclyl or 5-6-membered heteroaryl optionally substituted by R 2a ;
  • R a , R 4 , R 5 , R 7 , R 8 and R 9 are independently selected from H, halogen, OH, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, the C 1 - C 6 alkyl or C 1 -C 6 alkoxy is optionally substituted by R 4a ;
  • R 8 , R 9 and their connected atoms together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, or R 4 , R 5 and their connected atoms together form a C 3 -C 6 cycloalkyl group.
  • R 6a is selected from H, C 1 -C 6 alkyl, phenyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, the C 1 -C 6 alkyl, phenyl, 4-7 membered heteroaryl Heterocyclyl or 5-6-membered heteroaryl is optionally further substituted by R 6b , or two R 6a on one N atom and the N to which it is connected together form a 4-7-membered heterocyclyl or 5-6-membered heteroaryl. , the 4-7-membered heterocyclyl or 5-6-membered heteroaryl is optionally further substituted by R 6b ;
  • Q is phenyl or 6-membered heteroaryl, and the phenyl or 6-membered heteroaryl is optionally substituted by R 10 ;
  • R 10 is selected from halogen, OH, NH 2 , CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl, the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 10a ;
  • R c and R 11 and their respective connected atoms together form a 4-7-membered heterocyclic group, and the 4-7-membered heterocyclic group is optionally substituted by R 11c ;
  • R 1 and R 2 are independently selected from H, halogen, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 cycloalkyl or 4-10 membered heterocyclyl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 Cycloalkyl or 4-10 membered heterocyclyl is optionally substituted by R 1a ,
  • R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 10 cycloalkyl group or a 4-10 membered heterocyclyl group, and the C 3 -C 10 cycloalkyl group or 4-10 membered heterocyclyl group is optionally R 1b substitution;
  • W is selected from (CR 12 R 13 ) k W 1 , and said W 1 is selected from 5-10-membered heteroaryl or 4-10-membered heterocyclyl.
  • the ring group is optionally substituted by R 14 , R 12 and R 13 are independently selected from H, halogen, OH, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, R 14 is selected from halogen, OH, NH 2 , NH (C 1 -C 6 alkyl), N (C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl, wherein The C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 14a ;
  • the group is optionally substituted by R 12a ;
  • R 8a and R 12a are independently selected from halogen, OH, CN, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, said C 1 -C 6 alkyl or C 1 -C 6 alkoxy
  • p and k are independently selected from 0 or 1.
  • Z 1 and Z 2 are independently selected from CH or N.
  • Z 1 and Z 2 are both CH.
  • Z 1 is CH and Z 2 is N.
  • Z 1 is N and Z 2 is CH.
  • Y 1 , Y 2 , Y 3 and Y 4 are each CR b .
  • Y 1 and Y 2 are independently selected from CR b or N, and Y 3 and Y 4 are both CR b .
  • Y 1 and Y 2 are both N and Y 3 and Y 4 are both CR b .
  • Y 1 is N and Y 2 , Y 3 and Y 4 are all CR b .
  • R b is selected from H, halogen, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 3 -C 6 cycloalkyl -O-, C 3 -C 6 cycloalkyl -NH-, 4-7 membered heterocyclyl -O-, 4-7 membered heterocycle -NH- or 5-10 membered heteroaryl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, 4-7 membered heterocyclyl Or 5-10 membered heteroaryl is optionally substituted by R 2a .
  • R b is selected from H, halogen, or optionally substituted by R 2a : methyl, ethoxy, NHCH 3 , NHEt, NH(i-Pr), pyrazolyl, cyclopropyl -O-, cyclobutyl-NH- or oxetanyl-O-.
  • R 2a is selected from halogen, OH, or C 1 -C 3 alkyl.
  • R 2a is selected from F, OH, or methyl.
  • R b is selected from H, F, CF 3 , ethoxy, OCH 2 CHF 2 , NHCH 3 , NHEt, NH(i-Pr), NHCH 2 CH 2 OH,
  • X is selected from
  • Ring B is selected from a 5-10 membered nitrogen-containing heteroaryl optionally substituted by R3 , a 4-7 membered monocyclic nitrogen-containing heterocyclyl, or a 6-10 membered nitrogen-containing heterocyclyl.
  • Ring B is selected from the following groups optionally substituted with R : pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, azepine cyclobutyl, tetrahydropyrrolyl, piperidyl, piperazinyl, morpholinyl, azepanyl,
  • Ring B is selected from the following groups optionally substituted with R : pyrazolyl, azetidinyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, azepine Cycloheptyl,
  • R 3a is selected from F, OH, or methoxy.
  • R 4a is selected from halogen, OH, or C 1 -C 3 alkoxy.
  • R 4a is selected from F.
  • p is selected from 1.
  • p is selected from 0.
  • Ring D is selected from the following groups optionally substituted by R: C3-C6 cycloalkyl , 5-6 membered heteroaryl, 4-7 membered monocyclic heterocyclyl, or 6- 10-membered heterocyclyl, and ring D is connected to L with a non-N atom.
  • Ring D is selected from the following groups optionally substituted by R : cyclopropyl, cyclobutyl, cyclopentyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, Pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl or isothiazolyl.
  • L is selected from bond , -NR7- , -NR7CH2- , -O-, or -CR8R9- .
  • R 7 , R 8 and R 9 are independently selected from H, C 1 -C 3 alkyl, or OH.
  • R7 is selected from H or methyl.
  • R 8 , R 9 are selected from H.
  • L is selected from bond, -NCH3- , -NHCH2- , -O-, or -CH2- .
  • X is selected from the following groups:
  • X is selected from
  • Q is phenyl or 6-membered heteroaryl optionally substituted with R 10 .
  • Q is phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, or triazinyl optionally substituted with R 10 .
  • Q is phenyl, pyridinyl, pyridazinyl, or pyrimidinyl optionally substituted with R 10 .
  • Q is phenyl optionally substituted with R 10 .
  • R 10 is selected from halogen, OH, CN , C 1 -C 6 alkyl, or C 1 -C 6 alkoxy.
  • the group is optionally substituted by R 10a .
  • R 10 is selected from halogen, C 1 -C 3 alkyl, or C 1 -C 3 alkoxy, optionally substituted by R 10a replaced.
  • R 10a is selected from halogen, OH, or C 1 -C 3 alkyl.
  • R 10a is selected from F or OH.
  • R10 is selected from F, Cl, methyl, methoxy, CH2OH , or CF3 .
  • Q is selected from Wherein Z 4 and Z 5 are independently selected from CR c R d , NR 11 , O, S or SO 2 , represents a single or double bond, and when When it is a double bond, m is 1, when When it is a single bond, m is 1 or 2.
  • R c and R 11 and the atom to which each is attached together form a 4-7 membered heterocyclyl group that is optionally substituted by R 11c .
  • Q is selected from m is 1 or 2.
  • Q is selected from m is 1.
  • Q is selected from
  • Q is selected from phenyl, Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
  • Q is selected from phenyl, Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
  • Q is selected from phenyl, Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
  • Q is selected from Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
  • Q is selected from Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
  • R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 8 cycloalkyl group or a 4-10 membered heterocyclyl group .
  • the ring group is optionally substituted by R 1b .
  • R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group
  • the ring group is optionally substituted by R 1b .
  • R 1 , R 2 and the atoms to which they are attached together form the following group optionally substituted by R 1b : cyclobutyl, spiro[2,3]hexyl, or oxetanyl.
  • R 1b is selected from halogen, CN, C 1 -C 3 alkyl or C 1 -C 3 alkoxy, any of which C 1 -C 3 alkyl or C 1 -C 3 alkoxy Selected to be replaced by R 1c .
  • R 1c is selected from halogen, OH, or CN.
  • R 1c is selected from CN.
  • R lb is selected from F, CN, methyl, methoxy, or CH 2 CN.
  • R 1 and R 2 are independently selected from H, halogen, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl, the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 1a , and the C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl may be a spiro ring, a bridged ring or a Ring form.
  • R 1 and R 2 are independently selected from H, halogen, CN, C 1 -C 3 alkyl, C 1 -C 3 alkoxy, or C 3 -C 6 cycloalkyl, the C 1 -C 3 alkyl, C 1 -C 3 alkoxy or C 3 -C 6 cycloalkyl is optionally substituted by R 1a .
  • R 1 and R 2 are independently selected from H, C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl, said C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl The group is optionally substituted by R 1a .
  • R 1 and R 2 are independently selected from H, methyl, or cyclobutyl, or R 1 , R 2 and the atoms to which they are connected together form the following group:
  • R 1 , R 2 and the atoms to which they are connected together form the following group:
  • R 1 , R 2 and the atoms to which they are connected together form the following group:
  • R 1 and R 2 are independently selected from H, methyl, or cyclobutyl.
  • W is selected from -(CR 12 R 13 )W 1 .
  • R 12 , R 13 are independently selected from H, halogen, OH, or methyl.
  • R 12 and R 13 are both H.
  • R 1 and R 12 and the atoms and bonds to which each is attached together form a C 3 -C 6 cycloalkyl group optionally substituted by R 12a .
  • R 12a is selected from C 1 -C 3 alkyl.
  • R 12a is selected from methyl.
  • R 1 and R 12 and their respective atoms and bonds to which they are attached together form
  • W is selected from W 1 .
  • W 1 is selected from 4-10 membered heterocyclyl optionally substituted with R 14 .
  • W 1 is selected from 5-10 membered heteroaryl optionally substituted with R 14 .
  • W 1 is selected from 5-membered heteroaryl optionally substituted with R 14 .
  • W is selected from the group consisting of pyrrole, thiophene, furan, pyrazole, imidazole, thiazole, isothiazole, thiadiazole, triazole, oxazole, isoxazoles, optionally substituted with R 14 Azole, oxadiazole,
  • W 1 is selected from the group consisting of triazole, oxazole, isoxazole, or oxadiazole optionally substituted with R 14 .
  • W 1 is selected from the following groups optionally substituted with R 14 :
  • R 14 is selected from halogen, OH, NH 2 , C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl, said C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl Alkyl is optionally substituted with R 14a .
  • R 14 is selected from OH, C 1 -C 3 alkyl, or C 3 -C 6 cycloalkyl, which C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl is optionally replaced by R 14a substituted.
  • R 14 is selected from methyl or cyclopropyl, which is optionally substituted with R 14a .
  • R 14a is selected from halogen, OH, or CN.
  • R 14a is selected from F.
  • R14 is selected from methyl, CHF2 , or cyclopropyl.
  • W is selected from the following groups:
  • W is selected from
  • compounds of formula (V) of the present application are selected from compounds of formula (VI):
  • j is selected from 0, 1, 2 or 3
  • Z 1 , Z 2 , Y 1 , Y 2 , Y 3 , Y 4 , X, W, R 1 , R 2 and R 10 are as in formula (V) definition.
  • compounds of formula (V) of the present application are selected from compounds of formula (VII):
  • Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Y 1 , Y 2 , Y 3 , Y 4 , X, W, R 1 , R 2 and m are as defined in formula (V).
  • compounds of formula (V) of the present application are selected from the following compounds:
  • the CBL-b inhibitor has the structure of the CBL-b inhibitor described in WO 2020/264398 A1, and the full text of WO 2020/264398 A1 is incorporated into this application by reference.
  • the CBL-b inhibitor has the structure of the CBL-b inhibitor described in WO 2020/210508 A1, and the full text of WO 2020/210508 A1 is incorporated into this application by reference.
  • the CBL-b inhibitor is a compound with the following chemical formula or a pharmaceutically acceptable salt thereof:
  • CBL-b inhibitors are formulated as oral or injectable formulations.
  • the pharmaceutical composition containing a CBL-b inhibitor of the present application can be prepared by combining the CBL-b inhibitor of the present application with appropriate pharmaceutically acceptable excipients, for example, it can be formulated into a solid, semi-solid, liquid or gaseous preparation. , such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.
  • Typical routes for administering the CBL-b inhibitor salts or pharmaceutical compositions thereof of the present application include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, Subcutaneous and intravenous administration.
  • the pharmaceutical composition containing a CBL-b inhibitor of the present application can be manufactured using methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, etc.
  • compositions of CBL-b inhibitors of the present application are in oral form.
  • the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These excipients enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc. for oral administration to patients.
  • Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets.
  • Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants or flavoring agents, etc.
  • the pharmaceutical compositions may also be suitable for parenteral administration as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.
  • the cancer or tumor is selected from hematological tumors or solid tumors. In some specific embodiments, the cancer or tumor is a solid tumor. In some specific embodiments, the solid tumor is selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, renal cell cancer, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcomas.
  • the tumor or cancer refers to a tumor or cancer in which the antigen molecule against which the chimeric antigen receptor is directed is expressed on the surface of tumor cells or cancer cells.
  • the tumor is a tumor that highly expresses Claudin18.2 (Claudin18.2+).
  • a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 60% of the tumor cells in the tumor cell population express Claudin18.2.
  • a tumor that highly expresses Claudin18.2 means that at least 70% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 80% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 90% of the tumor cells in the tumor cell population express Claudin18.2.
  • a tumor that highly expresses Claudin18.2 means that at least 95% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 98% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 99% of the tumor cells in the tumor cell population express Claudin18.2. The same applies to the antigen molecules BCMA, GPRC5D and other tumor antigen molecules involved in this application.
  • the immune effector cells are selected from the following group: (1) T cells expressing chimeric antigen receptors; (2) NK cells expressing chimeric antigen receptors; (3) Chimeric antigen receptor expressing T cells Antigen receptor T cells and NK cells expressing chimeric antigen receptors.
  • 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 NK cells Cell-expressed chimeric antigen receptors bind the same or different antigens or epitopes.
  • 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.
  • the antigen can be Claudin18.2, BCMA or GPRC5D alone, a combination of Claudin18.2, BCMA and GPRC5D, or Claudin18.2, BCMA or GPRC5D.
  • the chimeric antigen receptor of the present application includes an antibody or antibody fragment targeting CLDN18.2 as the antigen-binding region, and the application also provides such an antibody or antibody fragment targeting CLDN18.2 separately. .
  • the VH or VL sequence of the antibody or antibody fragment targeting CLDN18.2 has at least 80% or at least 85% similarity with the sequence shown in the following SEQ ID NO: 8 or SEQ ID NO: 9 , at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical:
  • VH sequence (SEQ ID NO:8):
  • VL sequence (SEQ ID NO:9):
  • the VH/VL shown in SEQ ID NO:8-9 has the following CDR:
  • the antibody or antibody fragment is a scFv that has at least 80%, at least 85%, at least 90%, or at least 95% similarity to the following SEQ ID NO: 16 or SEQ ID NO: 17 sequence. %, at least 96%, at least 97%, at least 98% or 100% identity:
  • the chimeric antigen receptor of the present application includes an antibody or antibody fragment targeting BCMA as the antigen-binding region, and the application also provides such an antibody or antibody fragment targeting BCMA separately.
  • the BCMA-targeting antibody or antibody fragment is a Nanobody or VHH. In some specific embodiments, the BCMA-targeting 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% identical:
  • the BCMA-targeting VHHs are connected in series via a linker.
  • the VHH shown in SEQ ID NO:18-19 above has the CDR shown below:
  • the present application further provides antibodies or antibody fragments targeting different sequence compositions of BCMA.
  • 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:
  • the VHH shown in SEQ ID NO:38 above has the CDR shown below:
  • the chimeric antigen receptor of the present application includes an antibody or antibody fragment targeting GPRC5D as the antigen-binding region, and the application also provides such an antibody or antibody fragment targeting GPRC5D separately.
  • 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% similarity with the following SEQ ID NO:42 sequence. %, at least 98% or 100% identity:
  • the VHH shown in SEQ ID NO:42 above has CDRs as shown:
  • the immune effector cells comprise T cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the T cells include a signal peptide, an antigen-binding region, a hinge region, a trans- membrane region and intracellular signaling structural region.
  • the chimeric antigen receptor includes a CD8 ⁇ signal peptide, an antigen-specific binding sequence, CD8 ⁇ or CD28 hinge region, CD8 ⁇ or CD28 transmembrane region, CD28 costimulatory domain and CD3 ⁇ .
  • the CD8 ⁇ signal peptide 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.
  • the hinge region is a CD8 alpha hinge region.
  • the CD8 alpha hinge region has a molecular weight of at least 80%, at least 85%, at least 90%, at least 95%, at least 96% compared to the following SEQ ID NO: 26 or SEQ ID NO: 37 sequence. Sequences that are %, at least 97%, at least 98% or 100% identical to:
  • the hinge region is a CD28 hinge region.
  • the CD28 hinge region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, compared to the following SEQ ID NO:27 sequence. Sequences with at least 98% or 100% identity:
  • the transmembrane region can be selected from the group consisting of CD8 ⁇ transmembrane region, CD28 transmembrane region and NKG2D transmembrane region.
  • the CD8 ⁇ transmembrane region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:28 sequence. , a sequence of at least 98% or 100% identity:
  • the CD28 transmembrane region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:29 sequence. , a sequence of at least 98% or 100% identity:
  • the NKG2D transmembrane region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:30 sequence. , a sequence of at least 98% or 100% identity:
  • the CD3 ⁇ has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% compared to the following SEQ ID NO:31 sequence. % or 100% identity of the sequence:
  • the costimulatory domain is from CD28, 4-1BB or 2B4.
  • the CD28 co-stimulatory domain has a protein content that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:32 sequence. %, at least 98% or 100% identical sequence:
  • the 4-1BB costimulatory domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, compared to the following SEQ ID NO:33 sequence. Sequences that are at least 97%, at least 98% or 100% identical:
  • the 2B4 co-stimulatory domain has a molecular weight of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:34 sequence. %, at least 98% or 100% identical sequence:
  • the chimeric antigen receptor is further linked to a cytokine or chemokine via a self-cleaving peptide.
  • the self-cleaving peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, compared to the following SEQ ID NO:35 sequence. Sequences with at least 98% or 100% identity:
  • the cytokine or chemokine is IL15.
  • the IL15 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% compared to the following SEQ ID NO:36 sequence. % or 100% identity of the sequence:
  • 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.
  • the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NO:2-4, or has at least 80%, at least Sequences that are 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.
  • the immune effector cells comprise NK cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the NK cells include signal peptides, antigen-binding regions or target binding sequences, and hinges. region, transmembrane region, costimulatory domain and CD3 ⁇ .
  • the chimeric antigen receptor further includes a cytokine or chemokine linked to the CD3 ⁇ via a self-cleaving peptide.
  • the cytokine is IL15.
  • the chimeric antigen receptor expressed by NK cells includes CD8 ⁇ signal peptide, antigen-binding region or target binding sequence, CD8 ⁇ hinge region, CD8 ⁇ or CD28 or NKG2D transmembrane region, CD28 or 2B4 or 4- 1BB costimulatory domain and CD3 ⁇ , and a cytokine or chemokine preferably linked to said CD3 ⁇ via a self-cleaving peptide.
  • the cytokine is IL15.
  • the NK cell-expressed chimeric antigen receptor comprises SEQ ID NOs: 1, 5-6, and 46 The sequence shown in any one of them, or comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% of the sequence of any one of SEQ ID NO: 1, 5-6 %, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical sequences.
  • the present application also provides a method for amplifying immune effector cells in vitro, which method includes: amplifying and culturing the immune effector cells in the presence of a CBL-b inhibitor.
  • the immune effector cells are T cells or NK cells.
  • the T cells or NK cells express chimeric antigen receptor T cells or chimeric antigen receptor expressing NK cells.
  • the concentration of the CBL-b inhibitor during the culture process is 0.1 nM ⁇ 10 ⁇ M, such as 1 ⁇ M.
  • the T cells or NK cells are transfected with a chimeric antigen receptor, and the CBL-b inhibitor is before or after transfection of a nucleic acid molecule expressing the chimeric antigen receptor, Add the CBL-b inhibitor.
  • the CBL-b inhibitor is added when the T cells or NK cells begin to be activated, or the CBL-b is added after the T cells or NK cells are activated and cultured for 4-7 days. Inhibitors.
  • 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 CD8 ⁇ signal peptide, a specific sexually binds to the antigen-binding region of Claudin18.2, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, CD28 costimulatory domain and CD3 ⁇ .
  • the chimeric antigen receptor further includes a cytokine or chemokine linked to the CD3 ⁇ via a self-cleaving peptide.
  • the cytokine is IL15.
  • the antigen binding region is in the form of a scFv.
  • the antigen-binding region is a scFv having a VH-linker peptide-VL structure.
  • the VH includes HCDR1-HCDR3 as shown in SEQ ID NO: 10-12
  • the VL includes LCDR1-LCDR3 as shown in SEQ ID NO: 13-15.
  • the VH has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least the sequence shown in SEQ ID NO:8 A sequence that is 95%, at least 96%, at least 97%, at least 98% or 100% identical, and the VL has at least 80%, at least 85%, at least 90%, at least 91% with the sequence shown in SEQ ID NO:9 , a sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical; most preferably, the scFv has a sequence identical to SEQ ID NO: 16 -17 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 % identity of the sequence.
  • the chimeric antigen receptor has the sequence shown in SEQ ID NO: 1 at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least Sequences that are 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical.
  • the present application also provides the use of a combination of the aforementioned NK cells and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2 in the preparation of drugs for the treatment of cancer or tumors.
  • the present application also provides a cellular drug for treating cancer or tumors, which includes the aforementioned NK cells and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2.
  • the present application also provides a method for treating cancer or tumors, the method comprising administering an effective amount of the aforementioned NK cells to a subject in need thereof, and optionally, administering a chimeric gene that expresses specific binding to Claudin18.2. T with antigen receptor cell.
  • the chimeric antigen receptor expressed by the T cell includes a CD8 ⁇ signal peptide, a target binding sequence, a CD8 ⁇ or CD28 hinge region, a CD8 ⁇ or CD28 transmembrane region, a CD28 costimulatory domain, and CD3 ⁇ .
  • the antigen-binding region is in the form of a scFv having a VH-linker-VL or VL-linker-VH structure.
  • the chimeric antigen receptor expressed by T cells comprises any sequence of SEQ ID NO:2-4 or has at least 80%, at least Sequences that are 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.
  • the application provides the use of any combination of the following (a)-(c) in the preparation of a medicament for treating tumors or cancer:
  • the present application provides a pharmaceutical combination product for treating cancer or tumors, which includes any one of the following (a)-(c):
  • the application provides a method of treating cancer or tumors, the method comprising administering an effective amount of immune effector cells expressing chimeric antigen receptors to a subject in need thereof, the method further comprising: (1) Before administration, use a CBL-b inhibitor to stimulate the immune effector cells, and/or (2) administer an effective amount of PDL1-IL15 fusion protein to the subject.
  • the antigen bound by the chimeric antigen receptor is independently selected from Claudin18.2, BCMA, GPRC5D or any combination thereof.
  • the antigen can be Claudin18.2, BCMA or GPRC5D alone, a combination of Claudin18.2, BCMA and GPRC5D, or Claudin18.2, BCMA or GPRC5D.
  • the chimeric antigen receptor comprises a target binding sequence that specifically binds BCMA and/or GPRC5D.
  • the antigen-binding region includes a target-binding sequence that specifically binds BCMA and GPRC5D and is in the form of a VHH.
  • the VHH includes a first set of HCDR1-HCDR3 having the sequences set forth in SEQ ID NO:39-41 and/or a second set of HCDR1-HCDR3 having the sequence set forth in SEQ ID NO:43-45 .
  • the VHH has the sequence shown in SEQ ID NO:38 and/or 42 or is at least 80%, at least 85%, at least 90%, at least 91%, at least 92% identical to SEQ ID NO:38 , a sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical and/or at least 80%, at least 85% identical to the sequence shown in SEQ ID NO:42 , a sequence that is 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.
  • having the sequence shown in SEQ ID NO:46 or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% with the sequence shown in SEQ ID NO:46 a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical.
  • the immune effector cells are selected from the group consisting of 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.
  • 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 e.g., NK cells, natural killer T (NKT) cells
  • Neutrophils e.g., neutrophils, neutrophils, and dendritic cells.
  • the immune effector cells are NK cells.
  • the PDL1-IL15 fusion protein has the sequence set forth in SEQ ID NO:47.
  • the tumor or cancer is 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.
  • a chimeric antigen receptor for NK cells or T cells is constructed, and the nucleic acid encoding the chimeric antigen receptor for NK cells is cloned into a retrovirus shuttle plasmid to encode the chimeric antigen for T cells.
  • the nucleic acid of the receptor is cloned into the lentiviral shuttle plasmid, and the virus is packaged through the packaging plasmid.
  • the detailed information on chimeric antigen receptor and plasmid construction and virus packaging is as follows:
  • Plasmid construction synthesize polynucleotides encoding chimeric antigen receptors, and add restriction sites and shuttle plasmids to both ends homologous sequences.
  • the shuttle plasmid was digested with restriction enzymes, and the linear plasmid was recovered and purified by agarose gel electrophoresis.
  • the polynucleotide synthesized in the above steps and the linearized vector were connected using recombinase 5 ⁇ In-FusionHD enzyme (TaKaRa, Cat#ST0344).
  • the reaction system was as follows: 2 ⁇ l polynucleotide fragment (50 ng/ ⁇ l) , 1 ⁇ l linearized plasmid (50ng/ ⁇ l), 2 ⁇ l 5 ⁇ HD In-Fusion enzyme, 5 ⁇ l ddH 2 O. After mixing, centrifuge briefly and react at 50°C for 15 minutes. Add 10 ⁇ l of the recombinant reaction product to 100 ⁇ l of bacterial competent cells, place on ice for 5 minutes, spread the transformed bacterial solution evenly on an LB plate containing 100 ⁇ g/ml ampicillin, and incubate upside down in a constant temperature incubator for 12-16 hours. . Randomly pick 3-5 clones from each plate for sequencing and identification.
  • Lentivirus preparation 293T cells (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, Cat#GNHu17) were inoculated into a 100mm culture dish and cultured in DMEM medium (Gibco) added with 10% FBS (Gibco, Cat#10099141). , Cat#10566016).
  • Retrovirus One day before virus packaging, 293T cells (purchased from ATCC) were inoculated into the culture dish at 1E7 cells/10cm. When transfecting cells, mix the packaging plasmid and the retrovirus shuttle plasmid and add them to ⁇ -MEM medium. Add them to another centrifuge tube containing ⁇ -MEM medium. HD transfection reagent (Promega, E2311). Add the diluted transfection reagent drop by drop on top of the diluted plasmid, mix well, and let stand at room temperature for 15 minutes. Add the mixture to the culture dish for culturing 293T cells, shake gently 10 times, mix well, and place it in an incubator for culture. Three days after cell transfection, harvest the virus. Transfer 10 ml of virus-containing culture supernatant into a 50 ml centrifuge tube, centrifuge at 4°C, 1250 rpm for 5 minutes to remove dead 293T cells, filter and concentrate for later use.
  • PBMC peripheral blood mononuclear cells
  • the 24-well plate was coated with RetroNectin reagent (Takara, T202) at a concentration of 7 ⁇ g/ml, 500 ⁇ l per well, and kept overnight at 4°C.
  • RetroNectin reagent Tikara, T202
  • NK cells On day 6, add the retrovirus packaging 18.2-CAR1(NK) to the 24-well plate, centrifuge at 2000g and 4-8°C for 60 minutes, and discard the upper virus liquid. Add activated NK cells to a 24-well plate at 3E5 cells/well, centrifuge at 400g for 5 minutes at room temperature, and then culture at 37°C and 5% CO2 .
  • NK cells were transfected with 18.2-CAR1 (NK), the in vitro amplification ability was effectively improved. On day 13 (i.e., the 7th day after transfection), the expansion fold exceeded 150 times.
  • NK cells On the 7th day after transfection, take a certain amount of NK cells, wash and dilute the cells with PBS to 2E6 cells/ml, add Fc receptor blocker (BioLegend, Cat#422302) at a ratio of 50 ⁇ l/mL cell dilution, and incubate at room temperature. 10 minutes, then transfer 100 ⁇ l per well to a 96-well FACS reaction plate. Add 100 ⁇ l of CLDN18.2-His-tagged protein (Acro, Cat#CL2-H5546) at a concentration of 2 ⁇ g/ ⁇ l to each well and incubate on ice for 20 minutes.
  • Fc receptor blocker BioLegend, Cat#422302
  • the NUGC4 stably transduced cell line NUGC4-luc which highly expresses human CLDN18.2 and bacterial luciferase (Luciferase), and the CHO-K1 stably transduced cell line CHOK1-18.2, which highly expresses human CLDN18.2, were constructed as target cells for the detection of 18.2- Killing effect of CAR1(NK) and UTNK on target cells.
  • 4h tumor cell killing experiment Plate target cells NUGC4-luc on a 96-well plate at 20,000 cells/well. Add 18.2-CAR1 (NK) cells or UTNK to co-culture according to the effect-to-target ratio (E:T) of 10:1, 2:1, 1:1 and 1:2 respectively. After 4 hours, samples were taken, firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added, and the bioluminescence value was read (PE EnSight microplate reader). The results are shown in Figure 2C. Compared with UTNK, 18.2-CAR1 (NK) cells show strong killing activity against NUGC4-luc cells in a short time. At a high E:T ratio, the killing efficiency of 18.2-CAR1 (NK) against NUGC4-luc cells reaches 100 %.
  • E:T effect-to-target ratio
  • mice combined immunodeficient mice
  • 5E6 NUGC4-luc cells in the logarithmic growth phase and in good growth status.
  • V (mm 3 ) 1/2 ⁇ (a ⁇ b 2 )
  • NK cells (2E7 cells/animal) were injected into the tail vein, and the day of NK cell injection was day 0 (D0).
  • the grouping of mice is shown in Table 2. Continuously observe and measure changes in tumor volume and body weight of the mice. Measure and record twice a week and calculate the tumor inhibition rate.
  • Tumor inhibition rate TGI (%) (tumor volume of mice in PBS group - tumor volume of mice in experimental group)/tumor volume of mice in PBS group ⁇ 100%.
  • the results of mouse tumor volume detection are shown in Figure 3A.
  • the G2 group showed no tumor inhibitory effect after injection of UTNK cells, and its tumor growth rate was consistent with that of the G1 group (PBS control group).
  • the G3 group showed a significant inhibitory effect on tumor growth starting from the 14th day after injection of 18.2-CAR1 (NK) cells.
  • the tumor inhibition rate of the G3 group reached 68% on the 21st day after injection (Figure 3C). Mice in the G3 group were found dead on the 22nd day after injection. The cause of death may be related to the higher injection dose, the rapid expansion of 18.2-CAR1 (NK) in the body and the inflammatory response caused by IL15 secretion.
  • mice in the G3 group decreased ( ⁇ 10%) on the 18th day, while the body weight of the mice in the other groups did not change significantly (as shown in Figure 3B).
  • IL-15 18.2-CAR1
  • IL-15 in the CAR structure is effectively expressed and secreted in animals and supports the sustained expansion of 18.2-CAR1 (NK) cells in solid tumor models.
  • NK 18.2-CAR1
  • the mice were dissected and organ samples were collected to quantitatively study the infiltration of NK cells in the spleen and tumors.
  • Figures 3F and 3G compared with the UTNK group, the 18.2-CAR1 (NK) group had smaller A high proportion of 18.2-CAR1 (NK) cells was found in mouse spleens and tumor tissues, indicating that 18.2-CAR1 (NK) cells that can secrete IL-15 successfully infiltrate into immune organs and have good tumor homing effects, which is closely related to the tumor control rate. The results are consistent.
  • mice combined immunodeficient mice
  • 5E5 NUGC4-luc cells in the logarithmic growth phase and in good growth status.
  • bioluminescence intensity (total flux) of mouse abdominal tumor cells was measured to evaluate tumor growth.
  • Mice with tumor cell fluorescence intensity of around 3E8 photons/second (p/s) in a specific abdominal region (Region of interesting, ROI) were selected according to the random number principle and randomly grouped.
  • UTNK and 18.2-CAR1 (NK) cells were injected into the tail vein with an injection volume of 200 ⁇ l/animal.
  • the day of NK cell injection was day 0 (D0).
  • the grouping of mice and the injection of NK cells are shown in Table 3. Continuously observe and measure abdominal tumor growth and body weight changes in mice.
  • FIG. 4A The results of bioluminescence intensity detection of mouse abdominal tumors are shown in Figure 4A.
  • the abdominal tumors grew rapidly in the PBS control group G1 and UTNK injection group G2.
  • the G3 group injected with 18.2-CAR1 (NK) cells effectively delayed tumor growth, but did not completely eliminate it.
  • Tumors, mice in each treatment group and the control group all died due to tumor growth on the 24th day after treatment.
  • the weight changes of mice in each group were not obvious and showed 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, respectively. reached 18% (Fig.
  • 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-(((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-3yl)cyclobutyl)phenyl)-7-(((S)-3-methyl Preparation of piperidin-1-yl)methyl)-9-
  • SFC Instrument: CAS-SH-ANA-SFC-G (Agilent 1260 with DAD detector)
  • column ChiralPak AS-3 column length 150mm, inner diameter 4.6mm, particle size 3 ⁇ m
  • mobile phase A CO2
  • mobile Phase B IPA
  • Step 4 (S)-6-fluoro-1-(3-(3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)benzene Preparation of methyl)-4-((3-methylpiperidin-1-yl)methyl)benzo[cd]indole-2(1H)-one (compound 3)
  • Eu-Ubquitin-UbcH5B was prepared by incubating Eu-Ubquitin (Cisbio) with UbcH5B (ENZO) and E1 (ENZO) at 37°C for 4 hours.
  • Eu-Ubquitin-UbcH5B is aliquoted and stored at -80°C.
  • CBL-b activity experiments were performed in 384-well plates (Perkin Elmer).
  • reaction buffer 50mM HEPES pH 7.0 (Gibco), 100mM NaCl (Sigma), 0.01% Triton X-100 (Sigma), 0.01% BSA (Sigma) and 1mM DTT (Invitrogen).
  • the 96-well cell plate (Corning) was coated with 2 ⁇ g/mL Anti-Human CD3 Clone OKT3 (BD) at 37°C for 4 hours. 3-fold gradient dilution of compound (final concentration 10 ⁇ M-4.6 nM, starting concentration 10 ⁇ M, 3-fold dilution, 8 points, the 8th point is 4.6 nM) was incubated with 220 ⁇ L 1.11E6/mL Jurkat T cells (ATCC) for 1 hour, add 5 ⁇ L 45 ⁇ g/mL Anti-Human CD28 Clone CD28.2 (BD), mix evenly, transfer 100 ⁇ L to the aforementioned CD3-coated cell plate, culture it in a 37°C cell incubator for 48 hours, collect the supernatant and use IL-2 ELISA Kit (BD) detects IL-2 release. EC50 was analyzed using Prism. The experimental results are shown in Table 6.
  • T cells were isolated from fresh PBMC using Stemcell Easy Sep Kit (Stemcell, Cat#19055). The isolated T cells were transferred to culture dishes pre-coated with 1 ⁇ g/ml CD3/CD28 antibody (MY-ebioscience-16-0289-85, MY-ebioscience-16-0037-85).
  • the medium components are X-VIVO15 (Lonza, Cat#BEBP02-054Q), 5% human AB serum (Gemini, Cat#100-512), 100U/ml penicillin-streptomycin (Gibco, Cat#15140-122) and 200IU/ml human IL2 (Beijing Shuanglu, Cat#S19991007). Cell counts were performed twice a week, and when the cell density reached 2.5E6 cells/ml, the cells were subcultured and expanded.
  • T cells Take the above-mentioned T cells in good condition and inoculate them into a 12-well cell culture plate pre-coated with 5 ⁇ g/ml recombinant human fibrin (Takara, Cat#T100B) at a rate of 1E6 cells/well.
  • Condensate Sigma-Aldrich, Cat#TR-1003
  • UTNK and 18.2-CAR1 (NK) cells were highly lethal to NUGC4-luc cells at 24 hours, but the killing was significantly reduced at 48 hours.
  • the combination group still maintained strong killing activity at 48 hours, which was basically the same as the 24-hour killing effect of the combination group, and was significantly better than the killing ability of the NK group alone at 48 hours.
  • the CBL-b inhibitor (CBL-bi-1, final concentration in culture medium was 1 ⁇ M) effectively increased the response of UTNK and 18.2-CAR1 (NK) cells to NUGC4-luc cells. killing ability, killing efficiency Both increased by about 16%.
  • NK cells or T cells at a final concentration of 1 ⁇ M. of CBL-bi-3, co-cultured. Take a sample after 72 hours, add firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655), and use a PE EnSight microplate reader to read the bioluminescence value i to calculate the killing rate.
  • 18.2-CAR1 (NK) and 18.2-CAR3 (T) were prepared according to Example 2 and Example 4, and CBL-bi-1 was synthesized according to Example 3 and used in the combination scheme of this example.
  • CI Cell Index
  • the 18.2-CAR3(T) cell group and the 18.2-CAR3(T) cell + CBL-bi-1 group have relatively weak killing effects on CHOK1-18.2 cells. There was no effect in the first round of killing. Completely control target cell growth. The killing ability of the 18.2-CAR1(NK) group was only maintained until the second round.
  • Example 7 Effect of using culture medium supplemented with CBL-b inhibitor on the activity of CAR-T cells
  • T cells Prepare 18.2-CAR2 (T) cells with reference to Example 4, and add 1 ⁇ M CBL-bi-1 or CBL-bi-2 to the culture medium throughout the entire process to stimulate T cells.
  • Flow cytometry was also used to detect the phenotype of T cells 9 days after transfection, and detect central memory T cells (CM, CD62L + CD45RO + ), effector memory T cells (Effective) of CD4 T cells and CD8 T cells.
  • Tumor cell killing experiment Plate target cells NUGC4-luc on a 96-well plate at 20,000 cells/well, and add 18.2-CAR2(T ) cells or UT cells were co-cultured. Samples were taken at 24 hours, the firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added, and the bioluminescence value was read using a PE EnSight microplate reader. The results are shown in Figure 7E. Compared with 18.2-CAR2(T) cells in the control group, adding CBL-bi-1 and CBL-bi-2 to the culture medium significantly improved the performance of 18.2-CAR2(T) cells against NUGC4-luc cells at a low E:T ratio. The killing effect.
  • NK cells expressing BCMA-CAR1 (NK) and BCMA-CAR2 (NK) were prepared with reference to Example 2, and on the 4th day (Day4) of K562 cell activation of NK cell expansion, 1 ⁇ M CBL-bi- was added to the culture medium 4. Stimulate NK cells. Use FACS to detect the CAR transfection efficiency on Day 15 of amplification culture:
  • BCMA and GPRC5D design and construct a dual-target chimeric antigen receptor targeting BCMA and GPRC5D, including CD8 ⁇ signal peptide (SP), anti-GPRC5D antibody and anti-BCMA, CD8 ⁇ hinge region, NKG2D-F transmembrane region, 2B4 costimulatory domain and CD3 ⁇ .
  • IL15 connected through the self-cleaving peptide P2A, in which BCMA adopts the VHH shown in SEQ ID NO:38, and GPRC5D adopts the VHH shown in SEQ ID NO:42.
  • the BCMA-GPRC5D CAR-NK structure is shown in Figure 9. The sequence is shown in Table 7.
  • BCMA-GPRC5D CAR-NK (BI-CAR21) is prepared through conventional plasmid construction, virus packaging, PBMC induction into NK cells, activation of NK cells, and viral infection to construct CAR-NK.
  • CBL-bi-4 (final concentration 1 ⁇ M) was added during the activation and transfection of NK cells.
  • the CAR positive rate of NK cells was detected by the chimeric antigen receptor expression rate detection method. The results showed that after adding CBL-b inhibitor, the CAR positive rate of NK cells increased.
  • the expression rate of BI-CAR21 was detected by BCMA, wherein, compared with the control without adding CBL-b inhibitor, after adding CBL-b inhibitor, the expression rate of BI-CAR was determined by 80% rose to 92.5%.
  • CAR-NK (BI-CAR21) targeting GPRC5D and BCMA with CBL-b inhibitors and/or PDL1-IL15
  • CBL-b inhibitors and/or PDL1-IL15 Multiple rounds of killing effect on NCI H929+10% NCI H929-hBCMA-KO cells after combined use.
  • the experimental design plan is shown in Table 8.
  • the CBL-b inhibitor and its addition method are shown in Section 9.2.
  • the PDL1-IL15 is added when the target cells and NK cells are incubated.
  • the NCI H929-hBCMA-KO cells are H929 cell lines in which BCMA is knocked out using conventional gene manipulation methods.
  • the target cells NCI H929 and NCI H929-hBCMA-KO were transferred into the luciferase gene to express luciferase.
  • the fluorescence intensity is detected by luciferase reporter gene detection reagent, which reflects the cell viability and the killing effect of NK cells.
  • the formula for calculating the kill rate is as follows:
  • Killing rate (target cell well reading value - test well reading value)/target cell well reading value ⁇ 100%.
  • sequence information of PDL1-IL15 is as follows:
  • Example 10 Anti-tumor efficacy test of CAR-NK targeting GPRC5D and BCMA combined with CBL-b inhibitor in Molp8-luc mouse tumor model
  • Antitumor efficacy testing was conducted using a mouse bone marrow cancer intravenous xenograft tumor model. details as follows:
  • BCMA-GPRC5D CAR-NK cells were prepared according to the method of Example 9.
  • Molp8-Luc cells expressing bacterial luciferase were constructed by conventional methods in this field and cultured using conventional methods. Subsequently, Molp8-Luc cells in the logarithmic growth phase and in good growth status were collected, and a total of 5 ⁇ 10 5 cells were inoculated into the tail veins of NPG mice (combined immunodeficient mice). On the first day after tumor inoculation, the mouse body weight and tumor fluorescence signal value were measured. Mice with a weight of about 21.81 to 27.25g were selected based on the random number principle, and the average value was 23.98g for random grouping.
  • CAR-NK cells 8 ⁇ 10 6 /animal
  • the injection volume was 200 ⁇ l/animal.
  • the CAR-NK cell injection diary was day 0 (D0).
  • the grouping of mice and the injection of CAR-NK cells are shown in Table 9. Continuously monitor tumor growth fluorescence signal ROI value and body weight changes with an IVIS intravital imager. Measure and record once a week and observe the survival rate of mice.
  • the BI-CAR21+CBL-b inhibitor treatment group was able to significantly inhibit the tumor growth burden of mice. Two of the animals' tumors were cured, and one animal had less tumor recurrence and a lower tumor burden. Alleviation; the BI-CAR21 treatment group can inhibit the growth rate of mouse tumors, but cannot control the tumor progression of animals in this group. All 5 animals in the group had tumor recurrence and growth, and 3 animals died; the photon quantity statistical results are shown in Figure 12C.
  • the survival rate was continuously monitored to 30 days. Due to the strong invasiveness of the tumor in this model and the rapid progression of the tumor, one animal died in the PBNK treatment group starting from the 24th day, and the mortality rate was 100% by the 28th day; BI-CAR21 treatment One animal died in the group starting from day 25, and the mortality rate was 60% on day 30; in the clinical observation, the overall condition of the animals in the BI-CAR21+CBL-b inhibitor treatment group was better, and no death occurred. This group showed durable treatment effect while ensuring better security (as shown in Figure 12E).

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Abstract

The present application relates to an engineered immune effector cell and combined use thereof with and/or other active substances (such as the PDL1-IL15 fusion protein). The combined use relates to the preparation of a drug for treating cancer or tumors, a method for treating cancer or tumors, a method for amplifying immune cells in vitro, and the like.

Description

工程化免疫效应细胞及其与CBL-b抑制剂联用的应用Engineered immune effector cells and their application in combination with CBL-b inhibitors
相关申请的引用References to related applications
本申请要求2022年5月9日提交的中国专利申请202210499532.9号和2023年3月24日提交的中国专利申请202310305627.7号的优先权，通过援引加入的方式将该这两篇申请的内容全部并入本文，用于所有目的。This application claims priority from Chinese Patent Application No. 202210499532.9 submitted on May 9, 2022 and Chinese Patent Application No. 202310305627.7 submitted on March 24, 2023, and the entire contents of these two applications are incorporated by reference. This article is for all purposes.
技术领域Technical field
本申请涉及生物工程和细胞治疗领域，具体而言，本申请提供了工程化免疫效应细胞及其与CBL-b抑制剂和/或其他活性物质(例如PDL1-IL15融合蛋白)的联合应用。The present application relates to the fields of bioengineering and cell therapy. Specifically, the present application provides engineered immune effector cells and their combined application with CBL-b inhibitors and/or other active substances (such as PDL1-IL15 fusion protein).
背景技术Background technique
现有的细胞治疗技术包括靶向肿瘤抗原的嵌合抗原受体(Chimeric Antigen Receptor，CAR)修饰的T细胞或NK细胞(CAR-T或CAR-NK)疗法。Existing cell therapy technologies include Chimeric Antigen Receptor (CAR)-modified T cells or NK cells (CAR-T or CAR-NK) therapy that target tumor antigens.
CAR-T疗法已经证实可以有效治疗B细胞恶性肿瘤和多发性骨髓瘤，但仍存在不足之处，包括：病人可能无法提供足够用于工程化的T细胞，自体CAR-T细胞制备周期长，CAR-T疗法可能引发细胞释放因子综合征(CRS)、神经毒性和自身免疫毒性，以及在治疗实体瘤方面效果不理想等。CAR-T therapy has been proven to be effective in treating B-cell malignancies and multiple myeloma, but there are still shortcomings, including: patients may not be able to provide enough T cells for engineering, and the production cycle of autologous CAR-T cells is long. CAR-T therapy may cause cell-releasing factor syndrome (CRS), neurotoxicity and autoimmune toxicity, as well as unsatisfactory results in the treatment of solid tumors.
与T细胞不同，自然杀伤细胞(Natural killer cell，NK)不需要通过MHC分子呈递抗原才能识别和杀死靶细胞，适合采用“现货”模式，使用健康捐献者的外周血或脐带血中的NK细胞直接制备。同时，NK细胞在同种异体环境中不会引起移植物抗宿主病(graft versus host disease，GVHD)，适合作为通用型细胞候选产品，而且不会引发CRS，具有更好的安全性。此外，NK细胞可以被单核细胞、巨噬细胞和树突状细胞等辅助细胞活化，从而分泌活性杀伤因子。CAR-NK细胞在体外体内研究中显示出对肿瘤细胞快速强效的杀伤效果，但是较差的持久性通常会导致药效短暂，无法彻底消除实体瘤和控制肿瘤复发。Unlike T cells, natural killer cells (NK) do not need to present antigens through MHC molecules to recognize and kill target cells. They are suitable for the "spot" mode, using NK from peripheral blood or umbilical cord blood of healthy donors. Cells were prepared directly. At the same time, NK cells do not cause graft versus host disease (GVHD) in an allogeneic environment and are suitable as universal cell product candidates. They do not cause CRS and have better safety. In addition, NK cells can be activated by auxiliary cells such as monocytes, macrophages, and dendritic cells to secrete active killing factors. CAR-NK cells have shown rapid and potent killing effect on tumor cells in in vitro and in vivo studies, but poor persistence usually results in short-lived drug effects and inability to completely eliminate solid tumors and control tumor recurrence.
Casitas B谱系淋巴瘤原癌基因-b(Casitas B-lineage lymphoma proto-oncogene-b，CBL-b)是一种负调节T细胞或NK细胞活化的E3连接酶，属于负调节免疫检查点。研究表明CBL-b基因在耗竭的T细胞中表达上调，CBL-b基因的敲除可恢复耗竭型T细胞或CAR-T细胞的效应功能。IL2和IL15激活NK细胞，显著上调NK细胞表面CBL-b的表达，CBL-b表达的上调反过来抑制被激活NK细胞对肿瘤细胞的杀伤作用，在NK细胞的激活过程中起到负反馈调节作用。可见，CBL-b是一个重要的免疫调节靶点。Casitas B-lineage lymphoma proto-oncogene-b (CBL-b) is an E3 ligase that negatively regulates T cell or NK cell activation and is a negative regulatory immune checkpoint. Studies have shown that the expression of CBL-b gene is up-regulated in exhausted T cells, and the knockout of CBL-b gene can restore the effector function of exhausted T cells or CAR-T cells. IL2 and IL15 activate NK cells and significantly upregulate the expression of CBL-b on the surface of NK cells. The upregulation of CBL-b expression in turn inhibits the killing effect of activated NK cells on tumor cells and plays a negative feedback regulation in the activation process of NK cells. effect. It can be seen that CBL-b is an important immune regulatory target.
本领域亟需为现有细胞治疗方法的一个或多个不足之处提供解决方案。There is an urgent need in the field to provide solutions to one or more of the shortcomings of current cell therapy approaches.
发明概述Summary of the invention
第一方面，本申请提供CBL-b抑制剂与免疫效应细胞的组合在制备用于治疗癌症或肿瘤的药物中的应用，所述免疫效应细胞表达或不表达嵌合抗原受体。 In a first aspect, the present application provides the use of a combination of a CBL-b inhibitor and an immune effector cell, the immune effector cell expressing or not expressing a chimeric antigen receptor, in the preparation of a medicament for treating cancer or tumors.
第二方面，本申请提供用于治疗癌症或肿瘤的药物组合产品，其包含CBL-b抑制剂与免疫效应细胞，所述免疫效应细胞表达或不表达嵌合抗原受体。In a second aspect, the present application provides a pharmaceutical combination product for treating cancer or tumors, which includes a CBL-b inhibitor and an immune effector cell that expresses or does not express a chimeric antigen receptor.
第三方面，本申请提供治疗癌症或肿瘤的方法，其中，所述方法包括向有需要的受试者给予有效量的CBL-b抑制剂和免疫效应细胞，所述免疫效应细胞表达或不表达嵌合抗原受体。In a third aspect, the present application provides a method of treating cancer or tumors, wherein the method includes administering to a subject in need thereof an effective amount of a CBL-b inhibitor and an immune effector cell that expresses or does not express chimeric antigen receptor.
第四方面，本申请提供体外扩增免疫效应细胞的方法，所述方法包括：在CBL-b抑制剂存在的条件下，扩增培养所述免疫效应细胞。In a fourth aspect, the present application provides a method for amplifying immune effector cells in vitro, which method includes: amplifying and culturing the immune effector cells in the presence of a CBL-b inhibitor.
第五方面，本申请提供工程化的NK细胞，其中，所述NK细胞表达特异性结合Claudin18.2的嵌合抗原受体，所述嵌合抗原受体包括CD8α信号肽、特异性结合Claudin18.2的抗原结合区、CD8α铰链区、CD8α跨膜区、CD28共刺激结构域和CD3ζ。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 and specifically binds to Claudin18.2. 2’s antigen-binding region, CD8α hinge region, CD8α transmembrane region, CD28 costimulatory domain and CD3ζ.
第六方面，本申请提供第五方面所述NK细胞与表达特异性结合Claudin18.2的嵌合抗原受体的T细胞的组合在制备用于治疗癌症或肿瘤的药物中的应用。In a sixth aspect, the present application provides the use of a combination of the NK cells described in the fifth aspect and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2 in the preparation of drugs for the treatment of cancer or tumors.
第七方面，本申请提供用于治疗癌症或肿瘤的细胞药物，其包含第五方面所述NK细胞与表达特异性结合Claudin18.2的嵌合抗原受体的T细胞。In a seventh aspect, the present application provides a cellular drug for treating cancer or tumors, which includes the NK cells described in the fifth aspect and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2.
第八方面，本申请提供治疗癌症或肿瘤的方法，所述方法包括向有需要的受试者给予有效量的第五方面所述NK细胞，并任选地，给予表达特异性结合Claudin18.2的嵌合抗原受体的T细胞。In an eighth aspect, the present application provides a method for treating cancer or tumors, the method comprising administering to a subject in need an effective amount of the NK cells described in the fifth aspect, and optionally, administering NK cells that express specific binding to Claudin18.2 of chimeric antigen receptor T cells.
第九方面，本申请提供以下(a)-(c)中任一项组合在制备用于治疗肿瘤或癌症的药物中的应用：In the ninth aspect, this application provides the use of any combination of the following (a)-(c) in the preparation of a drug for treating tumors or cancer:
(a)表达嵌合抗原受体的免疫效应细胞和CBL-b抑制剂，(a) Immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,
(b)表达嵌合抗原受体的免疫效应细胞与PDL1-IL15融合蛋白，(b) Immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,
(c)表达嵌合抗原受体的免疫效应细胞、CBL-b抑制剂和PDL1-IL15融合蛋白。(c) Immune effector cells expressing chimeric antigen receptor, CBL-b inhibitor and PDL1-IL15 fusion protein.
第十方面，本申请提供用于治疗癌症或肿瘤的药物组合产品，其包含以下(a)-(c)中任一项：In a tenth aspect, the present application provides a pharmaceutical combination product for treating cancer or tumors, which includes any one of the following (a)-(c):
(a)表达嵌合抗原受体的免疫效应细胞和CBL-b抑制剂，(a) Immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,
(b)表达嵌合抗原受体的免疫效应细胞与PDL1-IL15融合蛋白，(b) Immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,
(c)表达嵌合抗原受体的免疫效应细胞、CBL-b抑制剂和PDL1-IL15融合蛋白。(c) Immune effector cells expressing chimeric antigen receptor, CBL-b inhibitor and PDL1-IL15 fusion protein.
第十一方面，本申请提供治疗癌症或肿瘤的方法，所述方法包括给予有需要的受试者有效量的表达嵌合抗原受体的免疫效应细胞，所述方法还包括：(1)在给药前，使用CBL-b抑制剂刺激所述免疫效应细胞，和/或(2)给予受试者有效量PDL1-IL15融合蛋白。In an eleventh aspect, the present application provides a method for treating cancer or tumors, the method comprising administering an effective amount of immune effector cells expressing chimeric antigen receptors to a subject in need thereof, the method further comprising: (1) Before administration, use a CBL-b inhibitor to stimulate the immune effector cells, and/or (2) administer an effective amount of PDL1-IL15 fusion protein to the subject.
附图简要说明Brief description of the drawings
图1显示本申请的不同示例性CAR-NK以及CAR-T质粒结构示意图。Figure 1 shows a schematic structural diagram of different exemplary CAR-NK and CAR-T plasmids of the present application.
图2A显示转染18.2-CAR1(NK)的NK细胞或未经转染的NK细胞(UTNK)的体外扩增结果。Figure 2A shows the in vitro expansion results of NK cells transfected with 18.2-CAR1 (NK) or untransfected NK cells (UTNK).
图2B显示18.2-CAR1(NK)和UTNK细胞的CAR转染效率的FACS检测结果。Figure 2B shows the FACS detection results of CAR transfection efficiency of 18.2-CAR1 (NK) and UTNK cells.
图2C显示18.2-CAR1(NK)和UTNK细胞对NUGC4-luc细胞4小时的杀伤作用，其中效 靶比(E:T)为分别为10:1、2:1、1:1和1:2。Figure 2C shows the killing effect of 18.2-CAR1 (NK) and UTNK cells on NUGC4-luc cells for 4 hours. The target ratios (E:T) are 10:1, 2:1, 1:1 and 1:2 respectively.
图2D显示18.2-CAR1(NK)和UTNK细胞在所示的不同效靶比下对CHOK1-18.2细胞系的连续随时间杀伤作用。Figure 2D shows the sequential killing effect of 18.2-CAR1 (NK) and UTNK cells on the CHOK1-18.2 cell line over time at the different efficacy-to-target ratios shown.
图2E显示18.2-CAR1(NK)和UTNK细胞在所示的不同效靶比下杀伤CHOK1-18.2细胞时的细胞因子IFN-γ分泌水平。Figure 2E shows the secretion levels of cytokine IFN-γ when 18.2-CAR1 (NK) and UTNK cells kill CHOK1-18.2 cells at the different efficacy-target ratios shown.
图3A显示NUGC4-luc小鼠皮下肿瘤模型中各组小鼠21天内的肿瘤生长曲线。Figure 3A shows the tumor growth curves of each group of mice within 21 days in the NUGC4-luc mouse subcutaneous tumor model.
图3B显示NUGC4-luc小鼠皮下肿瘤模型中各组小鼠21天内的体重变化率。Figure 3B shows the body weight change rate of mice in each group within 21 days in the NUGC4-luc mouse subcutaneous tumor model.
图3C显示NUGC4-luc小鼠皮下肿瘤模型中各组小鼠21天内的肿瘤抑制率(TGI％)。Figure 3C shows the tumor inhibition rate (TGI%) of each group of mice within 21 days in the NUGC4-luc mouse subcutaneous tumor model.
图3D显示NUGC4-luc小鼠皮下肿瘤模型中各组小鼠在第5天、第9天、第13天和第18天外周血中NK细胞数量的FACS检测结果。Figure 3D shows the FACS detection 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.
图3E显示NUGC4-luc小鼠皮下肿瘤模型中各组小鼠在第5天、第9天、第13天和18天外周血血清中人IL-15细胞因子分泌检测结果。Figure 3E shows the detection results of human IL-15 cytokine secretion in peripheral blood serum of each group of mice in the NUGC4-luc mouse subcutaneous tumor model on days 5, 9, 13 and 18.
图3F显示NUGC4-luc小鼠皮下肿瘤模型中各组小鼠在第22天解剖后NK细胞在肿瘤组织淋巴细胞中占比的FACS检测结果。Figure 3F shows the FACS detection results of the proportion of NK cells in tumor tissue lymphocytes after dissection of mice in each group of mice in the NUGC4-luc mouse subcutaneous tumor model on day 22.
图3G显示NUGC4-luc小鼠皮下肿瘤模型中各组小鼠在第22天解剖后脾脏和肿瘤组织中CAR拷贝数量的PCR检测结果。Figure 3G shows the PCR detection results of CAR copy number in the spleen and tumor tissue of each group of mice in the NUGC4-luc mouse subcutaneous tumor model after dissection on day 22.
图4A显示NUGC4-luc小鼠腹腔肿瘤模型中各组小鼠在20天内腹腔区域肿瘤细胞生长荧光强度曲线。Figure 4A shows the fluorescence intensity curve of tumor cell growth in the abdominal area of mice in each group of mice in the NUGC4-luc mouse abdominal tumor model within 20 days.
图4B显示NUGC4-luc小鼠腹腔肿瘤模型中各组小鼠在20天内的体重变化率。Figure 4B shows the body weight change rate of mice in each group within 20 days in the NUGC4-luc mouse abdominal tumor model.
图4C显示NUGC4-luc小鼠腹腔肿瘤模型中各组小鼠在第5天、第12天和第19天外周血中NK细胞在淋巴细胞中占比的FACS检测结果。Figure 4C shows the FACS detection results of the proportion of NK cells in lymphocytes in the peripheral blood of each group of mice in the NUGC4-luc mouse abdominal tumor model on days 5, 12 and 19.
图4D显示NUGC4-luc小鼠腹腔肿瘤模型中各组小鼠在第5天、第12天和第19天外周血血清中人IL-15细胞因子分泌检测结果。Figure 4D shows the detection results of human IL-15 cytokine secretion in peripheral blood serum of each group of mice in the NUGC4-luc mouse abdominal tumor model on days 5, 12 and 19.
图5A显示18.2-CAR1(NK)，18.2-CAR3(T)，以及18.2-CAR1(NK)与18.2-CAR3(T)联用对NUGC4-luc细胞24小时和48小时的杀伤作用，其中E:T为NK细胞：靶细胞比例＝1：2，T细胞：靶细胞＝1：4。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 at 24 hours and 48 hours, where E: T is the ratio of NK cells: target cells = 1:2, T cells: target cells = 1:4.
图5B显示18.2-CAR1(NK)和CBL-bi-1联合使用对NUGC4-luc细胞24小时的杀伤作用；E:T为NK细胞：靶细胞比例＝1：6。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: target cells = 1:6.
图5C显示18.2-CAR1(NK)与CBL-bi-3联用，以及18.2-CAR4(T)与CBL-bi-3联用对NUGC4-luc细胞的多轮连续杀伤作用，其中E:T为NK细胞：靶细胞比例＝1：24，T细胞：靶细胞比例＝1：16。Figure 5C shows the multiple rounds of continuous killing of NUGC4-luc cells by the combination of 18.2-CAR1(NK) and CBL-bi-3, and the combination of 18.2-CAR4(T) and CBL-bi-3, where E:T is NK cell: target cell ratio = 1:24, T cell: target cell ratio = 1:16.
图6显示18.2-CAR1(NK)，18.2-CAR3(T)，CBL-bi-1，以及两者联用或者三者联用对CHOK1-18.2细胞系的多轮连续实时杀伤作用。Figure 6 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 three on the CHOK1-18.2 cell line.
图7A显示FACS检测的18.2-CAR2(T)细胞在添加CBL-bi-1和CBL-bi-2培养扩增9天后的CAR转染效率。 Figure 7A shows the CAR transfection efficiency of 18.2-CAR2(T) cells detected by FACS after culture and expansion for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
图7B显示FACS检测的18.2-CAR2(T)细胞在添加CBL-bi-1和CBL-bi-2培养扩增9天后的CD4细胞记忆表型的表达水平。Figure 7B shows the expression level of CD4 cell memory phenotype of 18.2-CAR2(T) cells detected by FACS after culture and expansion for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
图7C显示FACS检测的18.2-CAR2(T)细胞在添加CBL-bi-1和CBL-bi-2培养扩增9天后的CD8细胞记忆表型的表达水平。Figure 7C shows the expression level of CD8 cell memory phenotype of 18.2-CAR2(T) cells detected by FACS after culture and expansion for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
图7D显示FACS检测的18.2-CAR2(T)细胞在添加CBL-bi-1和CBL-bi-2培养扩增9天后的CD4和CD8细胞CD69表达水平。Figure 7D shows the CD69 expression levels of CD4 and CD8 cells in 18.2-CAR2(T) cells detected by FACS after culture and expansion for 9 days with the addition of CBL-bi-1 and CBL-bi-2.
图7E显示18.2-CAR2(T)细胞在添加CBL-bi-1和CBL-bi-2培养扩增9天后对NUGC4-luc细胞24小时的杀伤作用，其中E:T为5:1、2:1、1:1、1:2。Figure 7E shows the killing effect of 18.2-CAR2(T) cells on NUGC4-luc cells for 24 hours after culture and expansion with the addition of CBL-bi-1 and CBL-bi-2 for 9 days, where E:T is 5:1, 2: 1, 1:1, 1:2.
图7F显示18.2-CAR2(T)细胞在添加CBL-bi-1和CBL-bi-2培养扩增9天后对NUGC4-luc细胞24小时的杀伤过程中T细胞表面表达CD107a水平和释放IFN-γ的水平，其中E:T为1:1。Figure 7F shows that 18.2-CAR2(T) cells killed NUGC4-luc cells for 24 hours after being cultured and expanded for 9 days with the addition of CBL-bi-1 and CBL-bi-2. The level of CD107a expressed on the T cell surface and the release of IFN-γ during the killing process of NUGC4-luc cells for 24 hours. level, where E:T is 1:1.
图7G显示18.2-CAR2(T)细胞在添加CBL-bi-1和CBL-bi-2培养扩增9天后，无靶标细胞加入的情况下培养24小时后T细胞表面表达CD107a水平和释放IFN-γ的水平。Figure 7G shows that 18.2-CAR2(T) cells were cultured and expanded with the addition of CBL-bi-1 and CBL-bi-2 for 9 days, and then cultured for 24 hours without the addition of target cells, expressing CD107a levels on the surface of T cells and releasing IFN- γ level.
图8显示FACS检测的BCMA-CAR1(NK)和BCMA-CAR2(NK)在添加CBL-bi-4的培养基中扩增15天后的CAR转染效率。Figure 8 shows the CAR transfection efficiency of BCMA-CAR1(NK) and BCMA-CAR2(NK) detected by FACS after amplification in culture medium supplemented with CBL-bi-4 for 15 days.
图9显示靶向BCMA和GPRC5D的双靶点嵌合抗原受体结构示意图。Figure 9 shows a schematic structural diagram of a dual-target chimeric antigen receptor targeting BCMA and GPRC5D.
图10显示BCMA-GPRC5D CAR-NK在添加或不添加CBL-bi的情况下的阳性率检测结果。Figure 10 shows the positive rate detection results of BCMA-GPRC5D CAR-NK with or without the addition of CBL-bi.
图11显示BCMA-GPRC5D CAR-NK与CBL-b抑制剂和/或PDL1-IL15联用对NCI H929+10％NCI H929-hBCMA-KO细胞的多轮杀伤实验结果。Figure 11 shows the results of multiple rounds of killing experiments on NCI H929+10% NCI H929-hBCMA-KO cells using BCMA-GPRC5D CAR-NK in combination with CBL-b inhibitors and/or PDL1-IL15.
图12A显示BCMA-GPRC5D CAR-NK与CBL-b抑制剂联合使用在Molp8-luc小鼠肿瘤模型中的抗肿瘤药效试验方案。Figure 12A shows the anti-tumor efficacy trial scheme of BCMA-GPRC5D CAR-NK combined with CBL-b inhibitor in Molp8-luc mouse tumor model.
图12B-12E分别显示BCMA-GPRC5D CAR-NK与CBL-b抑制剂联合使用在Molp8-luc小鼠肿瘤模型中的肿瘤生长荧光信号ROI值(图12B和12C)、体重变化(图12D)、小鼠生存率(图12E)，其中图12B中BI-CAR21-CBL-b抑制剂代表表9中BI-CAR21治疗组，即，仅BI-CAR21，不添加CBL-b抑制剂的治疗组。Figures 12B-12E respectively show the tumor growth fluorescence signal ROI value (Figure 12B and 12C), body weight changes (Figure 12D), and Mouse survival rate (Figure 12E), where the BI-CAR21-CBL-b inhibitor in Figure 12B represents the BI-CAR21 treatment group in Table 9, that is, the treatment group with only BI-CAR21 and no CBL-b inhibitor.
发明详细描述Detailed description of the invention
除非本申请另外定义，与本申请相关的科学和技术术语应具有本领域普通技术人员所理解的含义。Unless otherwise defined herein, scientific and technical terms related to this application shall have the meanings understood by those of ordinary skill in the art.
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。Furthermore, unless otherwise indicated herein, singular terms herein shall include the plural form and plural terms shall include the singular form. More specifically, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的组合物， 均落入前述“一种组合物”的范围内。The terms "comprises,""comprises," and "having" are used interchangeably herein and are intended to indicate an inclusive nature, meaning that there may be elements other than those listed. At the same time, it should be understood that the descriptions of "including", "including" and "having" are used in this article, and the solution of "consisting of" is also provided. For example, "a composition including A and B" should be understood as the following technical solution: a composition composed of A and B, and a composition containing other components in addition to A and B, All fall within the scope of the aforementioned “a composition”.
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。The term "and/or" when used herein includes the meaning of "and", "or" and "all or any other combination of elements linked by the applicable term".
尽管本申请的广义范围所示的数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。Notwithstanding the numerical ranges and parameter approximations set forth in the broad scope of this application, the numerical values set forth in the specific examples are set forth as accurately as possible. Any numerical values, however, are inherently bound to contain certain errors resulting from the standard deviation found in their respective measurements. Additionally, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range documented as "1 to 10" shall be deemed to include any and all subranges between the minimum value of 1 and the maximum value of 10, inclusive; that is, all subranges beginning with a minimum value of 1 or greater , such as 1 to 6.1, and a subrange ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any reference referred to as "incorporated herein" shall be understood to be incorporated in its entirety.
“嵌合抗原受体(chimeric antigen receptor，CAR)”"Chimeric antigen receptor (CAR)"
嵌合抗原受体是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工免疫效应细胞表面受体，其至少包含：(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链；(2)锚定CAR进入免疫效应细胞的跨膜结构域；和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。在一些具体的实施方式中，任选地，所述嵌合抗原受体的胞外结构域还包括信号肽和/或铰链区，和/或所述嵌合抗原受体的胞内结构域还包括共刺激结构域。Chimeric antigen receptors refer to artificial immune effector cell surface receptors engineered to be expressed on immune effector cells and specifically bind to antigens, which at least include: (1) an extracellular antigen-binding domain, such as a variable recombination domain of an antibody; chain or light chain; (2) a transmembrane domain that anchors the CAR into immune effector cells; and (3) an intracellular signaling domain. CARs are able to utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner. In some specific embodiments, optionally, the extracellular domain of the chimeric antigen receptor further includes a signal peptide and/or a hinge region, and/or the intracellular domain of the chimeric antigen receptor further Includes costimulatory domain.
如本文所用，嵌合抗原受体语境下的术语“信号肽”是指蛋白或多肽中用于引导所述蛋白或多肽进入分泌路径，转移至细胞膜和/或细胞表面的片段。信号肽的示例为CD8α信号肽。As used herein, the term "signal peptide" in the context of a chimeric antigen receptor refers to the fragment of a protein or polypeptide that serves to guide the protein or polypeptide into the secretory pathway, translocation to the cell membrane and/or cell surface. An example of a signal peptide is the CD8α signal peptide.
如本文所用，嵌合抗原受体语境下的术语“抗原结合区”或“靶点结合序列”二者可互换使用，是指负责结合CAR的靶标(例如肿瘤细胞上的肿瘤抗原)的部分。抗原结合区的形式可以类似抗体的结构，例如scFv。As used herein, the terms "antigen-binding region" or "target-binding sequence" in the context of chimeric antigen receptors are used interchangeably and refer to the region responsible for binding to the target of the CAR (e.g., a tumor antigen on a tumor cell) part. The antigen-binding region may be in a form similar to the structure of an antibody, such as scFv.
如本文所用，“特异性结合”是指抗原结合分子(例如抗体及其片段、抗原的天然配体及天然变体的变体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约10E-6M或更低、约10E-7M或更低约10E-8M或更低、约10E-9M或更低、约10E-10M或更低、10E-11M或更低或10E-12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。As used herein, "specifically binds" means that an antigen-binding molecule (e.g., an antibody and fragments thereof, a natural ligand of an antigen, and a variant of a natural variant) specifically binds an antigen and substantially the same antigen, typically with high affinity, but Does not bind unrelated antigens with high affinity. Affinity is usually reflected by the equilibrium dissociation constant (KD), where a lower KD indicates higher affinity. Taking antibodies as an example, high affinity usually refers to having about 10E-6M or less, about 10E-7M or less, about 10E-8M or less, about 10E-9M or less, about 10E-10M or less, 10E -11M or lower or 10E-12M or lower KD. KD is calculated as follows: KD=Kd/Ka, where Kd represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis determination.
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非 抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。The term "antibody" is used herein in its broadest sense and refers to a polypeptide that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen. or peptide combinations. "Antibody" herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity. "Antibody" herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds, which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds, which contain, 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 supports may also include non- Antibody-derived scaffolds, such as scaffold proteins known in the art for grafting CDRs, include but are not limited to tenascin, fibronectin, peptide aptamers, and the like.
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。"Antibody" herein includes a typical "quadruple chain antibody", which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to such a polypeptide chain, which It 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 in the direction from the N end to the C end; and, 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 composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction. A polypeptide chain composed of a polypeptide chain (CL); heavy chains and heavy chains and heavy chains and light chains are connected by disulfide bonds to form a "Y"-shaped structure. Because the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, their antigenicity is also different. Based on this, the "immunoglobulins" in this article can be divided into five categories, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain and δ chain respectively. , γ chain, α chain and ε chain. The same type of Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. IgA can be divided into IgA1 and IgA2. Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等产生的重链抗体(heavy-chain antibodies，HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor，IgNAR)。"Antibodies" herein also include antibodies that do not contain light chains, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Alpaca ( Heavy-chain antibodies (HCAbs) produced by Vicugna pacos and others, as well as immunoglobulin neoantigen receptors (Ig new antigen receptor, IgNAR) discovered in sharks and other cartilaginous fishes.
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。The "antibodies" herein can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camelids and llamas. , ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (such as shark).
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2、scFv和VHH。"Antigen-binding fragment" and "antibody fragment" are used interchangeably herein. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have the ability to bind Antigen capabilities. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, scFv and VHH.
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此，本文术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段，和重链的VH域和第一恒定域(CH1)的抗体片段。Fab’片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同，包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫醇基团的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’)2片段。Papain digestion of intact antibodies generates two identical antigen-binding fragments, termed "Fab" fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ). Thus, the term "Fab fragment" herein refers to a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy 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 domain carry free thiol groups. Pepsin treatment produces an F(ab')2 fragment with two antigen binding sites (two Fab fragments) and part of the Fc region.
本文术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如，Bird等人，Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein  Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising VL and VH domains 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 provided 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 described by Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL of scFv, forming a disulfide-linked Fv (dsFv).
本文术语“纳米抗体”是指骆驼等体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。The term "nanobody" in this article refers to the natural heavy chain antibody lacking the light chain that exists in camels and other bodies. Cloning its variable region can obtain a single domain antibody 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.
本文术语“VHH结构域”、“单域抗体”(single domain antibody，sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。The terms "VHH domain" and "single domain antibody" (single domain antibody, sdAb) in this article have the same meaning and can be used interchangeably. They refer to the variable region of a cloned heavy chain antibody, which is constructed from only one heavy chain variable region. A single-domain antibody is the smallest fully functional antigen-binding fragment. Usually, after obtaining a heavy chain antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构，每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。The term "variable region" herein refers to the region of the heavy or light chain of an antibody involved in enabling the antibody to bind to the antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR", and "light chain variable region" is used interchangeably. ” can be used interchangeably with “VL” and “LCVR”. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. 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 in this article, and usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region. Among them, the heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The term "framework region" or "FR region" is used interchangeably and refers to those amino acid residues other than CDRs in the heavy or light chain variable region of an antibody. Usually, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。For further description of CDR, refer to 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 M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003) ; and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDR" herein can be annotated and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but are not limited to AbRSA website (http://cao.labshare. cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. cgi#results). CDRs herein include overlaps and subsets of differently defined amino acid residues.
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。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).
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。 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).
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。The term "IMGT numbering system" in this article generally refers to the 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.
如本文所用，嵌合抗原受体语境下的术语“铰链区”一般是指作用为连接跨膜区和抗原结合区的任何寡肽或多肽。具体地，铰链区用来为抗原结合区提供更大的灵活性和可及性。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。As used herein, the term "hinge region" in the context of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that serves to connect the transmembrane region and the antigen-binding region. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antigen-binding region. The hinge region may be derived in whole or in part from a natural molecule, 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 completely synthetic hinge sequence.
如本文所用，嵌合抗原受体语境下的术语“跨膜(TM)区”是指能够使嵌合抗原受体在免疫效应细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫效应细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。As used herein, the term "transmembrane (TM) region" in the context of a chimeric antigen receptor refers to one that enables the chimeric antigen receptor to be expressed on the surface of immune effector cells (e.g., lymphocytes, NK cells, or NKT cells), and a polypeptide structure that guides the cellular response of immune effector cells against target cells. The transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain enables signaling when the chimeric antigen receptor binds to the target antigen.
如本文所用，嵌合抗原受体语境下的术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。胞内信号传导结构域负责在抗原结合结构域结合抗原以后的细胞内初级信号传递，从而导致免疫效应细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达CAR的免疫效应细胞的正常的效应功能的至少一种。示例性的胞内信号传导结构域包括CD3ζ。As used herein, the term "intracellular signaling domain" in the context of a chimeric antigen receptor refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specified function. The intracellular signaling domain is responsible for primary intracellular signal transmission after the antigen-binding domain binds the antigen, leading to the 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 cell in which the CAR is expressed. Exemplary intracellular signaling domains include CD3ζ.
如本文所使用，嵌合抗原受体语境下的术语“共刺激结构域”是指共刺激分子的细胞内信号传导结构域。共刺激分子是除抗原受体或Fc受体之外的细胞表面分子，所述细胞表面分子提供在与抗原结合后使T淋巴细胞有效活化和起作用所需的第二信号。共刺激结构域可以来自CD28、4-1BB或2B4，但不限于此。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 effective activation and function of T lymphocytes upon binding to an antigen. The costimulatory domain can be from CD28, 4-1BB or 2B4, but is not limited thereto.
如本文所用的术语“免疫效应细胞”或“效应细胞”是指参与免疫应答例如促进免疫效应子应答的细胞。免疫效应细胞的例子包括T细胞、例如α/βT细胞和γ/δT细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和髓系来源的吞噬细胞、以及TIL(肿瘤浸润淋巴细胞)等。免疫效应细胞或效应细胞包含编码嵌合抗原受体的核酸和/或表达嵌合抗原受体，根据免疫效应细胞或效应细胞的类型不同，为CAR-T、CAR-NK、CAR-M等等。The term "immune effector cell" or "effector cell" as used herein refers to cells that participate in an immune response, such as promoting an immune effector response. Examples of immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and myeloid-derived phagocytes, and TILs (tumor infiltrating lymphocytes) etc. Immune effector cells or effector cells contain nucleic acids encoding chimeric antigen receptors and/or express chimeric antigen receptors. Depending on the type of immune effector cells or effector cells, they are CAR-T, CAR-NK, CAR-M, etc. .
如本文所用，术语“百分比(％)序列一致性”和“百分比(％)序列同一性”可互换，是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。 As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the alignment of sequences and the introduction of gaps, if necessary, to achieve maximum percent sequence identity ( For example, for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and nonhomologous sequences may be ignored for comparison purposes) followed by the amino acid (or nucleotide) of the candidate sequence ) residues are identical to the amino acid (or nucleotide) residues of the reference sequence. For the purpose of determining percent sequence identity, alignment can be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% decrease in to 100% sequence identity. The length of the candidate sequences aligned for comparison purposes may 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.
如本文所用，“载体(vector)”是包含分离的核酸并且可以用于将分离的核酸递送至细胞内部的物质组成。许多载体是本领域已知的，包括但不限于线性多核苷酸，与离子或两亲性化合物相关的多核苷酸，质粒和病毒。因此，术语“载体”包括自主复制质粒或病毒。该术语还应解释为包括有助于核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物，脂质体等。病毒载体的实例包括但不限于腺病毒载体，腺相关病毒载体，逆转录病毒载体等。As used herein, a "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid 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, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.
本文术语“药物”是指用以预防、治疗及诊断疾病的物质，包括“药物组合物”或“药物组合”。The term "drug" herein refers to substances used to prevent, treat and diagnose diseases, including "pharmaceutical compositions" or "drug combinations".
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本申请的目的。例如，所述药物组合物中所含的成分(例如根据本申请的CAR-免疫细胞)可以以整体施用于个体，或者分开施用于个体。当所述药物组合物中所含的成分分开地施用于个体时，所述成分可以同时或依次施用于个体。根据本申请的药物组合物可以包括细胞培养的常规组分，以保持CAR-免疫细胞的活性。药学可接受的载体还可以包括水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。根据本申请的药物组合物或者药物制剂可通过任何适宜的途径施用，例如静脉内施用、皮内、皮下、肌内注射等。根据本申请的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。The term "pharmaceutical composition" herein refers to a preparation that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain unacceptable toxicity to the subject administered the pharmaceutical composition of additional ingredients. Pharmaceutical compositions include combinations that are separated in time and/or space, so long as they can act together to achieve the purposes of the present application. For example, the components contained in the pharmaceutical composition (eg, CAR-immune cells according to the present application) can be administered to the individual as a whole, or separately. When the ingredients contained in the pharmaceutical composition are administered to the individual separately, the ingredients may be administered to the individual simultaneously or sequentially. Pharmaceutical compositions 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, aqueous buffer solutions, isotonic saline solutions such as PBS (phosphate buffer saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, 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 suitable route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc. The compositions according to the present application may contain wetting agents, emulsifiers or buffer substances as additives.
本文所用的术语“组合”或“药物组合”或“药物组合产品”是指非固定组合，其中活性剂和至少一种另外的活性剂可以同时或在时间间隔内单独施用，特别是在这些时间间隔允许组合配偶体显示合作(例如，协同)效应的情况下。术语“非固定组合”意指活性成分(例如，一种活性剂和至少一种另外的活性剂)均作为分开的实体同时或在没有特定时间限制的情况下顺序地施用于患者，其中这种施用提供了患者体内两种化合物的治疗有效水平。The term "combination" or "pharmaceutical combination" or "pharmaceutical combination" as used herein refers to a non-fixed combination in which the active agent and at least one further active agent may be administered simultaneously or separately at intervals of time, in particular at these times Spacing allows for situations where the combination partners display cooperative (eg, synergistic) effects. The term "non-fixed combination" means that the active ingredients (e.g., one active agent and at least one additional active agent) are administered to a patient simultaneously or sequentially without specific time limits, as separate entities, wherein such Administration provides therapeutically effective levels of both compounds in the patient.
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without multiple toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
术语“药学上可接受的盐”是指药学上可接受的酸或碱的盐，包括化合物与无机酸或有机酸形成的盐，以及化合物与无机碱或有机碱形成的盐。The term "pharmaceutically acceptable salts" refers to salts of pharmaceutically acceptable acids or bases, including salts of compounds with inorganic or organic acids, and salts of compounds with inorganic or organic bases.
术语“药学上可接受的辅料或赋形剂”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。The term "pharmaceutically acceptable excipients or excipients" refers to those excipients that have no obvious irritating effect on the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.
如本文所用，术语“受试者”、“对象”、“个体”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、牛、绵羊、马和野牛等。As used herein, the terms "subject," "subject," "individual," and "patient" refer to an organism undergoing treatment for a particular disease or disorder (eg, cancer or 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 domestic cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.
如本文所用，术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传 染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。As used herein, the term "treatment" refers to surgical or therapeutic treatment with the purpose of preventing, slowing down (reducing) undesirable physiological changes or pathologies in the subject, such as cell proliferative disorders (e.g., cancer). or pass progression of infectious diseases). Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable. Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented. When referring to terms such as slow down, alleviation, weakening, alleviation, alleviation, their meanings also include elimination, disappearance, non-occurrence, etc.
如本文所用，术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。As used herein, the term "effective amount" refers to an amount of a therapeutic agent that is effective when administered alone or in combination with another therapeutic agent to a cell, tissue or subject to prevent or alleviate the symptoms of a disease or the progression of the disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions. When the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。The term "cancer" herein refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The term "tumor" or "tumor" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" as used herein are not mutually exclusive.
本文中表示连接位点。in this article Indicates the connection site.
本文中，由实线和虚线描绘的键表示单键或双键。In this article, bonds depicted by solid and dashed lines Represents a single or double bond.
本文中消旋体或者对映体纯的化合物的图示法来自Maehr，J.Chem.Ed.1985，62:114-120。除非另有说明，用楔形键和虚楔键表示一个立体中心的绝对构型，用黑实键和虚键表示一个立体中心的相对构型(如脂环化合物的顺反构型)。The schematic representation of racemic or enantiopure compounds herein is taken from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise stated, wedge and virtual wedge bonds are used Represents the absolute configuration of a stereocenter, using black real and imaginary bonds. Represents the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).
术语“互变异构体”是指因分子中某一原子在两个位置迅速移动而产生的官能团异构体。本申请化合物可表现出互变异构现象。互变异构的化合物可以存在两种或多种可相互转化的种类。互变异构体一般以平衡形式存在，尝试分离单一互变异构体时通常产生一种混合物，其理化性质与化合物的混合物是一致的。平衡的位置取决于分子内的化学特性。例如，在很多脂族醛和酮如乙醛中，酮型占优势；而在酚中，烯醇型占优势。本申请包含化合物的所有互变异构形式。The term "tautomer" refers to a functional group isomer 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 can exist in two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually yield a mixture whose physical and chemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form is dominant; in phenols, the enol form is dominant. This application encompasses all tautomeric forms of the compounds.
术语“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体，包括顺反异构体、对映异构体和非对映异构体。The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.
本申请的化合物可以具有不对称原子如碳原子、硫原子、氮原子、磷原子或不对称双键，因此本申请的化合物可以存在特定的几何或立体异构体形式。特定的几何或立体异构体形式可以是顺式和反式异构体、E型和Z型几何异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，以及其外消旋混合物或其它混合物，例如对映异构体或非对映体富集的混合物，以上所有这些异构体以及它们的混合物都属于本申请化合物的定义范围之内。烷基等取代基中可存在另外的不对称碳原子、不对称硫原子、不对称氮原子或不对称磷原子，所有取代基中涉及到的这些异构体以及它们的混合物，也均包括在本申请化合物的定义范围之内。本申请的含有不对称原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来，光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性 试剂合成。The compounds of the present application may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so the compounds of the present application may exist in specific geometric or stereoisomer forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S) )-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic or other mixtures thereof, such as enantiomers or diastereomers Enriched mixtures, all of the above isomers and their mixtures are within the scope of the definition of compounds in this application. There may be additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms in substituents such as alkyl groups. These isomers and their mixtures involved in all substituents are also included in Within the scope of the definition of compounds in this application. The compounds of the present application containing asymmetric atoms can be isolated in optically active pure form or in the racemic form. The optically active pure form can be resolved from the racemic mixture, or by using chiral starting materials or chiral forms. Reagent synthesis.
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence state of the specific atom is normal and the substituted compound is stable. When the substituent is oxo (i.e. =O), it means that two hydrogen atoms are replaced, and oxo does not occur on aromatic groups.
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，是指乙基可以是未被取代的(CH2CH3)、单取代的(CH2CH2F、CH2CH2Cl等)、多取代的(CHFCH2F、CH2CHF2、CHFCH2Cl、CH2CHCl2等)或完全被取代的(CF2CF3、CF2CCl3、CCl2CCl3等)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both the occurrence and absence of the stated event or circumstance. For example, the ethyl group is "optionally" substituted by halogen, which means that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (CH 2 CH 2 F, CH 2 CH 2 Cl, etc.), or polysubstituted. (CHFCH 2 F, CH 2 CHF 2 , CHFCH 2 Cl, CH 2 CHCl 2, etc.) or completely substituted (CF 2 CF 3 , CF 2 CCl 3 , CCl 2 CCl 3, etc.). It will be understood by those skilled in the art that any substitution or substitution pattern that is sterically impossible and/or cannot be synthesized will not be introduced for any group containing one or more substituents.
当任何变量(例如R1a、R2a)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。例如，如果一个基团被2个R1a所取代，则每个R1a都有独立的选项。When any variable (eg, R 1a , R 2a ) occurs more than once in the composition or structure of a compound, its definition in each instance is independent. For example, if a group is substituted by 2 R 1a , there is a separate option for each R 1a .
当一个连接基团的数量为0时，比如-(CR13R14)0-，表示该连接基团为键。When the number of a linking group is 0, such as -(CR 13 R 14 ) 0 -, it means that the linking group is a bond.
当其中一个变量选自化学键或不存在时，表示其连接的两个基团直接相连，比如中L代表键时表示该结构实际上是 When one of the variables is selected from a chemical bond or is absent, it means that the two groups to which it is connected are directly connected, e.g. When L stands for key, it means that the structure is actually
当本文中涉及到的连接基团若没有指明其连接方向，则其连接方向是任意的。例如当结构单元中的L选自“-NR7CH2-”时，此时L既可以按照与从左到右的方向连接环D构成“环D-NR7CH2-”，也可以按照从右到左的方向连接环D构成“环D-CH2NR7-”。When the linking group mentioned in this article does not specify the direction of connection, the direction of connection is arbitrary. For example, when the structural unit When L in is selected from "-NR 7 CH 2 -", L can be connected to ring D in the direction from left to right to form "ring D-NR 7 CH 2 -", or it can be connected in the direction from right to left The direction connects ring D to form "ring D-CH 2 NR 7 -".
当一个取代基的键交叉连接到一个环上的两个原子时，这种取代基可以与这个环上的任意原子相键合。例如，结构单元表示R10可在苯环上的位置1、2、3中任一处发生取代。When a substituent's bond is cross-linked to two atoms on a ring, the substituent can be bonded to any atom on the ring. For example, structural unit It means that R 10 can be substituted at any one of positions 1, 2, and 3 on the benzene ring.
本文中的Cm-Cn是指具有m-n范围中的整数个碳原子。例如“C1-C6”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子或6个碳原子。 Cm - Cn as used herein refers to having an integer number of carbon atoms in the range of mn. For example, "C 1 -C 6 " means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
术语“烷基”是指通式为CnH2n+1的烃基，该烷基可以是直链或支链的。术语“C1-C6烷基”应理解为表示具有1、2、3、4、5或6个碳原子的直链或支链饱和一价烃基。所述烷基的具体实例包括但不限于甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1,2-二甲基丙基、新戊基、1,1-二甲基丙基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、2,3-二甲基丁基、1,3-二甲基丁基或1,2-二甲基丁基等。术语“C1-C3烷基”可理解为表示具有1至3个碳原子的直链或支链饱和一价烃基。所述“C1-C6烷基”可以包含“C1-C3烷基”。 The term "alkyl" refers to a hydrocarbon group of the general formula C n H 2n+1 , which alkyl group may be straight or branched. The term "C 1 -C 6 alkyl" is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical 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-di Methylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc. The term "C 1 -C 3 alkyl" is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1 to 3 carbon atoms. The "C 1 -C 6 alkyl group" may include "C 1 -C 3 alkyl group".
术语“烷氧基”是指直链或支链醇类失去羟基上的氢原子产生的一价基团，可理解为“烷基氧基”或“烷基-O-”。术语“C1-C6烷氧基”可理解为“C1-C6烷基氧基”或“C1-C6烷基-O-”；术语“C1-C3烷氧基”可理解为“C1-C3烷基氧基”或“C1-C3烷基-O-”。所述“C1-C6烷氧基”可以进一步包含“C1-C3烷氧基”。The term "alkoxy" refers to a monovalent group produced by losing a hydrogen atom on a hydroxyl group of a straight-chain or branched alcohol, and can be understood as "alkyloxy" or "alkyl-O-". The term "C 1 -C 6 alkoxy" is understood to mean "C 1 -C 6 alkyloxy" or "C 1 -C 6 alkyl-O-"; the term "C 1 -C 3 alkoxy" It can be understood as "C 1 -C 3 alkyloxy" or "C 1 -C 3 alkyl-O-". The "C 1 -C 6 alkoxy group" may further include "C 1 -C 3 alkoxy group".
术语“烯基”是指由碳原子和氢原子组成的直链或支链的且具有至少一个双键的一价不饱和脂肪族烃基。术语“C2-C4烯基”应理解为表示直链或支链的不饱和一价烃基，其包含一个或多个双键并且具有2、3或4个碳原子，“C2-C4烯基”优选C2或C3烯基。应理解，在所述烯基包含多于一个双键的情况下，所述双键可相互分离或共轭。所述烯基的具体实例包括但不限于乙烯基、烯丙基、(E)-2-甲基乙烯基、(Z)-2-甲基乙烯基、(E)-丁-2-烯基、(Z)-丁-2-烯基、(E)-丁-1-烯基、(Z)-丁-1-烯基、异丙烯基、2-甲基丙-2-烯基、1-甲基丙-2-烯基、2-甲基丙-1-烯基、(E)-1-甲基丙-1-烯基或(Z)-1-甲基丙-1-烯基等。The term "alkenyl" refers to a linear or branched monovalent unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one double bond. The term "C 2 -C 4 alkenyl" is understood to mean a straight-chain or branched unsaturated monovalent hydrocarbon radical containing one or more double bonds and having 2, 3 or 4 carbon atoms, "C 2 -C " Alkenyl" is preferably C 2 or C 3 alkenyl. It will be understood that where the alkenyl group contains more than one double bond, the double bonds may be isolated or conjugated to each other. 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 wait.
术语“炔基”是指由碳原子和氢原子组成的直链或支链的具有至少一个三键的一价不饱和脂肪族烃基。术语“C2-C4炔基”可理解为表示直链或支链的不饱和一价烃基，其包含一个或多个三键并且具有2、3或4个碳原子。“C2-C4炔基”的实例包括但不限于乙炔基(-C≡CH)、丙炔基(-C≡CCH3、-CH2C≡CH)、丁-1-炔基、丁-2-炔基或丁-3-炔基。“C2-C4炔基”可以包含“C2-C3炔基”，“C2-C3炔基”实例包括乙炔基(-C≡CH)、丙-1-炔基(-C≡CCH3)、丙-2-炔基(炔丙基)。The term "alkynyl" refers to a linear or branched monovalent unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one triple bond. The term "C 2 -C 4 alkynyl" is understood to mean a straight-chain or branched unsaturated monovalent hydrocarbon radical which contains one or more triple bonds and has 2, 3 or 4 carbon atoms. Examples of "C 2 -C 4 alkynyl" include, but are not limited to, ethynyl (-C≡CH), propynyl (-C≡CCH 3 , -CH 2 C≡CH), but-1-ynyl, butyl -2-alkynyl or but-3-ynyl. "C 2 -C 4 alkynyl" may include "C 2 -C 3 alkynyl", and examples of "C 2 -C 3 alkynyl" include ethynyl (-C≡CH), prop-1-ynyl (-C ≡CCH 3 ), prop-2-ynyl (propargyl).
术语“环烷基”是指完全饱和的且以单环、并环、桥环或螺环等形式存在的碳环。除非另有指示，该碳环通常为3至10元环。术语“C3-C10环烷基”应理解为表示饱和的一价单环、并环、螺环或桥环，其具有3～10个碳原子。所述环烷基的具体实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基、环癸基，降冰片基(双环[2.2.1]庚基)、双环[2.2.2]辛基、金刚烷基、螺[4.5]癸烷基等。术语“C3-C10环烷基”可以包含“C3-C8环烷基”，“C3-C8环烷基”可以包含“C3-C6环烷基”，“C3-C6环烷基”可以包含“C3-C4环烷基”。术语“C3-C6环烷基”可理解为表示饱和的一价单环或双环烃环，其具有3～6个碳原子，具体实例包括但不限于环丙基、环丁基、环戊基或环己基等。The term "cycloalkyl" refers to a fully saturated carbocyclic ring that exists in the form of a single ring, a branched ring, a bridged ring or a spiro ring. Unless otherwise indicated, the carbocyclic ring is generally 3 to 10 membered. The term "C 3 -C 10 cycloalkyl" is understood to mean a saturated monovalent monocyclic, paracyclic, 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, etc. The term "C 3 -C 10 cycloalkyl" may include "C 3 -C 8 cycloalkyl", "C 3 -C 8 cycloalkyl" may include "C 3 -C 6 cycloalkyl", "C 3 -C 6 cycloalkyl" may include "C 3 -C 4 cycloalkyl". The term "C 3 -C 6 cycloalkyl" can be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring with 3 to 6 carbon atoms. Specific examples include but are not limited to cyclopropyl, cyclobutyl, cyclopropyl, cyclobutyl, Pentyl or cyclohexyl, etc.
术语“环烷基氧基”可理解为“环烷基-O-”。The term "cycloalkyloxy" is understood to mean "cycloalkyl-O-".
术语“环烯基”是指含有至少一个碳-碳双键的非芳族单环或多环烃基。“C3-C6环烯基”是指具有3-6个碳原子作为环原子且包含至少一个碳-碳双键的非芳族环状烃。C3-C6环烯基的具体实例包括但不限于环丙烯基、环丁烯基、环戊烯基、环戊二烯基、环己烯基、环己二烯基等。The term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical containing at least one carbon-carbon double bond. "C 3 -C 6 cycloalkenyl" refers to a non-aromatic cyclic hydrocarbon having 3 to 6 carbon atoms as ring atoms and containing at least one carbon-carbon double bond. Specific examples of C 3 -C 6 cycloalkenyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.
术语“杂环基”是指完全饱和的或部分饱和的(整体上不是具有芳香性的杂芳族)一价单环、并环、螺环或桥环基团，其环原子中含有1-5个杂原子或杂原子团(即含有杂原子的原子团)，所述“杂原子或杂原子团”包括但不限于氮原子(N)、氧原子(O)、硫原子(S)、磷原子(P)、硼原子(B)、-S(＝O)2-、-S(＝O)-以及任选被取代的-NH-、-S(＝O)(＝NH)-、-C(＝O)NH-、-C(＝NH)-、-S(＝O)2NH-、S(＝O)NH-或-NHC(＝O)NH-等。术语“4-10元杂环基”是指环原子数目为4、5、6、7、8、9或10的杂环基，且其环原子中含有1-5个独立选自上文所述的杂原子或杂原子团。术语“4-10元含氮杂环基”是指其环原子中至少含1个N原子的4-10元杂环基，“4-10元含氮杂环基”包括“6-10元含氮杂环基”。术语“4-7元单环含氮杂环基”是指单环形式的且环原子中至少含1个N原子的4-7元杂环基。“4-10元杂环基”包括“6-10元杂环基”，“6-10 元杂环基”进一步包括“4-7元杂环基”，其中，4元杂环基的具体实例包括但不限于氮杂环丁烷基或氧杂环丁烷基；5元杂环基的具体实例包括但不限于四氢呋喃基、二氧杂环戊烯基、吡咯烷基、咪唑烷基、吡唑烷基、吡咯啉基、4,5-二氢噁唑基或2,5-二氢-1H-吡咯基；6元杂环基的具体实例包括但不限于四氢吡喃基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基、三噻烷基、四氢吡啶基或4H-[1,3,4]噻二嗪基；7元杂环基的具体实例包括但不限于二氮杂环庚烷基；8元杂环基的具体实例包括但不限于6,7-二氢-5H-吡咯并[2,1-c][1,2,4]三唑基。所述杂环基还可以是双环基，其中，5,5元双环基的具体实例包括但不限于六氢环戊并[c]吡咯-2(1H)-基；5,6元双环基的具体实例包括但不限于六氢吡咯并[1,2-a]吡嗪-2(1H)-基、5,6,7,8-四氢-[1,2,4]三唑并[4,3-a]吡嗪基或5,6,7,8-四氢咪唑并[1,5-a]吡嗪基。任选地，所述杂环基可以是上述4-7元杂环基的苯并稠合环基，具体实例包括但不限于二氢异喹啉基等。“4-10元杂环基”可以包含“5-10元杂环基”、“4-7元杂环基”、“5-7元杂环基”、“5-6元杂环基”、“6-8元杂环基”、“4-10元杂环烷基”、“5-10元杂环烷基”、“4-7元杂环烷基”、“5-6元杂环烷基”、“6-8元杂环烷基”等范围，“4-7元杂环基”进一步可以包含“4-6元杂环基”、“5-7元杂环基”、“5-6元杂环基”、“4-7元杂环烷基”、“4-6元杂环烷基”、“5-6元杂环烷基”等范围。本申请中尽管有些双环类杂环基部分地含有一个苯环或一个杂芳环，但所述杂环基整体上仍是无芳香性的。The term "heterocyclyl" refers to a fully saturated or partially saturated (not aromatic heteroaromatic as a whole) monovalent monocyclic, paracyclic, spirocyclic or bridged cyclic group whose ring atoms contain 1- 5 heteroatoms or heteroatom groups (i.e. atomic groups containing heteroatoms), the "heteroatoms or heteroatom groups" include but are not limited to nitrogen atoms (N), oxygen atoms (O), sulfur atoms (S), phosphorus atoms ( P), boron atom (B), -S(=O) 2 -, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C( =O)NH-, -C(=NH)-, -S(=O) 2 NH-, S(=O)NH- or -NHC(=O)NH-, etc. The term "4-10 membered heterocyclyl" refers to a heterocyclyl with a number of ring atoms of 4, 5, 6, 7, 8, 9 or 10, and its ring atoms contain 1 to 5 independently selected from the above. of heteroatoms or heteroatom groups. The term "4-10-membered nitrogen-containing heterocyclic group" refers to a 4-10-membered heterocyclic group containing at least 1 N atom in its ring atom, and "4-10-membered nitrogen-containing heterocyclic group" includes "6-10-membered nitrogen-containing heterocyclic group". Nitrogen-containing heterocyclic group". The term "4-7 membered monocyclic nitrogen-containing heterocyclyl group" refers to a 4-7 membered heterocyclyl group in the form of a monocyclic ring and containing at least 1 N atom in the ring atom. "4-10 membered heterocyclyl" includes "6-10 membered heterocyclyl", "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; 5-membered heterocyclyl Specific examples include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydroxazolyl or 2,5-dihydroxazolyl. Hydrogen-1H-pyrrolyl; specific examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianiyl Alkyl, tetrahydropyridyl or 4H-[1,3,4]thiadiazinyl; specific examples of 7-membered heterocyclyl include but are not limited to diazacycloheptyl; specific examples of 8-membered heterocyclyl Including but not limited to 6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazolyl. The heterocyclic group can also be a bicyclic group, wherein, 5,5-membered Specific examples of bicyclic groups include, but are not limited to, hexahydrocyclopenta[c]pyrrole-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 can be a benzo-fused cyclic group of the above 4-7 membered heterocyclic group. Specific examples include but are not limited to dihydrogen Isoquinolinyl, etc. "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" and other ranges, "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. .Although some bicyclic heterocyclic groups in this application partially contain a benzene ring or a heteroaromatic ring, the heterocyclic groups are still non-aromatic as a whole.
术语“杂环基氧基”可理解为“杂环基-O-”。The term "heterocyclyloxy" is understood to mean "heterocyclyl-O-".
术语“杂环烷基”是指完全饱和的且以单环、并环、桥环或螺环等形式存在的一价环状基团，其环的环原子中含有1-5个杂原子或杂原子团(即含有杂原子的原子团)，所述“杂原子或杂原子团”包括但不限于氮原子(N)、氧原子(O)、硫原子(S)、磷原子(P)、硼原子(B)、-S(＝O)2-、-S(＝O)-以及任选被取代的-NH-、-S(＝O)(＝NH)-、-C(＝O)NH-、-C(＝NH)-、-S(＝O)2NH-、S(＝O)NH-或-NHC(＝O)NH-等。术语“4-10元杂环烷基”是指环原子数目为4、5、6、7、8、9或10的杂环烷基，且其环原子中含有1-5个独立选自上文所述的杂原子或杂原子团。“4-10元杂环烷基”包括“4-7元杂环烷基”，其中，4元杂环烷基的具体实例包括但不限于吖丁啶基、噁丁环基或噻丁环基；5元杂环烷基的具体实例包括但不限于四氢呋喃基、四氢噻吩基、吡咯烷基、异噁唑烷基、噁唑烷基、异噻唑烷基、噻唑烷基、咪唑烷基或四氢吡唑基；6元杂环烷基的具体实例包括但不限于哌啶基、四氢吡喃基、四氢噻喃基、吗啉基、哌嗪基、1,4-噻噁烷基、1,4-二氧六环基、硫代吗啉基、1,3-二噻烷基或1,4-二噻烷基；7元杂环烷基的具体实例包括但不限于氮杂环庚烷基、氧杂环庚烷基或硫杂环庚烷基。The term "heterocycloalkyl" refers to a monovalent cyclic group that is fully saturated and exists in the form of a single ring, a branched ring, a bridged ring or a spiro ring, and the ring atoms of the ring contain 1-5 heteroatoms or Heteroatom groups (i.e. atomic groups containing heteroatoms), the "heteroatoms or heteroatom groups" include but are not limited to nitrogen atoms (N), oxygen atoms (O), sulfur atoms (S), phosphorus atoms (P), boron atoms (B), -S(=O) 2- , -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH- , -C(=NH)-, -S(=O) 2 NH-, S(=O)NH- or -NHC(=O)NH-, etc. The term "4-10 membered heterocycloalkyl" refers to a heterocycloalkyl group with a number of ring atoms of 4, 5, 6, 7, 8, 9 or 10, and its ring atoms contain 1 to 5 independently selected from the above The heteroatom or heteroatom group. "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 thibutanyl; Specific examples of 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl or tetrahydrofuranyl. Hydropyrazolyl; specific examples of 6-membered heterocycloalkyl include but are not limited to piperidyl, 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 aza Cycloheptyl, oxeptanyl or thieptanyl.
术语“芳基”是指具有共轭的π电子体系的全碳单环或稠合多环的芳香环基团。芳基可以具有6-14个碳原子或6-10个碳原子。术语“C6-C10芳基”应理解为具有6～10个碳原子的一价芳香性的单环或双环基团。特别是具有6个碳原子的环(“C6芳基”)，例如苯基；或者具有10个碳原子的环(“C10芳基”)，例如萘基。The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. Aryl groups can have 6-14 carbon atoms or 6-10 carbon atoms. The term "C 6 -C 10 aryl" is understood to mean a monovalent aromatic monocyclic or bicyclic radical having 6 to 10 carbon atoms. In particular, rings with 6 carbon atoms ("C 6 aryl"), for example phenyl; or rings with 10 carbon atoms ("C 10 aryl"), for example naphthyl.
术语“芳基氧基”可理解为“芳基-O-”。The term "aryloxy" is understood to mean "aryl-O-".
术语“杂芳基”是指具有芳香性的单环或稠合多环体系，其中含有至少一个选自N、O、S的环原子，其余环原子为C的芳香环基。术语“5-10元杂芳基”应理解为包括这样的一价单环或双环芳族环系：其具有5、6、7、8、9或10个环原子，特别是5或6或9或10个环原子，且其包含1、2、3、4或5个，优选1、2或3个独立选自N、O和S的杂原子。特别地，杂芳基选自噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑 基、噁二唑基、三唑基或噻二唑基等以及它们的苯并衍生物，例如苯并呋喃基、苯并噻吩基、苯并噻唑基、苯并噁唑基、苯并异噁唑基、苯并咪唑基、苯并三唑基、吲唑基、吲哚基或异吲哚基等；或吡啶基、哒嗪基、嘧啶基、吡嗪基或三嗪基等以及它们的苯并衍生物，例如喹啉基、喹唑啉基或异喹啉基等；或吖辛因基、吲嗪基、嘌呤基等以及它们的苯并衍生物；或噌啉基、酞嗪基、喹唑啉基、喹喔啉基、萘啶基、蝶啶基、咔唑基、吖啶基、吩嗪基、吩噻嗪基或吩噁嗪基等。术语“5-10元含氮杂芳基”是指其环原子中至少含1个N原子的5-10元杂芳基，“5-10元含氮杂芳基”包含“5-6元含氮杂芳基”。术语“5-6元杂芳基”指具有5或6个环原子的芳族环系，且其包含1、2或3个，优选1或2个独立选自N、O和S的杂原子。术语“6元杂芳基”指具有6个环原子的芳族环系，且其包含1、2或3个，优选1或2个N作为杂原子。术语“5-10元杂芳基”包含“5-6元杂芳基”。The term "heteroaryl" refers to an aromatic monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, and 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 it contains 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 the group consisting of thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl base, oxadiazolyl, triazolyl or thiadiazolyl, etc. and their benzo derivatives, such as benzofuryl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisox Azolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl or isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl, etc. and their Benzo derivatives, such as quinolinyl, quinazolinyl or isoquinolinyl, etc.; or azocinyl, indolinyl, purinyl, etc. and their benzo derivatives; or cinnolinyl, phthalazinyl , quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl or phenoxazinyl, etc. The term "5-10-membered nitrogen-containing heteroaryl" refers to a 5-10-membered heteroaryl containing at least 1 N atom in its ring atom, 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 which contains 1, 2 or 3, preferably 1 or 2 N as heteroatoms. The term "5-10 membered heteroaryl" includes "5-6 membered heteroaryl".
术语“杂芳基氧基”可理解为“杂芳基-O-”。The term "heteroaryloxy" is understood to mean "heteroaryl-O-".
术语“卤”或“卤素”是指氟(F)、氯(Cl)、溴(Br)或碘(I)。The term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
术语“羟基”是指-OH基团。The term "hydroxy" refers to the -OH group.
术语“氰基”是指-CN基团。The term "cyano" refers to the -CN group.
术语“巯基”是指-SH基团。The term "mercapto" refers to the -SH group.
术语“氨基”是指-NH2基团。The term "amino" refers to the -NH group .
术语“硝基”是指-NO2基团。The term "nitro" refers to the -NO2 group.
本申请还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本申请化合物。可结合到本申请化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为2H、3H、11C、13C、14C、13N、15N、15O、17O、18O、31P、32P、35S、18F、123I、125I和36Cl等。The present application also includes compounds of the present application that are the same as those described herein, but are isotopically labeled in which one or more atoms are replaced by an atom having an atomic weight or mass number different from that typically found in nature. Examples of isotopes that may 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 respectively N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
某些同位素标记的本申请化合物(例如用3H及14C标记)可用于化合物和/或底物组织分布分析中。氚化(即3H)和碳-14(即14C)同位素对于由于它们易于制备和可检测性是尤其优选的。正电子发射同位素，诸如15O、13N、11C和18F可用于正电子发射断层扫描(PET)研究以测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本申请化合物。Certain isotopically labeled compounds of the present application (eg, labeled with 3 H and 14 C) can be used in compound and/or substrate tissue distribution analysis. Tritiated (ie 3 H) and carbon-14 (ie 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 substituting an isotopically labeled reagent for a non-isotopically labeled reagent by following procedures similar to those disclosed in the Schemes and/or Examples below.
本申请的发明人经过大量研发和测试，开发出工程化免疫效应细胞及其与和/或其他活性物质(例如PDL1-IL15融合蛋白)的联合应用，联合应用涉及用于治疗癌症或肿瘤的药物制备、治疗癌症或肿瘤的方法、体外扩增免疫细胞的方法等。After extensive research and development and testing, the inventor of the present application has developed engineered immune effector cells and their combined application with and/or other active substances (such as PDL1-IL15 fusion protein). The combined application involves drugs for the treatment of cancer or tumors. Methods for preparing and treating cancer or tumors, methods for amplifying immune cells in vitro, etc.
在第一方面，本申请提供CBL-b抑制剂与免疫效应细胞的组合在制备用于治疗癌症或肿瘤的药物中的应用，所述免疫效应细胞表达或不表达嵌合抗原受体。In a first aspect, the present application provides the use of a combination of a CBL-b inhibitor and immune effector cells, which express or do not express chimeric antigen receptors, in the preparation of a medicament for the treatment of cancer or tumors.
在第二方面，本申请提供用于治疗癌症或肿瘤的药物组合产品，其包含CBL-b抑制剂与免疫效应细胞，所述免疫效应细胞表达或不表达嵌合抗原受体。In a second aspect, the present application provides a pharmaceutical combination product for treating cancer or tumors, which includes a CBL-b inhibitor and immune effector cells that express or do not express chimeric antigen receptors.
在第三方面，本申请提供治疗癌症或肿瘤的方法，其中，所述方法包括向有需要的受试者给予有效量的CBL-b抑制剂和免疫效应细胞，所述免疫效应细胞表达或不表达嵌合抗原受体。In a third aspect, the application provides a method of treating cancer or tumors, wherein the method comprises administering to a subject in need thereof an effective amount of a CBL-b inhibitor and an immune effector cell that expresses or does not Expresses chimeric antigen receptor.
以下针对CBL-b抑制剂、免疫效应细胞、嵌合抗原受体、癌症或肿瘤所描述的具体实施方式适用于第一至第三方面，并且在不存在矛盾的情况下，也适用于本申请的任何方面所述 的发明的各个实施方案。The specific embodiments described below for CBL-b inhibitors, immune effector cells, chimeric antigen receptors, cancers or tumors are applicable to the first to third aspects, and, in the absence of contradiction, are also applicable to this application any aspect of various embodiments of the invention.
在一些具体的实施方式中，所述CBL-b抑制剂为式(I)化合物或其药学上可接受的盐，
In some specific embodiments, the CBL-b inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,
其中，in,
选自以下情况中的任一种：i)C＝C-A3，所述A3选自CR11aR11b、NR12、O或S；ii)A1-C＝A3，所述A1选自C或N，A3选自CR11c或N；iii)A1-A2-A3，所述A1和A2独立地选自C(R11)n或N，A3选自CR11aR11b、NR12、O或S； Selected from any one of the following situations: i) C=CA 3 , the A 3 is selected from CR 11a R 11b , NR 12 , O or S; ii) A 1 -C=A 3 , the A 1 is selected from From C or N, A 3 is selected from CR 11c or N; iii) A 1 -A 2 -A 3 , said A 1 and A 2 are independently selected from C(R 11 ) n or N, A 3 is selected from CR 11a R 11b , NR 12 , O or S;
n选自0或1；n is selected from 0 or 1;
R11a、R11b、R11c、R11、R12彼此独立地选自H、卤素、OH、CN、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基或C3-C6环烷基氧基，其中所述的C1-C6烷基、C1-C6烷氧基、C3-C6环烷基或C3-C6环烷基氧基任选被R11d取代；R 11a , R 11b , R 11c , R 11 , R 12 are independently selected from H, halogen, OH, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) ) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 cycloalkyloxy, wherein the C 1 -C 6 alkyl Base, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 cycloalkyloxy is optionally substituted by R 11d ;
Q环选自苯基、5-6元杂芳基或5-7元杂环基，所述苯基、5-6元杂芳基或5-7元杂环基任选被R10取代；Q ring is selected from phenyl, 5-6-membered heteroaryl or 5-7-membered heterocyclyl, and the phenyl, 5-6-membered heteroaryl or 5-7-membered heterocyclyl is optionally substituted by R 10 ;
R10选自卤素、＝O、OH、NH2、NO2、CN、C1-C6烷基、C2-C4烯基、C2-C4炔基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烷基氧基、C3-C6环烷基-NH-、4-7元杂环基、4-7元杂环基氧基或4-7元杂环基-NH-，所述NH2、C1-C6烷基、C2-C4烯基、C2-C4炔基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烷基氧基、C3-C6环烷基-NH-、4-7元杂环基、4-7元杂环基氧基或4-7元杂环基-NH-任选被R10a取代；R 10 is selected from halogen, =O, OH, NH 2 , NO 2 , CN, C 1 -C 6 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, C 1 -C 6 alkoxy base, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyloxy, C 3 -C 6 cycloalkyl -NH-, 4-7 membered heterocyclyl, 4-7 membered heterocyclyloxy base or 4-7 membered heterocyclic group -NH-, the NH 2 , C 1 -C 6 alkyl group, C 2 -C 4 alkenyl group, C 2 -C 4 alkynyl group, C 1 -C 6 alkoxy group , C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyloxy, C 3 -C 6 cycloalkyl-NH-, 4-7 membered heterocyclyl, 4-7 membered heterocyclyloxy Or 4-7 membered heterocyclyl-NH- is optionally substituted by R 10a ;
Y1、Y2、Y3和Y4独立选自CRb或N；Y 1 , Y 2 , Y 3 and Y 4 are independently selected from CR b or N;
X选自卤素、CN、OH、COOH、CONH2、C1-C6烷基、C1-C6烷氧基、 其中C1-C6烷基或C1-C6烷氧基任选被Re取代，环B选自任选被R3取代的以下基团：4-10元含氮杂环基或5-10元含氮杂芳基，环D选自任选被R6取代的以下基团：C3-C10环烷基、4-10元杂环基、苯基或5-10元杂芳基，并且环D以非N原子与L相连，L选自键、 -NR7-、-NR7CH2-、-O-、-C(＝O)-、-C(＝O)NH-或-CR8R9-；X is selected from halogen, CN, OH, COOH, CONH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, Wherein C 1 -C 6 alkyl or C 1 -C 6 alkoxy is optionally substituted by R e , and ring B is selected from the following groups optionally substituted by R 3 : 4-10 membered nitrogen-containing heterocyclyl or 5 -10-membered nitrogen-containing heteroaryl, ring D is selected from the following groups optionally substituted by R 6 : C 3 -C 10 cycloalkyl, 4-10 membered heterocyclyl, phenyl or 5-10 membered heteroaryl base, and ring D is connected to L with a non-N atom, and L is selected from the group consisting of bonds, -NR 7 -, -NR 7 CH 2 -, -O-, -C(=O)-, -C(=O)NH- or -CR 8 R 9 -;
Rb选自H、卤素、OH、CN、C1-C6烷基、C1-C6烷氧基、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、NHC(O)(C1-C6烷基)、NHC(O)-O(C1-C6烷基)、N(C1-C6烷基)C(O)-O(C1-C6烷基)、NHS(O)2(C1-C6烷基)、C3-C6环烷基、C3-C6环烷基-O-、C3-C6环烷基-NH-、N(C3-C6环烷基)2、NHC(O)-C3-C6环烷基、NHS(O)2-C3-C6环烷基、4-7元杂环基、4-7元杂环基氧基、4-7元杂环基-NH-、N(4-7元杂环基)2、NHC(O)-4-7元杂环基、NHS(O)2-4-7元杂环基、C6-C10芳基、C6-C10芳基氧基、C6-C10芳基-NH-、N(C6-C10芳基)2、NHC(O)-C6-C10芳基、NHS(O)2-C6-C10芳基、5-10元杂芳基、5-10元杂芳基氧基或5-10元杂芳基-NH-、N(5-10元杂芳基)2、NHC(O)-5-10元杂芳基、NHS(O)2-5-10元杂芳基，其中所述的C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、4-7元杂环基、C6-C10芳基或5-10元杂芳基任选被R2a取代；R b is selected from H, halogen, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 Alkyl) 2 , NHC(O)(C 1 -C 6 alkyl), NHC(O)-O(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl)C(O)- O(C 1 -C 6 alkyl), NHS(O) 2 (C 1 -C 6 alkyl), C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl-O-, C 3 - C 6 cycloalkyl-NH-, N(C 3 -C 6 cycloalkyl) 2 , NHC(O)-C 3 -C 6 cycloalkyl, NHS(O) 2 -C 3 -C 6 cycloalkyl , 4-7 membered heterocyclyl, 4-7 membered heterocyclyloxy, 4-7 membered heterocyclyl-NH-, N(4-7 membered heterocyclyl) 2 , NHC(O)-4-7 1-membered heterocyclyl, NHS(O) 2 -4-7-membered heterocyclyl, C 6 -C 10 aryl, C 6 -C 10 aryloxy, C 6 -C 10 aryl-NH-, N( C 6 -C 10 aryl) 2 , NHC(O)-C 6 -C 10 aryl, NHS(O) 2 -C 6 -C 10 aryl, 5-10 membered heteroaryl, 5-10 membered hetero Aryloxy or 5-10-membered heteroaryl-NH-, N(5-10-membered heteroaryl) 2 , NHC(O)-5-10-membered heteroaryl, NHS(O) 2 -5-10 1-membered heteroaryl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, 4-7 membered heterocyclyl, C 6 -C 10 aryl Or the 5-10 membered heteroaryl group is optionally substituted by R 2a ;
或者两个Rb与其相连的C原子共同形成C3-C6环烯基、苯基、4-7元杂环基或5-6元杂芳基，所述C3-C6环烯基、苯基、4-7元杂环基或5-6元杂芳基任选被R2a取代；Or two R b and the C atom to which they are connected together form a C 3 -C 6 cycloalkenyl group, a phenyl group, a 4-7 membered heterocyclyl group or a 5-6 membered heteroaryl group, and the C 3 -C 6 cycloalkenyl group , phenyl, 4-7-membered heterocyclyl or 5-6-membered heteroaryl optionally substituted by R 2a ;
R4、R5、R7、R8和R9彼此独立地选自H、卤素、OH、C1-C6烷基或C1-C6烷氧基，所述C1-C6烷基或C1-C6烷氧基任选被R4a取代；R 4 , R 5 , R 7 , R 8 and R 9 are independently selected from H, halogen, OH, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, the C 1 -C 6 alkyl The base or C 1 -C 6 alkoxy group is optionally substituted by R 4a ;
或者R8、R9及其相连的原子一起形成C3-C6环烷基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选进一步被R8a取代；Or R 8 , R 9 and their connected atoms together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group is optional further replaced by R 8a ;
或者R4、R5及其相连的原子一起形成C3-C6环烷基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选进一步被R8a取代；或者当p取自2时，两个R4及其相连的原子一起形成C3-C6环烷基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选进一步被R8a取代；Or R 4 , R 5 and their connected atoms together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group is optional Further substituted by R 8a ; or when p is taken from 2, two R 4 and its connected atoms together form a C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl group, the C 3 -C 6 ring Alkyl or 4-7 membered heterocyclyl is optionally further substituted by R 8a ;
或者R4、R5一起形成＝O；Or R 4 and R 5 together form =O;
R3和R6独立地选自卤素、CN、＝O、OH、NO2、C1-C6烷基、OR6a、SR6a、N(R6a)2、S(O)2R6a、S(O)2N(R6a)2、S(O)R6a、S(O)N(R6a)2、C(O)R6a、C(O)OR6a、C(O)N(R6a)2、C(O)N(R6a)OR6a、OC(O)R6a、OC(O)N(R6a)2、N(R6a)C(O)OR6a、N(R6a)C(O)R6a、N(R6a)C(O)N(R6a)2、N(R6a)C(NR6a)N(R6a)2、N(R6a)S(O)2N(R6a)2、N(R6a)S(O)2R6a、C3-C10环烷基、4-7元杂环基、6-10元芳基或5-10元杂芳基，其中C1-C6烷基、C3-C10环烷基、4-7元杂环基、C6-C10元芳基或5-10元杂芳基任选进一步被R3a取代；R 3 and R 6 are independently selected from halogen, CN, =O, OH, NO 2 , C 1 -C 6 alkyl, OR 6a , SR 6a , N(R 6a ) 2 , S(O) 2 R 6a , S(O) 2 N(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 ) 2 N(R 6a ) 2 , N(R 6a )S(O) 2 R 6a , C 3 -C 10 cycloalkyl, 4-7 membered heterocyclyl, 6-10 membered aryl group or 5-10 membered Heteroaryl, wherein C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl, 4-7 membered heterocyclyl, C 6 -C 10 membered aryl or 5-10 membered heteroaryl is optionally further R 3a substitution;
每一个R6a独立地选自H、C1-C6烷基、苯基、4-7元杂环基或5-6元杂芳基，所述C1-C6烷基、苯基、4-7元杂环基或5-6元杂芳基任选进一步被R6b取代，或者一个N原子上的2个R6a与其相连的N一起形成4-7元杂环基或5-6元杂芳基，所述4-7元杂环基或5-6元杂芳基任选进一步被R6b取代；Each R 6a is independently selected from H, C 1 -C 6 alkyl, phenyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, the C 1 -C 6 alkyl, phenyl, The 4-7-membered heterocyclyl or 5-6-membered heteroaryl is optionally further substituted by R 6b , or the two R 6a on one N atom and the N to which it is connected together form a 4-7-membered heterocyclyl or 5-6 One-membered heteroaryl, the 4-7-membered heterocyclyl or 5-6-membered heteroaryl is optionally further substituted by R 6b ;
R3a、R4a、R6b和Re独立选自卤素、OH、CN、＝O、C1-C6烷基、NH2、NH(C1-C6烷基)、 N(C1-C6烷基)2、COOH或C1-C6烷氧基，其中所述的C1-C6烷基或C1-C6烷氧基进一步任选被Rf取代；R 3a , R 4a , R 6b and Re are independently selected from halogen, OH, CN, =O, C 1 -C 6 alkyl, NH 2 , NH (C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , COOH or C 1 -C 6 alkoxy, wherein the C 1 -C 6 alkyl or C 1 -C 6 alkoxy is further optionally substituted by R f ;
Rf选自卤素、OH、＝O、NH2、NH(C1-C6烷基)或N(C1-C6烷基)2；R f is selected from halogen, OH, =O, NH 2 , NH(C 1 -C 6 alkyl) or N(C 1 -C 6 alkyl) 2 ;
R11d、R2a和R10a独立选自卤素、OH、CN、＝O、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、卤代C1-C6烷基、C1-C6烷氧基或卤代C1-C6烷氧基；R 11d , R 2a and R 10a are independently selected from halogen, OH, CN, =O, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, halogenated C 1 -C 6 alkyl, C 1 -C 6 alkoxy or halogenated C 1 -C 6 alkoxy;
R1和R2独立选自H、卤素、CN、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、C1-C6烷氧基、C3-C10环烷基或4-10元杂环基，其中所述的C1-C6烷基、C1-C6烷氧基、C3-C10环烷基或4-10元杂环基任选被R1a取代，R 1 and R 2 are independently selected from H, halogen, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 cycloalkyl or 4-10 membered heterocyclyl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 Cycloalkyl or 4-10 membered heterocyclyl is optionally substituted by R 1a ,
或者R1、R2与其连接的原子共同形成C3-C10环烷基或4-10元杂环基，所述C3-C10环烷基或4-10元杂环基任选被R1b取代；Or R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 10 cycloalkyl group or a 4-10 membered heterocyclyl group, and the C 3 -C 10 cycloalkyl group or 4-10 membered heterocyclyl group is optionally R 1b substitution;
或者R1、Rb及其各自相连的原子和键共同形成C3-C6环烯基或4-7元杂环基，所述C3-C6环烯基或4-7元杂环基任选被R1d取代；Or R 1 , R b and their respective connected atoms and bonds together form a C 3 -C 6 cycloalkenyl group or a 4-7 membered heterocyclic group, and the C 3 -C 6 cycloalkenyl group or 4-7 membered heterocyclic group The group is optionally substituted by R 1d ;
R1a和R1b独立选自卤素、OH、CN、＝O、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基或C1-C6烷氧基，所述C1-C6烷基或C1-C6烷氧基任选进一步被R1c取代；R 1a and R 1b are independently selected from halogen, OH, CN, =O, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl Or C 1 -C 6 alkoxy group, the C 1 -C 6 alkyl group or C 1 -C 6 alkoxy group is optionally further substituted by R 1c ;
R1c选自卤素、OH、CN、＝O、NH2或COOH；R 1c is selected from halogen, OH, CN, =O, NH 2 or COOH;
W选自(CR13R14)kW1；所述W1选自5-10元杂芳基或4-10元杂环基，所述5-10元杂芳基、4-10元杂环基任选被R15取代；R13、R14独立选自H、卤素、OH、C1-C6烷基或C1-C6烷氧基；R15选自卤素、OH、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、C3-C10环烷基或4-7元杂环基，其中所述的C1-C6烷基、C3-C10环烷基或4-7元杂环基任选被R15a取代；W is selected from (CR 13 R 14 ) k W 1 ; said W 1 is selected from 5-10-membered heteroaryl or 4-10-membered heterocyclyl, and said 5-10-membered heteroaryl, 4-10-membered heterocyclic group The ring group is optionally substituted by R 15 ; R 13 and R 14 are independently selected from H, halogen, OH, C 1 -C 6 alkyl or C 1 -C 6 alkoxy; R 15 is selected from halogen, OH, NH 2 , NH (C 1 -C 6 alkyl), N (C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl, wherein The C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 15a ;
或者R1与R13及其各自相连的原子和键共同形成C3-C6环烷基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选被R13a取代；Or R 1 and R 13 and their respective connected atoms and bonds together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclic group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclic group The group is optionally substituted by R 13a ;
R8a、R13a和R1d独立地选自卤素、OH、CN、C1-C6烷基或C1-C6烷氧基，所述C1-C6烷基或C1-C6烷氧基任选进一步被卤素取代；R15a选自卤素、＝O、OH、CN或C1-C6烷基；R 8a , R 13a and R 1d are independently selected from halogen, OH, CN, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, said C 1 -C 6 alkyl or C 1 -C 6 The alkoxy group is optionally further substituted by halogen; R 15a is selected from halogen, =O, OH, CN or C 1 -C 6 alkyl;
k选自0或1；k is selected from 0 or 1;
p选自0、1或2。p is selected from 0, 1 or 2.
在一些实施方案中，选自：i)C＝C-A3，所述A3选自CR11aR11b、NR12、O或S；或ii)A1-C＝A3，所述A1选自C或N，A3选自CR11c或N。In some embodiments, Selected from: i) C=CA 3 , said A 3 is selected from CR 11a R 11b , NR 12 , O or S; or ii) A 1 -C=A 3 , said A 1 is selected from C or N, A 3 is selected from CR 11c or N.
在一些实施方案中，选自A1-C＝A3，所述A1选自C或N，A3选自CR11c或N。In some embodiments, Selected from A 1 -C=A 3 , said A 1 is selected from C or N, and A 3 is selected from CR 11c or N.
在一些实施方案中，选自A1-C＝A3，所述A1选自N，A3选自CR11c或N。 In some embodiments, Selected from A 1 -C=A 3 , said A 1 is selected from N, and A 3 is selected from CR 11c or N.
在一些实施方案中，选自C＝C-A3，所述A3选自CR11aR11b、NR12、O或S。In some embodiments, Selected from C=CA 3 , said A 3 is selected from CR 11a R 11b , NR 12 , O or S.
在一些实施方案中，选自C＝C-A3，所述A3选自NR12、O或S。In some embodiments, Selected from C=CA 3 , said A 3 is selected from NR 12 , O or S.
在一些实施方案中，选自A1-A2-A3，所述A1和A2独立地选自C(R11)n或N，A3选自NR12、O或S。In some embodiments, Selected from A 1 -A 2 -A 3 , said A 1 and A 2 are independently selected from C(R 11 ) n or N, and A 3 is selected from NR 12 , O or S.
在一些实施方案中，选自A1-A2-A3，所述A1选自N，A2选自C(R11)n，A3选自NR12。In some embodiments, Selected from A 1 -A 2 -A 3 , A 1 is selected from N, A 2 is selected from C(R 11 ) n , and A 3 is selected from NR 12 .
在一些实施方案中，R11a、R11b、R11c、R11、R12彼此独立地选自H、卤素、OH、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基或C3-C6环烷基氧基，其中所述的C1-C6烷基、C1-C6烷氧基、C3-C6环烷基或C3-C6环烷基氧基任选被R11d取代。In some embodiments, R 11a , R 11b , R 11c , R 11 , R 12 are independently selected from H, halogen, OH, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 - C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 cycloalkyloxy, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl or C 3 -C 6 cycloalkyloxy is optionally substituted by R 11d .
在一些实施方案中，R11a、R11b、R11c、R11、R12彼此独立地选自H、卤素、OH、NH2、C1-C6烷基或C3-C6环烷基，所述C1-C6烷基或C3-C6环烷基任选被R11d取代。In some embodiments, R 11a , R 11b , R 11c , R 11 , R 12 are independently selected from H, halogen, OH, NH 2 , C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl. , the C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl group is optionally substituted by R 11d .
在一些实施方案中，R11d选自卤素、OH、NH2、C1-C6烷基或卤代C1-C6烷基。In some embodiments, R 11d is selected from halogen, OH, NH 2 , C 1 -C 6 alkyl, or haloC 1 -C 6 alkyl.
在一些实施方案中，n选自0。In some embodiments, n is selected from 0.
在一些实施方案中，R11c选自H、卤素或C1-C6烷基，所述C1-C6烷基任选被R11d取代。In some embodiments, R 11c is selected from H, halogen, or C 1 -C 6 alkyl optionally substituted with R 11d .
在一些实施方案中，R11c选自H或卤素。In some embodiments, R 11c is selected from H or halogen.
在一些实施方案中，R11c选自F。In some embodiments, R 11c is selected from F.
在一些实施方案中，R12选自H、卤素、OH、C1-C6烷基或C3-C6环烷基，所述C1-C6烷基或C3-C6环烷基任选被R11d取代。In some embodiments, R 12 is selected from H, halogen, OH, C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl, said C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl The group is optionally substituted by R 11d .
在一些实施方案中，R12选自H或C1-C3烷基。In some embodiments, R 12 is selected from H or C 1 -C 3 alkyl.
在一些实施方案中，R12选自H或甲基。In some embodiments, R 12 is selected from H or methyl.
在一些实施方案中，R12选自甲基。In some embodiments, R 12 is selected from methyl.
在一些实施方案中，选自以下情况中的一种：i)C＝C-A3，所述A3选自N-CH3、O或S；ii)A1-C＝A3，所述A1选自N，A3选自N或C-F；iii)A1-A2-A3，所述A1选自N，A2选自C，A3选自N-CH3。In some embodiments, Selected from one of the following situations: i) C=CA 3 , the A 3 is selected from N-CH 3 , O or S; ii) A 1 -C=A 3 , the A 1 is selected from N, A 3 is selected from N or CF; iii) A 1 -A 2 -A 3 , A 1 is selected from N, A 2 is selected from C, and A 3 is selected from N-CH 3 .
在一些实施方案中，选自A1-C＝A3，所述A1选自N，A3选自N。In some embodiments, Selected from A 1 -C=A 3 , said A 1 is selected from N, and A 3 is selected from N.
在一些实施方案中，Q环选自任选被R10取代的以下基团：苯基、吡啶基、嘧啶基、吡唑基、咪唑基、噁唑基、异噁唑基、噻唑基、异噻唑基或5-7元杂环基，所述5-7元杂环基包含1或2个N作为杂原子。 In some embodiments, the Q ring is selected from the following groups optionally substituted with R 10 : phenyl, pyridyl, pyrimidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, iso Thiazolyl or 5-7 membered heterocyclyl group containing 1 or 2 N as heteroatoms.
在一些实施方案中，Q环选自任选被R10取代的以下基团：苯基、 In some embodiments, the Q ring is selected from the following groups optionally substituted with R 10 : phenyl,
在一些实施方案中，Q环选自任选被R10取代的 In some embodiments, the Q ring is selected from optionally substituted with R 10
在一些实施方案中，R10选自卤素、＝O、OH、NH2、CN、C1-C6烷基、C2-C4烯基、C2-C4炔基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烷基-O-、C3-C6环烷基-NH-、4-7元杂环基、4-7元杂环基氧基或4-7元杂环基-NH-，所述NH2、C1-C6烷基、C2-C4烯基、C2-C4炔基、C1-C6烷氧基或C3-C6环烷基任选被R10a取代。In some embodiments, R 10 is selected from halogen, =O, OH, NH 2 , CN, C 1 -C 6 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl -O-, C 3 -C 6 cycloalkyl -NH-, 4-7 membered heterocyclyl, 4-7 membered Heterocyclyloxy or 4-7 membered heterocyclyl -NH-, the NH 2 , C 1 -C 6 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, C 1 -C 6 alkoxy or C 3 -C 6 cycloalkyl is optionally substituted by R 10a .
在一些实施方案中，R10选自卤素、＝O、NH2、C1-C6烷基、C2-C4炔基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烷基-O-或C3-C6环烷基-NH-，所述NH2、C1-C6烷基、C2-C4炔基、C1-C6烷氧基或C3-C6环烷基任选被R10a取代。In some embodiments, R 10 is selected from halogen, =O, NH 2 , C 1 -C 6 alkyl, C 2 -C 4 alkynyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl base, C 3 -C 6 cycloalkyl -O- or C 3 -C 6 cycloalkyl -NH-, the NH 2 , C 1 -C 6 alkyl, C 2 -C 4 alkynyl, C 1 - C 6 alkoxy or C 3 -C 6 cycloalkyl is optionally substituted by R 10a .
在一些实施方案中，R10选自卤素、＝O、NH2、C1-C6烷基、C2-C4炔基、C1-C6烷氧基、C3-C6环烷基，所述NH2、C1-C6烷基、C2-C4炔基、C1-C6烷氧基或C3-C6环烷基任选被R10a取代。In some embodiments, R 10 is selected from halogen, =O, NH 2 , C 1 -C 6 alkyl, C 2 -C 4 alkynyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl group, the NH 2 , C 1 -C 6 alkyl group, C 2 -C 4 alkynyl group, C 1 -C 6 alkoxy group or C 3 -C 6 cycloalkyl group is optionally substituted by R 10a .
在一些实施方案中，R10a选自卤素、OH、NH2、C1-C6烷基、卤代C1-C6烷基、C1-C6烷氧基或卤代C1-C6烷氧基。In some embodiments, R 10a is selected from halogen, OH, NH 2 , C 1 -C 6 alkyl, haloC 1 -C 6 alkyl, C 1 -C 6 alkoxy, or haloC 1 -C 6 alkoxy.
在一些实施方案中，R10a选自卤素、OH、C1-C6烷基或卤代C1-C6烷基。In some embodiments, R 10a is selected from halogen, OH, C 1 -C 6 alkyl, or haloC 1 -C 6 alkyl.
在一些实施方案中，R10a选自F或CH3。In some embodiments, R 10a is selected from F or CH 3 .
在一些实施方案中，R10选自＝O、F、Cl、Br、CN、甲基、乙基、乙炔基、CF3、CHF2、甲氧基、N(CH3)2、环丙基、环丙基-O-或环丙基-NH-。In some embodiments, R10 is selected from =O, F, Cl, Br, CN, methyl, ethyl, ethynyl, CF3, CHF2 , methoxy, N( CH3 ) 2 , cyclopropyl , cyclopropyl-O- or cyclopropyl-NH-.
在一些实施方案中，R10选自＝O、F、Cl、乙炔基、CF3、CHF2、甲氧基、N(CH3)2、环丙基、环丙基-O-或环丙基-NH-。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 Base -NH-.
在一些实施方案中，R10选自＝O、F、Cl、Br、CN、甲基、乙基、乙炔基、CF3、甲氧基、N(CH3)2或环丙基。In some embodiments, R10 is selected from =O, F, Cl, Br, CN, methyl, ethyl, ethynyl, CF3 , methoxy, N( CH3 ) 2 , or cyclopropyl.
在一些实施方案中，R10选自＝O、F、Cl、乙炔基、CF3、甲氧基、N(CH3)2或环丙基。In some embodiments, R10 is selected from =O, F, Cl, ethynyl, CF3 , methoxy, N( CH3 ) 2 , or cyclopropyl.
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，Y1、Y2独立选自CRb或N，Y3和Y4独立选自CRb。In some embodiments, Y 1 , Y 2 are independently selected from CR b or N, and Y 3 and Y 4 are independently selected from CR b .
在一些实施方案中，Y1、Y2、Y3和Y4均为CRb。In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 are each CR b .
在一些实施方案中，Y1和Y2均为N，Y3和Y4独立选自CRb。In some embodiments, Y 1 and Y 2 are both N and Y 3 and Y 4 are independently selected from CR b .
在一些实施方案中，Y1为N，Y2、Y3和Y4独立选自CRb。In some embodiments, Y 1 is N and Y 2 , Y 3 and Y 4 are independently selected from CR b .
在一些实施方案中，Rb选自H、卤素、OH、C1-C6烷基、C1-C6烷氧基、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基-O-、C3-C6环烷基-NH-、4-7元杂环基-O-、4-7元杂环基-NH-或5-10元杂芳基，其中所述的C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、4-7元杂环基或5-10元杂芳基任选被R2a取代。In some embodiments, R b is selected from H, halogen, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 3 -C 6 cycloalkyl -O-, C 3 -C 6 cycloalkyl -NH-, 4-7 membered heterocyclyl -O-, 4-7 membered heterocycle -NH- or 5-10 membered heteroaryl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, 4-7 membered heterocyclyl Or 5-10 membered heteroaryl is optionally substituted by R 2a .
在一些实施方案中，Rb选自H、卤素或任选被R2a取代的以下基团：甲基、乙氧基、NHCH3、NHEt、NH(i-Pr)、吡唑基、环丙基-O-、环丁基-NH-或氧杂环丁基-O-。In some embodiments, R b is selected from H, halogen, or optionally substituted by R 2a : methyl, ethoxy, NHCH 3 , NHEt, NH(i-Pr), pyrazolyl, cyclopropyl -O-, cyclobutyl-NH- or oxetanyl-O-.
在一些实施方案中，R2a选自卤素、OH、＝O、C1-C3烷基或C1-C3烷氧基。In some embodiments, R 2a is selected from halogen, OH, =O, C 1 -C 3 alkyl, or C 1 -C 3 alkoxy.
在一些实施方案中，R2a选自卤素、OH或C1-C3烷基。In some embodiments, R 2a is selected from halogen, OH, or C 1 -C 3 alkyl.
在一些实施方案中，R2a选自F、OH或甲基。In some embodiments, R 2a is selected from F, OH, or methyl.
在一些实施方案中，Rb选自H、F、CF3、OEt、NHCH3、NHEt、NH(i-Pr)、 In some embodiments, R b is selected from H, F, CF 3 , OEt, NHCH 3 , NHEt, NH(i-Pr),
在一些实施方案中，Rb、R1及其各自相连的原子和键共同形成C3-C6环烯基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选被R1d取代。In some embodiments, R b , R 1 and their respective connected atoms and bonds together form a C 3 -C 6 cycloalkenyl or 4-7 membered heterocyclyl group, the C 3 -C 6 cycloalkyl or 4 -7-membered heterocyclyl optionally substituted by R 1d .
在一些实施方案中，Rb、R1及其各自相连的原子和键共同形成任选被R1d取代的C3-C6环烯基。In some embodiments, R b , R 1 and their respective atoms and bonds to which they are attached together form a C 3 -C 6 cycloalkenyl group optionally substituted by R 1d .
在一些实施方案中，R1d选自卤素、OH、C1-C3烷基或卤代C1-C3烷基。In some embodiments, R 1d is selected from halogen, OH, C 1 -C 3 alkyl, or haloC 1 -C 3 alkyl.
在一些实施方案中，Rb、R1及其各自相连的原子和键共同形成环戊烯基。 In some embodiments, R b , R 1 and their respective atoms and bonds to which they are attached together form cyclopentenyl.
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，X选自或任选被Re取代的C1-C6烷基。In some embodiments, X is selected from Or C 1 -C 6 alkyl optionally substituted by Re .
在一些实施方案中，X选自 In some embodiments, X is selected from
在一些实施方案中，X选自 In some embodiments, X is selected from
在一些实施方案中，环B选自任选被R3取代的5-10元含氮杂芳基、4-7元单环含氮杂环基或6-10元含氮杂环基。In some embodiments, Ring B is selected from a 5-10 membered nitrogen-containing heteroaryl optionally substituted by R3 , a 4-7 membered monocyclic nitrogen-containing heterocyclyl, or a 6-10 membered nitrogen-containing heterocyclyl.
在一些实施方案中，环B选自任选被R3取代的以下基团：四氢吡咯基、哌啶基、哌嗪基、 吗啉基、 In some embodiments, Ring B is selected from the following groups optionally substituted by R : tetrahydropyrrolyl, piperidinyl, piperazinyl, Morpholinyl,
在一些实施方案中，环B选自任选被R3取代的以下基团：四氢吡咯基、哌啶基、哌嗪基、吗啉基、 In some embodiments, Ring B is selected from the following groups optionally substituted by R : tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl,
在一些实施方案中，R3选自卤素、OH、＝O、CN、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C6-C10芳基或5-10元杂芳基，所述C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C6-C10芳基或5-10元杂芳基任选进一步被R3a取代。In some embodiments, R 3 is selected from halogen, OH, =O, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, C 6 -C 10 Aryl or 5-10 membered heteroaryl, the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, C 6 -C 10 aryl or 5-10 The heteroaryl group is optionally further substituted with R 3a .
在一些实施方案中，R3选自卤素、OH、＝O、C1-C3烷基、C3-C6环烷基或苯基，所述C1-C3烷基、C3-C6环烷基或苯基任选进一步被R3a取代。In some embodiments, R 3 is selected from halogen, OH, =O, C 1 -C 3 alkyl, C 3 -C 6 cycloalkyl, or phenyl, said C 1 -C 3 alkyl, C 3 - C 6 cycloalkyl or phenyl is optionally further substituted by R 3a .
在一些实施方案中，R3a选自卤素、OH、＝O或C1-C3烷氧基。In some embodiments, R 3a is selected from halogen, OH, =O, or C 1 -C 3 alkoxy.
在一些实施方案中，R3a选自F。In some embodiments, R 3a is selected from F.
在一些实施方案中，R3选自＝O、OH、F、甲基、CF3、环丙基或苯基。In some embodiments, R3 is selected from =O, OH, F, methyl, CF3 , cyclopropyl, or phenyl.
在一些实施方案中，R4、R5独立选自H、卤素、OH或任选被R4a取代的C1-C3烷基；或者R4、R5及其相连的原子一起形成任选被R8a取代的C3-C6环烷基；或者R4、R5一起形成＝O；或者当p取自2时，两个R4及其相连的原子一起形成任选被R8a取代的C3-C6环烷基。In some embodiments, R 4 and R 5 are independently selected from H, halogen, OH or C 1 -C 3 alkyl optionally substituted by R 4a ; or R 4 , R 5 and the atoms to which they are attached together form an optional C 3 -C 6 cycloalkyl substituted by R 8a ; or R 4 and R 5 together form =O; or when p is taken from 2, two R 4 and its connected atoms together form optionally substituted by R 8a C 3 -C 6 cycloalkyl.
在一些实施方案中，R4、R5独立选自H、卤素、OH或任选被R4a取代的C1-C3烷基；或者R4、R5及其相连的原子一起形成任选被R8a取代的C3-C6环烷基；或者R4、R5一起形成＝O。In some embodiments, R 4 and R 5 are independently selected from H, halogen, OH or C 1 -C 3 alkyl optionally substituted by R 4a ; or R 4 , R 5 and the atoms to which they are attached together form an optional C 3 -C 6 cycloalkyl substituted by R 8a ; or R 4 and R 5 together form =O.
在一些实施方案中，R4a选自卤素、OH或C1-C3烷氧基。In some embodiments, R 4a is selected from halogen, OH, or C 1 -C 3 alkoxy.
在一些实施方案中，R4a选自F或OH。In some embodiments, R 4a is selected from F or OH.
在一些实施方案中，R8a选自卤素、OH或C1-C3烷基。In some embodiments, R 8a is selected from halogen, OH, or C 1 -C 3 alkyl.
在一些实施方案中，R4、R5独立选自H、甲基、羟甲基或CF3，或者R4、R5及其相连的原子一起形成环丙基，或者R4、R5一起形成＝O。In some embodiments, R 4 and R 5 are independently selected from H, methyl, hydroxymethyl or CF 3 , or R 4 , R 5 and the atoms to which they are attached together form a cyclopropyl group, or R 4 and R 5 together Form=O.
在一些实施方案中，p选自1。In some embodiments, p is selected from 1.
在一些实施方案中，p选自0。 In some embodiments, p is selected from 0.
在一些实施方案中，p选自2，且两个R4及其相连的原子一起形成任选被R8a取代的C3-C6环烷基。In some embodiments, p is selected from 2, and two R 4 and the atoms to which they are attached together form a C 3 -C 6 cycloalkyl group optionally substituted by R 8a .
在一些实施方案中，p选自2，且两个R4及其相连的原子一起形成环丙基或环丁基。In some embodiments, p is selected from 2, and two R 4 and the atoms to which they are attached together form cyclopropyl or cyclobutyl.
在一些实施方案中，选自如下基团： In some embodiments, Selected from the following groups:
在一些实施方案中，选自如下基团： In some embodiments, Selected from the following groups:
在一些实施方案中，环D选自任选被R6取代的以下基团：C3-C6环烷基、5-6元杂芳基、4-7元单环含氮杂环基或6-10元含氮杂环基，并且环D以非N原子与L相连。In some embodiments, Ring D is selected from the following groups optionally substituted by R: C3-C6 cycloalkyl , 5-6 membered heteroaryl, 4-7 membered monocyclic nitrogen-containing heterocyclyl, or 6-10 membered nitrogen-containing heterocyclic group, and ring D is connected to L with a non-N atom.
在一些实施方案中，环D选自任选被R6取代的以下基团：环丙基、环丁基、环戊基、四氢吡咯基、哌啶基、哌嗪基、吡唑基、咪唑基、噁唑基、异噁唑基、三唑基、噻唑基、异噻唑基或吡啶基。In some embodiments, Ring D is selected from the following groups optionally substituted by R: cyclopropyl , cyclobutyl, cyclopentyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, Pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl, isothiazolyl or pyridyl.
在一些实施方案中，环D选自任选被R6取代的以下基团：环戊基、哌啶基、吡啶基、 In some embodiments, Ring D is selected from the following groups optionally substituted by R : cyclopentyl, piperidyl, pyridyl,
在一些实施方案中，R6选自卤素、OH、CN、＝O或任选被R3a取代的C1-C3烷基。In some embodiments, R 6 is selected from halogen, OH, CN, =O, or C 1 -C 3 alkyl optionally substituted by R 3a .
在一些实施方案中，R6选自卤素、＝O、OH或C1-C3烷基。In some embodiments, R6 is selected from halogen, =O, OH, or Ci - C3 alkyl.
在一些实施方案中，R6选自F或甲基。In some embodiments, R6 is selected from F or methyl.
在一些实施方案中，L选自键、-NR7-、-NR7CH2-、-O-或-CR8R9-。In some embodiments, L is selected from bond , -NR7- , -NR7CH2- , -O-, or -CR8R9- .
在一些实施方案中，R7、R8和R9独立选自H、卤素、C1-C3烷基或OH。In some embodiments, R 7 , R 8 and R 9 are independently selected from H, halogen, C 1 -C 3 alkyl, or OH.
在一些实施方案中，R7、R8和R9独立选自H或卤素。In some embodiments, R 7 , R 8 and R 9 are independently selected from H or halogen.
在一些实施方案中，R7选自H。In some embodiments, R is selected from H.
在一些实施方案中，R8、R9选自H或F。In some embodiments, R 8 , R 9 are selected from H or F.
在一些实施方案中，L选自键、-NH-、-NHCH2-、-O-、-C(F)2-或-CH2-。In some embodiments, L is selected from the group consisting of bond, -NH-, -NHCH2- , -O-, -C(F) 2- , or -CH2- .
在一些实施方案中，选自如下基团： In some embodiments, Selected from the following groups:
在一些实施方案中，X选自任选被Re取代的C1-C6烷基。In some embodiments, X is selected from C 1 -C 6 alkyl optionally substituted by Re .
在一些实施方案中，Re选自卤素、OH、C1-C3烷基、NH2、NH(C1-C3烷基)、N(C1-C3烷基)2或C1-C3烷氧基，其中所述的C1-C3烷基或C1-C3烷氧基进一步任选被Rf取代。In some embodiments, Re is selected from halogen, OH, C 1 -C 3 alkyl, NH 2 , NH(C 1 -C 3 alkyl), N(C 1 -C 3 alkyl) 2 or C 1 -C 3 alkoxy group, wherein the C 1 -C 3 alkyl group or C 1 -C 3 alkoxy group is further optionally substituted by R f .
在一些实施方案中，Re选自C1-C3烷基、N(C1-C3烷基)2或C1-C3烷氧基，其中所述的C1-C3烷基或C1-C3烷氧基进一步任选被Rf取代。In some embodiments, Re is selected from C 1 -C 3 alkyl, N(C 1 -C 3 alkyl) 2 or C 1 -C 3 alkoxy, wherein said C 1 -C 3 alkyl Or the C 1 -C 3 alkoxy group is further optionally substituted by R f .
在一些实施方案中，Rf选自卤素、OH或N(C1-C6烷基)2。In some embodiments, R f is selected from halogen, OH, or N(C 1 -C 6 alkyl) 2 .
在一些实施方案中，Rf选自N(CH3)2。In some embodiments, R f is selected from N(CH 3 ) 2 .
在一些实施方案中，Re选自N(CH3)2、CH2N(CH3)2或 In some embodiments, Re is selected from N(CH 3 ) 2 , CH 2 N(CH 3 ) 2 or
在一些实施方案中，X选自 In some embodiments, X is selected from
在一些实施方案中，X选自如下基团： In some embodiments, X is selected from the following groups:
在一些实施方案中，X选自如下基团： In some embodiments, X is selected from the following groups:
在一些实施方案中，X选自 In some embodiments, X is selected from
在一些实施方案中，X选自 In some embodiments, X is selected from
在一些实施方案中，R1、R2与其连接的原子共同形成C3-C8环烷基或4-10元杂环基，所述C3-C8环烷基或4-10元杂环基任选被R1b取代。In some embodiments, R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 8 cycloalkyl group or a 4-10 membered heterocyclyl group . The ring group is optionally substituted by R 1b .
在一些实施方案中，R1、R2与其连接的原子共同形成C3-C6环烷基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选被R1b取代。In some embodiments, R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group The ring group is optionally substituted by R 1b .
在一些实施方案中，R1、R2与其连接的原子共同形成任选被R1b取代的如下基团：环丁基、螺[2,3]己基或氧杂环丁基。In some embodiments, R 1 , R 2 and the atoms to which they are attached together form the following group optionally substituted by R 1b : cyclobutyl, spiro[2,3]hexyl, or oxetanyl.
在一些实施方案中，R1b选自卤素、OH、CN、＝O、NH2、C1-C3烷基或C1-C3烷氧基，所 述C1-C3烷基或C1-C3烷氧基任选被R1c取代。In some embodiments, R 1b is selected from halogen, OH, CN, =O, NH 2 , C 1 -C 3 alkyl, or C 1 -C 3 alkoxy, so The C 1 -C 3 alkyl or C 1 -C 3 alkoxy group is optionally substituted by R 1c .
在一些实施方案中，R1b选自卤素、CN、C1-C3烷基或C1-C3烷氧基，所述C1-C3烷基或C1-C3烷氧基任选被R1c取代。In some embodiments, R 1b is selected from halogen, CN, C 1 -C 3 alkyl or C 1 -C 3 alkoxy, any of which C 1 -C 3 alkyl or C 1 -C 3 alkoxy Selected to be replaced by R 1c .
在一些实施方案中，R1c选自卤素、OH或CN。In some embodiments, R 1c is selected from halogen, OH, or CN.
在一些实施方案中，R1c选自CN。In some embodiments, R 1c is selected from CN.
在一些实施方案中，R1b选自F、CN、甲基、甲氧基或CH2CN。In some embodiments, R lb is selected from F, CN, methyl, methoxy, or CH 2 CN.
在一些实施方案中，R1和R2独立选自H、卤素、CN、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、C1-C6烷氧基、C3-C8环烷基或4-7元杂环基，所述C1-C6烷基、C1-C6烷氧基、C3-C8环烷基或4-7元杂环基任选被R1a取代，且所述C3-C8环烷基或4-7元杂环基可以是螺环、桥环或并环形式的。In some embodiments, R 1 and R 2 are independently selected from H, halogen, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl, the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 1a , and the C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl may be a spiro ring, a bridged ring or a Ring form.
在一些实施方案中，R1和R2独立选自H、卤素、CN、C1-C3烷基、C1-C3烷氧基或C3-C6环烷基，所述C1-C3烷基、C1-C3烷氧基或C3-C6环烷基任选被R1a取代。In some embodiments, R 1 and R 2 are independently selected from H, halogen, CN, C 1 -C 3 alkyl, C 1 -C 3 alkoxy, or C 3 -C 6 cycloalkyl, the C 1 -C 3 alkyl, C 1 -C 3 alkoxy or C 3 -C 6 cycloalkyl is optionally substituted by R 1a .
在一些实施方案中，R1和R2独立选自H或C1-C3烷基，所述C1-C3烷基任选被R1a取代。In some embodiments, R 1 and R 2 are independently selected from H or C 1 -C 3 alkyl, which C 1 -C 3 alkyl is optionally substituted with R 1a .
在一些实施方案中，R1a选自卤素、OH、CN、＝O、NH2、C1-C3烷基或C1-C3烷氧基。In some embodiments, R 1a is selected from halogen, OH, CN, =O, NH 2 , C 1 -C 3 alkyl, or C 1 -C 3 alkoxy.
在一些实施方案中，R1和R2独立选自H或甲基，或者R1、R2与其连接的原子共同形成如下基团： In some embodiments, R 1 and R 2 are independently selected from H or methyl, or R 1 , R 2 and the atoms to which they are connected together form the following group:
在一些实施方案中，R1、R2与其连接的原子共同形成如下基团： In some embodiments, R 1 , R 2 and the atoms to which they are connected together form the following group:
在一些实施方案中，R1、R2与其连接的原子共同形成如下基团： In some embodiments, R 1 , R 2 and the atoms to which they are connected together form the following group:
在一些实施方案中，R1和R2独立选自H或甲基。In some embodiments, R 1 and R 2 are independently selected from H or methyl.
在一些实施方案中，W选自-(CR13R14)W1。In some embodiments, W is selected from -(CR 13 R 14 )W 1 .
在一些实施方案中，R13、R14独立选自H、卤素、OH或甲基。In some embodiments, R 13 , R 14 are independently selected from H, halogen, OH, or methyl.
在一些实施方案中，R13、R14均为H。 In some embodiments, R 13 and R 14 are both H.
在一些实施方案中，R1与R13及其各自相连的原子和键共同形成任选被R13a取代的C3-C6环烷基。In some embodiments, R 1 and R 13 and the atoms and bonds to which each is attached together form a C 3 -C 6 cycloalkyl group optionally substituted by R 13a .
在一些实施方案中，R13a选自C1-C3烷基。In some embodiments, R 13a is selected from C 1 -C 3 alkyl.
在一些实施方案中，R13a选自甲基。In some embodiments, R 13a is selected from methyl.
在一些实施方案中，R1与R13及其各自相连的原子和键共同形成 In some embodiments, R 1 and R 13 and their respective atoms and bonds to which they are attached together form
在一些实施方案中，W选自W1。In some embodiments, W is selected from W 1 .
在一些实施方案中，W1选自任选被R15取代的5-10元杂芳基或6-10元杂环基。In some embodiments, W 1 is selected from 5-10 membered heteroaryl or 6-10 membered heterocyclyl optionally substituted by R 15 .
在一些实施方案中，W1选自任选被R15取代的5-10元杂芳基。In some embodiments, W 1 is selected from 5-10 membered heteroaryl optionally substituted with R 15 .
在一些实施方案中，W1选自任选被R15取代的5元杂芳基或8元杂环基。In some embodiments, W 1 is selected from 5-membered heteroaryl or 8-membered heterocyclyl optionally substituted with R 15 .
在一些实施方案中，W1选自任选被R15取代的5元杂芳基。In some embodiments, W 1 is selected from 5-membered heteroaryl optionally substituted with R 15 .
在一些实施方案中，W1选自任选被R15取代的如下基团：吡咯基、噻吩基、呋喃基、吡唑基、咪唑基、噻唑基、异噻唑基、噻二唑基、三氮唑基、噁唑基、异噁唑基、噁二唑基或6,7-二氢-5H-吡咯并[2,1-c][1,2,4]三唑基。In some embodiments, W is selected from the following groups optionally substituted with R : pyrrolyl, thienyl, furyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tris Azolyl, oxazolyl, isoxazolyl, oxadiazolyl or 6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazolyl.
在一些实施方案中，W1选自任选被R15取代的如下基团：吡咯基、噻吩基、呋喃基、吡唑基、咪唑基、噻唑基、异噻唑基、噻二唑基、三氮唑基、噁唑基、异噁唑基或噁二唑基。In some embodiments, W is selected from the following groups optionally substituted with R : pyrrolyl, thienyl, furyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tris Azolyl, oxazolyl, isoxazolyl or oxadiazolyl.
在一些实施方案中，W1选自任选被R15取代的如下基团：三氮唑基、噁唑基、异噁唑基、噁二唑基或6,7-二氢-5H-吡咯并[2,1-c][1,2,4]三唑基。In some embodiments, W is selected from the group consisting of triazolyl, oxazolyl, isoxazolyl, oxadiazolyl, or 6,7-dihydro-5H-pyrrole, optionally substituted with R 15 And [2,1-c][1,2,4]triazolyl.
在一些实施方案中，W1选自任选被R15取代的如下基团：三氮唑基、噁唑基、异噁唑基或噁二唑基。In some embodiments, W 1 is selected from the group consisting of triazolyl, oxazolyl, isoxazolyl, or oxadiazolyl, optionally substituted with R 15 .
在一些实施方案中，R15选自卤素、OH、NH2、C1-C3烷基或C3-C6环烷基，所述C1-C3烷基或C3-C6环烷基任选被R15a取代。In some embodiments, R 15 is selected from halogen, OH, NH 2 , C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl, said C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl Alkyl is optionally substituted with R 15a .
在一些实施方案中，R15选自OH、C1-C3烷基或C3-C6环烷基，所述C1-C3烷基或C3-C6环烷基任选被R15a取代。In some embodiments, R 15 is selected from OH, C 1 -C 3 alkyl, or C 3 -C 6 cycloalkyl, which C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl is optionally replaced by R 15a substituted.
在一些实施方案中，R15选自甲基或环丙基，所述甲基或环丙基任选被R15a取代。In some embodiments, R 15 is selected from methyl or cyclopropyl, which is optionally substituted with R 15a .
在一些实施方案中，R15a选自卤素、OH或CN。In some embodiments, R 15a is selected from halogen, OH, or CN.
在一些实施方案中，R15a选自F。In some embodiments, R 15a is selected from F.
在一些实施方案中，R15选自甲基、CHF2或环丙基。In some embodiments, R15 is selected from methyl, CHF2 , or cyclopropyl.
在一些实施方案中，W1选自如下基团： In some embodiments, W is selected from the following groups:
在一些实施方案中，W1选自如下基团： In some embodiments, W is selected from the following groups:
在一些实施方案中，W选自 In some embodiments, W is selected from
在一些实施方案中，本申请的式(I)化合物选自式(II)化合物：
In some embodiments, compounds of formula (I) of the present application are selected from compounds of formula (II):
其中，Y1、Y2、Y3、Y4、X、Q、W、R1和R2如上文所定义。Among them, Y 1 , Y 2 , Y 3 , Y 4 , X, Q, W, R 1 and R 2 are as defined above.
在一些实施方案中，本申请的式(I)化合物选自式(III)化合物：
In some embodiments, compounds of formula (I) of the present application are selected from compounds of formula (III):
其中，Z1、Z2、Z3独立地选自CH、CR10或N；R10、Y1、Y2、Y3、Y4、X、W、R1和R2如上文所定义。Wherein, Z 1 , Z 2 , Z 3 are 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 are as defined above.
在一些实施方案中，本申请的式(I)化合物选自式(IV)化合物：
In some embodiments, compounds of Formula (I) of the present application are selected from compounds of Formula (IV):
其中R10、Y1、Y2、Y3、Y4、X、W、R1和R2如上文所定义。在一些实施方案中，本申请的式(I)化合物选自以下化合物：





wherein R 10 , Y 1 , Y 2 , Y 3 , Y 4 , X, W, R 1 and R 2 are as defined above. In some embodiments, compounds of formula (I) of the present application are selected from the following compounds:





本申请还提供一种CBL-b抑制剂以及所述CBL-b抑制剂在本申请各个方面的发明中的应用，所述CBL-b抑制剂为式(V)化合物或其药学上可接受的盐，
This application also provides a CBL-b inhibitor and the application of the CBL-b inhibitor in the inventions of various aspects of the application. The CBL-b inhibitor is a compound of formula (V) or a pharmaceutically acceptable compound thereof. Salt,
其中，in,
Z1和Z2独立选自CRa或N；Z 1 and Z 2 are independently selected from CR a or N;
Z3选自C、CH或N；Z 3 is selected from C, CH or N;
Y1、Y2、Y3和Y4独立选自CRb或N；Y 1 , Y 2 , Y 3 and Y 4 are independently selected from CR b or N;
X选自卤素、CN、OH、COOH、CONH2、C1-C6烷基、C1-C6烷氧基、 其中C1-C6烷基或C1-C6烷氧基任选被Re取代，环B选自任选被R3取代的以下基团：4-10元含氮杂环基或5-10元含氮杂芳基，环D选自任选被R6取代的以下基团：C3-C10环烷基、4-10元杂环基、苯基或5-10元杂芳基，并且环D以非N原子与L相连，L选自键、-NR7-、-NR7CH2-、-O-、-C(＝O)-、-C(＝O)NH-或-CR8R9-；X is selected from halogen, CN, OH, COOH, CONH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, Wherein C 1 -C 6 alkyl or C 1 -C 6 alkoxy is optionally substituted by R e , and ring B is selected from the following groups optionally substituted by R 3 : 4-10 membered nitrogen-containing heterocyclyl or 5 -10-membered nitrogen-containing heteroaryl, ring D is selected from the following groups optionally substituted by R 6 : C 3 -C 10 cycloalkyl, 4-10 membered heterocyclyl, phenyl or 5-10 membered heteroaryl group, and ring D is connected to L with a non-N atom, and L is selected from the group consisting of bonds, -NR 7 -, -NR 7 CH 2 -, -O-, -C(=O)-, -C(=O)NH- or -CR 8 R 9 -;
Rb选自H、卤素、OH、CN、C1-C6烷基、C1-C6烷氧基、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、NHC(O)(C1-C6烷基)、NHS(O)2(C1-C6烷基)、C3-C6环烷基、C3-C6环烷基-O-、C3-C6环烷基-NH-、N(C3-C6环烷基)2、NHC(O)-C3-C6环烷基、NHS(O)2-C3-C6环烷基、4-7元杂环 基、4-7元杂环基氧基、4-7元杂环基-NH-、N(4-7元杂环基)2、NHC(O)-4-7元杂环基、NHS(O)2-4-7元杂环基、C6-C10芳基、C6-C10芳基氧基、C6-C10芳基-NH-、N(C6-C10芳基)2、NHC(O)-C6-C10芳基、NHS(O)2-C6-C10芳基、5-10元杂芳基、5-10元杂芳基氧基或5-10元杂芳基-NH-、N(5-10元杂芳基)2、NHC(O)-5-10元杂芳基、NHS(O)2-5-10元杂芳基，其中所述的C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、4-7元杂环基、C6-C10芳基或5-10元杂芳基任选被R2a取代；R b is selected from H, halogen, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 Alkyl) 2 , NHC(O)(C 1 -C 6 alkyl), NHS(O) 2 (C 1 -C 6 alkyl), C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl Base -O-, C 3 -C 6 cycloalkyl -NH-, N(C 3 -C 6 cycloalkyl) 2 , NHC(O)-C 3 -C 6 cycloalkyl, NHS(O) 2 - C 3 -C 6 cycloalkyl, 4-7 membered heterocycle base, 4-7-membered heterocyclyloxy group, 4-7-membered heterocyclyl-NH-, N(4-7-membered heterocyclyl) 2 , NHC(O)-4-7-membered heterocyclyl, NHS( O) 2-4-7 membered heterocyclyl, C 6 -C 10 aryl, C 6 -C 10 aryloxy, C 6 -C 10 aryl-NH-, N(C 6 -C 10 aryl ) 2 , NHC(O)-C 6 -C 10 aryl, NHS(O) 2 -C 6 -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, where The above-mentioned C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group, C 3 -C 6 cycloalkyl group, 4-7 membered heterocyclyl group, C 6 -C 10 aryl group or 5-10 membered heteroaromatic group The group is optionally substituted by R 2a ;
或者两个Rb与其相连的C原子共同形成C3-C6环烯基、苯基、4-7元杂环基或5-6元杂芳基，所述C3-C6环烯基、苯基、4-7元杂环基或5-6元杂芳基任选被R2a取代；Or two R b and the C atom to which they are connected together form a C 3 -C 6 cycloalkenyl group, a phenyl group, a 4-7 membered heterocyclyl group or a 5-6 membered heteroaryl group, and the C 3 -C 6 cycloalkenyl group , phenyl, 4-7-membered heterocyclyl or 5-6-membered heteroaryl optionally substituted by R 2a ;
Ra、R4、R5、R7、R8和R9彼此独立地选自H、卤素、OH、C1-C6烷基或C1-C6烷氧基，所述C1-C6烷基或C1-C6烷氧基任选被R4a取代；R a , R 4 , R 5 , R 7 , R 8 and R 9 are independently selected from H, halogen, OH, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, the C 1 - C 6 alkyl or C 1 -C 6 alkoxy is optionally substituted by R 4a ;
或者R8、R9及其相连的原子一起形成C3-C6环烷基或4-7元杂环基，或者R4、R5及其相连的原子一起形成C3-C6环烷基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选进一步被R8a取代，或者R4、R5一起形成＝O；Either R 8 , R 9 and their connected atoms together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, or R 4 , R 5 and their connected atoms together form a C 3 -C 6 cycloalkyl group. base or 4-7 membered heterocyclyl group, the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group is optionally further substituted by R 8a , or R 4 and R 5 together form =O;
R3和R6独立地选自卤素、CN、＝O、NO2、C1-C6烷基、OR6a、SR6a、N(R6a)2、S(O)2R6a、S(O)2N(R6a)2、S(O)R6a、S(O)N(R6a)2、C(O)R6a、C(O)OR6a、C(O)N(R6a)2、C(O)N(R6a)OR6a、OC(O)R6a、OC(O)N(R6a)2、N(R6a)C(O)OR6a、N(R6a)C(O)R6a、N(R6a)C(O)N(R6a)2、N(R6a)C(NR6a)N(R6a)2、N(R6a)S(O)2N(R6a)2、N(R6a)S(O)2R6a、C3-C10环烷基、4-7元杂环基、6-10元芳基或5-10元杂芳基，其中C1-C6烷基、C3-C10环烷基、4-7元杂环基、6-10元芳基或5-10元杂芳基任选进一步被R3a取代；R 3 and R 6 are independently selected from halogen, CN, =O, NO 2 , C 1 -C 6 alkyl, OR 6a , SR 6a , N(R 6a ) 2 , S(O) 2 R 6a , S( O) 2 N(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) 2 N(R 6a ) 2 , N(R 6a )S(O) 2 R 6a , C 3 -C 10 cycloalkyl, 4-7 membered heterocyclyl, 6-10 membered aryl or 5-10 membered heteroaryl Base, wherein C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl, 4-7 membered heterocyclyl, 6-10 membered aryl or 5-10 membered heteroaryl is optionally further substituted by R 3a ;
R6a选自H、C1-C6烷基、苯基、4-7元杂环基或5-6元杂芳基，所述C1-C6烷基、苯基、4-7元杂环基或5-6元杂芳基任选进一步被R6b取代，或者一个N原子上的2个R6a与其相连的N一起形成4-7元杂环基或5-6元杂芳基，所述4-7元杂环基或5-6元杂芳基任选进一步被R6b取代；R 6a is selected from H, C 1 -C 6 alkyl, phenyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, the C 1 -C 6 alkyl, phenyl, 4-7 membered heteroaryl Heterocyclyl or 5-6-membered heteroaryl is optionally further substituted by R 6b , or two R 6a on one N atom and the N to which it is connected together form a 4-7-membered heterocyclyl or 5-6-membered heteroaryl. , the 4-7-membered heterocyclyl or 5-6-membered heteroaryl is optionally further substituted by R 6b ;
R3a、R4a、R6b和Re独立选自卤素、OH、CN、＝O、NH2、COOH或C1-C6烷氧基；R 3a , R 4a , R 6b and R e are independently selected from halogen, OH, CN, =O, NH 2 , COOH or C 1 -C 6 alkoxy;
Q为苯基或6元杂芳基，所述苯基或6元杂芳基任选被R10取代；Q is phenyl or 6-membered heteroaryl, and the phenyl or 6-membered heteroaryl is optionally substituted by R 10 ;
或者Q为如下所示基团：其中Z4和Z5独立选自CRcRd、NR11、O、S、或S(＝O)2，代表单键或双键，并且当为双键时，m为1，当为单键时m为1或2；Or Q is a group as shown below: wherein Z 4 and Z 5 are independently selected from CR c R d , NR 11 , O, S, or S(=O) 2 , represents a single or double bond, and when When it is a double bond, m is 1, when When it is a single key, m is 1 or 2;
R10选自卤素、OH、NH2、CN、C1-C6烷基、C1-C6烷氧基、C3-C10环烷基或4-7元杂环基，所述C1-C6烷基、C1-C6烷氧基、C3-C10环烷基或4-7元杂环基任选被R10a取代； R 10 is selected from halogen, OH, NH 2 , CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl, the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 10a ;
R2a和R10a独立选自卤素、OH、CN、＝O、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、卤代C1-C6烷基、C1-C6烷氧基或卤代C1-C6烷氧基；R 2a and R 10a are independently selected from halogen, OH, CN, =O, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl , halogenated C 1 -C 6 alkyl, C 1 -C 6 alkoxy or halogenated C 1 -C 6 alkoxy;
Rc、Rd和R11独立选自H、卤素、OH、CN、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基或C1-C6烷氧基，其中所述的C1-C6烷基或C1-C6烷氧基任选被R11a取代，或者Rc、Rd共同形成＝O，或者Rc、Rd与其相连的原子共同形成C3-C10环烷基或4-7元杂环基，所述C3-C10环烷基或4-7元杂环基任选被R11b取代；R c , R d and R 11 are independently selected from H, halogen, OH, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 Alkyl or C 1 -C 6 alkoxy, wherein the C 1 -C 6 alkyl or C 1 -C 6 alkoxy is optionally substituted by R 11a , or R c and R d together form =O, Or R c , R d and the atoms to which they are connected together form a C 3 -C 10 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 10 cycloalkyl group or 4-7 membered heterocyclyl group is optionally R 11b substitution;
或者Rc与R11及其各自相连的原子共同形成4-7元杂环基，所述4-7元杂环基任选被R11c取代；Or R c and R 11 and their respective connected atoms together form a 4-7-membered heterocyclic group, and the 4-7-membered heterocyclic group is optionally substituted by R 11c ;
R11a、R11b和R11c独立地选自卤素、OH、＝O、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2或C1-C6烷基；R 11a , R 11b and R 11c are independently selected from halogen, OH, =O, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 or C 1 -C 6 alkyl;
R1和R2独立选自H、卤素、CN、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、C1-C6烷氧基、C3-C10环烷基或4-10元杂环基，其中所述的C1-C6烷基、C1-C6烷氧基、C3-C10环烷基或4-10元杂环基任选被R1a取代，R 1 and R 2 are independently selected from H, halogen, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 cycloalkyl or 4-10 membered heterocyclyl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 10 Cycloalkyl or 4-10 membered heterocyclyl is optionally substituted by R 1a ,
或者R1、R2与其连接的原子共同形成C3-C10环烷基或4-10元杂环基，所述C3-C10环烷基或4-10元杂环基任选被R1b取代；Or R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 10 cycloalkyl group or a 4-10 membered heterocyclyl group, and the C 3 -C 10 cycloalkyl group or 4-10 membered heterocyclyl group is optionally R 1b substitution;
R1a和R1b独立选自卤素、OH、CN、＝O、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基或C1-C6烷氧基，所述C1-C6烷基或C1-C6烷氧基任选进一步被R1c取代；R 1a and R 1b are independently selected from halogen, OH, CN, =O, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl Or C 1 -C 6 alkoxy group, the C 1 -C 6 alkyl group or C 1 -C 6 alkoxy group is optionally further substituted by R 1c ;
R1c选自卤素、OH、CN、＝O、NH2或COOH；R 1c is selected from halogen, OH, CN, =O, NH 2 or COOH;
W选自(CR12R13)kW1，所述W1选自5-10元杂芳基或4-10元杂环基，所述5-10元杂芳基、4-10元杂环基任选被R14取代，R12、R13独立选自H、卤素、OH、C1-C6烷基或C1-C6烷氧基，R14选自卤素、OH、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、C3-C10环烷基或4-7元杂环基，其中所述的C1-C6烷基、C3-C10环烷基或4-7元杂环基任选被R14a取代；W is selected from (CR 12 R 13 ) k W 1 , and said W 1 is selected from 5-10-membered heteroaryl or 4-10-membered heterocyclyl. The ring group is optionally substituted by R 14 , R 12 and R 13 are independently selected from H, halogen, OH, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, R 14 is selected from halogen, OH, NH 2 , NH (C 1 -C 6 alkyl), N (C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl, wherein The C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 14a ;
或者R1与R12及其各自相连的原子和键共同形成C3-C6环烷基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选被R12a取代；Or R 1 and R 12 and their respective connected atoms and bonds together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclic group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclic group The group is optionally substituted by R 12a ;
R8a和R12a独立地选自卤素、OH、CN、C1-C6烷基或C1-C6烷氧基，所述C1-C6烷基或C1-C6烷氧基任选进一步被卤素取代，R14a选自卤素、＝O、OH、CN或C1-C6烷基；R 8a and R 12a are independently selected from halogen, OH, CN, C 1 -C 6 alkyl or C 1 -C 6 alkoxy, said C 1 -C 6 alkyl or C 1 -C 6 alkoxy Optionally further substituted by halogen, R 14a is selected from halogen, =O, OH, CN or C 1 -C 6 alkyl;
p和k独立地选自0或1。p and k are independently selected from 0 or 1.
在一些实施方案中，Z1和Z2独立选自CH或N。In some embodiments, Z 1 and Z 2 are independently selected from CH or N.
在一些实施方案中，Z1和Z2均为CH。In some embodiments, Z 1 and Z 2 are both CH.
在一些实施方案中，Z1为CH，Z2为N。In some embodiments, Z 1 is CH and Z 2 is N.
在一些实施方案中，Z1为N，Z2为CH。 In some embodiments, Z 1 is N and Z 2 is CH.
在一些实施方案中，Y1、Y2、Y3和Y4均为CRb。In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 are each CR b .
在一些实施方案中，Y1和Y2独立选自CRb或N，Y3和Y4均为CRb。In some embodiments, Y 1 and Y 2 are independently selected from CR b or N, and Y 3 and Y 4 are both CR b .
在一些实施方案中，Y1和Y2均为N，Y3和Y4均为CRb。In some embodiments, Y 1 and Y 2 are both N and Y 3 and Y 4 are both CR b .
在一些实施方案中，Y1为N，Y2、Y3和Y4均为CRb。In some embodiments, Y 1 is N and Y 2 , Y 3 and Y 4 are all CR b .
在一些实施方案中，Rb选自H、卤素、OH、C1-C6烷基、C1-C6烷氧基、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基-O-、C3-C6环烷基-NH-、4-7元杂环基-O-、4-7元杂环基-NH-或5-10元杂芳基，其中所述的C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、4-7元杂环基或5-10元杂芳基任选被R2a取代。In some embodiments, R b is selected from H, halogen, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 3 -C 6 cycloalkyl -O-, C 3 -C 6 cycloalkyl -NH-, 4-7 membered heterocyclyl -O-, 4-7 membered heterocycle -NH- or 5-10 membered heteroaryl, wherein the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, 4-7 membered heterocyclyl Or 5-10 membered heteroaryl is optionally substituted by R 2a .
在一些实施方案中，Rb选自H、卤素或任选被R2a取代的以下基团：甲基、乙氧基、NHCH3、NHEt、NH(i-Pr)、吡唑基、环丙基-O-、环丁基-NH-或氧杂环丁基-O-。In some embodiments, R b is selected from H, halogen, or optionally substituted by R 2a : methyl, ethoxy, NHCH 3 , NHEt, NH(i-Pr), pyrazolyl, cyclopropyl -O-, cyclobutyl-NH- or oxetanyl-O-.
在一些实施方案中，R2a选自卤素、OH、＝O、C1-C3烷基或C1-C3烷氧基。In some embodiments, R 2a is selected from halogen, OH, =O, C 1 -C 3 alkyl, or C 1 -C 3 alkoxy.
在一些实施方案中，R2a选自卤素、OH或C1-C3烷基。In some embodiments, R 2a is selected from halogen, OH, or C 1 -C 3 alkyl.
在一些实施方案中，R2a选自F、OH或甲基。In some embodiments, R 2a is selected from F, OH, or methyl.
在一些实施方案中，Rb选自H、F、CF3、乙氧基、OCH2CHF2、NHCH3、NHEt、NH(i-Pr)、NHCH2CH2OH、 In some embodiments, R b is selected from H, F, CF 3 , ethoxy, OCH 2 CHF 2 , NHCH 3 , NHEt, NH(i-Pr), NHCH 2 CH 2 OH,
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，选自 In some embodiments, Selected from
在一些实施方案中，X选自 In some embodiments, X is selected from
在一些实施方案中，环B选自任选被R3取代的5-10元含氮杂芳基、4-7元单环含氮杂环基或6-10元含氮杂环基。In some embodiments, Ring B is selected from a 5-10 membered nitrogen-containing heteroaryl optionally substituted by R3 , a 4-7 membered monocyclic nitrogen-containing heterocyclyl, or a 6-10 membered nitrogen-containing heterocyclyl.
在一些实施方案中，环B选自任选被R3取代的以下基团：吡唑基、咪唑基、三唑基、噁唑基、异噁唑基、噻唑基、异噻唑基、氮杂环丁基、四氢吡咯基、哌啶基、哌嗪基、吗啉基、氮杂环庚基、 In some embodiments, Ring B is selected from the following groups optionally substituted with R : pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, azepine cyclobutyl, tetrahydropyrrolyl, piperidyl, piperazinyl, morpholinyl, azepanyl,
在一些实施方案中，环B选自任选被R3取代的以下基团：吡唑基、氮杂环丁基、四氢吡咯基、哌啶基、哌嗪基、吗啉基、氮杂环庚基、 In some embodiments, Ring B is selected from the following groups optionally substituted with R : pyrazolyl, azetidinyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, azepine Cycloheptyl,
在一些实施方案中，R3选自卤素、OH、＝O、CN、C1-C6烷基、C1-C6烷氧基、6-10元芳基或5-10元杂芳基，所述C1-C6烷基、C1-C6烷氧基、6-10元芳基或5-10元杂芳基任选进一步被R3a取代。In some embodiments, R 3 is selected from halogen, OH, =O, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, 6-10 membered aryl or 5-10 membered heteroaryl , the C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group, 6-10 membered aryl group or 5-10 membered heteroaryl group is optionally further substituted by R 3a .
在一些实施方案中，R3选自卤素、OH、＝O、CN、C1-C3烷基、C1-C3烷氧基或苯基，所述C1-C3烷基、C1-C3烷氧基或苯基任选进一步被R3a取代。In some embodiments, R 3 is selected from halogen, OH, =O, CN, C 1 -C 3 alkyl, C 1 -C 3 alkoxy, or phenyl, said C 1 -C 3 alkyl, C 1 -C 3 alkoxy or phenyl is optionally further substituted by R 3a .
在一些实施方案中，R3a选自卤素、OH、＝O或C1-C3烷氧基。 In some embodiments, R 3a is selected from halogen, OH, =O, or C 1 -C 3 alkoxy.
在一些实施方案中，R3a选自F、OH或甲氧基。In some embodiments, R 3a is selected from F, OH, or methoxy.
在一些实施方案中，R3选自＝O、OH、F、CN、甲基、CF3、羟甲基、甲氧基、或苯基。In some embodiments, R3 is selected from =O, OH, F, CN, methyl, CF3 , hydroxymethyl, methoxy, or phenyl.
在一些实施方案中，R4、R5独立选自H、卤素、OH或任选被R4a取代的C1-C3烷基，或者R4、R5一起形成＝O。In some embodiments, R 4 , R 5 are independently selected from H, halogen, OH, or C 1 -C 3 alkyl optionally substituted by R 4a , or R 4 , R 5 together form =O.
在一些实施方案中，R4a选自卤素、OH或C1-C3烷氧基。In some embodiments, R 4a is selected from halogen, OH, or C 1 -C 3 alkoxy.
在一些实施方案中，R4a选自F。In some embodiments, R 4a is selected from F.
在一些实施方案中，R4、R5独立选自H、甲基、CF3或乙基，或者R4、R5一起形成＝O。In some embodiments, R 4 , R 5 are independently selected from H, methyl, CF 3 or ethyl, or R 4 , R 5 together form =O.
在一些实施方案中，p选自1。In some embodiments, p is selected from 1.
在一些实施方案中，p选自0。In some embodiments, p is selected from 0.
在一些实施方案中，选自如下基团： In some embodiments, Selected from the following groups:
在一些实施方案中，环D选自任选被R6取代的以下基团：C3-C6环烷基、5-6元杂芳基、4-7元单环杂环基或6-10元杂环基，并且环D以非N原子与L相连。 In some embodiments, Ring D is selected from the following groups optionally substituted by R: C3-C6 cycloalkyl , 5-6 membered heteroaryl, 4-7 membered monocyclic heterocyclyl, or 6- 10-membered heterocyclyl, and ring D is connected to L with a non-N atom.
在一些实施方案中，环D选自任选被R6取代的以下基团：环丙基、环丁基、环戊基、四氢吡咯基、哌啶基、哌嗪基、吗啉基、吡唑基、咪唑基、噁唑基、异噁唑基、三唑基、噻唑基或异噻唑基。In some embodiments, Ring D is selected from the following groups optionally substituted by R : cyclopropyl, cyclobutyl, cyclopentyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, Pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl or isothiazolyl.
在一些实施方案中，R6选自卤素、OH、CN、＝O或任选被R3a取代的C1-C3烷基。In some embodiments, R 6 is selected from halogen, OH, CN, =O, or C 1 -C 3 alkyl optionally substituted by R 3a .
在一些实施方案中，R6选自卤素、＝O、OH或C1-C3烷基。In some embodiments, R6 is selected from halogen, =O, OH, or Ci - C3 alkyl.
在一些实施方案中，R6选自F、＝O或甲基。In some embodiments, R is selected from F, =O, or methyl.
在一些实施方案中，L选自键、-NR7-、-NR7CH2-、-O-或-CR8R9-。In some embodiments, L is selected from bond , -NR7- , -NR7CH2- , -O-, or -CR8R9- .
在一些实施方案中，R7、R8和R9独立选自H、C1-C3烷基或OH。In some embodiments, R 7 , R 8 and R 9 are independently selected from H, C 1 -C 3 alkyl, or OH.
在一些实施方案中，R7选自H或甲基。In some embodiments, R7 is selected from H or methyl.
在一些实施方案中，R8、R9选自H。In some embodiments, R 8 , R 9 are selected from H.
在一些实施方案中，L选自键、-NCH3-、-NHCH2-、-O-或-CH2-。In some embodiments, L is selected from bond, -NCH3- , -NHCH2- , -O-, or -CH2- .
在一些实施方案中，选自如下基团： In some embodiments, Selected from the following groups:
在一些实施方案中，X选自如下基团： In some embodiments, X is selected from the following groups:
在一些实施方案中，X选自 In some embodiments, X is selected from
在一些实施方案中，Q为任选被R10取代的苯基或6元杂芳基。In some embodiments, Q is phenyl or 6-membered heteroaryl optionally substituted with R 10 .
在一些实施方案中，Q为任选被R10取代的以下基团：苯基、吡啶基、哒嗪基、嘧啶基、吡嗪基或三嗪基。In some embodiments, Q is phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, or triazinyl optionally substituted with R 10 .
在一些实施方案中，Q为任选被R10取代的以下基团：苯基、吡啶基、哒嗪基或嘧啶基。In some embodiments, Q is phenyl, pyridinyl, pyridazinyl, or pyrimidinyl optionally substituted with R 10 .
在一些实施方案中，Q为任选被R10取代的苯基。In some embodiments, Q is phenyl optionally substituted with R 10 .
在一些实施方案中，R10选自卤素、OH、CN、C1-C6烷基或C1-C6烷氧基，所述C1-C6烷基或C1-C6烷氧基任选被R10a取代。In some embodiments, R 10 is selected from halogen, OH, CN , C 1 -C 6 alkyl, or C 1 -C 6 alkoxy. The group is optionally substituted by R 10a .
在一些实施方案中，R10选自卤素、C1-C3烷基或C1-C3烷氧基，所述C1-C3烷基或C1-C3烷氧基任选被R10a取代。In some embodiments, R 10 is selected from halogen, C 1 -C 3 alkyl, or C 1 -C 3 alkoxy, optionally substituted by R 10a replaced.
在一些实施方案中，R10a选自卤素、OH或C1-C3烷基。In some embodiments, R 10a is selected from halogen, OH, or C 1 -C 3 alkyl.
在一些实施方案中，R10a选自F或OH。In some embodiments, R 10a is selected from F or OH.
在一些实施方案中，R10选自F、Cl、甲基、甲氧基、CH2OH或CF3。 In some embodiments, R10 is selected from F, Cl, methyl, methoxy, CH2OH , or CF3 .
在一些实施方案中，Q选自其中Z4和Z5独立选自CRcRd、NR11、O、S或SO2，代表单键或双键，并且当为双键时，m为1，当为单键时，m为1或2。In some embodiments, Q is selected from Wherein Z 4 and Z 5 are independently selected from CR c R d , NR 11 , O, S or SO 2 , represents a single or double bond, and when When it is a double bond, m is 1, when When it is a single bond, m is 1 or 2.
在一些实施方案中，Rc、Rd和R11独立选自H、卤素、OH、NH2或C1-C6烷基，所述C1-C6烷基任选被R11a取代，或者Rc、Rd共同形成＝O，或者Rc、Rd与其相连的原子共同形成C3-C6环烷基，所述C3-C6环烷基任选被R11b取代。In some embodiments, R c , R d and R 11 are independently selected from H, halogen, OH, NH 2 or C 1 -C 6 alkyl optionally substituted with R 11a , Either R c and R d together form =O, or R c , R d and the atoms to which they are connected together form a C 3 -C 6 cycloalkyl group, and the C 3 -C 6 cycloalkyl group is optionally substituted by R 11b .
在一些实施方案中，Rc、Rd和R11独立选自H、卤素、OH或任选被R11a取代的C1-C3烷基，或者Rc、Rd共同形成＝O，或者Rc、Rd与其相连的原子共同形成任选被R11b取代的C3-C4环烷基。In some embodiments, R c , R d and R 11 are independently selected from H, halogen, OH, or C 1 -C 3 alkyl optionally substituted by R 11a , or R c , R d together form =O, or R c , R d and the atoms to which they are connected together form a C 3 -C 4 cycloalkyl group optionally substituted by R 11b .
在一些实施方案中，Rc与R11及其各自相连的原子共同形成4-7元杂环基，所述4-7元杂环基任选被R11c取代。In some embodiments, R c and R 11 and the atom to which each is attached together form a 4-7 membered heterocyclyl group that is optionally substituted by R 11c .
在一些实施方案中，R11a、R11b和R11c独立选自卤素、OH、＝O、NH2或C1-C3烷基。In some embodiments, R 11a , R 11b and R 11c are independently selected from halogen, OH, =O, NH 2 or C 1 -C 3 alkyl.
在一些实施方案中，R11a、R11b和R11c独立选自卤素、OH或＝O。In some embodiments, R 11a , R 11b and R 11c are independently selected from halogen, OH, or =O.
在一些实施方案中，Rc、Rd和R11独立选自H、卤素或甲基，或者Rc、Rd共同形成＝O，或者Rc、Rd与其相连的原子共同形成环丙基。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 and the atoms to which they are attached together form cyclopropyl .
在一些实施方案中，Rc和Rd二者及其相连的原子共同形成环丙基。In some embodiments, both R c and R d and the atoms to which they are attached together form cyclopropyl.
在一些实施方案中，Z4和Z5独立选自CH2、CF2、CHCH3、C(CH3)2、NCH3、C＝O、O、S、S(＝O)2或或者Z4-Z5共同形成 In some embodiments, Z 4 and Z 5 are independently selected from CH 2 , CF 2 , CHCH 3 , C(CH 3 ) 2 , NCH 3 , C=O, O, S, S(=O) 2 or Or Z 4 -Z 5 together form
在一些实施方案中，Q选自m为1或2。In some embodiments, Q is selected from m is 1 or 2.
在一些实施方案中，Q选自m为1。In some embodiments, Q is selected from m is 1.
在一些实施方案中，Q选自 In some embodiments, Q is selected from
在一些实施方案中，Q选自苯基、 其中a代表Q与母核中的6元环共用的键，b代表Q与母核中的5元环共用的键。In some embodiments, Q is selected from phenyl, Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
在一些实施方案中，Q选自苯基、 其中a代表Q与母核中的6元环共用的键，b代表Q与母核中的5元环共用的键。In some embodiments, Q is selected from phenyl, Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
在一些实施方案中，Q选自苯基、 其中a代表Q与母核中的6元环共用的键，b代表Q与母核中的5元环共用的键。 In some embodiments, Q is selected from phenyl, Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
在一些实施方案中，Q选自 其中a代表Q与母核中的6元环共用的键，b代表Q与母核中的5元环共用的键。In some embodiments, Q is selected from Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
在一些实施方案中，Q选自 其中a代表Q与母核中的6元环共用的键，b代表Q与母核中的5元环共用的键。In some embodiments, Q is selected from Where a represents the bond shared by Q and the 6-membered ring in the parent nucleus, and b represents the bond shared by Q and the 5-membered ring in the parent nucleus.
在一些实施方案中，R1、R2与其连接的原子共同形成C3-C8环烷基或4-10元杂环基，所述C3-C8环烷基或4-10元杂环基任选被R1b取代。In some embodiments, R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 8 cycloalkyl group or a 4-10 membered heterocyclyl group . The ring group is optionally substituted by R 1b .
在一些实施方案中，R1、R2与其连接的原子共同形成C3-C6环烷基或4-7元杂环基，所述C3-C6环烷基或4-7元杂环基任选被R1b取代。In some embodiments, R 1 and R 2 and the atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the C 3 -C 6 cycloalkyl group or 4-7 membered heterocyclyl group The ring group is optionally substituted by R 1b .
在一些实施方案中，R1、R2与其连接的原子共同形成任选被R1b取代的如下基团：环丁基、螺[2,3]己基或氧杂环丁基。In some embodiments, R 1 , R 2 and the atoms to which they are attached together form the following group optionally substituted by R 1b : cyclobutyl, spiro[2,3]hexyl, or oxetanyl.
在一些实施方案中，R1b选自卤素、OH、CN、＝O、NH2、C1-C3烷基或C1-C3烷氧基，所述C1-C3烷基或C1-C3烷氧基任选被R1c取代。In some embodiments, R 1b is selected from halogen, OH, CN, =O, NH 2 , C 1 -C 3 alkyl, or C 1 -C 3 alkoxy, the C 1 -C 3 alkyl or C 1 -C 3 alkoxy is optionally substituted by R 1c .
在一些实施方案中，R1b选自卤素、CN、C1-C3烷基或C1-C3烷氧基，所述C1-C3烷基或C1-C3烷氧基任选被R1c取代。In some embodiments, R 1b is selected from halogen, CN, C 1 -C 3 alkyl or C 1 -C 3 alkoxy, any of which C 1 -C 3 alkyl or C 1 -C 3 alkoxy Selected to be replaced by R 1c .
在一些实施方案中，R1c选自卤素、OH或CN。In some embodiments, R 1c is selected from halogen, OH, or CN.
在一些实施方案中，R1c选自CN。In some embodiments, R 1c is selected from CN.
在一些实施方案中，R1b选自F、CN、甲基、甲氧基或CH2CN。 In some embodiments, R lb is selected from F, CN, methyl, methoxy, or CH 2 CN.
在一些实施方案中，R1和R2独立选自H、卤素、CN、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C1-C6烷基、C1-C6烷氧基、C3-C8环烷基或4-7元杂环基，所述C1-C6烷基、C1-C6烷氧基、C3-C8环烷基或4-7元杂环基任选被R1a取代，且所述C3-C8环烷基或4-7元杂环基可以是螺环、桥环或并环形式的。In some embodiments, R 1 and R 2 are independently selected from H, halogen, CN, NH 2 , NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl, the C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by R 1a , and the C 3 -C 8 cycloalkyl or 4-7 membered heterocyclyl may be a spiro ring, a bridged ring or a Ring form.
在一些实施方案中，R1和R2独立选自H、卤素、CN、C1-C3烷基、C1-C3烷氧基或C3-C6环烷基，所述C1-C3烷基、C1-C3烷氧基或C3-C6环烷基任选被R1a取代。In some embodiments, R 1 and R 2 are independently selected from H, halogen, CN, C 1 -C 3 alkyl, C 1 -C 3 alkoxy, or C 3 -C 6 cycloalkyl, the C 1 -C 3 alkyl, C 1 -C 3 alkoxy or C 3 -C 6 cycloalkyl is optionally substituted by R 1a .
在一些实施方案中，R1和R2独立选自H、C1-C3烷基或C3-C6环烷基，所述C1-C3烷基或C3-C6环烷基任选被R1a取代。In some embodiments, R 1 and R 2 are independently selected from H, C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl, said C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl The group is optionally substituted by R 1a .
在一些实施方案中，R1a选自卤素、OH、CN、＝O、NH2、C1-C3烷基或C1-C3烷氧基。In some embodiments, R 1a is selected from halogen, OH, CN, =O, NH 2 , C 1 -C 3 alkyl, or C 1 -C 3 alkoxy.
在一些实施方案中，R1和R2独立选自H、甲基或环丁基，或者R1、R2与其连接的原子共同形成如下基团： In some embodiments, R 1 and R 2 are independently selected from H, methyl, or cyclobutyl, or R 1 , R 2 and the atoms to which they are connected together form the following group:
在一些实施方案中，R1、R2与其连接的原子共同形成如下基团： In some embodiments, R 1 , R 2 and the atoms to which they are connected together form the following group:
在一些实施方案中，R1、R2与其连接的原子共同形成如下基团： In some embodiments, R 1 , R 2 and the atoms to which they are connected together form the following group:
在一些实施方案中，R1和R2独立选自H、甲基或环丁基。In some embodiments, R 1 and R 2 are independently selected from H, methyl, or cyclobutyl.
在一些实施方案中，W选自-(CR12R13)W1。In some embodiments, W is selected from -(CR 12 R 13 )W 1 .
在一些实施方案中，R12、R13独立选自H、卤素、OH或甲基。In some embodiments, R 12 , R 13 are independently selected from H, halogen, OH, or methyl.
在一些实施方案中，R12、R13均为H。In some embodiments, R 12 and R 13 are both H.
在一些实施方案中，R1与R12及其各自相连的原子和键共同形成任选被R12a取代的C3-C6环烷基。In some embodiments, R 1 and R 12 and the atoms and bonds to which each is attached together form a C 3 -C 6 cycloalkyl group optionally substituted by R 12a .
在一些实施方案中，R12a选自C1-C3烷基。In some embodiments, R 12a is selected from C 1 -C 3 alkyl.
在一些实施方案中，R12a选自甲基。 In some embodiments, R 12a is selected from methyl.
在一些实施方案中，R1与R12及其各自相连的原子和键共同形成 In some embodiments, R 1 and R 12 and their respective atoms and bonds to which they are attached together form
在一些实施方案中，W选自W1。In some embodiments, W is selected from W 1 .
在一些实施方案中，W1选自任选被R14取代的4-10元杂环基。In some embodiments, W 1 is selected from 4-10 membered heterocyclyl optionally substituted with R 14 .
在一些实施方案中，W1选自任选被R14取代的5-10元杂芳基。In some embodiments, W 1 is selected from 5-10 membered heteroaryl optionally substituted with R 14 .
在一些实施方案中，W1选自任选被R14取代的5元杂芳基。In some embodiments, W 1 is selected from 5-membered heteroaryl optionally substituted with R 14 .
在一些实施方案中，W1选自任选被R14取代的如下基团：吡咯、噻吩、呋喃、吡唑、咪唑、噻唑、异噻唑、噻二唑、三氮唑、噁唑、异噁唑、噁二唑、 In some embodiments, W is selected from the group consisting of pyrrole, thiophene, furan, pyrazole, imidazole, thiazole, isothiazole, thiadiazole, triazole, oxazole, isoxazoles, optionally substituted with R 14 Azole, oxadiazole,
在一些实施方案中，W1选自任选被R14取代的如下基团：三氮唑、噁唑、异噁唑或噁二唑。In some embodiments, W 1 is selected from the group consisting of triazole, oxazole, isoxazole, or oxadiazole optionally substituted with R 14 .
在一些实施方案中，W1选自任选被R14取代的如下基团： In some embodiments, W 1 is selected from the following groups optionally substituted with R 14 :
在一些实施方案中，R14选自卤素、OH、NH2、C1-C3烷基或C3-C6环烷基，所述C1-C3烷基或C3-C6环烷基任选被R14a取代。In some embodiments, R 14 is selected from halogen, OH, NH 2 , C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl, said C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl Alkyl is optionally substituted with R 14a .
在一些实施方案中，R14选自OH、C1-C3烷基或C3-C6环烷基，所述C1-C3烷基或C3-C6环烷基任选被R14a取代。In some embodiments, R 14 is selected from OH, C 1 -C 3 alkyl, or C 3 -C 6 cycloalkyl, which C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl is optionally replaced by R 14a substituted.
在一些实施方案中，R14选自甲基或环丙基，所述甲基或环丙基任选被R14a取代。In some embodiments, R 14 is selected from methyl or cyclopropyl, which is optionally substituted with R 14a .
在一些实施方案中，R14a选自卤素、OH或CN。In some embodiments, R 14a is selected from halogen, OH, or CN.
在一些实施方案中，R14a选自F。In some embodiments, R 14a is selected from F.
在一些实施方案中，R14选自甲基、CHF2或环丙基。In some embodiments, R14 is selected from methyl, CHF2 , or cyclopropyl.
在一些实施方案中，W1选自如下基团： In some embodiments, W is selected from the following groups:
在一些实施方案中，W选自 In some embodiments, W is selected from
在一些实施方案中，本申请的式(V)化合物选自式(VI)化合物：
In some embodiments, compounds of formula (V) of the present application are selected from compounds of formula (VI):
其中，j选自0、1、2或3，Z1、Z2、Y1、Y2、Y3、Y4、X、W、R1、R2和R10如式(V)中所定义。Among them, j is selected from 0, 1, 2 or 3, Z 1 , Z 2 , Y 1 , Y 2 , Y 3 , Y 4 , X, W, R 1 , R 2 and R 10 are as in formula (V) definition.
在一些实施方案中，本申请的式(V)化合物选自式(VII)化合物：
In some embodiments, compounds of formula (V) of the present application are selected from compounds of formula (VII):
其中，Z1、Z2、Z3、Z4、Z5、Y1、Y2、Y3、Y4、X、W、R1、R2和m如式(V)中所定义。Among them, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Y 1 , Y 2 , Y 3 , Y 4 , X, W, R 1 , R 2 and m are as defined in formula (V).
在一些实施方案中，本申请的式(V)化合物选自以下化合物：






In some embodiments, compounds of formula (V) of the present application are selected from the following compounds:






在一些具体的实施方式中，所述CBL-b抑制剂具有WO 2020/264398 A1所述CBL-b抑制剂的结构，将WO 2020/264398 A1的全文通过援引加入的方式并入本申请。In some specific embodiments, the CBL-b inhibitor has the structure of the CBL-b inhibitor described in WO 2020/264398 A1, and the full text of WO 2020/264398 A1 is incorporated into this application by reference.
在一些具体的实施方式中，所述CBL-b抑制剂具有WO 2020/210508 A1所述CBL-b抑制剂的结构，将WO 2020/210508 A1的全文通过援引加入的方式并入本申请。In some specific embodiments, the CBL-b inhibitor has the structure of the CBL-b inhibitor described in WO 2020/210508 A1, and the full text of WO 2020/210508 A1 is incorporated into this application by reference.
在一些具体的实施方式中，所述CBL-b抑制剂为具有如下化学式的化合物或其药学上可接受的盐：

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

在一些具体的实施方式中，CBL-b抑制剂被配制为口服或注射制剂。In some specific embodiments, CBL-b inhibitors are formulated as oral or injectable formulations.
本申请的含CBL-b抑制剂的药物组合物可通过将本申请的CBL-b抑制剂与适宜的药学上可接受的辅料组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等。The pharmaceutical composition containing a CBL-b inhibitor of the present application can be prepared by combining the CBL-b inhibitor of the present application with appropriate pharmaceutically acceptable excipients, for example, it can be formulated into a solid, semi-solid, liquid or gaseous preparation. , such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.
给予本申请CBL-b抑制剂盐或其药物组合物的典型途径包括但不限于口服、直肠、局部、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。Typical routes for administering the CBL-b inhibitor salts or pharmaceutical compositions thereof of the present application include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, Subcutaneous and intravenous administration.
本申请的含CBL-b抑制剂的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、乳化法、冷冻干燥法等。The pharmaceutical composition containing a CBL-b inhibitor of the present application can be manufactured using methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, etc.
在一些实施方案中，本申请CBL-b抑制剂的药物组合物是口服形式。对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的辅料混合，来配制该药物组合物。这些辅料能使本申请的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。可以通过常规的混合、填充或压片方法来制备固体口服组合物。例如，可通过下述方法获得：将所述的活性化合物与固体辅料混合，任选地碾磨所得的混合物，如果需要则加入其它合适的辅料，然后将该混合物加工成颗粒，得到了片 剂或糖衣剂的核心。适合的辅料包括但不限于：粘合剂、稀释剂、崩解剂、润滑剂、助流剂或矫味剂等。药物组合物还可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。In some embodiments, pharmaceutical compositions of CBL-b inhibitors of the present application are in oral form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These excipients enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc. for oral administration to patients. Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets. The core of the agent or sugar coating. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants or flavoring agents, etc. The pharmaceutical compositions may also be suitable for parenteral administration as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.
在一些具体的实施方式中，所述癌症或肿瘤选自血液瘤或实体瘤。在一些具体的实施方式中，所述癌症或肿瘤为实体瘤。在一些具体的实施方式中，所述实体瘤选自胃癌、肝癌、结直肠癌、肾细胞癌、乳腺癌、肺癌、卵巢癌、皮肤癌、膀胱癌、肝癌、前列腺癌、宫颈癌、胰腺癌和肉瘤。In some specific embodiments, the cancer or tumor is selected from hematological tumors or solid tumors. In some specific embodiments, the cancer or tumor is a solid tumor. In some specific embodiments, the solid tumor is selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, renal cell cancer, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcomas.
在免疫效应细胞表达嵌合抗原受体的一些实施方案中，所述肿瘤或癌症是指肿瘤细胞或癌细胞表面表达所述嵌合抗原受体所针对的抗原分子的肿瘤或癌症。以本申请涉及的Claudin18.2为例，在一些实施方案中，肿瘤为高表达Claudin18.2(Claudin18.2+)的肿瘤。在一些实施方案中，高表达Claudin18.2(Claudin18.2+)的肿瘤是指肿瘤细胞群体中至少60％的肿瘤细胞表达Claudin18.2。在一些实施方案中，高表达Claudin18.2(Claudin18.2+)的肿瘤是指肿瘤细胞群体中至少70％的肿瘤细胞表达Claudin18.2。在一些实施方案中，高表达Claudin18.2(Claudin18.2+)的肿瘤是指肿瘤细胞群体中至少80％的肿瘤细胞表达Claudin18.2。在一些实施方案中，高表达Claudin18.2(Claudin18.2+)的肿瘤是指肿瘤细胞群体中至少90％的肿瘤细胞表达Claudin18.2。在一些实施方案中，高表达Claudin18.2(Claudin18.2+)的肿瘤是指肿瘤细胞群体中至少95％的肿瘤细胞表达Claudin18.2。在一些实施方案中，高表达Claudin18.2(Claudin18.2+)的肿瘤是指肿瘤细胞群体中至少98％的肿瘤细胞表达Claudin18.2。在一些实施方案中，高表达Claudin18.2(Claudin18.2+)的肿瘤是指肿瘤细胞群体中至少99％的肿瘤细胞表达Claudin18.2。对于本申请涉及的抗原分子BCMA、GPRC5D以及其他的肿瘤抗原分子也同样适用。In some embodiments where the immune effector cells express the chimeric antigen receptor, the tumor or cancer refers to a tumor or cancer in which the antigen molecule against which the chimeric antigen receptor is directed is expressed on the surface of tumor cells or cancer cells. Taking Claudin18.2 involved in this 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+) means that at least 60% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 70% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 80% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 90% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 95% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 98% of the tumor cells in the tumor cell population express Claudin18.2. In some embodiments, a tumor that highly expresses Claudin18.2 (Claudin18.2+) means that at least 99% of the tumor cells in the tumor cell population express Claudin18.2. The same applies to the antigen molecules BCMA, GPRC5D and other tumor antigen molecules involved in this application.
在一些具体的实施方式中，所述免疫效应细胞选自下组：(1)表达嵌合抗原受体的T细胞；(2)表达嵌合抗原受体的NK细胞；(3)表达嵌合抗原受体的T细胞和表达嵌合抗原受体的NK细胞。In some specific embodiments, the immune effector cells are selected from the following group: (1) T cells expressing chimeric antigen receptors; (2) NK cells expressing chimeric antigen receptors; (3) Chimeric antigen receptor expressing T cells Antigen receptor T cells and NK cells expressing chimeric antigen receptors.
在一些具体的实施方式中，所述免疫效应细胞包含表达嵌合抗原受体的T细胞和/或表达嵌合抗原受体的NK细胞，并且T细胞表达的嵌合抗原受体和所述NK细胞表达的嵌合抗原受体结合相同或不同的抗原或抗原表位。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 NK cells Cell-expressed chimeric antigen receptors bind the same or different antigens or epitopes.
在一些具体的实施方式中，所述免疫效应细胞表达嵌合抗原受体，并且所述嵌合抗原受体所结合的抗原独立地选自Claudin18.2、BCMA、GPRC5D或它们的任意组合。例如，抗原可以为单独的Claudin18.2、BCMA或GPRC5D，Claudin18.2、BCMA与GPRC5D的两两组合，或Claudin18.2、BCMA或GPRC5D三者。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 combination of Claudin18.2, BCMA and GPRC5D, or Claudin18.2, BCMA or GPRC5D.
在一些具体的实施方式中，本申请的嵌合抗原受体包含靶向CLDN18.2的抗体或抗体片段作为抗原结合区，并且本申请也单独提供这样的靶向CLDN18.2的抗体或抗体片段。In some specific embodiments, the chimeric antigen receptor of the present application includes an antibody or antibody fragment targeting CLDN18.2 as the antigen-binding region, and the application also provides such an antibody or antibody fragment targeting CLDN18.2 separately. .
在一些具体的实施方式中，所述靶向CLDN18.2的抗体或抗体片段的VH或VL序列与下述SEQ ID NO:8或SEQ ID NO:9所示序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性：In some specific embodiments, the VH or VL sequence of the antibody or antibody fragment targeting CLDN18.2 has at least 80% or at least 85% similarity with the sequence shown in the following SEQ ID NO: 8 or SEQ ID NO: 9 , at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical:
VH序列(SEQ ID NO:8)：
VH sequence (SEQ ID NO:8):
VL序列(SEQ ID NO:9):
VL sequence (SEQ ID NO:9):
根据Kabat编码系统，SEQ ID NO:8-9所示VH/VL具有如下所示的CDR：
According to the Kabat coding system, the VH/VL shown in SEQ ID NO:8-9 has the following CDR:
在一些具体的实施方式中，所述抗体或抗体片段为scFv，所述scFv与下述SEQ ID NO:16或SEQ ID NO:17序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性：
In some specific embodiments, the antibody or antibody fragment is a scFv that has at least 80%, at least 85%, at least 90%, or at least 95% similarity to the following SEQ ID NO: 16 or SEQ ID NO: 17 sequence. %, at least 96%, at least 97%, at least 98% or 100% identity:
在一些具体的实施方式中，本申请的嵌合抗原受体包含靶向BCMA的抗体或抗体片段作为抗原结合区，并且本申请也单独提供这样的靶向BCMA的抗体或抗体片段。In some specific embodiments, the chimeric antigen receptor of the present application includes an antibody or antibody fragment targeting BCMA as the antigen-binding region, and the application also provides such an antibody or antibody fragment targeting BCMA separately.
在一些具体的实施方式中，所述靶向BCMA的抗体或抗体片段为纳米抗体或VHH。在一些具体的实施方式中，所述靶向BCMA的抗体或抗体片段与下述SEQ ID NO:18或SEQ ID NO:19序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性：
In some specific embodiments, the BCMA-targeting antibody or antibody fragment is a Nanobody or VHH. In some specific embodiments, the BCMA-targeting 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% identical:
在一些具体的实施方式中，所述靶向BCMA的VHH通过连接子串联。In some specific embodiments, the BCMA-targeting VHHs are connected in series via a linker.
根据IMGT编码系统，上述SEQ ID NO:18-19所示的VHH具有如下所示的CDR：
According to the IMGT coding system, the VHH shown in SEQ ID NO:18-19 above has the CDR shown below:
在一些具体的实施方式中，本申请进一步提供靶向BCMA的不同序列组成的抗体或抗体片段。In some specific embodiments, the present application further provides antibodies or antibody fragments targeting different sequence compositions of BCMA.
在一些具体的实施方式中，所述BCMA的抗体或抗体片段为纳米抗体或VHH。在一些具体的实施方式中，所述BCMA的抗体或抗体片段与下述SEQ ID NO:38序列相比具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性：
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:
根据Kabat编码系统，上述SEQ ID NO:38所示的VHH具有如下所示的CDR：
According to the Kabat encoding system, the VHH shown in SEQ ID NO:38 above has the CDR shown below:
在一些具体的实施方式中，本申请的嵌合抗原受体包含靶向GPRC5D的抗体或抗体片段作为抗原结合区，并且本申请也单独提供这样的靶向GPRC5D的抗体或抗体片段。In some specific embodiments, the chimeric antigen receptor of the present application includes an antibody or antibody fragment targeting GPRC5D as the antigen-binding region, and the application also provides such an antibody or antibody fragment targeting GPRC5D separately.
在一些具体的实施方式中，所述靶向GPRC5D的抗体或抗体片段为纳米抗体或VHH。在一些具体的实施方式中，所述靶向GPRC5D的抗体或抗体片段与下述SEQ ID NO:42序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性：
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% similarity with the following SEQ ID NO:42 sequence. %, at least 98% or 100% identity:
根据Kabat编码系统，上述SEQ ID NO:42所示的VHH具有如有所示的CDR：
According to the Kabat encoding system, the VHH shown in SEQ ID NO:42 above has CDRs as shown:
在一些具体的实施方式中，所述免疫效应细胞包含表达嵌合抗原受体的T细胞，并且所述T细胞表达的所述嵌合抗原受体包括信号肽、抗原结合区、铰链区、跨膜区和胞内信号传导结构区。In some specific embodiments, the immune effector cells comprise T cells expressing chimeric antigen receptors, and the chimeric antigen receptors expressed by the T cells include a signal peptide, an antigen-binding region, a hinge region, a trans- membrane region and intracellular signaling structural region.
在一些具体的实施方式中，所述嵌合抗原受体包括CD8α信号肽、抗原特异性结合序列、 CD8α或CD28铰链区、CD8α或CD28跨膜区、CD28共刺激结构域和CD3ζ。In some specific embodiments, the chimeric antigen receptor includes a CD8α signal peptide, an antigen-specific binding sequence, CD8α or CD28 hinge region, CD8α or CD28 transmembrane region, CD28 costimulatory domain and CD3ζ.
在一些具体的实施方式中，所述CD8α信号肽与下述SEQ ID NO:7具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性。
In some specific embodiments, the CD8α signal peptide 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.
在一些具体的实施方式中，所述铰链区为CD8α铰链区。在一些具体的实施方式中，所述CD8α铰链区具有与下述SEQ ID NO:26或SEQ ID NO:37序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the hinge region is a CD8 alpha hinge region. In some specific embodiments, the CD8 alpha hinge region has a molecular weight of at least 80%, at least 85%, at least 90%, at least 95%, at least 96% compared to the following SEQ ID NO: 26 or SEQ ID NO: 37 sequence. Sequences that are %, at least 97%, at least 98% or 100% identical to:
在一些具体的实施方式中，所述铰链区为CD28铰链区。在一些具体的实施方式中，所述CD28铰链区具有与下述SEQ ID NO:27序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the hinge region is a CD28 hinge region. In some specific embodiments, the CD28 hinge region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, compared to the following SEQ ID NO:27 sequence. Sequences with at least 98% or 100% identity:
在一些具体的实施方式中，跨膜区可选自CD8α跨膜区、CD28跨膜区和NKG2D跨膜区。In some specific embodiments, the transmembrane region can be selected from the group consisting of CD8α transmembrane region, CD28 transmembrane region and NKG2D transmembrane region.
在一些具体的实施方式中，所述CD8α跨膜区具有与下述SEQ ID NO:28序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the CD8α transmembrane region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:28 sequence. , a sequence of at least 98% or 100% identity:
在一些具体的实施方式中，所述CD28跨膜区具有与下述SEQ ID NO:29序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the CD28 transmembrane region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:29 sequence. , a sequence of at least 98% or 100% identity:
在一些具体的实施方式中，所述NKG2D跨膜区具有与下述SEQ ID NO:30序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the NKG2D transmembrane region has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:30 sequence. , a sequence of at least 98% or 100% identity:
在一些具体的实施方式中，所述CD3ζ具有与下述SEQ ID NO:31序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the CD3ζ has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% compared to the following SEQ ID NO:31 sequence. % or 100% identity of the sequence:
在一些具体的实施方式中，所述共刺激结构域来自CD28、4-1BB或2B4。In some specific embodiments, the costimulatory domain is from CD28, 4-1BB or 2B4.
在一些具体的实施方式中，所述CD28共刺激结构域具有与下述SEQ ID NO:32序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the CD28 co-stimulatory domain has a protein content that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:32 sequence. %, at least 98% or 100% identical sequence:
在一些具体的实施方式中，所述4-1BB共刺激结构域具有与下述SEQ ID NO:33序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the 4-1BB costimulatory domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, compared to the following SEQ ID NO:33 sequence. Sequences that are at least 97%, at least 98% or 100% identical:
在一些具体的实施方式中，所述2B4共刺激结构域具有与下述SEQ ID NO:34序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the 2B4 co-stimulatory domain has a molecular weight of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% compared to the following SEQ ID NO:34 sequence. %, at least 98% or 100% identical sequence:
在一些具体的实施方式中，所述嵌合抗原受体还通过自裂解肽连接细胞因子或趋化因子。在一些具体的实施方式中，所述自裂解肽具有与下述SEQ ID NO:35序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
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 at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, compared to the following SEQ ID NO:35 sequence. Sequences with at least 98% or 100% identity:
在一些具体的实施方式中，所述细胞因子或趋化因子为IL15。在一些具体的实施方式中，所述IL15具有与下述SEQ ID NO:36序列相比，至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列：
In some specific embodiments, the cytokine or chemokine is IL15. In some specific embodiments, the IL15 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% compared to the following SEQ ID NO:36 sequence. % or 100% identity of the sequence:
在一些具体的实施方式中，所述抗原结合区或靶点结合序列的形式为具有VH-连接子-VL或VL-连接子-VH结构的scFv。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.
在一些具体的实施方式中，所述嵌合抗原受体包含SEQ ID NO:2-4任一项所示序列，或包含与SEQ ID NO:2-4任一项序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列。In some specific embodiments, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NO:2-4, or has at least 80%, at least Sequences that are 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.
在一些具体的实施方式中，所述免疫效应细胞包含表达嵌合抗原受体的NK细胞，并且所述NK细胞表达的嵌合抗原受体包括信号肽、抗原结合区或靶点结合序列、铰链区、跨膜区、共刺激结构域和CD3ζ。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 signal peptides, antigen-binding regions or target binding sequences, and hinges. region, transmembrane region, costimulatory domain and CD3ζ.
在一些具体的实施方式中，所述嵌合抗原受体还包括通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子。在一些具体的实施方式中，细胞因子为IL15。In some specific embodiments, the chimeric antigen receptor further includes a cytokine or chemokine linked to the CD3ζ via a self-cleaving peptide. In some specific embodiments, the cytokine is IL15.
在一些具体的实施方式中，NK细胞表达的嵌合抗原受体包括CD8α信号肽、抗原结合区或靶点结合序列、CD8α铰链区、CD8α或CD28或NKG2D跨膜区、CD28或2B4或4-1BB共刺激结构域和CD3ζ，以及优选通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子。在一些具体的实施方式中，细胞因子为IL15。In some specific embodiments, the chimeric antigen receptor expressed by NK cells includes CD8α signal peptide, antigen-binding region or target binding sequence, CD8α hinge region, CD8α or CD28 or NKG2D transmembrane region, CD28 or 2B4 or 4- 1BB costimulatory domain and CD3ζ, and a cytokine or chemokine preferably linked to said CD3ζ via a self-cleaving peptide. In some specific embodiments, the cytokine is IL15.
在一些具体的实施方式中，NK细胞表达的嵌合抗原受体包含SEQ ID NO:1、5-6和46 任一项所示序列，或包含与SEQ ID NO:1、5-6任一项序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列。In some specific embodiments, the NK cell-expressed chimeric antigen receptor comprises SEQ ID NOs: 1, 5-6, and 46 The sequence shown in any one of them, or comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% of the sequence of any one of SEQ ID NO: 1, 5-6 %, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical sequences.
在第四方面，本申请还提供一种体外扩增免疫效应细胞的方法，所述方法包括：在CBL-b抑制剂存在的条件下，扩增培养所述免疫效应细胞。In a fourth aspect, the present application also provides a method for amplifying immune effector cells in vitro, which method includes: amplifying and culturing the immune effector cells in the presence of a CBL-b inhibitor.
在一些具体的实施方式中，所述免疫效应细胞为T细胞或NK细胞。在一些具体的实施方式中，所述T细胞或NK细胞表达嵌合抗原受体的T细胞或表达嵌合抗原受体的NK细胞。In some specific embodiments, the immune effector cells are T cells or NK cells. In some specific embodiments, the T cells or NK cells express chimeric antigen receptor T cells or chimeric antigen receptor expressing NK cells.
在一些具体的实施方式中，所述CBL-b抑制剂在培养过程中的浓度为0.1nM～10μM，例如1μM。In some specific embodiments, the concentration of the CBL-b inhibitor during the culture process is 0.1 nM ~ 10 μM, such as 1 μM.
在一些具体的实施方式中，所述T细胞或NK细胞被转染嵌合抗原受体，并且所述CBL-b抑制剂在转染表达所述嵌合抗原受体的核酸分子之前或之后，添加所述CBL-b抑制剂。In some specific embodiments, the T cells or NK cells are transfected with a chimeric antigen receptor, and the CBL-b inhibitor is before or after transfection of a nucleic acid molecule expressing the chimeric antigen receptor, Add the CBL-b inhibitor.
在一些具体的实施方式中，在所述T细胞或NK细胞开始激活时添加所述CBL-b抑制剂，或在所述T细胞或NK细胞激活培养4-7天后，添加所述CBL-b抑制剂。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 is added after the T cells or NK cells are activated and cultured for 4-7 days. Inhibitors.
在第五方面，本申请还提供一种工程化的NK细胞，其中，所述NK细胞表达特异性结合Claudin18.2的嵌合抗原受体，所述嵌合抗原受体包括CD8α信号肽、特异性结合Claudin18.2的抗原结合区、CD8α铰链区、CD8α跨膜区、CD28共刺激结构域和CD3ζ。在一些具体的实施方式中，所述嵌合抗原受体还包括通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子。在一些具体的实施方式中，细胞因子为IL15。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, a specific Sexually binds to the antigen-binding region of Claudin18.2, CD8α hinge region, CD8α transmembrane region, CD28 costimulatory domain and CD3ζ. In some specific embodiments, the chimeric antigen receptor further includes a cytokine or chemokine linked to the CD3ζ via a self-cleaving peptide. In some specific embodiments, the cytokine is IL15.
在一些具体的实施方式中，所述抗原结合区为scFv的形式。在一些具体的实施方式中，所述抗原结合区为具有VH-连接肽-VL结构的scFv。在一些具体的实施方式中，所述VH包括如SEQ ID NO:10-12所示的HCDR1-HCDR3，所述VL包括如SEQ ID NO:13-15所示的LCDR1-LCDR3。在一些具体的实施方式中，所述VH具有与SEQ ID NO:8所示序列至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列，所述VL具有与SEQ ID NO:9所示序列至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列；最优选地，所述scFv具有与SEQ ID NO:16-17所示序列至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列。In some specific embodiments, the antigen binding region is in the form of a scFv. In some specific embodiments, the antigen-binding region is a scFv having a VH-linker peptide-VL structure. In some specific embodiments, the VH includes HCDR1-HCDR3 as shown in SEQ ID NO: 10-12, and the VL includes LCDR1-LCDR3 as shown in SEQ ID NO: 13-15. In some specific embodiments, the VH has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least the sequence shown in SEQ ID NO:8 A sequence that is 95%, at least 96%, at least 97%, at least 98% or 100% identical, and the VL has at least 80%, at least 85%, at least 90%, at least 91% with the sequence shown in SEQ ID NO:9 , a sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical; most preferably, the scFv has a sequence identical to SEQ ID NO: 16 -17 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 % identity of the sequence.
在一些具体的实施方式中，所述嵌合抗原受体具有如SEQ ID NO:1所示序列至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列。In some specific embodiments, the chimeric antigen receptor has the sequence shown in SEQ ID NO: 1 at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least Sequences that are 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical.
在第六方面，本申请还提供前述NK细胞与表达特异性结合Claudin18.2的嵌合抗原受体的T细胞的组合在制备用于治疗癌症或肿瘤的药物中的应用。In a sixth aspect, the present application also provides the use of a combination of the aforementioned NK cells and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2 in the preparation of drugs for the treatment of cancer or tumors.
在第七方面，本申请还提供用于治疗癌症或肿瘤的细胞药物，其包含前述NK细胞与表达特异性结合Claudin18.2的嵌合抗原受体的T细胞。In a seventh aspect, the present application also provides a cellular drug for treating cancer or tumors, which includes the aforementioned NK cells and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2.
在第八方面，本申请还提供治疗癌症或肿瘤的方法，所述方法包括向有需要的受试者给予有效量的前述NK细胞，并任选地，给予表达特异性结合Claudin18.2的嵌合抗原受体的T 细胞。In an eighth aspect, the present application also provides a method for treating cancer or tumors, the method comprising administering an effective amount of the aforementioned NK cells to a subject in need thereof, and optionally, administering a chimeric gene that expresses specific binding to Claudin18.2. T with antigen receptor cell.
在一些具体的实施方式中，T细胞表达的嵌合抗原受体包括CD8α信号肽、靶点结合序列、CD8α或CD28铰链区、CD8α或CD28跨膜区、CD28共刺激结构域和CD3ζ。在一些具体的实施方式中，所述抗原结合区的形式为具有VH-连接子-VL或VL-连接子-VH结构的scFv。In some specific embodiments, the chimeric antigen receptor expressed by the T cell includes 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.
在一些具体的实施方式中，T细胞表达的所述嵌合抗原受体包含SEQ ID NO:2-4任一项序列或与SEQ ID NO:2-4任一项序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列。In some specific embodiments, the chimeric antigen receptor expressed by T cells comprises any sequence of SEQ ID NO:2-4 or has at least 80%, at least Sequences that are 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.
在第九方面，本申请提供以下(a)-(c)中任一项组合在制备用于治疗肿瘤或癌症的药物中的应用：In a ninth aspect, the application provides the use of any combination of the following (a)-(c) in the preparation of a medicament for treating tumors or cancer:
(a)表达嵌合抗原受体的免疫效应细胞和CBL-b抑制剂，(a) Immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,
(b)表达嵌合抗原受体的免疫效应细胞与PDL1-IL15融合蛋白，(b) Immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,
(c)表达嵌合抗原受体的免疫效应细胞、CBL-b抑制剂和PDL1-IL15融合蛋白。(c) Immune effector cells expressing chimeric antigen receptor, CBL-b inhibitor and PDL1-IL15 fusion protein.
在第十方面，本申请提供用于治疗癌症或肿瘤的药物组合产品，其包含以下(a)-(c)中任一项：In a tenth aspect, the present application provides a pharmaceutical combination product for treating cancer or tumors, which includes any one of the following (a)-(c):
(a)表达嵌合抗原受体的免疫效应细胞和CBL-b抑制剂，(a) Immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,
(b)表达嵌合抗原受体的免疫效应细胞与PDL1-IL15融合蛋白，(b) Immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,
(c)表达嵌合抗原受体的免疫效应细胞、CBL-b抑制剂和PDL1-IL15融合蛋白。(c) Immune effector cells expressing chimeric antigen receptor, CBL-b inhibitor and PDL1-IL15 fusion protein.
在第十一方面，本申请提供治疗癌症或肿瘤的方法，所述方法包括给予有需要的受试者有效量的表达嵌合抗原受体的免疫效应细胞，所述方法还包括：(1)在给药前，使用CBL-b抑制剂刺激所述免疫效应细胞，和/或(2)给予受试者有效量PDL1-IL15融合蛋白。In an eleventh aspect, the application provides a method of treating cancer or tumors, the method comprising administering an effective amount of immune effector cells expressing chimeric antigen receptors to a subject in need thereof, the method further comprising: (1) Before administration, use a CBL-b inhibitor to stimulate the immune effector cells, and/or (2) administer an effective amount of PDL1-IL15 fusion protein to the subject.
在一些具体的实施方式中，所述嵌合抗原受体所结合的抗原独立地选自Claudin18.2、BCMA、GPRC5D或它们的任意组合。例如，抗原可以为单独的Claudin18.2、BCMA或GPRC5D，Claudin18.2、BCMA与GPRC5D的两两组合，或Claudin18.2、BCMA或GPRC5D三者。在一些实施方案中，所述嵌合抗原受体包含特异性结合BCMA和/或GPRC5D的靶点结合序列。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 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 BCMA and/or GPRC5D.
在一些具体的实施方式中，所述抗原结合区包含特异性结合BCMA和GPRC5D的靶点结合序列，并且为VHH的形式。In some specific embodiments, the antigen-binding region includes a target-binding sequence that specifically binds BCMA and GPRC5D and is in the form of a VHH.
在一些具体的实施方式中，所述VHH包括具有SEQ ID NO:39-41所示序列的第一组HCDR1-HCDR3和/或SEQ ID NO:43-45所示序列的第二组HCDR1-HCDR3。In some specific embodiments, the VHH includes a first set of HCDR1-HCDR3 having the sequences set forth in SEQ ID NO:39-41 and/or a second set of HCDR1-HCDR3 having the sequence set forth in SEQ ID NO:43-45 .
在一些具体的实施方式中，所述VHH具有SEQ ID NO:38和/或42所示序列或与SEQ ID NO:38至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列和/或与SEQ ID NO:42所示序列至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列。 In some specific embodiments, the VHH has the sequence shown in SEQ ID NO:38 and/or 42 or is at least 80%, at least 85%, at least 90%, at least 91%, at least 92% identical to SEQ ID NO:38 , a sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical and/or at least 80%, at least 85% identical to the sequence shown in SEQ ID NO:42 , a sequence that is 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.
在一些具体的实施方式中，具有SEQ ID NO:46所示序列或与SEQ ID NO:46所示序列至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或100％同一性的序列。In some specific embodiments, having the sequence shown in SEQ ID NO:46 or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% with the sequence shown in SEQ ID NO:46 , a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% identical.
在一些实施方案中，所述免疫效应细胞选自T细胞(例如细胞毒性T细胞、辅助T细胞、肿瘤浸润T细胞)、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、嗜中性粒细胞、巨噬细胞和树突状细胞。在一些实施方案中，所述免疫效应细胞为NK细胞。In some embodiments, the immune effector cells are selected from the group consisting of 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.
在一些实施方案中，所述PDL1-IL15融合蛋白具有如SEQ ID NO:47所示的序列。In some embodiments, the PDL1-IL15 fusion protein has the sequence set forth in SEQ ID NO:47.
在一些实施方案中，所述肿瘤或癌症为B细胞淋巴瘤。在一些实施方案中，所述肿瘤或癌症为多发性骨髓瘤(MM)。在一些实施方案中，所述多发性骨髓瘤为难治性或复发性多发性骨髓瘤。In some embodiments, the tumor or cancer is 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.
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。It should be understood that the above detailed description is only to enable those skilled in the art to understand the contents of the present application more clearly, and is not intended to be limiting in any respect. Those skilled in the art will be able to make various modifications and changes to the described embodiments.
实施例Example
下面结合具体实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。The present application will be further described below in conjunction with specific embodiments. The advantages and features of the present application will become clearer with the description. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
本申请实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本申请的精神和范围下可以对本申请技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。The embodiments of this application are only exemplary and do not constitute any limitation on the scope of this application. Those skilled in the art should understand that the details and forms of the technical solution of the present application can be modified or replaced without departing from the spirit and scope of the present application, but these modifications and substitutions all fall within the protection scope of the present application.
实施例1嵌合抗原受体的构建和病毒包装Example 1 Construction of chimeric antigen receptor and virus packaging
参照图1，构建用于NK细胞或T细胞的嵌合抗原受体，将编码用于NK细胞的嵌合抗原受体的核酸克隆至逆转录病毒穿梭质粒，编码用于T细胞的嵌合抗原受体的核酸克隆至慢病毒穿梭质粒，并通过包装质粒进行病毒包装。嵌合抗原受体及质粒构建、病毒包装的具体信息如下所示：Referring to Figure 1, a chimeric antigen receptor for NK cells or T cells is constructed, and the nucleic acid encoding the chimeric antigen receptor for NK cells is cloned into a retrovirus shuttle plasmid to encode the chimeric antigen for T cells. The nucleic acid of the receptor is cloned into the lentiviral shuttle plasmid, and the virus is packaged through the packaging plasmid. The detailed information on chimeric antigen receptor and plasmid construction and virus packaging is as follows:
表1.嵌合抗原受体中各元件的氨基酸序列

Table 1. Amino acid sequence of each element in the chimeric antigen receptor

质粒构建：合成编码嵌合抗原受体的多聚核苷酸，并在其两端增加酶切位点和穿梭质粒 同源序列。使用限制性内切酶对穿梭质粒进行酶切，琼脂糖凝胶电泳回收纯化线性质粒。将以上步骤中合成的多聚核苷酸和线性化载体通过重组酶5×In-FusionHD酶(TaKaRa，Cat#ST0344)进行连接，反应体系如下：2μl多聚核苷酸片段(50ng/μl)，1μl线性化质粒(50ng/μl)，2μl 5×HD In-Fusion酶，5μl ddH2O。混匀后短暂离心，50℃反应15分钟。取10μl重组反应产物加入到100μl细菌感受态细胞中，冰上放置5分钟，将转化菌液均匀涂布在含有100μg/ml氨苄青霉素的LB平板上，在恒温培养箱中倒置培养12-16小时。随机从每个平板上挑取3-5个克隆进行测序鉴定。将测序正确的菌液转接于100ml含100μg/ml氨苄青霉素的LB液体培养基中，37℃培养过夜，用MN无内毒素质粒中抽试剂盒(MN，Cat#740420.50)进行质粒抽提，定量后用无内毒超纯水稀释至1000ng/μl。Plasmid construction: synthesize polynucleotides encoding chimeric antigen receptors, and add restriction sites and shuttle plasmids to both ends homologous sequences. The shuttle plasmid was digested with restriction enzymes, and the linear plasmid was recovered and purified by agarose gel electrophoresis. The polynucleotide synthesized in the above steps and the linearized vector were connected using recombinase 5×In-FusionHD enzyme (TaKaRa, Cat#ST0344). The reaction system was as follows: 2 μl polynucleotide fragment (50 ng/μl) , 1μl linearized plasmid (50ng/μl), 2μl 5×HD In-Fusion enzyme, 5μl ddH 2 O. After mixing, centrifuge briefly and react at 50°C for 15 minutes. Add 10 μl of the recombinant reaction product to 100 μl of bacterial competent cells, place on ice for 5 minutes, spread the transformed bacterial solution evenly on an LB plate containing 100 μg/ml ampicillin, and incubate upside down in a constant temperature incubator for 12-16 hours. . Randomly pick 3-5 clones from each plate for sequencing and identification. Transfer the correctly sequenced bacterial liquid into 100 ml of LB liquid culture medium containing 100 μg/ml ampicillin, culture it at 37°C overnight, and use the MN endotoxin-free plasmid extraction kit (MN, Cat#740420.50) to extract the plasmid. After quantification, dilute to 1000ng/μl with endotoxic-free ultrapure water.
慢病毒制备：将293T细胞(中国科学院典型培养物保藏委员会细胞库，Cat#GNHu17)接种至100mm培养皿中培养，培养基为加入10％FBS(Gibco，Cat#10099141)的DMEM培养基(Gibco，Cat#10566016)。待细胞融合度达到70％左右时进行质粒转染：取表达嵌合抗原受体的慢病毒穿梭质粒，与慢病毒包装质粒(pRRE、pRSV-Rev和pVSV-G)混合后加入1.2ml Opti-MEM培养基(Thermofisher Scientific，Cat#31985070)，加入35μl Fugene HD(Promega，Cat#04709691001)，混匀后室温孵育15分钟。最后将混合物加入293T细胞，37℃，5％CO2条件下培养2天，收集上清，0.45μm滤膜过滤，浓缩备用。Lentivirus preparation: 293T cells (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, Cat#GNHu17) were inoculated into a 100mm culture dish and cultured in DMEM medium (Gibco) added with 10% FBS (Gibco, Cat#10099141). , Cat#10566016). When the cell confluence reaches about 70%, carry out plasmid transfection: take the lentiviral shuttle plasmid expressing the chimeric antigen receptor, mix it with the lentiviral packaging plasmid (pRRE, pRSV-Rev and pVSV-G) and add 1.2ml Opti- MEM medium (Thermofisher Scientific, Cat#31985070), add 35 μl Fugene HD (Promega, Cat#04709691001), mix and incubate at room temperature for 15 minutes. Finally, the mixture was added to 293T cells and cultured for 2 days at 37°C and 5% CO2 . The supernatant was collected, filtered with a 0.45 μm filter, and concentrated for later use.
逆转录病毒制备：病毒包装前一天，293T细胞(购自ATCC)按1E7个/10cm接种至培养皿。转染细胞时，将包装质粒和逆转录病毒穿梭质粒混合后加入α-MEM培养基中，在另一含α-MEM培养基的离心管内加入HD转染试剂(Promega，E2311)。稀释后的转染试剂逐滴加入稀释的质粒上方，混匀，室温静置15分钟，将混合物加入到培养293T细胞的培养皿内，轻晃10次、混匀，放入培养箱培养。细胞转染3天后，收获病毒，将10ml含病毒培养上清转入50ml离心管，4℃，1250rpm，离心5分钟，去除死亡的293T细胞，过滤后浓缩备用。Preparation of retrovirus: One day before virus packaging, 293T cells (purchased from ATCC) were inoculated into the culture dish at 1E7 cells/10cm. When transfecting cells, mix the packaging plasmid and the retrovirus shuttle plasmid and add them to α-MEM medium. Add them to another centrifuge tube containing α-MEM medium. HD transfection reagent (Promega, E2311). Add the diluted transfection reagent drop by drop on top of the diluted plasmid, mix well, and let stand at room temperature for 15 minutes. Add the mixture to the culture dish for culturing 293T cells, shake gently 10 times, mix well, and place it in an incubator for culture. Three days after cell transfection, harvest the virus. Transfer 10 ml of virus-containing culture supernatant into a 50 ml centrifuge tube, centrifuge at 4°C, 1250 rpm for 5 minutes to remove dead 293T cells, filter and concentrate for later use.
实施例2靶向CLDN18.2的CAR-NK的制备和功能评价Example 2 Preparation and functional evaluation of CAR-NK targeting CLDN18.2
1.CAR-NK细胞的制备1. Preparation of CAR-NK cells
取健康捐献者的新鲜外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)，室温，500g离心7分钟，弃上清，按照Human NK Cell分离试剂盒(Stemcell,17955)分离NK细胞。分离的NK细胞用K562细胞激活并转染CAR：Take fresh peripheral blood mononuclear cells (PBMC) from healthy donors, centrifuge them at 500g for 7 minutes at room temperature, discard the supernatant, and isolate NK cells according to the Human NK Cell isolation kit (Stemcell, 17955). Isolated NK cells were activated with K562 cells and transfected with CAR:
第0天，用AO/PI计数，按NK:K562＝1:2混合细胞，以2ml/孔将混合细胞添加至Non-Treated 6孔板(培养基为含200IU/ml人IL-2的NK细胞培养基(Miltenyi Biotec，130-114-429))，在37℃、5％CO2条件下培养。On day 0, count using AO/PI, mix cells at NK:K562=1:2, add mixed cells to Non-Treated 6-well plate at 2ml/well (the culture medium is NK containing 200IU/ml human IL-2 Cell culture medium (Miltenyi Biotec, 130-114-429)), cultured at 37°C, 5% CO2 .
第4天，每孔添加3ml培养基。On day 4, add 3 ml of culture medium to each well.
第5天，用浓度为7μg/ml RetroNectin试剂(Takara，T202)包被24孔板，每孔500μl，4℃过夜。On the 5th day, the 24-well plate was coated with RetroNectin reagent (Takara, T202) at a concentration of 7 μg/ml, 500 μl per well, and kept overnight at 4°C.
第6天，添加包装18.2-CAR1(NK)的逆转录病毒到24孔板中，2000g，4-8℃离心60分钟，弃去上层病毒液。按3E5个/孔将激活的NK细胞添加至24孔板，400g室温离心5分钟后，在37℃,5％CO2条件下培养。On day 6, add the retrovirus packaging 18.2-CAR1(NK) to the 24-well plate, centrifuge at 2000g and 4-8°C for 60 minutes, and discard the upper virus liquid. Add activated NK cells to a 24-well plate at 3E5 cells/well, centrifuge at 400g for 5 minutes at room temperature, and then culture at 37°C and 5% CO2 .
第7天，将转染后的NK转移到Non-Treated 6孔板中继续培养6-7天。 On day 7, transfer the transfected NK to a Non-Treated 6-well plate and continue culturing for 6-7 days.
检测NK细胞在培养过程中的增殖情况，结果如图2A所示。与UTNK相比，NK细胞转染18.2-CAR1(NK)后，体外扩增能力得到有效提高，第13天(即转染后第7天)，其扩增倍数超过150倍。The proliferation of NK cells during culture was detected, and the results are shown in Figure 2A. Compared with UTNK, after NK cells were transfected with 18.2-CAR1 (NK), the in vitro amplification ability was effectively improved. On day 13 (i.e., the 7th day after transfection), the expansion fold exceeded 150 times.
2.CAR转染效率的检测2. Detection of CAR transfection efficiency
转染后第7天取一定量NK细胞，洗涤后用PBS稀释细胞至2E6个/ml，按50μl/mL细胞稀释液的比例加入Fc受体阻断剂(BioLegend，Cat#422302)，室温孵育10分钟，然后按每孔100μl转移至96孔FACS反应板中。每孔加入100μl浓度为2μg/μl的CLDN18.2-His标签蛋白(Acro，Cat#CL2-H5546)，冰上孵育20分钟。FACS缓冲液离心洗涤2次，每孔加入100μl THETM His标签抗体(Genscript，Cat#A00612)，冰上孵育20分钟。FACS缓冲液离心洗涤3次，100μl FACS缓冲液重悬细胞，用流式仪(BD，CANTOII)进行检测和分析结果。结果如图2B所示，18.2-CAR1(NK)的表达率达88.5％，显示良好的病毒转染效率。On the 7th day after transfection, take a certain amount of NK cells, wash and dilute the cells with PBS to 2E6 cells/ml, add Fc receptor blocker (BioLegend, Cat#422302) at a ratio of 50 μl/mL cell dilution, and incubate at room temperature. 10 minutes, then transfer 100 μl per well to a 96-well FACS reaction plate. Add 100 μl of CLDN18.2-His-tagged protein (Acro, Cat#CL2-H5546) at a concentration of 2 μg/μl to each well and incubate on ice for 20 minutes. Centrifuge and wash twice with FACS buffer, add 100μl THETM His tag antibody (Genscript, Cat#A00612) to each well, and incubate on ice for 20 minutes. Centrifuge and wash 3 times with FACS buffer, resuspend the cells in 100 μl FACS buffer, and use a flow cytometer (BD, CANTOII) to detect and analyze the results. The results are shown in Figure 2B. The expression rate of 18.2-CAR1(NK) reached 88.5%, indicating good virus transfection efficiency.
3.CAR-NK细胞对高表达CLDN18.2胃癌肿瘤细胞杀伤效果的检测3. Detection of the killing effect of CAR-NK cells on gastric cancer tumor cells with high expression of CLDN18.2
构建高表达人CLDN18.2和细菌荧光素酶(Luciferase)的NUGC4稳转株NUGC4-luc、高表达人CLDN18.2的CHO-K1稳转细胞株CHOK1-18.2作为靶细胞，用于检测18.2-CAR1(NK)和UTNK对靶细胞的杀伤效果。The NUGC4 stably transduced cell line NUGC4-luc, which highly expresses human CLDN18.2 and bacterial luciferase (Luciferase), and the CHO-K1 stably transduced cell line CHOK1-18.2, which highly expresses human CLDN18.2, were constructed as target cells for the detection of 18.2- Killing effect of CAR1(NK) and UTNK on target cells.
4h肿瘤细胞杀伤实验：按20000个/孔将靶细胞NUGC4-luc铺在96孔板上。分别按10:1、2:1、1:1和1:2的效靶比(E:T)，加入18.2-CAR1(NK)细胞或UTNK共培养。4小时后取样，加入萤火虫荧光素酶底物D-荧光素(Abcam，Cat#ab143655)，读取生物荧光值(PE EnSight酶标仪)，结果如图2C所示。相较UTNK，18.2-CAR1(NK)细胞对NUGC4-luc细胞在短时间内显示出强杀伤活性，在高E:T比时，18.2-CAR1(NK)对NUGC4-luc细胞的杀伤效率达到100％。4h tumor cell killing experiment: Plate target cells NUGC4-luc on a 96-well plate at 20,000 cells/well. Add 18.2-CAR1 (NK) cells or UTNK to co-culture according to the effect-to-target ratio (E:T) of 10:1, 2:1, 1:1 and 1:2 respectively. After 4 hours, samples were taken, firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added, and the bioluminescence value was read (PE EnSight microplate reader). The results are shown in Figure 2C. Compared with UTNK, 18.2-CAR1 (NK) cells show strong killing activity against NUGC4-luc cells in a short time. At a high E:T ratio, the killing efficiency of 18.2-CAR1 (NK) against NUGC4-luc cells reaches 100 %.
实时细胞杀伤分析实验：按20000个细胞/孔在E-plate 96孔板中加入靶细胞CHOK1-18.2。将E-Plate 96孔板置于实时无标记细胞分析仪(Agilent，Cat#RTCA MP)进行培养。第二天，加入相应效靶比的NK细胞，继续培养3天。培养过程中，每隔15分钟检测细胞指数(Cell Index，CI)，记录靶细胞生长情况，结果如图2D所示，18.2-CAR1(NK)细胞在E:T＝4:1，2:1，1:1时，对CHOK1-18.2展示出100％杀伤效率，但UTNK在高E:T时，对CHOK1-18.2无明显杀伤效果。NK细胞和靶细胞共培养72小时后，使用ELISA试剂盒(BD OptEIATM Human IFN-γELISA Set，Cat No#.555142)检测细胞培养基中人干扰素γ(IFN-γ)分泌水平。实验结果如图2E所示：在4个E:T比实验组中，18.2-CAR1(NK)细胞的IFN-γ分泌量均显著高于UTNK对照组，并且呈现显著的剂量依赖关系。Real-time cell killing analysis experiment: Add target cells CHOK1-18.2 to the E-plate 96-well plate at 20,000 cells/well. The E-Plate 96-well plate was placed on a real-time label-free cell analyzer (Agilent, Cat#RTCA MP) for culture. The next day, NK cells with corresponding target ratio were added and cultured for 3 days. During the culture process, the Cell Index (CI) was detected every 15 minutes and the growth of the target cells was recorded. The results are shown in Figure 2D. 18.2-CAR1 (NK) cells were at E:T=4:1, 2:1. , at 1:1, it showed 100% killing efficiency against CHOK1-18.2, but at high E:T, UTNK had no obvious killing effect against CHOK1-18.2. After NK cells and target cells were co-cultured for 72 hours, an ELISA kit (BD OptEIA TM Human IFN-γ ELISA Set, Cat No. 555142) was used to detect the secretion level of human interferon γ (IFN-γ) in the cell culture medium. The experimental results are shown in Figure 2E: In the four E:T ratio experimental groups, the IFN-γ secretion of 18.2-CAR1 (NK) cells was significantly higher than that of the UTNK control group, and showed a significant dose-dependent relationship.
4.CLDN18.2-NK细胞对NUGC4-luc小鼠皮下肿瘤模型抗肿瘤药效的检测4. Detection of anti-tumor efficacy of CLDN18.2-NK cells on NUGC4-luc mouse subcutaneous tumor model
使用小鼠胃癌皮下移植瘤模型评估18.2-CAR1(NK)细胞的抗肿瘤效果：NPG小鼠(联合免疫缺陷小鼠)皮下接种5E6个处于对数生长期且生长状态良好的NUGC4-luc细胞。接种后第7天测量小鼠肿瘤的长短径a和b，计算小鼠肿瘤体积V(mm3)＝1/2×(a×b2)，根据随机数原则选择肿瘤体积在50mm3左右的小鼠随机分组。接种肿瘤后第8天，尾静脉注射NK细胞(2E7个/只)，NK细胞注射日为第0天(D0)。小鼠分组情况见表2。持续观察并测量小鼠肿瘤体积及体重变化。每周测量记录2次并计算肿瘤抑制率，抑瘤率TGI(％)＝(PBS组小鼠肿瘤体积-实验组小鼠肿瘤体积)/PBS组小鼠肿瘤体积×100％。The mouse gastric cancer subcutaneous transplant tumor model was used to evaluate the anti-tumor effect of 18.2-CAR1 (NK) cells: NPG mice (combined immunodeficient mice) were subcutaneously inoculated with 5E6 NUGC4-luc cells in the logarithmic growth phase and in good growth status. On the 7th day after inoculation, measure the long and short diameters a and b of the mouse tumors, calculate the mouse tumor volume V (mm 3 ) = 1/2 × (a × b 2 ), and select tumors with a volume of about 50 mm 3 based on the random number principle. Mice were randomly divided into groups. On the 8th day after tumor inoculation, NK cells (2E7 cells/animal) were injected into the tail vein, and the day of NK cell injection was day 0 (D0). The grouping of mice is shown in Table 2. Continuously observe and measure changes in tumor volume and body weight of the mice. Measure and record twice a week and calculate the tumor inhibition rate. Tumor inhibition rate TGI (%) = (tumor volume of mice in PBS group - tumor volume of mice in experimental group)/tumor volume of mice in PBS group × 100%.
表2.体内抗肿瘤实验分组情况
Table 2. Grouping status of anti-tumor experiments in vivo
小鼠肿瘤体积检测结果如图3A所示，G2组在注射UTNK细胞后没有显示肿瘤抑制作用，其肿瘤生长速度与G1组(PBS对照组)的生长速度一致。G3组注射18.2-CAR1(NK)细胞后从第14天开始显示出明显抑制体内肿瘤生长的作用，注射后第21天G3组的肿瘤抑制率达68％(图3C)。G3组小鼠在注射后第22天发现死亡，导致死亡原因可能与较高的注射剂量以及18.2-CAR1(NK)在体内的迅速扩增和IL15分泌引起的炎症反应相关。G3组小鼠体重在第18天出现下降(<10％)，其他各组小鼠体重变化不明显(如图3B所示)。为检验分泌型IL-15对18.2-CAR1(NK)细胞的体内扩增和肿瘤归巢作用，在注射细胞后第5、9、13和18天检测小鼠外周血中NK数量以及血清中的IL-15细胞因子分泌量。如图3D所示，G2组UTNK注射小鼠外周血中人NK细胞(CD56+CD3-)没有发现任何扩增现象，而G3组18.2-CAR1(NK)细胞注射小鼠外周血中NK数量逐渐升高，在第18天达到峰值，达到1E4个NK细胞/25μl血样。与此相对应，G2组小鼠血清中人IL-15分泌一直处于基线，而G3组小鼠血清中人IL-15分泌量逐渐升高，在第13天达到峰值，达到350pg/ml(图3E)。以上结果表明，CAR结构中IL-15在动物体内有效表达和分泌，并支持18.2-CAR1(NK)细胞在实体肿瘤模型中持续扩增。注射NK细胞后第22天，解剖小鼠并收集脏器样本，定量研究NK细胞在脾脏和肿瘤中的浸润，如图3F和3G所示，相比UTNK组，18.2-CAR1(NK)组小鼠脾脏和肿瘤组织中均发现高比例18.2-CAR1(NK)细胞，说明可分泌IL-15的18.2-CAR1(NK)细胞成功浸润到免疫器官并有良好的肿瘤归巢作用，与肿瘤控制率结果一致。The results of mouse tumor volume detection are shown in Figure 3A. The G2 group showed no tumor inhibitory effect after injection of UTNK cells, and its tumor growth rate was consistent with that of the G1 group (PBS control group). The G3 group showed a significant inhibitory effect on tumor growth starting from the 14th day after injection of 18.2-CAR1 (NK) cells. The tumor inhibition rate of the G3 group reached 68% on the 21st day after injection (Figure 3C). Mice in the G3 group were found dead on the 22nd day after injection. The cause of death may be related to the higher injection dose, the rapid expansion of 18.2-CAR1 (NK) in the body and the inflammatory response caused by IL15 secretion. The body weight of the mice in the G3 group decreased (<10%) on the 18th day, while the body weight of the mice in the other groups did not change significantly (as shown in Figure 3B). To examine the in vivo expansion and tumor homing effects of secreted IL-15 on 18.2-CAR1 (NK) cells, the number of NK in the peripheral blood of mice and the amount of NK in the serum were measured on days 5, 9, 13 and 18 after injection of cells. IL-15 cytokine secretion. As shown in Figure 3D, no amplification of human NK cells (CD56 + CD3 - ) was found in the peripheral blood of UTNK-injected mice in the G2 group, while the number of NK cells in the peripheral blood of the 18.2-CAR1 (NK) cell-injected mice in the G3 group gradually increased. It increased and reached the peak on the 18th day, reaching 1E4 NK cells/25μl blood sample. Correspondingly, the secretion of human IL-15 in the serum of mice in the G2 group has always been at baseline, while the secretion of human IL-15 in the serum of mice in the G3 group gradually increased, reaching a peak on the 13th day, reaching 350pg/ml (Figure 3E). The above results show that IL-15 in the CAR structure is effectively expressed and secreted in animals and supports the sustained expansion of 18.2-CAR1 (NK) cells in solid tumor models. On the 22nd day after the injection of NK cells, the mice were dissected and organ samples were collected to quantitatively study the infiltration of NK cells in the spleen and tumors. As shown in Figures 3F and 3G, compared with the UTNK group, the 18.2-CAR1 (NK) group had smaller A high proportion of 18.2-CAR1 (NK) cells was found in mouse spleens and tumor tissues, indicating that 18.2-CAR1 (NK) cells that can secrete IL-15 successfully infiltrate into immune organs and have good tumor homing effects, which is closely related to the tumor control rate. The results are consistent.
5.CLDN18.2-NK细胞对NUGC4-luc小鼠肿瘤腹腔模型抗肿瘤药效的检测5. Detection of anti-tumor efficacy of CLDN18.2-NK cells on NUGC4-luc mouse abdominal tumor model
使用小鼠胃癌腹腔接种肿瘤模型评价18.2-CAR1(NK)细胞的抗肿瘤效果：NPG小鼠(联合免疫缺陷小鼠)腹腔接种5E5个处于对数生长期且生长状态良好的NUGC4-luc细胞。接种后第10天测量小鼠腹腔肿瘤细胞生物荧光强度(total flux)，评估肿瘤生长情况。根据随机数原则选择特定腹部位(Region of Interesting,ROI)肿瘤细胞荧光强度在3E8光子/秒(p/s)左右的小鼠进行随机分组。接种肿瘤后第10天，尾静脉注射UTNK和18.2-CAR1(NK)细胞，注射体积为200μl/只，NK细胞注射日为第0天(D0)。小鼠分组与NK细胞注射情况见表3。持续观察并测量小鼠腹腔肿瘤生长及体重变化。The mouse gastric cancer tumor model was intraperitoneally inoculated to evaluate the anti-tumor effect of 18.2-CAR1 (NK) cells: NPG mice (combined immunodeficient mice) were intraperitoneally inoculated with 5E5 NUGC4-luc cells in the logarithmic growth phase and in good growth status. On the 10th day after inoculation, the bioluminescence intensity (total flux) of mouse abdominal tumor cells was measured to evaluate tumor growth. Mice with tumor cell fluorescence intensity of around 3E8 photons/second (p/s) in a specific abdominal region (Region of Interesting, ROI) were selected according to the random number principle and randomly grouped. On the 10th day after tumor inoculation, UTNK and 18.2-CAR1 (NK) cells were injected into the tail vein with an injection volume of 200 μl/animal. The day of NK cell injection was day 0 (D0). The grouping of mice and the injection of NK cells are shown in Table 3. Continuously observe and measure abdominal tumor growth and body weight changes in mice.
表3.体内腹腔模型抗肿瘤实验分组情况
Table 3. Grouping of anti-tumor experiments in the in vivo abdominal cavity model
小鼠腹腔肿瘤生物光强度检测结果如图4A所示，PBS对照组G1和UTNK注射组G2中腹腔肿瘤生长迅速。而注射18.2-CAR1(NK)细胞的G3组有效延迟肿瘤生长，但未彻底消除 肿瘤，各治疗组小鼠与对照组均在治疗后第24天出现肿瘤生长引起的死亡。各组小鼠体重变化不明显，呈缓慢增长趋势(图4B)。G3组18.2-CAR1(NK)细胞注射小鼠外周血中NK细胞比例和血清中人IL-15分泌量逐渐升高，在第19天达到峰值，分别为NK细胞在外周血淋巴细胞中占比达18％(图4C)，人IL-15分泌量均值达500pg/ml(图4D)。结果表明降低18.2-CAR1(NK)剂量有效控制IL-15分泌和CAR-NK细胞迅速扩增引起的炎症反应和毒性，但是由于18.2-CAR1(NK)的扩增水平较低，无法长效控制肿瘤生长和延长动物生存率。The results of bioluminescence intensity detection of mouse abdominal tumors are shown in Figure 4A. The abdominal tumors grew rapidly in the PBS control group G1 and UTNK injection group G2. The G3 group injected with 18.2-CAR1 (NK) cells effectively delayed tumor growth, but did not completely eliminate it. Tumors, mice in each treatment group and the control group all died due to tumor growth on the 24th day after treatment. The weight changes of mice in each group were not obvious and showed 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, respectively. reached 18% (Fig. 4C), and the average secretion amount of human IL-15 reached 500pg/ml (Fig. 4D). The results show that reducing the dose of 18.2-CAR1(NK) effectively controls 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), long-term control cannot be achieved. Tumor growth and prolonged animal survival.
实施例3CBL-b抑制剂的合成Example 3 Synthesis of CBL-b inhibitor
(一)合成下表所示化合物(1) Synthesis of the compounds shown in the table below
表4 CBL-b抑制剂的代号和化学式结构

Table 4 Code names and chemical formula structures of CBL-b inhibitors

1.CBL-bi-1及其同分异构体的合成路线
1.Synthetic route of CBL-bi-1 and its isomers
步骤1：(S)-三氟(3-甲基哌啶-1-基)甲基硼酸钾(2B)的制备Step 1: Preparation of (S)-potassium trifluoro(3-methylpiperidin-1-yl)methylborate (2B)
往(S)-3-甲基哌啶盐酸盐(2A，540mg,3.98mmol)、(溴甲基)三氟硼酸钾(960mg,4.78mmol)、碳酸钾(605mg,4.38mmol)以及碘化钾(67mg,0.40mmol)中加入无水四氢呋喃(10mL)，抽换氮气3次。混合物在氮气保护下加热至回流并搅拌12小时。反应液冷却至室温，加入100mL丙酮，过滤，固体用丙酮洗，滤液蒸干得到胶状标题化合物2B粗品420mg，该粗品直接用于下一步反应。MS m/z(ESI):162.1[M-KF+H]+。To (S)-3-methylpiperidine hydrochloride (2A, 540mg, 3.98mmol), potassium (bromomethyl)trifluoroborate (960mg, 4.78mmol), potassium carbonate (605mg, 4.38mmol) and potassium iodide ( 67 mg, 0.40 mmol), add anhydrous tetrahydrofuran (10 mL), and replace nitrogen three times. The mixture was heated to reflux under nitrogen and stirred for 12 hours. The reaction solution was cooled to room temperature, 100 mL of acetone was added, filtered, the solid was washed with acetone, and the filtrate was evaporated to dryness to obtain 420 mg of a colloidal crude product of the title compound 2B, which was directly used in the next step of the reaction. MS m/z(ESI):162.1[M-KF+H]+.
步骤2：7-溴-9-(三氟甲基)-4H-吡啶[1,2-a]嘧啶-4-酮(2D)的制备Step 2: Preparation of 7-bromo-9-(trifluoromethyl)-4H-pyridine[1,2-a]pyrimidin-4-one (2D)
往5-溴-3-(三氟甲基)吡啶基-2-胺(2C，8.00g,33.2mmol)以及5-(甲氧基亚甲基)-2,2-二甲基-1,3-二氧六环-4,6-二酮(7.42g,39.8mmol)的混合物中加入1,2-二氯苯(100mL)中，混合物加热至110℃，搅拌4小时，然后温度升至200℃，继续搅拌3小时。反应液降至室温，直接进行正相柱层析(石油醚:乙酸乙酯＝3:1)，得到固体标题化合物2D(4.9g)。MS m/z(ESI):293.1/295.1[M+H]+。To 5-bromo-3-(trifluoromethyl)pyridyl-2-amine (2C, 8.00g, 33.2mmol) and 5-(methoxymethylene)-2,2-dimethyl-1, To the mixture of 3-dioxane-4,6-dione (7.42g, 39.8mmol) was added 1,2-dichlorobenzene (100mL), the mixture was heated to 110°C, stirred for 4 hours, and then the temperature was raised to 200°C and continue stirring for 3 hours. The reaction solution was cooled to room temperature and directly subjected to normal phase column chromatography (petroleum ether:ethyl acetate=3:1) to obtain the title compound 2D (4.9g) as a solid. MS m/z(ESI):293.1/295.1[M+H]+.
步骤3：7-[[(3S)-3-甲基-1-哌啶基]甲基]-9-(三氟甲基)吡啶[1,2-a]嘧啶-4-酮(2E)的制备Step 3: 7-[[(3S)-3-methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyridin[1,2-a]pyrimidin-4-one (2E) Preparation
往7-溴-9-(三氟甲基)-4H-吡啶[1,2-a]嘧啶基-4-酮(2D，410mg,1.40mmol)、(S)-三氟(3-甲基哌啶-1-基)甲基硼酸钾(2B，460mg,2.10mmol)、氯(2-二环己基膦基-2',4',6'-三异丙基-1,1'-联苯基)[2-(2'-氨基-1,1'-联苯)]钯(II)(110mg,0.14mmol)、2-二环己基膦-2',4',6'-三异丙基联苯(110mg,0.14mmol)以及碳酸钾(133mg,4.20mmol)的混合物中加入1,4-二氧六环(20mL)和水(5mL)，抽换氮气3次，然后在氮气保护下加热至100℃，搅拌4小时。反应液冷却至室温， 旋蒸除去溶剂，残留物进行反相柱层析(色谱柱：快速硅胶柱，乙腈：水＝60：40)，得到固体标题化合物2E(210mg)。MS m/z(ESI):326.1[M+H]+。To 7-bromo-9-(trifluoromethyl)-4H-pyridin[1,2-a]pyrimidinyl-4-one (2D, 410 mg, 1.40 mmol), (S)-trifluoro(3-methyl) Potassium piperidin-1-yl)methylborate (2B, 460 mg, 2.10 mmol), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-bi Phenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (110 mg, 0.14 mmol), 2-dicyclohexylphosphine-2',4',6'-triiso Add 1,4-dioxane (20 mL) and water (5 mL) to the mixture of propylbiphenyl (110 mg, 0.14 mmol) and potassium carbonate (133 mg, 4.20 mmol), replace the nitrogen gas 3 times, and then place under nitrogen protection Heat to 100°C and stir for 4 hours. The reaction solution was cooled to room temperature, The solvent was removed by rotary evaporation, and the residue was subjected to reversed-phase column chromatography (column: Fast silica gel column, acetonitrile: water = 60:40), to obtain the title compound 2E (210 mg) as a solid. MS m/z(ESI):326.1[M+H]+.
步骤4：3-碘-7-[[(3S)-3-甲基-1-哌啶基]甲基]-9-(三氟甲基)吡啶[1,2-a]嘧啶-4-酮(2F)的制备Step 4: 3-iodo-7-[[(3S)-3-methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyridine[1,2-a]pyrimidine-4- Preparation of Ketone (2F)
将7-[[(3S)-3-甲基-1-哌啶基]甲基]-9-(三氟甲基)吡啶[1,2-a]嘧啶基-4-酮(2E，210mg,0.66mmol)溶于乙腈(10Ml)，加入碘(197mg,0.77mmol)以及硝酸铈铵(342mg,0.65mmol)混合物在室温下搅拌12小时。过量的碘用饱和亚硫酸钠水溶液淬灭，二氯甲烷萃取，有机相用无水硫酸镁干燥，过滤，蒸干，得固体状标题化合物2F粗品(240mg)，粗品直接用于下一步反应。MS m/z(ESI):452.0[M+H]+。7-[[(3S)-3-methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyridin[1,2-a]pyrimidinyl-4-one (2E, 210 mg , 0.66mmol) was dissolved in acetonitrile (10Ml), iodine (197mg, 0.77mmol) and ceric ammonium nitrate (342mg, 0.65mmol) were added and the mixture was stirred at room temperature for 12 hours. Excess iodine was quenched with saturated sodium sulfite aqueous solution, extracted with dichloromethane, and the organic phase was dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness to obtain crude title compound 2F (240 mg) as a solid, which was directly used in the next reaction. MS m/z(ESI):452.0[M+H]+.
步骤5：3-[3-[3-甲基-1-(4-甲基-1,2,4-三氮唑-3-基)环丁基]苯基]-7-[[(3S)-3-甲基-1-哌啶基]甲基]-9-(三氟甲基)吡啶[1,2-a]嘧啶-4-酮(化合物2)、3-(3-((1S,3R)-3-甲基-1-(4-甲基-4H-1,2,4-三氮唑-3-基)环丁基)苯基)-7-(((S)-3-甲基哌啶-1-基)甲基)-9-(三氟甲基)-4H-吡啶并[1,2-a]嘧啶-4-酮和3-(3-((1R,3S)-3-甲基-1-(4-甲基-4H-1,2,4-三氮唑-3基)环丁基)苯基)-7-(((S)-3-甲基哌啶-1-基)甲基)-9-(三氟甲基)-4H-吡啶并[1,2-a]嘧啶-4-酮的制备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-(((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-3yl)cyclobutyl)phenyl)-7-(((S)-3-methyl Preparation of piperidin-1-yl)methyl)-9-(trifluoromethyl)-4H-pyrido[1,2-a]pyrimidin-4-one
向3-碘-7-[[(3S)-3-甲基-1-哌啶基]甲基]-9-(三氟甲基)吡啶[1,2-a]嘧啶基-4-酮(2F，240mg,0.53mmol)、4-甲基-3-[3-甲基-1-[3-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)苯基]环丁基]-1,2,4-三氮唑(188mg,0.53mmol)以及碳酸钾(221mg,1.60mmol)的二氧六环/水(25mL，4：1)溶液中，加入[1,1'-双(二苯基膦)二茂铁]二氯化钯(39mg,0.53mmol)，所得混合物在氮气保护下加热至80℃，搅拌4小时。反应液冷却至室温，旋蒸除去溶剂，残留物先直接进行正相柱层析(甲醇:二氯甲烷＝1:10)，然后再进行反相柱层析(色谱柱：快速硅胶柱，乙腈：水＝75：25)，得到固体CBL-bi-5(130mg)。MS m/z(ESI):551.0[M+H]+。将化合物2进行SFC(仪器：CAS-SH-ANA-SFC-G(Agilent 1260 with DAD detector)；柱子：ChiralPak AS-3柱长150mm，内径4.6mm,粒径3μm；流动相A：CO2,流动相B：IPA(含0.05％DEA)；梯度：流动相B从5％到40％用时4.5分钟，然后用5％流动相B洗脱1.5分钟；流速：2.5mL/min；柱温：40℃；自动背压调节器(ABPR)：100bar)分离，得异构体固体CBL-bi-1(tR＝4.52min,70mg)和CBL-bi-6(tR＝4.69min,15mg)。To 3-iodo-7-[[(3S)-3-methyl-1-piperidinyl]methyl]-9-(trifluoromethyl)pyridin[1,2-a]pyrimidinyl-4-one (2F, 240mg, 0.53mmol), 4-methyl-3-[3-methyl-1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxetane) Pentaborane-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) Add [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (39 mg, 0.53 mmol) to the solution. The resulting mixture was heated to 80°C under nitrogen protection 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 subjected to reversed phase column chromatography (chromatographic column: Fast silica gel column, acetonitrile: water = 75:25), obtained solid CBL-bi-5 (130 mg). MS m/z(ESI):551.0[M+H]+. Compound 2 was subjected to SFC (instrument: CAS-SH-ANA-SFC-G (Agilent 1260 with DAD detector); column: ChiralPak AS-3 column length 150mm, inner diameter 4.6mm, particle size 3μm; mobile phase A: CO2, mobile Phase B: IPA (containing 0.05% DEA); gradient: mobile phase B from 5% to 40% in 4.5 minutes, then eluted with 5% mobile phase B for 1.5 minutes; flow rate: 2.5mL/min; column temperature: 40°C ; Automatic back pressure regulator (ABPR): 100bar) was separated to obtain isomer solids CBL-bi-1 (tR=4.52min, 70mg) and CBL-bi-6 (tR=4.69min, 15mg).
CBL-bi-1：CBL-bi-1：
MS m/z(ESI):551.2[M+H]+；MS m/z(ESI):551.2[M+H] + ;
1H 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)。 1 H NMR (400MHz, CDCl 3 ) 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).
CBL-bi-6：CBL-bi-6:
MS m/z(ESI):551.2[M+H]+；MS m/z(ESI):551.2[M+H] + ;
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)。 1 H NMR (400MHz, CDCl 3 ) 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).
2.CBL-bi-2的合成路线
2.Synthetic route of CBL-bi-2
步骤1：(S)-三氟(3-甲基哌啶-1-基)甲基硼酸钾(3B)的制备Step 1: Preparation of (S)-potassium trifluoro(3-methylpiperidin-1-yl)methylborate (3B)
向(S)-3-甲基哌啶盐酸盐(3A，540mg,3.98mmol)、(溴甲基)三氟硼酸钾(960mg,4.78mmol)、碳酸钾(605mg,4.38mmol)以及碘化钾(67mg,0.40mmol)中加入无水四氢呋喃(10mL)，抽换氮气3次。混合物在氮气保护下加热至回流并搅拌12小时。反应液冷却至室温，加入100mL丙酮，过滤，固体用丙酮洗，把滤液蒸干得到浅黄色胶状标题化合物粗品420mg，该粗品直接用于下一步反应。To (S)-3-methylpiperidine hydrochloride (3A, 540mg, 3.98mmol), potassium (bromomethyl)trifluoroborate (960mg, 4.78mmol), potassium carbonate (605mg, 4.38mmol) and potassium iodide ( 67 mg, 0.40 mmol), add anhydrous tetrahydrofuran (10 mL), and replace nitrogen three times. The mixture was heated to reflux under nitrogen and stirred for 12 hours. The reaction solution was cooled to room temperature, 100 mL of acetone was added, filtered, the solid was washed with acetone, and the filtrate was evaporated to dryness to obtain 420 mg of the crude title compound as a light yellow gum, which was directly used in the next step of the reaction.
MS m/z(ESI):162.1[M-KF+H]+。MS m/z(ESI):162.1[M-KF+H] + .
步骤2：4-溴-6-氟苯并[cd]吲哚-2(1H)-酮(3C)的制备Step 2: Preparation of 4-bromo-6-fluorobenzo[cd]indole-2(1H)-one (3C)
室温下，将4-溴苯并[cd]吲哚-2(1H)-酮(1J，0.2g,0.8mmol)溶解于N,N-二甲基甲酰胺(5mL)中，依次加入乙酸(48.4mg,0.8mmol)和1-氯甲基-4-氟-1,4-二氮杂双环[2.2.2]辛烷二(四氟硼酸)盐(Selectflour)(428.4mg,1.2mmol)，体系加热到50℃，反应8小时后，冷却至室温，加入乙酸乙酯(50ml)，并用水(30ml x3)洗涤，有机相用无水硫酸钠干燥，过滤，浓缩。所得残余物经反相硅胶柱纯化(色谱柱：快速硅胶柱，水:乙腈＝95:5-5:95)冻干后得固体标题化合物(40.0mg,收率19％)。Dissolve 4-bromobenzo[cd]indole-2(1H)-one (1J, 0.2g, 0.8mmol) in N,N-dimethylformamide (5mL) at room temperature, and add acetic acid ( 48.4 mg, 0.8 mmol) and 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tetrafluoroborate) salt (Selectflour) (428.4 mg, 1.2 mmol), The system was heated to 50°C, and after 8 hours of reaction, it was cooled 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 residue obtained was purified by reverse phase silica gel column (chromatography column: Fast silica gel column, water: acetonitrile = 95:5-5:95) was freeze-dried to obtain the solid title compound (40.0 mg, yield 19%).
MS m/z(ESI):266[M+H]+。MS m/z(ESI):266[M+H] + .
步骤3：(S)-6-氟-4-((3-甲基哌啶-1-基)甲基)苯并[cd]吲哚-2(1H)-酮(3D)的制备Step 3 Preparation of: (S)-6-fluoro-4-((3-methylpiperidin-1-yl)methyl)benzo[cd]indole-2(1H)-one (3D)
室温下，将化合物4-溴-6-氟苯并[cd]吲哚-2(1H)-酮(3C，40.0mg,150.0μmol)、(S)-三氟((3-甲基哌啶-1-基)甲基)硼酸钾(98.6mg,450μmol)、XphosPdG2(23.6mg,30.1μmol)、Xphos(28.7mg,60.1μmol)和碳酸钾(83.1mg,601.4μmol)加入到反应管中，置换氩气，加入1,4-二氧六环(2mL)和水(0.5mL)，体系加热到100℃，反应2小时，冷却至室温，过滤，所得残余物经反相硅胶柱纯化(色谱柱：快速硅胶柱，水:乙腈＝95：5-5:95)冻干后得固体标题化合物(20mg)。At room temperature, compound 4-bromo-6-fluorobenzo[cd]indole-2(1H)-one (3C, 40.0 mg, 150.0 μmol), (S)-trifluoro((3-methylpiperidine) Potassium -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 the reaction tube. Replace the argon gas, add 1,4-dioxane (2mL) and water (0.5mL), heat the system to 100°C, react for 2 hours, cool to room temperature, filter, and the residue obtained is purified by a reversed-phase silica gel column (chromatography column: Fast silica gel column, water:acetonitrile=95:5-5:95), the solid title compound (20 mg) was obtained after lyophilization.
MS m/z(ESI):299[M+H]+。MS m/z(ESI):299[M+H] + .
步骤4：(S)-6-氟-1-(3-(3-甲基-1-(4-甲基-4H-1,2,4-三唑-3-基)环丁基)苯基)-4-((3-甲基哌啶-1-基)甲基)苯并[cd]吲哚-2(1H)-酮(化合物3)的制备Step 4: (S)-6-fluoro-1-(3-(3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)benzene Preparation of methyl)-4-((3-methylpiperidin-1-yl)methyl)benzo[cd]indole-2(1H)-one (compound 3)
室温下，将(S)-6-氟-4-((3-甲基哌啶-1-基)甲基)苯并[cd]吲哚-2(1H)-酮(3D，20.0mg,66.6μmol)、3-(1-(3-碘苯基)-3-甲基环丁基)-4-甲基-4H-1,2,4-三唑(1G，23.5mg,66.6μmol)、碳酸钾(18.4mg，133.2μmol)和碘化亚铜(2.5mg，13.3μmol)加入反应管，置换氩气，加入无水N,N-二甲基甲酰胺(2ml)和(1R,2R)-(-)-N,N'-二甲基-1,2-环己二胺(4.7mg，33.3μmol),体系加热到 100℃，反应2小时，冷却至室温，过滤，所得残余物经反相硅胶柱纯化(色谱柱：快速硅胶柱，水:乙腈＝95:5-5:95)冻干后得固体标题化合物(6.9mg)。(S)-6-fluoro-4-((3-methylpiperidin-1-yl)methyl)benzo[cd]indole-2(1H)-one (3D, 20.0 mg, 66.6μmol), 3-(1-(3-iodophenyl)-3-methylcyclobutyl)-4-methyl-4H-1,2,4-triazole (1G, 23.5mg, 66.6μmol) , potassium carbonate (18.4mg, 133.2μmol) and copper iodide (2.5mg, 13.3μmol) were added to the reaction tube, replaced with argon, and added anhydrous N,N-dimethylformamide (2ml) and (1R,2R )-(-)-N,N'-dimethyl-1,2-cyclohexanediamine (4.7mg, 33.3μmol), the system was heated to 100°C, react for 2 hours, cool to room temperature, filter, and the residue obtained is purified by a reversed-phase silica gel column (chromatographic column: Fast silica gel column, water: acetonitrile = 95:5-5:95), the solid title compound (6.9 mg) was obtained after lyophilization.
MS m/z(ESI):524.0[M+H]+；MS m/z(ESI):524.0[M+H] + ;
1H-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). 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).
3.CBL-bi-3的合成路线参见专利申请WO 2020/264398 A1。3. For the synthetic route of CBL-bi-3, please refer to the patent application WO 2020/264398 A1.
4.CBL-bi-4的合成路线参见专利申请WO 2020/210508 A1。4. For the synthetic route of CBL-bi-4, please refer to the patent application WO 2020/210508 A1.
(二)检测表4所示化合物的生物学活性(2) Test the biological activity of the compounds shown in Table 4
1.检测化合物CBL-bi-1、CBL-bi-5、CBL-bi-6和CBL-bi-2对CBL-b蛋白与UbcH5B-Ub相互作用的抑制1. Detect the inhibition of the interaction between CBL-b protein and UbcH5B-Ub by compounds CBL-bi-1, CBL-bi-5, CBL-bi-6 and CBL-bi-2
实验方法：Eu-Ubquitin(Cisbio)与UbcH5B(ENZO)，E1(ENZO)在37℃孵育4小时制备Eu-Ubquitin-UbcH5B。Eu-Ubquitin-UbcH5B分装，于-80℃保存。CBL-b活性实验在384孔板(Perkin Elmer)中进行。100nL 3倍梯度稀释化合物(终浓度为10μM-0.5nM，起始浓度10μM，进行3倍稀释，10个点，第10个点是0.5nM)与5μL 50nM Biotin-CBL-b蛋白(Sigma)在室温孵育1小时，反应缓冲液为50mM HEPES pH 7.0(Gibco),100mM NaCl(Sigma),0.01％Triton X-100(Sigma),0.01％BSA(Sigma)以及lmM DTT(Invitrogen)。在反应板中加入5μL Src混合液(40nM Src(R&D),2mM ATP(Sigma)，10mM MgCl2(Sigma))，室温孵育3小时。在反应板中加入10μL检测液(12.5nM Strepdividin-XL665(Cisbio)，500nM Eu-Ubquitin-UbcH5B，120nM EDTA(Invitrogen)，0.004％BSA(Sigma))，室温孵育过夜，在Envision(Perkin Elmer)上读取HTRF信号(665nm/615nm)。使用IDBS XLfit计算IC50。实验结果如表5所示。Experimental method: Eu-Ubquitin-UbcH5B was prepared by incubating Eu-Ubquitin (Cisbio) with UbcH5B (ENZO) and E1 (ENZO) at 37°C for 4 hours. Eu-Ubquitin-UbcH5B is aliquoted and stored at -80°C. CBL-b activity experiments were performed in 384-well plates (Perkin Elmer). 100 nL 3-fold gradient dilution compound (final concentration 10 μM-0.5 nM, starting concentration 10 μM, 3-fold dilution, 10 points, the 10th point is 0.5 nM) and 5 μL 50 nM Biotin-CBL-b protein (Sigma) in Incubate at room temperature for 1 hour. The reaction buffer is 50mM HEPES pH 7.0 (Gibco), 100mM NaCl (Sigma), 0.01% Triton X-100 (Sigma), 0.01% BSA (Sigma) and 1mM DTT (Invitrogen). Add 5 μL of Src mixture (40 nM Src (R&D), 2mM ATP (Sigma), 10mM MgCl 2 (Sigma)) to the reaction plate, and incubate at room temperature for 3 hours. Add 10 μL of detection solution (12.5 nM Strepdividin-XL665 (Cisbio), 500 nM Eu-Ubquitin-UbcH5B, 120 nM EDTA (Invitrogen), 0.004% BSA (Sigma)) to the reaction plate, incubate at room temperature overnight, and incubate on Envision (Perkin Elmer) Read HTRF signal (665nm/615nm). Calculate IC50 using IDBS XLfit. The experimental results are shown in Table 5.
表5化合物抑制CBL-b蛋白与UbcH5B-Ub相互作用的IC50
Table 5 IC 50 of compounds inhibiting the interaction between CBL-b protein and UbcH5B-Ub
2.检测化合物CBL-bi-1和CBL-bi-2对Jurkat T细胞IL-2释放激活作用2. Detect the activating effect of compounds CBL-bi-1 and CBL-bi-2 on the release of IL-2 in Jurkat T cells
96孔细胞板(Corning)使用2μg/mL Anti-Human CD3 Clone OKT3(BD)37℃包被4小时。3倍梯度稀释化合物(终浓度为10μM-4.6nM，起始浓度10μM，进行3倍稀释，8个点，第8个点是4.6nM)与220μL 1.11E6/mL Jurkat T细胞(ATCC)孵育1小时，加入5μL 45μg/mL Anti-Human CD28 Clone CD28.2(BD)，混合均匀，转移100μL至前述CD3包被细胞板中，37℃细胞培养箱培养48小时，收上清使用IL-2 ELISA试剂盒(BD)检测IL-2释放量。使用Prism分析EC50。实验结果如表6所示。The 96-well cell plate (Corning) was coated with 2 μg/mL Anti-Human CD3 Clone OKT3 (BD) at 37°C for 4 hours. 3-fold gradient dilution of 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, add 5 μL 45 μg/mL Anti-Human CD28 Clone CD28.2 (BD), mix evenly, transfer 100 μL to the aforementioned CD3-coated cell plate, culture it in a 37°C cell incubator for 48 hours, collect the supernatant and use IL-2 ELISA Kit (BD) detects IL-2 release. EC50 was analyzed using Prism. The experimental results are shown in Table 6.
表6化合物激活Jurkat T细胞释放IL2的EC50
Table 6 EC 50 of compounds activating Jurkat T cells to release IL2
实施例4 CLDN18.2 CAR-T制备Example 4 CLDN18.2 CAR-T preparation
使用Stemcell Easy Sep Kit试剂盒(Stemcell,Cat#19055)从新鲜PBMC中分离T细胞。分离后的T细胞转移至预先用1μg/ml CD3/CD28抗体(MY-ebioscience-16-0289-85,MY-ebioscience-16-0037-85)包被的培养皿中培养。培养基组分为X-VIVO15(Lonza，Cat#BEBP02-054Q)、5％人AB血清(Gemini，Cat#100-512)、100U/ml青霉素-链霉素(Gibco，Cat#15140-122)和200IU/ml人IL2(北京双鹭，Cat#S19991007)。每周进行2次细胞计数，当细胞密度到2.5E6个/ml时传代扩培。T cells were isolated from fresh PBMC using Stemcell Easy Sep Kit (Stemcell, Cat#19055). The isolated T cells were transferred to culture dishes pre-coated with 1 μg/ml CD3/CD28 antibody (MY-ebioscience-16-0289-85, MY-ebioscience-16-0037-85). The medium components are X-VIVO15 (Lonza, Cat#BEBP02-054Q), 5% human AB serum (Gemini, Cat#100-512), 100U/ml penicillin-streptomycin (Gibco, Cat#15140-122) and 200IU/ml human IL2 (Beijing Shuanglu, Cat#S19991007). Cell counts were performed twice a week, and when the cell density reached 2.5E6 cells/ml, the cells were subcultured and expanded.
取上述状态良好的T细胞，按1E6个/孔接种到预先用5μg/ml重组人纤维蛋白(Takara，Cat#T100B)包被的12孔细胞培养板，添加50μl浓缩慢病毒和10μg/ml聚凝胺(Sigma-Aldrich，Cat#TR-1003)，4℃下1000g离心1小时。离心后在37℃，5％CO2条件下培养，每周计数2次，当细胞密度达到2.5E6个/ml时传代扩培。Take the above-mentioned T cells in good condition and inoculate them into a 12-well cell culture plate pre-coated with 5 μg/ml recombinant human fibrin (Takara, Cat#T100B) at a rate of 1E6 cells/well. Add 50 μl of concentrated lentivirus and 10 μg/ml polypeptide. Condensate (Sigma-Aldrich, Cat#TR-1003), centrifuged at 1000 g for 1 hour at 4°C. After centrifugation, culture at 37°C and 5% CO2 , count twice a week, and subculture when the cell density reaches 2.5E6 cells/ml.
实施例5 CLDN18.2 CAR-T和CLDN18.2 CAR-NK联用、CLDN18.2 CAR-T与CBL-b抑制剂联合使用，以及CLDN18.2 CAR-NK与CBL-b抑制剂联合使用的体外杀伤功能性评价Example 5 Combination of CLDN18.2 CAR-T and CLDN18.2 CAR-NK, combination of CLDN18.2 CAR-T and CBL-b inhibitor, and combination of CLDN18.2 CAR-NK and CBL-b inhibitor In vitro killing functional evaluation
按照实施例2和实施例4制备18.2-CAR1(NK)细胞和18.2-CAR3(T)细胞，按照实施例3合成CBL-bi-1和CBL-bi-3，用于本实施例的联用方案。Prepare 18.2-CAR1 (NK) cells and 18.2-CAR3 (T) cells according to Example 2 and Example 4, synthesize CBL-bi-1 and CBL-bi-3 according to Example 3, and use them in combination in this example. plan.
1.检测CLDN18.2 CAR-T和NK细胞联用对肿瘤细胞的杀伤作用1. Detect the killing effect of CLDN18.2 CAR-T and NK cells combined on tumor cells
按20000个细胞/孔将靶细胞NUGC4-luc铺在96孔板上，按照NK细胞:靶细胞比例＝1:2，T细胞:靶细胞＝1:4，在96孔板中加入NK细胞和T细胞，与靶细胞共培养。在24小时和48小时分别取样，加入萤火虫荧光素酶底物D-荧光素(Abcam，Cat#ab143655)，使用PE EnSight酶标仪读取生物荧光值，结果如图5A所示，与单独使用NK细胞(UTNK或18.2-CAR1(NK)细胞)或CAR-T细胞相比，联用组显示更强的杀伤效果。另外，UTNK和18.2-CAR1(NK)细胞在24小时对NUGC4-luc细胞杀伤较强，但48小时杀伤显著降低。而联用组在48小时仍维持强杀伤活力，与联用组的24小时杀伤效果基本相当，明显优于单独使用NK组在48h的杀伤能力。Plate the target cells NUGC4-luc on a 96-well plate at 20,000 cells/well. According to the ratio of NK cells: target cells = 1:2, T cells: target cells = 1:4, add NK cells and T cells, co-cultured with target cells. Take samples at 24 hours and 48 hours respectively, add firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655), and use the PE EnSight microplate reader to read the bioluminescence value. The results are shown in Figure 5A, which is the same as when used alone. Compared with NK cells (UTNK or 18.2-CAR1 (NK) cells) or CAR-T cells, the combination group showed a stronger killing effect. In addition, UTNK and 18.2-CAR1 (NK) cells were highly lethal to NUGC4-luc cells at 24 hours, but the killing was significantly reduced at 48 hours. The combination group still maintained strong killing activity at 48 hours, which was basically the same as the 24-hour killing effect of the combination group, and was significantly better than the killing ability of the NK group alone at 48 hours.
2.检测CBL-b抑制剂与NK细胞联用对肿瘤细胞的杀伤作用2. Detect the killing effect of CBL-b inhibitors combined with NK cells on tumor cells
按20000个细胞/孔将靶细胞NUGC4-luc铺在96孔板上，按照NK细胞:靶细胞比例＝1:6加入NK细胞，同时加入终浓度为1μM的CBL-bi-1，共培养24小时后，取样，加入萤火虫荧光素酶底物D-荧光素(Abcam，Cat#ab143655)，使用PE EnSight酶标仪读取生物荧光值，结果如图5B所示。在NK细胞:靶细胞比例＝1:6时，CBL-b抑制剂(CBL-bi-1，在培养基的终浓度为1μM)有效提高UTNK和18.2-CAR1(NK)细胞对NUGC4-luc细胞杀伤能力，杀伤效率 均提高16％左右。Plate the target cells NUGC4-luc on a 96-well plate at 20,000 cells/well, add NK cells according to the ratio of NK cells: target cells = 1:6, and add CBL-bi-1 with a final concentration of 1 μM, and co-culture for 24 After an hour, a sample was taken, the firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added, and the bioluminescence value was read using a PE EnSight microplate reader. The results are shown in Figure 5B. At the NK cell:target cell ratio = 1:6, the CBL-b inhibitor (CBL-bi-1, final concentration in culture medium was 1 μM) effectively increased the response of UTNK and 18.2-CAR1 (NK) cells to NUGC4-luc cells. killing ability, killing efficiency Both increased by about 16%.
3.检测CBL-b抑制剂与CLDN18.2 CART或CLDN18.2 CARNK细胞联用的多轮杀伤作用3. Detect the multiple rounds of killing effects of CBL-b inhibitors combined with CLDN18.2 CART or CLDN18.2 CARNK cells
将NUGC4-luc按40000个/孔铺在96孔板上，按照NK细胞：靶细胞比例＝1：24或T细胞:靶细胞＝1:16将NK细胞或T细胞，同时加入终浓度为1μM的CBL-bi-3，共培养。72小时后取样，加入萤火虫荧光素酶底物D-荧光素(Abcam，Cat#ab143655)，使用PE EnSight酶标仪读取生物荧光值i计算杀伤率。将共培育72小时的细胞重悬，取1/2体积细胞悬浮液再与相同数量新鲜NUGC4-luc细胞共培育进行下一轮杀伤实验。以此类推，共完成3轮杀伤。结果如图5C所示，CBL-bi-3有效提高18.2-CAR1(NK)细胞和对NUGC4-luc细胞杀伤能力，并且显著延长杀伤持久性至第三轮。相似地，CBL-bi-3和18.2-CAR4(T)细胞联合使用有效延长CAR-T杀伤能力，在效靶比较低的形况下，联用组的杀伤效率在第三轮达到100％。Plate NUGC4-luc on a 96-well plate at 40,000 cells/well. Add NK cells or T cells according to the ratio of NK cells: target cells = 1:24 or T cells: target cells = 1:16. At the same time, add NK cells or T cells at a final concentration of 1 μM. of CBL-bi-3, co-cultured. Take a sample after 72 hours, add firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655), and use a PE EnSight microplate reader to read the bioluminescence value i to calculate the killing rate. The cells cultured for 72 hours were resuspended, and 1/2 volume of the cell suspension was taken and co-cultured with the same number of fresh NUGC4-luc cells for the next round of killing experiments. By analogy, a total of 3 rounds of killing were completed. The results are shown in Figure 5C. CBL-bi-3 effectively improved the killing ability of 18.2-CAR1 (NK) cells and NUGC4-luc cells, and significantly extended the killing persistence to the third round. Similarly, the combined use of CBL-bi-3 and 18.2-CAR4(T) cells effectively prolonged the killing ability of CAR-T. In the case of low target efficacy, the killing efficiency of the combination group reached 100% in the third round.
以上结果说明CLDN18.2 CAR-T和CLDN18.2 CAR-NK联合使用，以及CLDN18.2 CAR-NK与CBL-b抑制剂联合使用可以克服CAR-NK细胞单独使用药效短暂的缺点；同时也说明CLDN18.2 CAR-T与CBL-b抑制剂联合使用可以降低CAR-T使用量提高细胞治疗的安全性。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 shortcomings of short-lived efficacy of CAR-NK cells alone; at the same time, This shows that the combined use of CLDN18.2 CAR-T and CBL-b inhibitors can reduce the dosage of CAR-T and improve the safety of cell therapy.
实施例6 CLDN18.2 CAR-T，CLDN18.2 CAR-NK，CBL-b抑制剂三者联合使用的体外杀伤功能性评价Example 6 In vitro killing functional evaluation of the combined use of CLDN18.2 CAR-T, CLDN18.2 CAR-NK, and CBL-b inhibitors
按照实施例2和实施例4制备18.2-CAR1(NK)和18.2-CAR3(T)，按照实施例3合成CBL-bi-1，用于本实施例的联用方案。18.2-CAR1 (NK) and 18.2-CAR3 (T) were prepared according to Example 2 and Example 4, and CBL-bi-1 was synthesized according to Example 3 and used in the combination scheme of this example.
实时细胞多轮连续杀伤分析实验：取状态良好的靶细胞CHOK1-18.2加入E-plate 96孔板，每孔20000个细胞。将E-Plate 96孔板放置于实时无标记细胞分析仪(Agilent，Cat#RTCA MP)进行培养。第二天，加入相应效靶比的CAR-NK，CAR-T细胞以及CBL-b抑制剂，E:T为NK细胞:靶细胞比例＝1:6，T细胞:靶细胞＝1:8。继续培养72小时。培养过程中，每隔15分钟检测细胞指数(Cell Index，CI)，记录靶细胞生长情况，72小时后取出1/2体积培养基，在相同孔中加入与起始数量相同的新鲜CHOK1-18.2细胞共培育进行下一轮杀伤实验。以此类推，共完成3轮杀伤。结果如图6所示，18.2-CAR3(T)细胞组和18.2-CAR3(T)细胞+CBL-bi-1组对于CHOK1-18.2细胞和的杀伤作用相对较弱，在第一轮杀伤中没有完全控制住靶细胞生长。18.2-CAR1(NK)组的杀伤能力仅维持到第二轮。而18.2-CAR1(NK)细胞+CBL-bi-1组，18.2-CAR1(NK)细胞+18.2-CAR3(T)细胞组，以及18.2-CAR1(NK)细胞+18.2-CAR3(T)细胞+CBL-bi-1组杀伤能力维持到第三轮。在第三轮杀伤实验中，仅有三者联用组依旧显示出对靶细胞的完全抑制作用。此结果说明CBL-b抑制剂可以有效活化NK和T细胞，表现出显著的协同性，进一步提高CAR-NK细胞+CAR-T细胞联用的杀伤功能抵抗多轮肿瘤细胞攻击，有效降低肿瘤复发。Real-time cell multi-round continuous killing analysis experiment: Take the target cells CHOK1-18.2 in good condition and add them to the E-plate 96-well plate, with 20,000 cells in each well. Place the E-Plate 96-well plate on a real-time label-free cell analyzer (Agilent, Cat#RTCA MP) for culture. The next day, add CAR-NK, CAR-T cells and CBL-b inhibitors with corresponding target ratios. E:T is the ratio of NK cells: target cells = 1:6, and T cells: target cells = 1:8. Continue culturing for 72 hours. During the culture process, detect the Cell Index (CI) every 15 minutes and record the growth of the target cells. After 72 hours, take out 1/2 volume of the culture medium and add the same amount of fresh CHOK1-18.2 to the same well as the starting amount. The cells were co-cultured for the next round of killing experiments. By analogy, a total of 3 rounds of killing were completed. The results are shown in Figure 6. The 18.2-CAR3(T) cell group and the 18.2-CAR3(T) cell + CBL-bi-1 group have relatively weak killing effects on CHOK1-18.2 cells. There was no effect in the first round of killing. Completely control target cell growth. The killing ability of the 18.2-CAR1(NK) group was only maintained until the second round. And 18.2-CAR1(NK) cells+CBL-bi-1 group, 18.2-CAR1(NK) cells+18.2-CAR3(T) cells group, and 18.2-CAR1(NK) cells+18.2-CAR3(T) cells+ The killing ability of the CBL-bi-1 group was maintained until the third round. In the third round of killing experiments, only the combination of the three groups still showed complete inhibition of target cells. This result shows that CBL-b inhibitors can effectively activate NK and T cells, show significant synergy, further improve the killing function of the combination of CAR-NK cells + CAR-T cells, resist multiple rounds of tumor cell attacks, and effectively reduce tumor recurrence. .
实施例7使用添加CBL-b抑制剂的培养基对于CAR-T细胞活性的影响Example 7 Effect of using culture medium supplemented with CBL-b inhibitor on the activity of CAR-T cells
参照实施例4制备18.2-CAR2(T)细胞，并全程在培养基中额外添加1μM CBL-bi-1或CBL-bi-2刺激T细胞。 Prepare 18.2-CAR2 (T) cells with reference to Example 4, and add 1 μM CBL-bi-1 or CBL-bi-2 to the culture medium throughout the entire process to stimulate T cells.
1.FACS检测CAR转染效率以及T细胞表型1. FACS detection of CAR transfection efficiency and T cell phenotype
参照实施例2所述CAR转染效率检测方法，在加入CD3/CD28抗体扩增培养的第9天(转染后的第7天)的18.2-CAR2(T)细胞中CAR表达情况。结果如图7A所示，含有CBL-bi-1和CBL-bi-2的培养条件有效提高CAR转染效率，相比18.2-CAR2(T)对照组，加入CBL-bi-1和CBL-bi-2组的T细胞CAR表达提高约10％。Referring to the CAR transfection efficiency detection method described in Example 2, the expression of CAR in 18.2-CAR2(T) cells on the 9th day (the 7th day after transfection) after CD3/CD28 antibody amplification culture was added. 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 addition of CBL-bi-1 and CBL-bi -T cell CAR expression increased by approximately 10% in group 2.
同样采用流式细胞术检测转染9天后T细胞的表型，检测CD4T细胞和CD8T细胞的中央记忆性T细胞(Central Memory T cell，CM,CD62L+CD45RO+)、效应记忆性T细胞(Effective Memory T Cell，EM,CD62L-CD45RO+)、干细胞样记忆T细胞(Stem cell-like memory T cell，SCM,CD62L+CD45RO-)和效应T细胞(Effector T cells，EF,CD62L-CD45RO-)的表达情况以及T细胞激活表型CD69的表达情况。如图7B-图7C所示，相比18.2-CAR2(T)对照组，CBL-bi-1和CBL-bi-2的添加未对18.2-CAR2(T)细胞记忆表型产生影响。如图7D所示，相比18.2-CAR2(T)对照组，在培养基中添加CBL-bi-1和CBL-bi-2显著提高CD4T和CD8T细胞的激活表型CD69的表达量。Flow cytometry was also used to detect the phenotype of T cells 9 days after transfection, and detect central memory T cells (CM, CD62L + CD45RO + ), effector memory T cells (Effective) of CD4 T cells and CD8 T cells. Memory T Cell, EM, CD62L - CD45RO + ), stem cell-like memory T cell (Stem cell-like memory T cell, SCM, CD62L + CD45RO - ) and effector T cells (EF, CD62L - CD45RO - ) Expression and T cell activation phenotype CD69 expression. As shown in Figure 7B-Figure 7C, compared with the 18.2-CAR2(T) control group, the addition of CBL-bi-1 and CBL-bi-2 did not affect the memory phenotype of 18.2-CAR2(T) cells. As shown in Figure 7D, compared with the 18.2-CAR2(T) control group, adding CBL-bi-1 and CBL-bi-2 to the culture medium significantly increased the expression of activation phenotype CD69 of CD4T and CD8T cells.
2.18.2-CAR2(T)细胞对高表达CLDN18.2胃癌肿瘤细胞杀伤效果的检测2.18.2-Detection of the killing effect of CAR2(T) cells on gastric cancer tumor cells with high expression of CLDN18.2
杀伤肿瘤细胞实验：按20000个/孔将靶细胞NUGC4-luc铺在96孔板上，按E:T比＝5:1、2:1、1:2、1:2加入18.2-CAR2(T)细胞或UT细胞共培养。在24小时取样，加入萤火虫荧光素酶底物D-荧光素(Abcam，Cat#ab143655)，使用PE EnSight酶标仪读取生物荧光值，结果如图7E所示。相较对照组的18.2-CAR2(T)细胞，在培养基中添加CBL-bi-1和CBL-bi-2明显提高18.2-CAR2(T)细胞在低E:T比下对NUGC4-luc细胞的杀伤作用。Tumor cell killing experiment: Plate target cells NUGC4-luc on a 96-well plate at 20,000 cells/well, and add 18.2-CAR2(T ) cells or UT cells were co-cultured. Samples were taken at 24 hours, the firefly luciferase substrate D-luciferin (Abcam, Cat#ab143655) was added, and the bioluminescence value was read using a PE EnSight microplate reader. The results are shown in Figure 7E. Compared with 18.2-CAR2(T) cells in the control group, adding CBL-bi-1 and CBL-bi-2 to the culture medium significantly improved the performance of 18.2-CAR2(T) cells against NUGC4-luc cells at a low E:T ratio. The killing effect.
细胞脱粒标志物CD107a表达和IFN-γ分泌检测：按20000个/孔将靶细胞NUGC4-luc铺于96孔板，按照E:T比＝1:1加入不同18.2-CAR2(T)细胞和UT细胞，向每孔加入1μl荧光标记的CD107a抗体(BioLegend，Cat.328620)，共同培养1小时后，向每孔加入1μl Monensin Solution(eBioscience Cat.No.00-4505)和1μl Brefeldin A Solution(eBioscience Cat.No.00-4506)。5小时后采用流式表面染色方法标记T细胞表面抗原。缓冲液洗涤一次，离心完全弃上清。加100μl Intracellular Fixation Buffer(eBioscience Cat.No.88-8824)，涡旋固定细胞，室温避光孵育20-60分钟。洗涤后向每孔加入100μl 1X Permeabilization Buffer(eBioscience Cat.No.88-8824)，加入IFN-γ抗体(BioLegend，Cat.506506)，室温避光孵育20-60分钟。使用1X Permeabilization Buffer洗涤2次，重悬于100μl FACS缓冲液，用FACS(CANTOII，BD)检测和分析结果。结果如图7F所示，相对于对照组CAR-T，在培养基中添加CBL-bi-1和CBL-bi-2明显提高18.2-CAR2(T)细胞内分泌IFN-γ和表面CD107a的表达。在无肿瘤细胞刺激的条件下，各组UT细胞以及各组18.2-CAR2(T)细胞内分泌IFN-γ和表面CD107a的表达均无明显变化(图7G)。Detection of cell degranulation marker CD107a expression and IFN-γ secretion: Plate target cells NUGC4-luc in a 96-well plate at 20,000 cells/well, and add different 18.2-CAR2 (T) cells and UT according to the E:T ratio = 1:1 For cells, add 1 μl of fluorescently labeled CD107a antibody (BioLegend, Cat.328620) to each well. After co-culture for 1 hour, add 1 μl of Monensin Solution (eBioscience Cat. No. 00-4505) and 1 μl of Brefeldin A Solution (eBioscience) to each well. Cat.No.00-4506). Flow cytometry surface staining method was used to label T cell surface antigens 5 hours later. Wash once with buffer, centrifuge and discard supernatant completely. Add 100 μl Intracellular Fixation Buffer (eBioscience Cat. No. 88-8824), vortex to fix the cells, and incubate at room temperature in the dark for 20-60 minutes. After washing, add 100 μl 1X Permeabilization Buffer (eBioscience Cat. No. 88-8824) to each well, add IFN-γ antibody (BioLegend, Cat. 506506), and incubate at room temperature in the dark for 20-60 minutes. Wash twice with 1X Permeabilization Buffer, resuspend in 100μl FACS buffer, detect and analyze the results with FACS (CANTOII, BD). The results are shown in Figure 7F. Compared with the control group CAR-T, adding CBL-bi-1 and CBL-bi-2 to the culture medium significantly increased the expression of endocrine IFN-γ and surface CD107a in 18.2-CAR2(T) cells. Under conditions without tumor cell stimulation, there were no significant changes in the expression of endocrine IFN-γ and surface CD107a in UT cells in each group and 18.2-CAR2(T) cells in each group (Figure 7G).
实施例8添加CBL-b抑制剂的培养基对于BCMA CAR-NK细胞活性的影响Example 8 Effect of culture medium adding CBL-b inhibitor on the activity of BCMA CAR-NK cells
参照实施例2制备表达BCMA-CAR1(NK)和BCMA-CAR2(NK)的NK细胞，并在K562细胞激活NK细胞扩增的第4天(Day4)，在培养基中添加1μM CBL-bi-4刺激NK细胞。采用FACS检测扩增培养的第15天(Day15)的CAR转染效率：NK cells expressing BCMA-CAR1 (NK) and BCMA-CAR2 (NK) were prepared with reference to Example 2, and on the 4th day (Day4) of K562 cell activation of NK cell expansion, 1 μM CBL-bi- was added to the culture medium 4. Stimulate NK cells. Use FACS to detect the CAR transfection efficiency on Day 15 of amplification culture:
用PBS缓冲液洗涤细胞2次，计数后取2E5个细胞，加入50μl Fc受体阻断剂(BioLegend， Cat#422302)室温孵育10分钟。加入100μl终浓度为2μg/ml的FITC-hBCMA-his(ACRO Biosystems，BCA-HF254)，4℃避光孵育1小时。用FACS缓冲液离心洗涤3次，用100μl FACS缓冲液悬浮细胞，用流式仪进行(BD，CANTOII)检测和分析结果。如图8所示，含有CBL-bi-4的培养条件可以有效提高CAR-NK细胞的CAR转染效率，相比BCMA-CAR1(NK)和BCMA-CAR2(NK)细胞对照组和UTNK组，加入CBL-bi-4组的NK细胞CAR表达均提高约15％。Wash the cells twice with PBS buffer, count 2E5 cells, and add 50 μl Fc receptor blocker (BioLegend, Cat#422302) and incubate at room temperature for 10 minutes. Add 100 μl of FITC-hBCMA-his (ACRO Biosystems, BCA-HF254) with a final concentration of 2 μg/ml and incubate at 4°C in the dark for 1 hour. Centrifuge and wash 3 times with FACS buffer, resuspend the cells in 100 μl FACS buffer, and detect and analyze the results using a flow cytometer (BD, CANTOII). As shown in Figure 8, 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 group and the UTNK group, The CAR expression of NK cells in the CBL-bi-4 group increased by about 15%.
实施例9靶向GPRC5D和BCMA的CAR-NK与CBL-b抑制剂和/或PDL1-IL15的联用Example 9 Combination of CAR-NK targeting GPRC5D and BCMA with CBL-b inhibitor and/or PDL1-IL15
9.1 BCMA-GPRC5D CAR-NK的构建9.1 Construction of BCMA-GPRC5D CAR-NK
设计构建靶向BCMA和GPRC5D的双靶点嵌合抗原受体包括CD8α信号肽(SP)、抗GPRC5D抗体和抗BCMA、CD8α铰链区、NKG2D-F跨膜区、2B4共刺激结构域和CD3ζ，以及通过自切割肽P2A连接的IL15，其中BCMA采用如SEQ ID NO:38所示的VHH、GPRC5D采用SEQ ID NO:42所示的VHH。BCMA-GPRC5D CAR-NK结构如图9所示。序列如表7所示。Design and construct a dual-target chimeric antigen receptor targeting BCMA and GPRC5D, including CD8α signal peptide (SP), anti-GPRC5D antibody and anti-BCMA, CD8α hinge region, NKG2D-F transmembrane region, 2B4 costimulatory domain and CD3ζ. And IL15 connected through the self-cleaving peptide P2A, in which BCMA adopts the VHH shown in SEQ ID NO:38, and GPRC5D adopts the VHH shown in SEQ ID NO:42. The BCMA-GPRC5D CAR-NK structure is shown in Figure 9. The sequence is shown in Table 7.
表7 BCMA-GPRC5D CAR氨基酸序列表
Table 7 BCMA-GPRC5D CAR amino acid sequence list
9.2 CBL-b抑制剂上调CAR阳性率9.2 CBL-b inhibitors increase CAR positivity rate
通过本领域常规的质粒的构建、病毒包装、PBMC诱导为NK细胞的方法及NK细胞的激活以及病毒侵染构建CAR-NK的方法，制备BCMA-GPRC5D CAR-NK(BI-CAR21)，同时在NK细胞的激活和转染过程中添加CBL-bi-4(终浓度1μM)。逆转录病毒转染NK细胞6天后，通过嵌合抗原受体表达率检测方法检测NK细胞的CAR阳性率。结果显示，添加CBL-b抑制剂后，NK细胞的CAR阳性率升高。示例性地，如图10所示，通过BCMA检测BI-CAR21的表达率，其中，与未添加CBL-b抑制剂的对照相比，添加CBL-b抑制剂后，BI-CAR的表达率由80％上升至92.5％。BCMA-GPRC5D CAR-NK (BI-CAR21) is prepared through conventional plasmid construction, virus packaging, PBMC induction into NK cells, activation of NK cells, and viral infection to construct CAR-NK. CBL-bi-4 (final concentration 1 μM) was added during the activation and transfection of NK cells. Six days after retrovirus transfection of NK cells, the CAR positive rate of NK cells was detected by the chimeric antigen receptor expression rate detection method. The results showed that after adding CBL-b inhibitor, the CAR positive rate of NK cells increased. Exemplarily, as shown in Figure 10, the expression rate of BI-CAR21 was detected by BCMA, wherein, compared with the control without adding CBL-b inhibitor, after adding CBL-b inhibitor, the expression rate of BI-CAR was determined by 80% rose to 92.5%.
9.3 CBL-b抑制剂和/或PDL1-IL15增强CAR-NK杀伤效果9.3 CBL-b inhibitors and/or PDL1-IL15 enhance the killing effect of CAR-NK
检测靶向GPRC5D和BCMA的CAR-NK(BI-CAR21)与CBL-b抑制剂和/或PDL1-IL15 联用后对NCI H929+10％NCI H929-hBCMA-KO细胞的多轮杀伤效果。试验设计方案参见表8，其中所述CBL-b抑制剂及其添加方式参见9.2部分，所述PDL1-IL15在靶细胞和NK细胞孵育时添加。其中，所述NCI H929-hBCMA-KO细胞为使用常规基因操作方法敲除BCMA的H929细胞系。Detection of CAR-NK (BI-CAR21) targeting GPRC5D and BCMA with CBL-b inhibitors and/or PDL1-IL15 Multiple rounds of killing effect on NCI H929+10% NCI H929-hBCMA-KO cells after combined use. The experimental design plan is shown in Table 8. The CBL-b inhibitor and its addition method are shown in Section 9.2. The PDL1-IL15 is added when the target cells and NK cells are incubated. Wherein, the NCI H929-hBCMA-KO cells are H929 cell lines in which BCMA is knocked out using conventional gene manipulation methods.
并且，所述靶细胞NCI H929、NCI H929-hBCMA-KO均转入荧光素酶基因，表达荧光素酶。通过荧光素酶报告基因检测试剂检测荧光强度，反映细胞活率和NK细胞的杀伤效果。杀伤率计算公式如下所示：Moreover, the target cells NCI H929 and NCI H929-hBCMA-KO were transferred into the luciferase gene to express luciferase. The fluorescence intensity is detected by luciferase reporter gene detection reagent, which reflects the cell viability and the killing effect of NK cells. The formula for calculating the kill rate is as follows:
杀伤率＝(靶细胞孔读值-试验孔读值)/靶细胞孔读值×100％。Killing rate = (target cell well reading value - test well reading value)/target cell well reading value × 100%.
多轮杀伤结果如图11所示，在培养过程中使用CBL-b抑制剂刺激NK细胞或在CAR-NK细胞杀伤时添加PDL1-IL15融合蛋白均能增强BCMA-GPRC5D CAR-NK的杀伤作用，而CBL-b抑制剂与PDL1-IL15的联用，可进一步增强BCMA-GPRC5D CAR-NK的杀伤作用，G5组在R5-1d仍保持较高的靶细胞杀伤率，明显高于G3组和G4组。The results of multiple rounds of killing are shown in Figure 11. Using CBL-b inhibitors to stimulate NK cells during the culture process 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 maintains a high target cell killing rate on R5-1d, which is significantly higher than the G3 group and G4 Group.
表8试验设计方案
Table 8 Experimental design plan
注：“/”表示不添加。Note: "/" means not to add.
PDL1-IL15的序列信息如下所示：
The sequence information of PDL1-IL15 is as follows:
实施例10.靶向GPRC5D和BCMA的CAR-NK与CBL-b抑制剂联合使用在Molp8-luc小鼠肿瘤模型中的抗肿瘤药效试验Example 10. Anti-tumor efficacy test of CAR-NK targeting GPRC5D and BCMA combined with CBL-b inhibitor in Molp8-luc mouse tumor model
使用小鼠骨髓癌静脉移植瘤模型进行抗肿瘤药效试验。具体如下： Antitumor efficacy testing was conducted using a mouse bone marrow cancer intravenous xenograft tumor model. details as follows:
参照实施例9方法制备BCMA-GPRC5D CAR-NK细胞。BCMA-GPRC5D CAR-NK cells were prepared according to the method of Example 9.
通过本领域常规的方法构建表达细菌荧光素酶(Luciferase)的Molp8-Luc细胞，并采用常规方法进行培养。随后，收集处于对数生长期且生长状态良好的Molp8-Luc细胞，于NPG小鼠(联合免疫缺陷小鼠)尾静脉共接种5×105个细胞。接种肿瘤后第1天测量小鼠体重和肿瘤荧光信号值，根据随机数原则选择体重在21.81～27.25g左右的小鼠，均值为23.98g进行随机分组。接种肿瘤后第1天，即分组当天尾静脉注射冷冻复苏制备CAR-NK细胞(8×106个/只)，注射体积为200μl/只，CAR-NK细胞注射日记为第0天(D0)。小鼠分组与CAR-NK细胞注射情况见表9。持续用IVIS活体成像仪监测肿瘤生长荧光信号ROI值及体重变化，每周测量记录1次并观察小鼠生存率。Molp8-Luc cells expressing bacterial luciferase (Luciferase) were constructed by conventional methods in this field and cultured using conventional methods. Subsequently, Molp8-Luc cells in the logarithmic growth phase and in good growth status were collected, and a total of 5 × 10 5 cells were inoculated into the tail veins of NPG mice (combined immunodeficient mice). On the first day after tumor inoculation, the mouse body weight and tumor fluorescence signal value were measured. Mice with a weight of about 21.81 to 27.25g were selected based on the random number principle, and the average value was 23.98g for random grouping. On the 1st day after tumor inoculation, that is, on the day of grouping, freeze-resuscitated CAR-NK cells (8×10 6 /animal) were injected into the tail vein. The injection volume was 200 μl/animal. The CAR-NK cell injection diary was day 0 (D0). . The grouping of mice and the injection of CAR-NK cells are shown in Table 9. Continuously monitor tumor growth fluorescence signal ROI value and body weight changes with an IVIS intravital imager. Measure and record once a week and observe the survival rate of mice.
表9.体内抗肿瘤实验分组情况
Table 9. Grouping status of anti-tumor experiments in vivo
小鼠肿瘤生长荧光信号检测结果如图12B所示，结果表明：The results of fluorescence signal detection of mouse tumor growth are shown in Figure 12B. The results show:
在注射CAR-NK细胞后的第29天，BI-CAR21+CBL-b抑制剂治疗组能够显著抑制小鼠肿瘤生长负荷，其中两只动物肿瘤出现治愈，1只动物肿瘤复发较少，肿瘤负荷减轻；BI-CAR21治疗组能够抑制小鼠肿瘤生长速度，但不能控制该组动物肿瘤进展，组内5只动物肿瘤全部复发增长且3只动物出现死亡；光子量统计结果如图12C所示。同时，BI-CAR21+CBL-b抑制剂治疗组实验过程中监测动物体重整体呈现上升趋势，未表现出给药后不良反应；BI-CAR21治疗组在实验中动物体重呈现波动变化趋势，因组内单只动物出现体重下降导致(如图12D所示)；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. Two of the animals' tumors were cured, and one animal had less tumor recurrence and a lower tumor burden. Alleviation; the BI-CAR21 treatment group can inhibit the growth rate of mouse tumors, but cannot control the tumor progression of animals in this group. All 5 animals in the group had tumor recurrence and growth, and 3 animals died; the photon quantity statistical results are shown in Figure 12C. At the same time, the body weight of the animals in the BI-CAR21+CBL-b inhibitor treatment group showed an overall upward trend during the experiment, and no adverse reactions were shown after administration; the body weight of the animals in the BI-CAR21 treatment group showed a fluctuating trend during the experiment. A single animal experienced weight loss (as shown in Figure 12D);
实验过程中生存率连续监测至30天，由于该模型肿瘤侵袭性强，肿瘤进展较快，PBNK治疗组从24天开始发生1只动物死亡，到28天死亡率为100％；BI-CAR21治疗组从25天开始发生1只动物死亡，到30天死亡率为60％；BI-CAR21+CBL-b抑制剂治疗组在临床观察中动物整体状态较佳，未发生死亡，该组表现持久治疗效果，同时保证了较好的安全性(如图12E所示)。 During the experiment, the survival rate was continuously monitored to 30 days. Due to the strong invasiveness of the tumor in this model and the rapid progression of the tumor, one animal died in the PBNK treatment group starting from the 24th day, and the mortality rate was 100% by the 28th day; BI-CAR21 treatment One animal died in the group starting from day 25, and the mortality rate was 60% on day 30; in the clinical observation, the overall condition of the animals in the BI-CAR21+CBL-b inhibitor treatment group was better, and no death occurred. This group showed durable treatment effect while ensuring better security (as shown in Figure 12E).

Claims (48)

  1. CBL-b抑制剂与免疫效应细胞的组合在制备用于治疗癌症或肿瘤的药物中的应用，所述免疫效应细胞表达或不表达嵌合抗原受体。Use of a combination of a CBL-b inhibitor and immune effector cells, which express or do not express chimeric antigen receptors, for the preparation of a medicament for the treatment of cancer or tumors.
  2. 用于治疗癌症或肿瘤的药物组合产品，其包含CBL-b抑制剂与免疫效应细胞，所述免疫效应细胞表达或不表达嵌合抗原受体。A pharmaceutical combination product for the treatment of cancer or tumors, which contains a CBL-b inhibitor and immune effector cells, the immune effector cells expressing or not expressing chimeric antigen receptors.
  3. 根据权利要求1所述的应用或权利要求2所述的药物组合产品，其中，所述免疫效应细胞选自下组：(1)表达嵌合抗原受体的T细胞；(2)表达嵌合抗原受体的NK细胞；(3)表达嵌合抗原受体的T细胞和表达嵌合抗原受体的NK细胞。The application according to claim 1 or the pharmaceutical combination product according to claim 2, wherein the immune effector cells are selected from the following group: (1) T cells expressing chimeric antigen receptors; (2) Chimeric antigen receptor expressing T cells NK cells with antigen receptors; (3) T cells expressing chimeric antigen receptors and NK cells expressing chimeric antigen receptors.
  4. 根据权利要求1-3中任一项所述的应用或药物组合产品，其中，所述CBL-b抑制剂为具有如下化学式的化合物或其药学上可接受的盐：

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

  5. 根据权利要求1-4任一项所述的应用或药物组合产品，其中，所述CBL-b抑制剂被配制为口服或注射制剂。The use or pharmaceutical combination product according to any one of claims 1-4, wherein the CBL-b inhibitor is formulated as an oral or injectable preparation.
  6. 根据权利要求1-5任一项所述的应用或药物组合产品，其中，所述癌症或肿瘤选自血液瘤或实体瘤，优选实体瘤，优选地，所述实体瘤选自胃癌、肝癌、结直肠癌、肾细胞癌、乳腺癌、肺癌、卵巢癌、皮肤癌、膀胱癌、肝癌、前列腺癌、宫颈癌、胰腺癌和肉瘤。The application or pharmaceutical combination product according to any one of claims 1 to 5, wherein the cancer or tumor is selected from hematological tumors or solid tumors, preferably solid tumors, preferably, the solid tumors are selected from gastric cancer, liver cancer, Colorectal cancer, renal cell cancer, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcoma.
  7. 根据权利要求1-6任一项所述的应用或药物组合产品，其中，所述免疫效应细胞包含表达嵌合抗原受体的T细胞和/或表达嵌合抗原受体的NK细胞，并且T细胞表达的嵌合抗原受体和所述NK细胞表达的嵌合抗原受体结合相同或不同的抗原或抗原表位。The application or pharmaceutical combination product according to any one of claims 1 to 6, wherein the immune effector cells comprise T cells expressing chimeric antigen receptors and/or NK cells expressing chimeric antigen receptors, and T The chimeric antigen receptor expressed by the cell and the chimeric antigen receptor expressed by the NK cell bind the same or different antigens or antigenic epitopes.
  8. 根据权利要求1-7任一项所述的应用或药物组合产品，其中，所述免疫效应细胞表达嵌合抗原受体，并且所述嵌合抗原受体所结合的抗原独立地选自Claudin18.2、BCMA、GPRC5D或它们的任意组合。The application or pharmaceutical combination product according to any one of claims 1 to 7, wherein the immune effector cells express chimeric antigen receptors, and the antigen bound by the chimeric antigen receptors is independently selected from Claudin18. 2. BCMA, GPRC5D or any combination thereof.
  9. 根据权利要求1-8任一项所述的应用或药物组合产品，其中，所述免疫效应细胞包含表达嵌合抗原受体的T细胞，并且所述T细胞表达的嵌合抗原受体包括信号肽、抗原结合区、铰链区、跨膜区和胞内信号传导结构区；The application 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 include signals Peptides, antigen-binding regions, hinge regions, transmembrane regions and intracellular signaling structural regions;
    优选地，所述嵌合抗原受体包括CD8α信号肽、抗原结合区、CD8α或CD28铰链区、CD8α或CD28跨膜区、CD28共刺激结构域和CD3ζ，优选地，所述抗原结合区的形式为具有VH-连接子-VL或VL-连接子-VH结构的scFv；Preferably, the chimeric antigen receptor includes a CD8α signal peptide, an antigen-binding region, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 costimulatory domain and CD3ζ. Preferably, the antigen-binding region is in the form It is an scFv having a VH-linker-VL or VL-linker-VH structure;
    优选地，所述嵌合抗原受体包含SEQ ID NO:2-4任一项所示序列，或包含与SEQ ID NO:2-4任一项所示序列具有至少80％同一性的序列。Preferably, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NO: 2-4, or a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO: 2-4.
  10. 根据权利要求1-9任一项所述的应用或药物组合产品，其中，所述免疫效应细胞包含表达嵌合抗原受体的NK细胞，并且所述NK细胞表达的嵌合抗原受体包括信号肽、抗原结合区、铰链区、跨膜区、共刺激结构域和CD3ζ；The application 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 include signals Peptides, antigen-binding regions, hinge regions, transmembrane regions, costimulatory domains and CD3ζ;
    优选地，所述嵌合抗原受体还包括通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子，所述细胞因子例如IL15；Preferably, the chimeric antigen receptor further includes a cytokine or chemokine, such as IL15, linked to the CD3ζ via a self-cleaving peptide;
    更优选地，NK细胞表达的嵌合抗原受体包括CD8α信号肽、抗原结合区、CD8α铰链区、CD8α或CD28或NKG2D跨膜区、CD28或2B4或4-1BB共刺激结构域和CD3ζ，以及优选 通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子，所述细胞因子例如IL15；More preferably, the chimeric antigen receptor expressed by the NK cell includes CD8α signal peptide, antigen-binding region, CD8α hinge region, CD8α or CD28 or NKG2D transmembrane region, CD28 or 2B4 or 4-1BB costimulatory domain and CD3ζ, and preferred A cytokine or chemokine, such as IL15, linked to said CD3ζ via a self-cleaving peptide;
    最优选地，NK细胞表达的嵌合抗原受体包含SEQ ID NO:1、5-6和46任一项所示序列，或包含与SEQ ID NO:1、5-6和46任一项所示序列具有至少80％同一性的序列。Most preferably, the chimeric antigen receptor expressed by NK cells includes the sequence shown in any one of SEQ ID NO:1, 5-6 and 46, or includes the same sequence as any one of SEQ ID NO:1, 5-6 and 46. Represents sequences with at least 80% identity.
  11. 一种治疗癌症或肿瘤的方法，其中，所述方法包括向有需要的受试者给予有效量的CBL-b抑制剂和免疫效应细胞，所述免疫效应细胞表达或不表达嵌合抗原受体。A method of treating cancer or tumors, wherein the method includes administering to a subject in need thereof an effective amount of a CBL-b inhibitor and an immune effector cell that expresses or does not express a chimeric antigen receptor .
  12. 根据权利要求11所述的方法，其中，所述免疫效应细胞选自下组：(1)表达嵌合抗原受体的T细胞；(2)表达嵌合抗原受体的NK细胞；(3)表达嵌合抗原受体的T细胞和表达嵌合抗原受体的NK细胞。The method according to claim 11, wherein the immune effector cells are selected from the following group: (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.
  13. 根据权利要求11-12任一项所述的方法，其中，所述CBL-b抑制剂为具有如下化学式或具有如下化学式的化合物的药学上可接受的盐：

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

  14. 根据权利要求11-13任一项所述的方法，其中，所述CBL-b抑制剂被配制为口服或注射制剂。The method of any one of claims 11-13, wherein the CBL-b inhibitor is formulated as an oral or injectable formulation.
  15. 根据权利要求11-14任一项所述的方法，其中，所述癌症或肿瘤选自血液瘤或实体瘤，优选实体瘤，优选地，所述实体瘤选自胃癌、肝癌、结直肠癌、肾细胞癌、乳腺癌、肺癌、卵巢癌、皮肤癌、膀胱癌、肝癌、前列腺癌、宫颈癌、胰腺癌和肉瘤。The method according to any one of claims 11 to 14, wherein the cancer or tumor is selected from the group consisting of hematological tumors or solid tumors, preferably solid tumors, preferably the solid tumors are selected from the group consisting of 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. 根据权利要求11-15任一项所述的方法，其中，所述免疫效应细胞包含表达嵌合抗原受体的T细胞和/或表达嵌合抗原受体的NK细胞，并且T细胞表达的所述嵌合抗原受体和NK细胞表达的所述嵌合抗原受体结合相同或不同的靶点。The method according to any one of claims 11 to 15, wherein the immune effector cells comprise T cells expressing chimeric antigen receptors and/or NK cells expressing chimeric antigen receptors, and the T cells express The chimeric antigen receptor and the chimeric antigen receptor expressed by NK cells bind the same or different targets.
  17. 根据权利要求11-16任一项所述的方法，其中，所述免疫效应细胞表达嵌合抗原受体，并且所述嵌合抗原受体所结合的抗原独立地选自Claudin18.2、BCMA、GPRC5D或它们的任意组合。The method according to any one of claims 11-16, wherein the immune effector cell expresses a chimeric antigen receptor, and the antigen bound by the chimeric antigen receptor is independently selected from the group consisting of Claudin18.2, BCMA, GPRC5D or any combination thereof.
  18. 根据权利要求11-17任一项所述的方法，其中，所述免疫效应细胞包含表达嵌合抗原受体的T细胞，并且T细胞表达的嵌合抗原受体包括信号肽、抗原结合区、铰链区、跨膜区和胞内信号传导结构区；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 include a signal peptide, an antigen-binding region, Hinge region, transmembrane region and intracellular signaling structural region;
    优选地，所述嵌合抗原受体包括CD8α信号肽、抗原结合区、CD8α或CD28铰链区、CD8α或CD28跨膜区、CD28共刺激结构域和CD3ζ，优选地，所述抗原结合区的形式为具有VH-连接子-VL或VL-连接子-VH结构的scFv；Preferably, the chimeric antigen receptor includes a CD8α signal peptide, an antigen-binding region, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 costimulatory domain and CD3ζ. Preferably, the antigen-binding region is in the form It is an scFv having a VH-linker-VL or VL-linker-VH structure;
    优选地，所述嵌合抗原受体包含SEQ ID NO:2-4任一项所示序列，或包含与SEQ ID NO:2-4任一项所示序列具有至少80％同一性的序列。Preferably, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NO: 2-4, or a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO: 2-4.
  19. 根据权利要求11-18任一项所述的方法，其中，所述免疫效应细胞包含表达嵌合抗原受体的NK细胞，并且NK细胞表达的嵌合抗原受体包括信号肽、抗原结合区、铰链区、跨膜区、共刺激结构域和CD3ζ；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 include a signal peptide, an antigen-binding region, Hinge region, transmembrane region, costimulatory domain and CD3ζ;
    优选地，所述嵌合抗原受体还包括通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子，所述细胞因子例如IL15；Preferably, the chimeric antigen receptor further includes a cytokine or chemokine, such as IL15, linked to the CD3ζ via a self-cleaving peptide;
    更优选地，NK细胞表达的嵌合抗原受体包括CD8α信号肽、抗原结合区、CD8α铰链区、CD8α或CD28或NKG2D跨膜区、CD28、2B4或4-1BB共刺激结构域和CD3ζ，以及优选通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子，所述细胞因子例如IL15；More preferably, the chimeric antigen receptor expressed by the NK cell includes the CD8α signal peptide, the antigen-binding region, the CD8α hinge region, the CD8α or CD28 or NKG2D transmembrane region, CD28, 2B4 or 4-1BB costimulatory domain and CD3ζ, and A cytokine or chemokine, such as IL15, preferably linked to said CD3ζ via a self-cleaving peptide;
    最优选地，NK细胞表达的嵌合抗原受体包含SEQ ID NO:1、5-6和46任一项所示序列，或包含与SEQ ID NO:1、5-6和46任一项所示序列具有至少80％同一性的序列。Most preferably, the chimeric antigen receptor expressed by NK cells includes the sequence shown in any one of SEQ ID NO:1, 5-6 and 46, or includes the same sequence as any one of SEQ ID NO:1, 5-6 and 46. Represents sequences with at least 80% identity.
  20. 一种体外扩增免疫效应细胞的方法，所述方法包括：在CBL-b抑制剂存在的条件下，扩增培养所述免疫效应细胞。 A method for amplifying immune effector cells in vitro, the method includes: amplifying and culturing the immune effector cells in the presence of a CBL-b inhibitor.
  21. 根据权利要求20所述的方法，其中，所述免疫效应细胞为T细胞或NK细胞，优选地，所述T细胞或NK细胞为表达嵌合抗原受体的T细胞或表达嵌合抗原受体的NK细胞。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 expressing chimeric antigen receptors. of NK cells.
  22. 根据权利要求20-21任一项所述的方法，其中，所述CBL-b抑制剂在培养过程中的浓度为0.1nM～10μM，例如1μM。The method according to any one of claims 20 to 21, wherein the concentration of the CBL-b inhibitor during the culture process is 0.1 nM ~ 10 μM, such as 1 μM.
  23. 根据权利要求21-22任一项所述的方法，其中，所述T细胞或NK细胞被转染嵌合抗原受体，并且在转染表达所述嵌合抗原受体的核酸分子之前或之后，添加所述CBL-b抑制剂。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 before or after transfection of a nucleic acid molecule expressing the chimeric antigen receptor , add the CBL-b inhibitor.
  24. 根据权利要求23所述的方法，其中，在所述T细胞或NK细胞开始激活时添加所述CBL-b抑制剂，或在所述T细胞或NK细胞激活培养4-7天后，添加所述CBL-b抑制剂。The method according to claim 23, wherein the CBL-b inhibitor is added when the T cells or NK cells start 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. CBL-b inhibitors.
  25. 根据权利要求20-24任一项所述的方法，其中，所述CBL-b抑制剂具有如下化学式或具有如下化学式的化合物的药学上可接受的盐：

    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 with the following chemical formula:

  26. 一种工程化的NK细胞，其中，所述NK细胞表达特异性结合Claudin18.2的嵌合抗原受体，所述嵌合抗原受体包括CD8α信号肽、特异性结合Claudin18.2的抗原结合区、CD8α铰链区、CD8α跨膜区、CD28共刺激结构域和CD3ζ，优选地，所述嵌合抗原受体还包括通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子，所述细胞因子例如IL15。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 and an antigen-binding region that specifically binds to Claudin18.2 , CD8α hinge region, CD8α transmembrane region, CD28 costimulatory domain and CD3ζ. Preferably, the chimeric antigen receptor also includes a cytokine or chemokine connected to the CD3ζ through a self-cleaving peptide, and the cell Factors such as IL15.
  27. 根据权利要求26所述的NK细胞，其中，所述抗原结合区为scFv的形式，优选为具有VH-连接肽-VL结构的scFv，更优选地，所述VH包括具有SEQ ID NO:10-12所示序列的HCDR1、HCDR2和HCDR3，所述VL包括具有SEQ ID NO:13-15所示序列的LCDR1、LCDR2和LCDR3；更优选地，所述VH具有SEQ ID NO:8所示序列或与SEQ ID NO:8所示序列至少80％同一性的序列，所述VL具有SEQ ID NO:9所示序列或与SEQ ID NO:9所示序列至少80％同一性的序列；最优选地，所述scFv具有SEQ ID NO:16或17所示序列或与SEQ ID NO:16或17所示序列至少80％同一性的序列。The NK cell according to claim 26, wherein the antigen-binding region is in the form of scFv, preferably a scFv with a VH-linking peptide-VL structure, and more preferably, the VH includes SEQ ID NO: 10- HCDR1, HCDR2 and HCDR3 of the sequence shown in 12, the VL includes LCDR1, LCDR2 and LCDR3 with the sequence shown in SEQ ID NO: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 a 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. 根据权利要求26-27任一项所述的NK细胞，其中，所述嵌合抗原受体具有SEQ ID NO:1所示序列或与SEQ ID NO:1所示序列至少80％同一性的序列。The NK cell according to any one of claims 26-27, wherein the chimeric antigen receptor has a 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. 权利要求26-28任一项所述NK细胞与表达特异性结合Claudin18.2的嵌合抗原受体的T细胞的组合在制备用于治疗癌症或肿瘤的药物中的应用。Use of the combination of NK cells described in any one of claims 26 to 28 and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2 in the preparation of drugs for the treatment of cancer or tumors.
  30. 用于治疗癌症或肿瘤的细胞药物，其包含权利要求26-28任一项所述NK细胞与表达特异性结合Claudin18.2的嵌合抗原受体的T细胞。Cell medicine for treating cancer or tumors, which comprises the NK cells of any one of claims 26-28 and T cells expressing chimeric antigen receptors that specifically bind to Claudin18.2.
  31. 根据权利要求29所述的应用或权利要求30所述的细胞药物，其中，所述T细胞表达的嵌合抗原受体包括CD8α信号肽、抗原结合区、CD8α或CD28铰链区、CD8α或CD28跨膜区、CD28共刺激结构域和CD3ζ，优选地，所述抗原结合区的形式为具有VH-连接子-VL或VL-连接子-VH结构的scFv；The application according to claim 29 or the cellular medicine according to claim 30, wherein the chimeric antigen receptor expressed by the T cell includes a CD8α signal peptide, an antigen-binding region, a CD8α or CD28 hinge region, a CD8α or a CD28 trans Membrane region, CD28 co-stimulatory domain and CD3ζ, preferably, the antigen-binding region is in the form of a scFv with a VH-linker-VL or VL-linker-VH structure;
    优选地，所述T细胞表达的嵌合抗原受体包括SEQ ID NO:2-4任一项序列或与SEQ ID NO:2-4任一项序列具有至少80％同一性的序列。Preferably, the chimeric antigen receptor expressed by the T cell includes any sequence of SEQ ID NO: 2-4 or a sequence having at least 80% identity with any sequence of SEQ ID NO: 2-4.
  32. 根据权利要求29-31任一项所述的应用或细胞药物，其中，所述癌症或肿瘤选自血液瘤或实体瘤，优选实体瘤，优选地，所述实体瘤选自胃癌、肝癌、结直肠癌、肾细胞癌、乳腺癌、肺癌、卵巢癌、皮肤癌、膀胱癌、肝癌、前列腺癌、宫颈癌、胰腺癌和肉瘤。The application or cellular medicine according to any one of claims 29 to 31, wherein the cancer or tumor is selected from the group consisting of hematological tumors or solid tumors, preferably solid tumors, and preferably the solid tumors are selected from the group consisting of gastric cancer, liver cancer, and lymph node cancer. Rectal cancer, renal cell cancer, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcoma.
  33. 一种治疗癌症或肿瘤的方法，所述方法包括向有需要的受试者给予有效量的权利要求26-28任一项所述的NK细胞，并任选地，给予表达特异性结合Claudin18.2的嵌合抗原受体的T细胞。A method of treating cancer or tumors, the method comprising administering to a subject in need an effective amount of the NK cells of any one of claims 26-28, and optionally, administering an NK cell that expresses specific binding to Claudin18. 2 chimeric antigen receptor T cells.
  34. 根据权利要求33所述的方法，其中，所述T细胞表达的嵌合抗原受体包括CD8α信号肽、抗原结合区、CD8α或CD28铰链区、CD8α或CD28跨膜区、CD28共刺激结构域和CD3ζ，优选地，所述抗原结合区的形式为具有VH-连接子-VL或VL-连接子-VH结构的scFv； The method of claim 33, wherein the chimeric antigen receptor expressed by the T cell includes a CD8α signal peptide, an antigen-binding region, a CD8α or CD28 hinge region, a CD8α or CD28 transmembrane region, a CD28 costimulatory domain, and CD3ζ, preferably, the antigen-binding region is in the form of a scFv with a VH-linker-VL or VL-linker-VH structure;
    优选地，所述T细胞表达的嵌合抗原受体包含SEQ ID NO:2-4任一项序列或与SEQ ID NO:2-4任一项序列具有至少80％同一性的序列。Preferably, the chimeric antigen receptor expressed by the T cell comprises any one of the sequences of SEQ ID NO: 2-4 or a sequence that is at least 80% identical to any one of the sequences of SEQ ID NO: 2-4.
  35. 根据权利要求33-34任一项所述的方法，其中所述癌症或肿瘤选自血液瘤或实体瘤，优选实体瘤，优选地，所述实体瘤选自胃癌、肝癌、结直肠癌、肾细胞癌、乳腺癌、肺癌、卵巢癌、皮肤癌、膀胱癌、肝癌、前列腺癌、宫颈癌、胰腺癌和肉瘤。The method according to any one of claims 33 to 34, wherein the cancer or tumor is selected from the group consisting of hematological tumors or solid tumors, preferably solid tumors. Preferably, the solid tumors are selected from the group consisting of gastric cancer, liver cancer, colorectal cancer, renal cancer, and gastric cancer. Cell carcinoma, breast cancer, lung cancer, ovarian cancer, skin cancer, bladder cancer, liver cancer, prostate cancer, cervical cancer, pancreatic cancer and sarcoma.
  36. 以下(a)-(c)中任一项组合在制备用于治疗肿瘤或癌症的药物中的应用：Use of any combination of the following (a)-(c) in the preparation of a medicament for the treatment of tumors or cancer:
    (a)表达嵌合抗原受体的免疫效应细胞和CBL-b抑制剂，(a) Immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,
    (b)表达嵌合抗原受体的免疫效应细胞与PDL1-IL15融合蛋白，(b) Immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,
    (c)表达嵌合抗原受体的免疫效应细胞、CBL-b抑制剂和PDL1-IL15融合蛋白。(c) Immune effector cells expressing chimeric antigen receptor, CBL-b inhibitor and PDL1-IL15 fusion protein.
  37. 用于治疗癌症或肿瘤的药物组合产品，其包含以下(a)-(c)中任一项：Pharmaceutical combination products for the treatment of cancer or tumors, which contain any one of the following (a)-(c):
    (a)表达嵌合抗原受体的免疫效应细胞和CBL-b抑制剂，(a) Immune effector cells expressing chimeric antigen receptors and CBL-b inhibitors,
    (b)表达嵌合抗原受体的免疫效应细胞与PDL1-IL15融合蛋白，(b) Immune effector cells expressing chimeric antigen receptors and PDL1-IL15 fusion protein,
    (c)表达嵌合抗原受体的免疫效应细胞、CBL-b抑制剂和PDL1-IL15融合蛋白。(c) Immune effector cells expressing chimeric antigen receptor, CBL-b inhibitor and PDL1-IL15 fusion protein.
  38. 根据权利要求36所述的应用或权利要求37所述的药物组合产品，其中，所述嵌合抗原受体结合一个或多个抗原或抗原表位，优选地，所述抗原独立地选自Claudin18.2、BCMA、GPRC5D或它们的任意组合；The use according to claim 36 or the pharmaceutical combination product according to claim 37, wherein the chimeric antigen receptor binds one or more antigens or antigenic epitopes, preferably, the antigens are independently selected from Claudin18 .2. BCMA, GPRC5D or any combination thereof;
    优选地，所述嵌合抗原受体包括CD8α信号肽、特异性结合BCMA和/或GPRC5D的抗原结合区、CD8α铰链区、CD8α跨膜区、CD28共刺激结构域和CD3ζ，优选地，所述嵌合抗原受体还包括通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子，所述细胞因子例如IL15；Preferably, the chimeric antigen receptor includes a CD8α signal peptide, an antigen-binding region that specifically binds BCMA and/or GPRC5D, a CD8α hinge region, a CD8α transmembrane region, a CD28 costimulatory domain and CD3ζ. Preferably, the Chimeric antigen receptors also include cytokines or chemokines, such as IL15, linked to the CD3ζ via a self-cleaving peptide;
    优选地，所述抗原结合区为VHH的形式，更优选地，所述VHH包括具有SEQ ID NO:39-41所示序列的第一组HCDR1-HCDR3和/或SEQ ID NO:43-45所示序列的第二组HCDR1-HCDR3，更优选地，所述VHH具有SEQ ID NO:38和/或42所示序列或与SEQ ID NO:38至少80％同一性的序列和/或与SEQ ID NO:42所示序列至少80％同一性的序列；Preferably, the antigen-binding region is in the form of a VHH. More preferably, the VHH includes the first group of HCDR1-HCDR3 having the sequences shown in SEQ ID NO: 39-41 and/or the sequences shown in SEQ ID NO: 43-45. The second group of HCDR1-HCDR3 with the sequence shown, more preferably, the VHH has the sequence shown in SEQ ID NO:38 and/or 42 or a sequence with at least 80% identity to SEQ ID NO:38 and/or with SEQ ID NO. A sequence with at least 80% identity to the sequence shown in NO:42;
    优选地，所述嵌合抗原受体具有SEQ ID NO:46所示序列或与SEQ ID NO:46所示序列至少80％同一性的序列。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. 根据权利要求36-38任一项所述的应用或药物组合产品，其中，所述免疫效应细胞选自T细胞(例如细胞毒性T细胞、辅助T细胞、肿瘤浸润T细胞)、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、嗜中性粒细胞、巨噬细胞和树突状细胞；优选地，所述免疫效应细胞为NK细胞。The application or pharmaceutical combination product according to any one of claims 36 to 38, wherein the immune effector cells are selected from T cells (such as 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. 根据权利要求36-39任一项所述的应用或药物组合产品，其中所述CBL-b抑制剂为具有如下化学式的化合物或其药学上可接受的盐：
    The application or pharmaceutical combination product according to any one of claims 36-39, wherein the CBL-b inhibitor is a compound with the following chemical formula or a pharmaceutically acceptable salt thereof:
  41. 根据权利要求36-40任一项所述的应用或药物组合产品，其中，所述PDL1-IL15融合蛋白具有如SEQ ID NO:47所示的序列。The application or pharmaceutical combination product according to any one of claims 36-40, wherein the PDL1-IL15 fusion protein has the sequence shown in SEQ ID NO: 47.
  42. 根据权利要求36-41任一项所述的应用或药物组合产品，其中，所述肿瘤或癌症为B细胞淋巴瘤；更优选为多发性骨髓瘤(MM)；最优选地，所述多发性骨髓瘤为难治性或复发性多发性骨髓瘤。The application or pharmaceutical combination product according to any one of claims 36-41, wherein the tumor or cancer is B-cell lymphoma; more preferably, it is multiple myeloma (MM); most preferably, the multiple myeloma (MM) Myeloma is refractory or relapsed multiple myeloma.
  43. 一种治疗癌症或肿瘤的方法，所述方法包括给予有需要的受试者有效量的表达嵌合 抗原受体的免疫效应细胞，所述方法还包括：(1)在给药前，使用CBL-b抑制剂刺激所述免疫效应细胞，和/或(2)给予受试者有效量PDL1-IL15融合蛋白。A method of treating cancer or tumors, the method comprising administering to a subject in need thereof an effective amount of an expression chimeric Immune effector cells of antigen receptors, the method further comprising: (1) using a CBL-b inhibitor to stimulate the immune effector cells before administration, and/or (2) administering an effective amount of PDL1-IL15 to the subject fusion protein.
  44. 根据权利要求43所述的方法，其中，所述嵌合抗原受体结合一个或多个抗原或抗原表位，优选地，所述抗原独立地选自Claudin18.2、BCMA、GPRC5D或它们的任意组合；The method according to claim 43, wherein the chimeric antigen receptor binds one or more antigens or antigenic epitopes, preferably, the antigen is independently selected from Claudin18.2, BCMA, GPRC5D or any of them combination;
    优选地，所述嵌合抗原受体包括CD8α信号肽、特异性结合BCMA和/或GPRC5D的抗原结合区、CD8α铰链区、CD8α跨膜区、CD28共刺激结构域和CD3ζ，优选地，所述嵌合抗原受体还包括通过自裂解肽连接至所述CD3ζ的细胞因子或趋化因子，所述细胞因子例如IL15；Preferably, the chimeric antigen receptor includes a CD8α signal peptide, an antigen-binding region that specifically binds BCMA and/or GPRC5D, a CD8α hinge region, a CD8α transmembrane region, a CD28 costimulatory domain and CD3ζ. Preferably, the Chimeric antigen receptors also include cytokines or chemokines, such as IL15, linked to the CD3ζ via a self-cleaving peptide;
    优选地，所述抗原结合区为VHH的形式，更优选地，所述VHH包括具有SEQ ID NO:39-41所示序列的第一组HCDR1-HCDR3和/或SEQ ID NO:43-45所示序列的第二组HCDR1-HCDR3，更优选地，所述VHH具有SEQ ID NO:38和/或42所示序列或与SEQ ID NO:38至少80％同一性的序列和/或与SEQ ID NO:42所示序列至少80％同一性的序列；Preferably, the antigen-binding region is in the form of a VHH. More preferably, the VHH includes the first group of HCDR1-HCDR3 having the sequences shown in SEQ ID NO: 39-41 and/or the sequences shown in SEQ ID NO: 43-45. The second group of HCDR1-HCDR3 with the sequence shown, more preferably, the VHH has the sequence shown in SEQ ID NO:38 and/or 42 or a sequence with at least 80% identity to SEQ ID NO:38 and/or with SEQ ID NO. A sequence with at least 80% identity to the sequence shown in NO:42;
    优选地，所述嵌合抗原受体具有SEQ ID NO:46所示序列或与SEQ ID NO:46所示序列至少80％同一性的序列。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. 根据权利要求43-44任一项所述的方法，其中，所述免疫效应细胞选自T细胞(例如细胞毒性T细胞、辅助T细胞、肿瘤浸润T细胞)、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、嗜中性粒细胞、巨噬细胞和树突状细胞；优选地，所述免疫效应细胞为NK细胞。The method according to any one of claims 43-44, wherein the immune effector cells are selected from T cells (such as 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. 根据权利要求43-45任一项所述的方法，其中，所述CBL-b抑制剂为具有如下化学式的化合物或其药学上可接受的盐：

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

  47. 根据权利要求43-46任一项所述的方法，其中，所述PDL1-IL15融合蛋白具有如SEQ ID NO:47所示的序列。The method according to any one of claims 43-46, wherein the PDL1-IL15 fusion protein has the sequence shown in SEQ ID NO:47.
  48. 根据权利要求43-47任一项所述的方法，其中，所述肿瘤或癌症为B细胞淋巴瘤；更优选为多发性骨髓瘤(MM)；最优选地，所述多发性骨髓瘤为难治性或复发性多发性骨髓瘤。 The method according to any one of claims 43-47, wherein the tumor or cancer is B-cell lymphoma; more preferably, it is multiple myeloma (MM); most preferably, the multiple myeloma is refractory to treatment. chronic or relapsing multiple myeloma.
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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108473957A (en) * 2015-04-17 2018-08-31 诺华股份有限公司 The method of the effect of improving Chimeric antigen receptor expression cell and amplification
WO2020038147A1 (en) * 2018-08-24 2020-02-27 深圳普瑞金生物药业有限公司 Anti-bcma single domain antibodies and application thereof
WO2020143631A1 (en) * 2019-01-07 2020-07-16 科济生物医药（上海）有限公司 Cellular immunotherapy combination
CN111848809A (en) * 2019-04-08 2020-10-30 上海健信生物医药科技有限公司 CAR molecule targeting Claudin18.2, immune cell modified by same and application
WO2021061870A1 (en) * 2019-09-24 2021-04-01 Nurix Therapeutics, Inc. Cbl inhibitors and compositions for expansion of immune cells
WO2021147928A1 (en) * 2020-01-21 2021-07-29 南京北恒生物科技有限公司 Immune cell comprising chimeric antigen receptor and use thereof
CN113234174A (en) * 2015-08-11 2021-08-10 南京传奇生物科技有限公司 Chimeric antigen receptors targeting BCMA and methods of use thereof
CN113549640A (en) * 2020-04-24 2021-10-26 阿斯利康(瑞典)有限公司 Compositions and methods for treating cancer with chimeric antigen receptors
CN113603788A (en) * 2014-01-15 2021-11-05 卡德门企业有限公司 Immunomodulator
CN114364670A (en) * 2019-06-26 2022-04-15 纽力克斯治疗公司 Substituted benzyl-triazoles for Cbl-b inhibition and further uses thereof
CN114364667A (en) * 2019-04-09 2022-04-15 紐力克斯治疗公司 3-substituted piperidines for Cbl-b inhibition and the use of Cbl-b inhibitors in combination with cancer vaccines and/or oncolytic viruses
WO2023036330A1 (en) * 2021-09-13 2023-03-16 先声再明医药有限公司 Tricyclic compound as cbl-b inhibitor

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113603788A (en) * 2014-01-15 2021-11-05 卡德门企业有限公司 Immunomodulator
CN108473957A (en) * 2015-04-17 2018-08-31 诺华股份有限公司 The method of the effect of improving Chimeric antigen receptor expression cell and amplification
CN113234174A (en) * 2015-08-11 2021-08-10 南京传奇生物科技有限公司 Chimeric antigen receptors targeting BCMA and methods of use thereof
WO2020038147A1 (en) * 2018-08-24 2020-02-27 深圳普瑞金生物药业有限公司 Anti-bcma single domain antibodies and application thereof
WO2020143631A1 (en) * 2019-01-07 2020-07-16 科济生物医药（上海）有限公司 Cellular immunotherapy combination
CN111848809A (en) * 2019-04-08 2020-10-30 上海健信生物医药科技有限公司 CAR molecule targeting Claudin18.2, immune cell modified by same and application
CN114364667A (en) * 2019-04-09 2022-04-15 紐力克斯治疗公司 3-substituted piperidines for Cbl-b inhibition and the use of Cbl-b inhibitors in combination with cancer vaccines and/or oncolytic viruses
CN114364670A (en) * 2019-06-26 2022-04-15 纽力克斯治疗公司 Substituted benzyl-triazoles for Cbl-b inhibition and further uses thereof
WO2021061870A1 (en) * 2019-09-24 2021-04-01 Nurix Therapeutics, Inc. Cbl inhibitors and compositions for expansion of immune cells
WO2021147928A1 (en) * 2020-01-21 2021-07-29 南京北恒生物科技有限公司 Immune cell comprising chimeric antigen receptor and use thereof
CN113549640A (en) * 2020-04-24 2021-10-26 阿斯利康(瑞典)有限公司 Compositions and methods for treating cancer with chimeric antigen receptors
WO2023036330A1 (en) * 2021-09-13 2023-03-16 先声再明医药有限公司 Tricyclic compound as cbl-b inhibitor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KUMAR JITENDRA, KUMAR RITESH, KUMAR SINGH AMIR, TSAKEM ELVICHE L, KATHANIA MAHESH, RIESE MATTHEW J, THEISS ARIANNE L, DAVILA MARCO: "Deletion of Cbl-b inhibits CD8 + T-cell exhaustion and promotes CAR T-cell function", JOURNAL FOR IMMUNOTHERAPY OF CANCER, vol. 9, no. 1, 14 December 2020 (2020-12-14), pages e001688, XP093008772, DOI: 10.1136/jitc-2020-001688 *
MARTOMO S., X-J. FENG, D. LU, Z. POLONSKAYA, X. LUNA, M.V. POYUROVSKY, K. MCCRACKEN, F. MIYARA, L. LI , S. AGGARWAL4, J. PATEL: "Anti-PD-L1/IL-15 fusion protein generates robust adaptive immune gene signatures in tumours leading to tumour inhibition and memory responses", vol. 30, 1 October 2019 (2019-10-01), pages v475, XP055886471, DOI: 10.1093/annonc/mdz253 *
YANG CUIQING, WANG YIFANG, LIU TINGTING, JIANG FUWEI, WANG CHAO, WANG QINGYANG, WANG QIN, LI ZHENGTAO, MAI WANSUN, YE GANG, TANG R: "Abstract 2899: Small molecule inhibitor of ubiquitin ligase CBL-B enhanced anti-tumor response of CAR-T and CAR-NK cell therapies", 1SHANGHAI SIMNOVA BIOTECHNOLOGY CO., LTD., SHANGHAI, CHINA;, vol. 83, no. 7_Supplement, 4 April 2023 (2023-04-04), pages 2899 - 2899, XP093106817, DOI: 10.1158/1538-7445.AM2023-2899 *

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