System for controlling immune cells

Abstract

The present invention relates to: a recombinant polypeptide comprising a bromodomain variant; a chimeric antigen receptor comprising the bromodomain variant in an intracellular signaling domain; a gene construct comprising a nucleotide sequence encoding the recombinant polypeptide or the chimeric antigen receptor; a cell or an immune cell, each expressing the recombinant polypeptide or the chimeric antigen receptor; a pharmaceutical composition for the prevention or treatment of cancer, comprising the immune cell; a composition for degrading the recombinant polypeptide or chimeric antigen receptor of the immune cell, the composition comprising a PROTAC compound as an active ingredient; and a kit for the prevention or treatment of cancer, comprising the pharmaceutical composition and the PROTAC compound.

Description

Immune cell regulatory system

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0096530, filed July 25, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a recombinant polypeptide comprising a variant of a bromodomain, a chimeric antigen receptor comprising the variant of the bromodomain in an intracellular signaling domain, a gene construct comprising a base sequence encoding the recombinant polypeptide or the chimeric antigen receptor, a cell or immune cell expressing the recombinant polypeptide or the chimeric antigen receptor, a pharmaceutical composition for preventing or treating cancer comprising the immune cell, a composition for degrading the recombinant polypeptide or the chimeric antigen receptor of the immune cell comprising a protac compound as an active ingredient, and a kit for preventing or treating cancer comprising the pharmaceutical composition and the protac compound.

[0004]

[0005] There is growing interest in gene therapy methods that utilize immune cells to strengthen or genetically modify them and then inject them back into the patient. For example, tumor-infiltrating lymphocytes (TIL), chimeric antigen receptors (CAR), and T-cell receptor (TCR) technologies are being studied.

[0006] A chimeric antigen receptor (CAR) is an artificial chimeric protein that fuses a single-chain antibody that recognizes a cancer cell surface antigen with a signaling domain that induces T cell activation. By introducing the gene encoding the chimeric antigen receptor into conventional peripheral blood T cells (peripheral blood T lymphocytes) that do not exhibit tumor reactivity, it becomes possible to mass-produce chimeric antigen receptor-expressing T cells (hereinafter referred to as "CAR-T") that express the chimeric antigen receptor. These CAR-T cells exhibit tumor reactivity, enabling them to induce cancer cell damage without relying on interaction with the major histocompatibility complex (MHC).

[0007] CAR-T cell therapy has gained significant attention, particularly in clinical trials targeting hematological malignancies, due to its dramatic efficacy. Specifically, CAR-T cell therapy, which utilizes antibodies that recognize the B-lymphocyte-derived hematological antigen CD19, demonstrated remarkable therapeutic efficacy in early clinical trials involving patients with acute lymphoblastic leukemia who had previously failed all other treatments: 90% of patients (27 out of 30) achieved complete remission within a month, with a six-month overall survival rate of 78%. Based on these results, two CD19 CAR-T cell therapies received FDA approval and were successfully commercialized in late 2017.

[0008] However, immunotherapies expressing chimeric antigen receptors, such as the above, can cause unexpected side effects, including cytokine release syndrome, neurotoxicity, and off-target drug effects. Therefore, there is a growing need to eliminate or modulate the activity of immune cells expressing chimeric antigen receptors, if necessary, to enhance the safety of immunotherapies expressing chimeric antigen receptors.

[0009] Furthermore, excessive expression of chimeric antigen receptors accelerates T cell exhaustion, thereby inhibiting the anticancer effects of CAR-T cells. Therefore, there is a pressing need for technologies that can mitigate T cell exhaustion by appropriately regulating chimeric antigen receptor expression, thereby enhancing the therapeutic efficacy of CAR-T.

[0010] Accordingly, the inventors of the present invention completed the present invention to solve the side effects and exhaustion phenomenon of immune cells expressing chimeric antigen receptors.

[0011]

[0012] One object of the present invention is to provide a recombinant polypeptide comprising a variant of a bromodomain.

[0013] One object of the present invention is to provide a chimeric antigen receptor (CAR) comprising a variant of a bromodomain in its intracellular domain.

[0014] Another object of the present invention is to provide a gene construct comprising a base sequence encoding the recombinant polypeptide or the chimeric antigen receptor.

[0015] Another object of the present invention is to provide a cell or immune cell expressing the recombinant polypeptide or chimeric antigen receptor.

[0016] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the immune cells.

[0017] Another object of the present invention is to provide a composition for degrading a recombinant polypeptide or chimeric antigen receptor of the immune cell, which comprises a protac compound as an active ingredient.

[0018] Another object of the present invention is to provide a kit for preventing or treating cancer, comprising the pharmaceutical composition and a protac compound.

[0019] To achieve the above object, one aspect of the present invention provides a recombinant polypeptide comprising a bromodomain variant. When a protac compound is used against a wild-type bromodomain, dozens of proteins containing bromodomains within cells, particularly proteins belonging to the BET family, are also degraded, which can result in severe cytotoxicity. Therefore, the present invention has constructed a system capable of degrading a recombinant polypeptide comprising a bromodomain variant using a protac compound that selectively acts only on the bromodomain variant.

[0020] Another aspect of the present invention provides a chimeric antigen receptor comprising an extracellular binding domain comprising an antigen binding site; a transmembrane domain; and an intracellular domain of a receptor comprising the recombinant polypeptide.

[0021] Another aspect of the present invention provides a genetic construct comprising a base sequence encoding the recombinant polypeptide or the chimeric antigen receptor.

[0022] Another aspect of the present invention provides a cell or immune cell expressing the recombinant polypeptide or chimeric antigen receptor.

[0023] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the immune cells.

[0024] Another aspect of the present invention provides a composition for degrading a recombinant polypeptide or chimeric antigen receptor of the immune cell, comprising a protac compound as an active ingredient.

[0025] Another aspect of the present invention provides a kit for preventing or treating cancer, comprising the pharmaceutical composition and a protac compound.

[0026] The recombinant polypeptide of the present invention contains a variant of a bromo domain, and can bind to a substance targeting the variant of the bromo domain. When the substance is a target protein decomposing agent such as a protease compound, the protease compound can recognize and bind to the variant of the bromo domain, and decompose the recombinant polypeptide containing the variant of the bromo domain.

[0027] In addition, the cell expressing the recombinant polypeptide of the present invention or a chimeric antigen receptor comprising the same expresses a recombinant polypeptide or a chimeric antigen receptor comprising a variant of a bromo domain, and the recombinant polypeptide or chimeric antigen receptor of the cell can bind to the protac compound and be degraded by the protac compound.

[0028] When the above cell is an immune cell and expresses a chimeric antigen receptor, it exhibits cytotoxic activity or cytokine secretion activity against cancer cells, and can be used for the prevention or treatment of cancer. In addition, the immune cell expresses a chimeric antigen receptor containing a variant of the bromo domain, and when a protac compound is treated to the immune cell, the protac compound can specifically target and bind to a recombinant polypeptide or chimeric antigen receptor containing a variant of the bromo domain of the immune cell, thereby degrading the recombinant polypeptide or chimeric antigen receptor containing a variant of the bromo domain, thereby having the effect of regulating the activity of the immune cell. In addition, depending on the treatment amount of the protac compound, the anticancer activity of the immune cell expressing the recombinant polypeptide or chimeric antigen receptor containing a variant of the bromo domain can be regulated to a desired degree. Specifically, lowering the dosage of the protac compound degrades the chimeric antigen receptor containing the variant of the bromo domain to an appropriate level, thereby suppressing the exhaustion of T cells caused by overexpression of the chimeric antigen receptor, thereby sustaining or increasing the anticancer activity of CAR-T cells. On the other hand, increasing the dosage of the protac compound degrades the chimeric antigen receptor containing the variant of the bromo domain to a high level, thereby reducing the side effects caused by the high activity of the CAR-T cells.

[0029] The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

[0030]

[0031] Figure 1 shows the results confirming that CAR is not degraded even when treated with lenalidomide and RIPK degrader 2 protease compound after expressing a CAR protein bound to the CK1a or RIPK2 domain.

[0032] Figure 2 shows a gene construct that includes the gene sequence of a bromo domain or a variant of the bromo domain of the present invention between the gene sequences of CD28 and CD3ζ.

[0033] Figure 3 shows the structures of ARV771, a wild-type bromo domain protac compound, and AGB-1, a protac compound for a bromo domain mutant. It also shows the binding affinity of ARV771 and AGB1 to BD2 WT or BD2 L387V. Furthermore, it shows the results of confirming the cytotoxicity of the AGB-1 protac compound in PBMCs and T cells. Through this, it was confirmed that AGB-1 has no cytotoxicity at all compared to ARV771.

[0034] Figure 4 shows the results of Western blotting to determine whether CAR is degraded when Jurkat cells expressing a CAR comprising a variant of the bromo domain of the present invention are treated with a protease compound. It was confirmed that AGB-1 degrades CAR approximately 10 times better than ARV771.

[0035] Figure 5 shows the results of confirming the destruction of CAR proteins by AGB-1 for other targets containing variants of the bromo domain.

[0036] Figure 6 shows the results of recovery of the destruction of the chimeric antigen receptor by AGB-1 by the proteasome inhibitor epoxomicin.

[0037] Figure 7 shows the results of confirming whether the IL-2 secretion activity of a Jurkat T cell line expressing a chimeric antigen receptor including a variant of the bromo domain of the present invention is reduced when the protac compound is treated on the same.

[0038] Figures 8a and 8b show the results of confirming the destruction of a chimeric antigen receptor present on the surface of a T cell when a protac compound is treated on a T cell expressing a chimeric antigen receptor including a variant of the bromo domain of the present invention, and the results of confirming whether the cytotoxic activity of the T cell is reduced.

[0039] Figure 9 shows the results of confirming whether the cytotoxic activity of NK cells is reduced when a KHYG-1 NK cell line expressing a chimeric antigen receptor including a variant of the bromo domain of the present invention is treated with a protac compound.

[0040] Figure 10 shows the results of confirming whether a CAR including a variant of the bromo domain of the immune cell of the present invention is degraded by AGB-1 and whether the activity of the immune cell of the present invention changes depending on the connection position of the gene of the variant of the bromo domain of the present invention and the chimeric antigen receptor gene.

[0041] Figures 11a and 11b show the results of confirming whether the anticancer activity of CAR-T cells is restored when the protac compound is removed after treating T cells expressing a chimeric antigen receptor including a variant of the bromo domain of the present invention with a protac compound.

[0042] Figure 12 shows the survival rate of mice after administering T cells expressing a chimeric antigen receptor including a variant of the bromo domain of the present invention to immunodeficient mice transplanted with tumor cells.

[0043] Figures 13 and 14 show the results of comparing the anticancer activity by observing the tumor volume of immunodeficient mice for about 122 days when only PBS (phosphate buffer saline) was treated, when only the CAR-BD2m T cells of the present invention were treated, and when the CAR-BD2m T cells of the present invention and AGB-1 were treated together to confirm the effect of improving the anticancer efficacy when the CAR-BD2m T cells of the present invention were used in combination with AGB-1.

[0044] Figure 15 shows that when treating CAR-BD2m T cells of the present invention with an AGB-1 protac compound, the activity of CAR-T cells can be suppressed or the anticancer activity of CAR-T cells can be enhanced depending on the treatment dose of AGB-1.

[0045]

[0046] Hereinafter, the present invention will be described in detail.

[0047]

[0048] 1. Recombinant polypeptide of the present invention

[0049] One aspect of the present invention provides a recombinant polypeptide comprising a variant of a bromodomain.

[0050] The term "polypeptide" as used herein means a molecule formed by multiple amino acid residues being linked to each other by peptide bonds.

[0051] The bromo domain above is a region that recognizes ethyl lysine of histones, and is a region that exists in nuclear proteins such as histone acetyltransferases (HATs) or chromatin remodeling factors. Like the function of Brahma / brm, a protein containing a bromo domain, the bromo domain is mainly found in proteins involved in transcriptional activation. In particular, it is known to be mainly found in histone acetylases (HATs) or chromatin remodeling complexes, which are proteins that promote transcriptional activation by changing the structure of chromatin or inducing modification.

[0052] The variant of the bromo domain of the present invention may mean that a mutation has been introduced into the amino acid sequence of the wild-type bromo domain.

[0053] The above mutation may be, but is not limited to, a mutation of amino acid deletion, insertion, inversion, substitution or sequence mixing.

[0054] Among the proteins having the above bromo domain, there is the BET (bromodomain and extra-terminal domain) family, and the BET family may include BRD2, BRD3, BRD4, and BRDT. The similarity of the amino acid sequences of the bromo domains possessed by BRD2, BRD3, BRD4, and BRDT, which belong to the BET family, is quite high.

[0055] The bromo domain may be bromo domain 1 (Bromodomain 1, BD1) or bromo domain 2 (Bromodomain 2, BD2) of any one protein selected from the group consisting of bromodomain-containing protein 2 (BRD2), bromodomain-containing protein 3 (BRD3), bromodomain-containing protein 4 (BRD4), and bromodomain-containing protein T (BRDT), and the variant of the bromo domain may be one in which a mutation is introduced into bromo domain 1 (Bromodomain 1, BD1) or bromo domain 2 (Bromodomain 2, BD2) of any one protein selected from the group consisting of bromodomain-containing protein 2 (BRD2), bromodomain-containing protein 3 (BRD3), bromodomain-containing protein 4 (BRD4), and bromodomain-containing protein T (BRDT).

[0056] The above bromo domain variant may be a variant in which a mutation is introduced into the amino acid sequence of the second bromo domain (hereinafter, BD2) included in BRD4 (Bromodomain-containing protein 4), and specifically, may be a variant in which a mutation is introduced into leucine (L), which is the 387th amino acid of BD2 of BRD4.

[0057] In one embodiment of the present invention, the variant of the bromo domain may include one in which the 387th leucine (L) of the BD2 is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), for example, one in which the 387th leucine (L) of the BD2 is substituted with alanine (A) or valine (V), and in particular, one in which the 387th leucine (L) of the BD2 is substituted with alanine (A) or valine (V).

[0058] In one embodiment of the present invention, the variant of the bromo domain may be an L387A variant or an L387V variant of BD2 of the BRD4, and may include an amino acid sequence of SEQ ID NO: 1.

[0059] In addition, the variant of the bromo domain may be a variant in which a mutation is introduced into the amino acid sequence of the first bromo domain (hereinafter, BD1) included in BRD4 (Bromodomain-containing protein 4), and specifically, a mutation may be introduced into leucine (L), which is the 94th amino acid of BD1 of the BRD4.

[0060] The variant of the bromo domain may include a variant in which the 94th leucine (L) of the BD1 of the BRD4 is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), for example, a variant in which the 94th leucine (L) of the BD1 is substituted with alanine (A) or valine (V), and in particular, a variant in which the 94th leucine (L) of the BD1 is substituted with alanine (A) or valine (V).

[0061] In one embodiment of the present invention, the variant of the bromo domain may be an L94V variant or an L94A variant of BD1 of the BRD4, and may include an amino acid sequence of SEQ ID NO: 2.

[0062] In addition, the variant of the bromo domain may be a variant in which a mutation is introduced into the amino acid sequence of the first bromo domain (hereinafter, BD1) included in BRD2 (Bromodomain-containing protein 2), and specifically, a mutation may be introduced into leucine (L), which is the 110th amino acid of BD1 of the BRD2.

[0063] The variant of the bromo domain may include a variant in which the 110th leucine (L) of the BD1 of the BRD2 is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), for example, a variant in which the 110th leucine (L) of the BD1 is substituted with alanine (A) or valine (V), and in particular, a variant in which the 110th leucine (L) of the BD1 is substituted with alanine (A) or valine (V).

[0064] In one embodiment of the present invention, the variant of the bromo domain may be an L110V variant or an L110A variant of BD1 of the BRD2, and may include an amino acid sequence of SEQ ID NO: 3.

[0065] In addition, the variant of the bromo domain may be a variant in which a mutation is introduced into the amino acid sequence of the second bromo domain (hereinafter, BD2) included in BRD2 (Bromodomain-containing protein 2), and specifically, a mutation may be introduced into leucine (L), which is the 383rd amino acid of BD2 of the BRD2.

[0066] The variant of the bromo domain may include a variant in which the 383rd leucine (L) of the BD2 of the BRD2 is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), for example, a variant in which the 383rd leucine (L) of the BD1 is substituted with alanine (A) or valine (V), and in particular, a variant in which the 383rd leucine (L) of the BD1 is substituted with alanine (A) or valine (V).

[0067] In one embodiment of the present invention, the variant of the bromo domain may be an L383V variant or an L383V variant of BD2 of the BRD2, and may include an amino acid sequence of SEQ ID NO: 4.

[0068] In addition, the variant of the bromo domain may be a variant in which a mutation is introduced into the amino acid sequence of the first bromo domain (hereinafter, BD1) included in BRD3 (Bromodomain-containing protein 3), and specifically, a mutation may be introduced into leucine (L), which is the 70th amino acid of BD1 of the BRD3.

[0069] The variant of the bromo domain may include a variant in which the 70th leucine (L) of BD1 of the BRD3 is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), for example, a variant in which the 70th leucine (L) of the BD1 is substituted with alanine (A) or valine (V), and in particular, a variant in which the 70th leucine (L) of the BD1 is substituted with alanine (A) or valine (V).

[0070] In one embodiment of the present invention, the variant of the bromo domain may be an L70V variant or an L70A variant of BD1 of the BRD3, and may include an amino acid sequence of SEQ ID NO: 5.

[0071] In addition, the variant of the bromo domain may be a variant in which a mutation is introduced into the amino acid sequence of the first bromo domain (hereinafter, BD2) included in BRD3 (Bromodomain-containing protein 3), and specifically, a mutation may be introduced into leucine (L), which is the 345th amino acid of BD2 of the BRD3.

[0072] The variant of the bromo domain may include a variant in which the 345th leucine (L) of the BD2 of the BRD3 is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), for example, a variant in which the 345th leucine (L) of the BD2 is substituted with alanine (A) or valine (V), and in particular, a variant in which the 345th leucine (L) of the BD1 is substituted with alanine (A) or valine (V).

[0073] In one embodiment of the present invention, the variant of the bromo domain may be an L345V variant or an L345A variant of BD2 of the BRD3, and may include an amino acid sequence of SEQ ID NO: 6.

[0074] In addition, the variant of the bromo domain may be a variant in which a mutation is introduced into the amino acid sequence of the first bromo domain (hereinafter, BD1) included in BRDT (Bromodomain-containing protein T), and specifically, a mutation may be introduced into leucine (L), which is the 63rd amino acid of BD1 of the BRDT.

[0075] The variant of the bromo domain may include a variant in which the 63rd leucine (L) of the BD1 of the BRDT is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), for example, a variant in which the 63rd leucine (L) of the BD1 is substituted with alanine (A) or valine (V), and in particular, a variant in which the 63rd leucine (L) of the BD1 is substituted with alanine (A) or valine (V).

[0076] In one embodiment of the present invention, the variant of the bromo domain may be an L63V variant or an L63A variant of BD1 of the BRDT, and may include an amino acid sequence of SEQ ID NO: 7.

[0077] In addition, the variant of the bromo domain may be a variant in which a mutation is introduced into the amino acid sequence of the first bromo domain (hereinafter, BD2) included in BRDT (Bromodomain-containing protein T), and specifically, a mutation may be introduced into leucine (L), which is the 306th amino acid of BD2 of the BRDT.

[0078] The variant of the bromo domain may include a variant in which the 306th leucine (L) of the BD2 of the BRDT is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), for example, a variant in which the 306th leucine (L) of the BD2 is substituted with alanine (A) or valine (V), and in particular, a variant in which the 306th leucine (L) of the BD1 is substituted with alanine (A) or valine (V).

[0079] In one embodiment of the present invention, the variant of the bromo domain may be an L306V variant or an L306A variant of BD1 of the BRDT, and may include an amino acid sequence of SEQ ID NO: 8.

[0080] Accordingly, the variant of the bromo domain of the present invention may be one in which a mutation is introduced into any one leucine (L) selected from the group consisting of the 63rd leucine (L) of BD1 of the BRDT, the 70th leucine (L) of BD1 of the BRD3, the 94th leucine (L) of BD1 of the BRD4, the 110th leucine (L) of BD1 of the BRD2, the 306th leucine (L) of BD2 of the BRDT, the 345th leucine (L) of BD2 of the BRD3, the 383rd leucine (L) of BD2 of the BRD2, and the 387th leucine (L) of BD2 of the BRD4, and specifically, the 63rd leucine (L) of BD1 of the BRDT, the 70th leucine (L) of BD1 of the BRD3, the 94th leucine (L) of BD1 of the BRD4 Any one leucine (L) selected from the group consisting of the 94th leucine (L) of the BD1 of the BRD2, the 110th leucine (L) of the BD2 of the BRDT, the 306th leucine (L) of the BD2 of the BRDT, the 345th leucine (L) of the BD2 of the BRD3, the 383rd leucine (L) of the BD2 of the BRD2, and the 387th leucine (L) of the BD2 of the BRD4 may be substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M), and in particular, the 63rd leucine (L) of the BD1 of the BRDT, the 70th leucine (L) of the BD1 of the BRD3, the 94th leucine (L) of the BD1 of the BRD4, the 110th leucine (L) of the BD1 of the BRD2, Any one leucine (L) selected from the group consisting of the 306th leucine (L) of BD2 of the BRDT, the 345th leucine (L) of BD2 of the BRD3, the 383rd leucine (L) of BD2 of the BRD2, and the 387th leucine (L) of BD2 of the BRD4 may be substituted with alanine (A) or valine (V).

[0081] In one embodiment of the present invention, the variant of the bromo domain may comprise any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 8.

[0082] The bromo domain variant of the present invention may bind to a protac compound, and the protac compound may selectively destroy a target, and the protac compound may be specifically AGB-1. When a protac compound for a wild-type bromo domain is used, dozens of proteins containing bromo domains within a cell, especially proteins belonging to the BET family, are also degraded, and therefore the protac compound may have high cytotoxicity toward normal cells. Therefore, the present invention sought to construct a system capable of degrading a polypeptide containing a bromo domain variant through a protac compound that selectively acts only on the bromo domain variant. A protac compound that selectively acts only on the bromo domain variant does not destroy other proteins within the cell, and thus can degrade only the recombinant polypeptide containing the bromo domain variant of the present invention without cytotoxicity.

[0083] In a specific embodiment of the present invention, the binding of a variant of the bromo domain in which a mutation was introduced into the 387th leucine (L) of BD2 of the BRD4 to the AGB-1 protac compound was analyzed, and as a result, it was confirmed that the variant of the bromo domain and the AGB-1 protac compound had high selectivity.

[0084] Meanwhile, the recombinant polypeptide containing the variant of the bromo domain may act as a cell therapeutic agent, and the cell therapeutic agent may refer to a cell therapeutic agent made by combining genetic information with cells, and may be, for example, an immune cell therapeutic agent or a stem cell therapeutic agent. Specifically, the recombinant polypeptide of the present invention may act as a cell therapeutic agent such as a tumor infiltrating lymphocyte (TIL), a chimeric antigen receptor (CAR), a T-cell receptor (TCR), or a stem cell therapeutic agent.

[0085]

[0086] 2. Chimeric antigen receptor of the present invention

[0087] Another aspect of the present invention provides a chimeric antigen receptor (CAR) comprising an intracellular domain comprising a recombinant polypeptide comprising a variant of the bromo domain.

[0088] The chimeric antigen receptor comprises an extracellular binding domain comprising an antigen binding site; a transmembrane domain; and an intracellular domain comprising a recombinant polypeptide comprising a variant of the bromo domain.

[0089] The description of the variant of the above bromo domain is the same as that described above, so it will not be described again.

[0090] In the present invention, the term "chimeric antigen receptor (CAR)" refers to an artificial chimeric protein that fuses a single-chain antibody that recognizes a cell surface antigen of a target cell with a signal transduction domain that induces T cell activation. Specifically, the chimeric antigen receptor may include an extracellular binding domain, a transmembrane domain, and / or an intracellular signaling domain. The chimeric antigen receptor is expressed on the surface of an immune cell and recognizes and binds to a specific antigen, for example, an antigen specifically expressed on the surface of a cancer cell, through an antigen binding site included in the extracellular domain, thereby inducing signal transduction within the immune cell and changing the activity of the immune cell, thereby inducing an immune response by targeting only a specific antigen.

[0091] The intracellular domain of the chimeric antigen receptor of the present invention may further include an intracellular signaling domain, and thus, the intracellular domain of the chimeric antigen receptor of the present invention may include a variant of the bromo domain and an intracellular signaling domain.

[0092] The above extracellular domain includes an antigen binding site that specifically binds to a target antigen of interest, but is not limited in type. The extracellular domain can be applied without limitation as long as it has a structure that can specifically recognize and bind to a biological molecule, such as a cell surface receptor, an oncoprotein, a lipid, a polysaccharide, or other cell surface target molecule, and may be, for example, a protein, a polypeptide, or a variant thereof having the above characteristics. The above "specifically binds" may mean binding to another molecule with a binding affinity greater than background binding, and for example, the extracellular domain may bind to the target antigen with an affinity or Ka (equilibrium dissociation constant of a specific binding interaction having units of 1 / M) of about 10-5 M or more for the target antigen. The affinity may be an equilibrium dissociation constant (Kd) of a specific binding interaction having units of M of 10-5 M to 10-13 M or less.

[0093] The above antigen binding site may specifically bind to an antigen expressed on both cancer cells and normal cells. Specifically, the antigen expressed on the cancer cells may not be a cancer cell-specific antigen, i.e., may not be a cancer-specific marker used to prevent, treat, improve, or diagnose cancer. Accordingly, the chimeric antigen receptor of the present invention can recognize and bind to cancer cells, thereby causing signal transduction in immune cells expressing the chimeric antigen receptor. The antigen binding site may recognize a tumor-associated antigen (TAA) expressed on the surface of cancer cells, but is not limited thereto.

[0094] The cell surface antigen of the cancer cell recognized by the above antigen binding site may be a biomolecule specifically expressed in the cancer cell and its precursor cells, a biomolecule newly confirmed to be expressed due to cancerization of the cell, or a biomolecule whose expression level in the cancer cell is increased compared to that in the normal cell, and may be, for example, CD19, CD20, EGFR, FITC, CD22, CD33, PSMA, GD2, EGFRvariant, ROR1, c-Met, HER2, CEA, mesothelin, GM2, CD7, CD10, CD30, CD34, CD38, CD41, CD44, CD74, CD79, CD79b, CD123 CD133, CD171, MUC16, MUC1, CS1(CD319), IL-13 Ra2, BCMA, LewisY, IgG kappa chain, Folate receptor-alpha, PSCA, EpCAM.

[0095] In one embodiment of the present invention, the chimeric antigen receptor (CAR) may comprise an antibody or antigen-binding fragment thereof that specifically binds to CD19.

[0096] In one embodiment of the present invention, the chimeric antigen receptor (CAR) may comprise an antibody or an antigen-binding fragment thereof that specifically binds to GD2.

[0097] In one embodiment of the present invention, the chimeric antigen receptor (CAR) may comprise an antibody or an antigen-binding fragment thereof that specifically binds to CD79b.

[0098] The above CD19 is a cluster of differentiation (CD) antigens assigned the number 19 to identify cell surface molecules according to immunophenotype. It is known that CD19 is expressed in most B cells and malignant tumor cells, providing an ideal target for these cancers.

[0099] The above GD2 is a glycolipid ganglioside found in ganglia, and it is known that the above GD2 is expressed in tumors of neural epithelial origin, including human neuroblastoma and melanoma.

[0100] The above CD79b is a cluster of cell surface antigens (Cluster of Differentiation; CD), which is assigned the number 79 to identify cell surface molecules according to immunophenotype, and it is known that the CD79 is expressed in most B cells and malignant tumor cells, providing an ideal target for these cancers.

[0101] The antigen binding site may be, but is not limited to, an antibody, an antigen binding fragment thereof, a ligand protein binding to an antigen, or a domain thereof. More specifically, the antigen binding site may be an antibody or an antigen binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a linear antibody, a single-domain antibody (sdAb), a natural ligand, and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), a nanobody, an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a synthelin), and in particular, a single chain variable fragment (scFv) of an antibody.

[0102] The above antibody refers to an immunoglobulin molecule that immunologically binds specifically to an epitope of an antigen and has reactivity. The antibody may include a monoclonal antibody, a polyclonal antibody, an antibody having a full-length chain structure (full-length antibody), a functional fragment having at least an antigen-binding function (antigen-binding fragment), and a recombinant antibody. Specifically, the antibody of the present invention may be a monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody refers to an antibody molecule having a single molecular composition obtained from a substantially identical antibody population, and such a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. The full-length antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain may be linked to a heavy chain by a disulfide bond. The above antibody comprises a heavy chain (HC) and a light chain (LC) polypeptide, and the heavy chain and light chain may include a variable region and a constant region.

[0103] The constant region is a site that mediates binding of the antibody to various types of cells of the immune system (such as T cells), host tissues including components of the complement system, etc. The constant region has the same function regardless of the type of antigen as long as it is an antibody of the same type derived from the same species, and the amino acid sequence constituting the constant region is also the same or has a high degree of similarity between antibodies. The constant region can be divided into a heavy chain constant region (which may be abbreviated as CH) and a light chain constant region (which may be abbreviated as CL). The heavy chain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ), and / or epsilon (ε) type, and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and / or alpha2 (α2) as subclasses. The light chain constant region has a kappa (κ) and lambda (λ) type. IgG has subtypes, including IgG1, IgG2, IgG3, and IgG4.

[0104] The variable region is an antibody portion having specificity for an antigen, and can be divided into a heavy chain variable region (which may be abbreviated as VH) and a light chain variable region (which may be abbreviated as VL). The variable region may include three CDRs (complementary-determining regions) and four FRs (framework regions). The CDRs may be ring-shaped portions involved in antigen recognition, and specificity for an antigen may be determined according to the amino acid sequence of the CDRs. The CDRs may be referred to as CDR1, CDR2, and CDR3 according to their order, and depending on which polypeptide among the heavy and light chains they are CDRs of, the heavy chain variable region may be referred to as CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region may be referred to as CDR-L1, CDR-L2, and CDR-L3. Likewise, FR can be referred to as FR-H1, FR-H2, FR-H3, FR-H4 for the heavy chain variable region, and FR-L1, FR-L2, FR-L3, FRL4 for the light chain variable region.

[0105] The above antigen-binding fragment refers to a meaningful fragment of an antibody that retains the antigen-binding function of the antibody. The above antigen-binding fragment may be referred to interchangeably with terms such as "fragment", "antibody fragment", etc., and the above antigen-binding fragment may be, but is not limited to, Fab, Fab', F(ab')2, Fv, etc.

[0106] The extracellular domain may further comprise at least one selected from the group consisting of a hinge domain and a spacer domain. The antigen binding site of the extracellular domain may be linked to the transmembrane domain via the hinge domain and / or the spacer domain.

[0107] The hinge domain may play an important role in positioning the extracellular domain, which allows the antigen binding site to be physically separated from the surface of an immune cell on which the chimeric antigen receptor is expressed, to enable appropriate cell / cell contact, binding of appropriate antigen / antigen binding site, and activation of the appropriate chimeric antigen receptor. The chimeric antigen receptor may comprise one or more hinge domains between the extracellular domain and the transmembrane domain. The hinge domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. The altered hinge region is (a) a naturally occurring hinge region having at most 30% amino acid change (e.g., at most 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region that is at least 10 amino acids long (e.g., at least 12, 13, 14 or 15 amino acids) having at most 30% amino acid change (e.g., at most 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions), or (c) a core hinge region (which is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or Refers to a portion of a naturally occurring hinge region comprising a sequence of amino acids (e.g., 15 amino acids in length). In certain embodiments, one or more cysteine ​​residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue, e.g., a cysteine.

[0108] The above spacer domain may be referred to as a linking domain.

[0109] The hinge domain or spacer domain may be a hinge region derived from an extracellular domain such as a Myc epitope, an alpha (α), beta (β) or zeta (ζ) chain of a cytotoxic receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD8, CD4, CD28 and CD7, but any hinge region that can connect the antigen binding site, the transmembrane domain and the intracellular signaling domain across the cell membrane may be used without limitation. In addition, it may be a wild-type hinge region from these molecules or may be modified.

[0110] The above transmembrane domain refers to a portion of the region that connects and fuses the extracellular domain and the intracellular signaling domain and plays a role in anchoring the chimeric antigen receptor to the plasma membrane of an immune cell. The transmembrane domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. The transmembrane domain may be, but is not limited to, the alpha (α), beta (β), or zeta (ζ) chain of the T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0111] The transmembrane domain may be attached to the extracellular domain via a linker. For example, the linker may be a short oligopeptide or polypeptide linker of 2 to 10 amino acids in length, such as, but not limited to, a glycine (G)-serine (S) doublet.

[0112] The above intracellular domain is a domain existing within a cell and may further include a signaling domain.

[0113] The above signal transduction domain is a region capable of transmitting a signal into a cell when the single-chain antibody recognizes a cell surface antigen of a cancer cell, and may include at least one or two or more selected from the group consisting of polypeptides of the intracellular domains of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ, and Fc Receptorassociated γ chain.

[0114] When the above signal transduction domain comprises two or more polypeptides, they may be connected via an oligopeptide linker or polypeptide linker consisting of 2 to 10 amino acids, and such linker sequence may be, for example, a glycine-serine continuous sequence.

[0115] In particular, the chimeric antigen receptor of the present invention comprises a variant of a bromodomain in the intracellular domain, and further comprises a signaling domain in the intracellular domain.

[0116]

[0117] 3. Gene construct of the present invention

[0118] Another aspect of the present invention provides a recombinant polypeptide comprising a variant of the bromo domain of the present invention or a genetic construct comprising a base sequence encoding a chimeric antigen receptor of the present invention.

[0119] The description of the variants, recombinant polypeptides and chimeric antigen receptors of the above bromo domain is the same as that described above, and therefore will not be described again.

[0120] The above genetic construct can be constructed by a recombinant DNA or recombinant RNA method known in the art.

[0121] The base sequence encoding the variant of the above bromo domain may be operably linked to at least a promoter so as to be properly transcribed in the delivered cell.

[0122] The above promoter may be any promoter that can function in eukaryotic cells. For efficient transcription of a nucleic acid encoding a variant of the bromo domain, the promoter may additionally include regulatory sequences, including a leader sequence, a polyadenylation sequence, a promoter, an enhancer, an upstream activating sequence, a signal peptide sequence, and a transcription terminator, as needed.

[0123] In one embodiment of the present invention, the base sequence encoding the recombinant polypeptide comprising the variant of the bromo domain may include the base sequence encoding the variant of the bromo domain, and the base sequence encoding the variant of the bromo domain may include the base sequence of SEQ ID NO: 12.

[0124] The term "operably linked" as described above means that the bond between base sequences is functionally related. Any base sequence is operably linked when any base sequence is positioned so as to have a functional relationship with another base sequence. For example, if any transcriptional regulatory sequence influences the transcription of a base sequence encoding a variant of the bromo domain, the transcriptional regulatory sequence is said to be operably linked to the base sequence encoding the variant of the bromo domain.

[0125] The above "base sequence encoding a chimeric antigen receptor" is not particularly limited as long as it is a nucleic acid encoding a polypeptide constituting the chimeric antigen receptor, and may include a nucleic acid encoding a single-chain antibody recognizing a cell surface antigen of a cancer cell, a cell membrane penetrating domain, and a polypeptide of an intracellular signal transduction domain.

[0126] Any nucleic acid can be included between the nucleic acid encoding the chimeric antigen receptor and the nucleic acid encoding the variant of the bromo domain, as long as each nucleic acid can be expressed, and can be linked via a linker sequence encoding, for example, a self-cleaving peptide (2A peptide) or an internal ribozyme entry site (IRES). By linking using such a sequence, the gene construct can efficiently express each nucleic acid.

[0127] The base sequence encoding the above chimeric antigen receptor can be produced by a known technique, such as a chemical synthesis method or a PCR amplification method, based on a base sequence encoding a polypeptide of a single-chain antibody, a cell membrane penetrating domain, and an intracellular signal transduction domain for a cell surface antigen of a cancer cell.

[0128] In addition, information on the base sequence encoding the polypeptide of the single-chain antibody, cell membrane penetrating domain, and intracellular signal transduction domain for the cell surface antigen of the cancer cell can be appropriately obtained by searching the known literature or databases such as NCBI (http: / www.ncbi.nlm.nih.gov / guide / ).

[0129] According to one embodiment of the present invention, in the gene construct of the present invention, the base sequence encoding the variant of the bromo domain is located between the nucleic acid encoding the CD28 and the base sequence encoding the CD3ζ. Specifically, the base sequence encoding the variant of the bromo domain is located downstream of the base sequence encoding the CD28, and the base sequence encoding the CD3ζ is located downstream of the base sequence encoding the variant of the bromo domain. In addition, a base sequence encoding CD19 scFv may be additionally linked upstream of the base sequence encoding the CD28. Accordingly, the base sequences encoding CD19 scFv, CD28, the variant of the bromo domain, and CD3ζ may be linked in that order, respectively.

[0130] According to one embodiment of the present invention, in the gene construct of the present invention, the base sequence encoding CD3ζ is located downstream of the base sequence encoding CD28, and the base sequence encoding a bromo domain variant is located downstream of the base sequence encoding CD3ζ. In addition, a base sequence encoding CD19 scFv may be additionally linked upstream of the base sequence encoding CD28. Accordingly, the base sequences encoding CD19 scFv, CD28, CD3ζ, and the bromo domain variant may be linked in that order.

[0131] The above genetic construct can be introduced into an immune cell to produce an immune cell expressing a recombinant polypeptide comprising a variant of the bromo domain of the present invention or the chimeric antigen receptor.

[0132] The above genetic construct can be included in a recombinant vector and introduced into immune cells.

[0133] The above recombinant vector can be used as an expression vector of a target polypeptide that can express the target polypeptide with high efficiency in an appropriate host cell when the encoding gene of the target polypeptide to be expressed is operably linked, and the recombinant vector can be expressed in the host cell. The host cell can be preferably a eukaryotic cell, and depending on the type of host cell, expression control sequences such as a promoter, terminator, enhancer, sequences for membrane targeting or secretion, etc. can be appropriately selected and variously combined according to the purpose. The vector can be a plasmid, lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, and bathynia virus, and any vector that can be used by a person skilled in the art to express a target protein in a host cell can be used without limitation.

[0134] The method for introducing the above recombinant vector into immune cells is not particularly limited, but may include a method of introducing by a known method such as a viral infection method, calcium phosphate method, lipofection method, microinjection method, or electroporation method.

[0135] The above viral infection method may be a method of producing a recombinant virus by transfecting the recombinant vector and packaging plasmid into a packaging cell, and infecting T cells with the recombinant virus.

[0136] Introduction of the recombinant vector into immune cells can be confirmed by examining the expression of the chimeric antigen receptor in immune cells by methods such as PCR, ELISA, Western blotting, flow cytometry, Northern blotting, Southern blotting, RT-PCR, etc.

[0137] Immune cells into which the above recombinant vector has been introduced can express a recombinant polypeptide comprising a variant of the bromo domain of the present invention or a chimeric antigen receptor comprising a variant of the bromo domain.

[0138]

[0139] 4. Application of the recombinant polypeptide or chimeric antigen receptor of the present invention

[0140] One aspect of the present invention provides a cell expressing a recombinant polypeptide comprising a variant of the bromo domain of the present invention or a chimeric antigen receptor of the present invention.

[0141] The description of the variant, recombinant polypeptide or chimeric antigen receptor of the above bromo domain is the same as that described above, and therefore will not be described again.

[0142] The above cells may be stem cells or immune cells.

[0143] The above stem cells are undifferentiated cells that can repeatedly divide and develop into different types of differentiated cells.

[0144] Cells expressing the recombinant polypeptide or chimeric antigen receptor of the present invention may act as cell therapeutic agents.

[0145] The above cell therapy agent may be, for example, an immunocyte therapy agent, a stem cell therapy agent, etc., and is not limited as long as it is used for the purpose of treatment, diagnosis, or prevention through a series of actions such as manipulating the biological characteristics of cells.

[0146] Specifically, the cells of the present invention may act as a cell therapeutic agent in a form combined with tumor infiltrating lymphocytes (TIL), chimeric antigen receptors (CAR), T-cell receptors (TCR), tumor-associated glycoprotein (TAG) antibodies, etc.

[0147] The above-mentioned immune cells are cells involved in the immune response, and include all cells directly or indirectly involved in the immune response, as well as their pre-differentiated cells. The pre-differentiated cells may be embryonic stem cells, adult stem cells, or induced pluripotent stem cells with self-renewal and differentiation capabilities.

[0148] The above immune cells may be human-derived immune cells or immune cells derived from non-human mammals such as dogs, cats, pigs, and mice. In addition, the immune cells may be isolated and purified from body fluids such as blood and bone marrow, tissues such as the spleen, thymus, and lymph nodes, or immune cells infiltrating cancer tissues such as primary tumors, metastatic tumors, and malignant ascites.

[0149] The above immune cells may be monocytes or macrophages.

[0150] The above immune cells may be CD3 positive cells, for example, T cells.

[0151] The above immune cells may be CD56 positive cells, for example, NK cells.

[0152] The above immune cells may be CD3 and CD56 double positive cells, and may be, for example, NKT (Natural Killer T) cells or CIK (Cytokine Induced Killer cells).

[0153] The above T cells may be at least one selected from the group consisting of αβT cells, γδT cells, CD8 positive T cells (e.g., CD8+ naive T cells, CD8+ effector T cells, central memory T cells, or effector memory T cells), CD4 positive T cells, natural killer T (NKT) cells, regulatory T cells (Treg), memory T cells, tumor-infiltrating T cells, lymphoid progenitor cells, hematopoietic stem cells, T cells isolated from peripheral blood mononuclear cells (PBMCs), etc.

[0154] An immune cell expressing a recombinant polypeptide comprising a variant of the bromo domain of the present invention or a chimeric antigen receptor of the present invention may be produced by introducing the genetic construct into an immune cell. The introduction of the genetic construct can be understood in the same manner as described above, and therefore will not be described again.

[0155] The immune cell expressing the chimeric antigen receptor of the present invention may express a chimeric antigen receptor comprising a variant of the bromo domain.

[0156] The variant of the above bromo domain can be combined with a Proteolysis-targeting chimera (PROTAC) compound, and the PROTAC compound can degrade the recombinant polypeptide or chimeric antigen receptor, thereby regulating the immune activity of the immune cells.

[0157] The above-mentioned Protac compound acts as a target protein degrader (TPD), and is a compound that induces the degradation of a target protein (induced proteolysis) by utilizing the ubiquitin-proteasome system (UPS), an endogenous protein degradation mechanism that cells use to degrade proteins. The Protac compound is characterized by the fact that even various pathological proteins that were previously considered difficult to understand or difficult to treat with drugs can be "controlled by removing the target protein" by utilizing the properties of this degradation-inducing technology. The Protac compound can be applied to the protein that is the target of degradation and all E3 ligases that have the ability to ubiquitine. The Protac compound can be used to degrade a target protein. The above protactic compound may include, for example, lenalidomide, CC885, RIPK degrader 2, ARV771, etc., and specifically, the above protactic compound may be used without limitation as long as it can remove a mutant protein of the bromo domain or a recombinant polypeptide containing the same as a target protein, and may be, for example, an AGB-1 protactic compound.

[0158] The above protactic compound may bind to a variant of the bromo domain expressed by the immune cells of the present invention, and may be capable of removing or destroying the variant of the bromo domain or a recombinant polypeptide containing the same as a target protein.

[0159] By binding the above protac compound to the variant of the bromo domain of the present invention, the protac compound can degrade the recombinant polypeptide or chimeric antigen receptor containing the variant of the bromo domain as a target protein, thereby regulating the immune activity of immune cells expressing the recombinant polypeptide or chimeric antigen receptor of the present invention.

[0160] The immune activity of immune cells expressing the above recombinant polypeptide or chimeric antigen receptor may be cytokine secretion activity or cytotoxic activity of the immune cells.

[0161] The above cytokine may refer to a protein (~5-20 kDa) that plays a role in cell signaling. The cytokines are released by cells and influence the behavior of the immune cells and / or other cells that release the cytokines. Non-limiting examples of cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor, monokines, and colony-stimulating factors. Cytokines can be produced by a wide range of cells, including, but not limited to, immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, and monocytes, endothelial cells, fibroblasts, and stromal cells. The cytokine may be an interferon, an interleukin, or a mutant thereof. Many interleukins are synthesized by NK cells, helper CD4 T lymphocytes, as well as monocytes, macrophages, and endothelial cells. The interleukins can promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Non-limiting examples of the interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36 or mutants thereof.

[0162] The above cytotoxic activity refers to the activity of immune cells to injure or kill cells, and may, for example, impair various functions of cells, including cell death, proliferation, and DNA synthesis.

[0163]

[0164] Another aspect of the present invention provides a pharmaceutical composition comprising the immune cells as an active ingredient.

[0165] In one embodiment, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating cancer.

[0166] The terms “cancer” and “tumor” may be used interchangeably and refer to or mean a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0167] The cancer includes not only angiogenic tumors but also tumors that are not angiogenic or are not yet substantially angiogenic. The cancer may include non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma) or solid tumors. Types of cancer include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, and benign and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0168] The above blood cancers are cancers of the blood or bone marrow. Examples of blood (or hematopoietic) cancers include leukemias including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, and myeloblastic, prolymphocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic lymphocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0169] The above-mentioned solid tumors are abnormal tissue masses that usually do not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, squamous cell carcinoma, rhabdomyosarcoma, rectal carcinoma, lymphoid malignancies, colon cancer, stomach cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, laryngopharyngeal cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumor, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (e.g., brainstem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, Schwannoma craniopharyogioma, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendroglioma, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0170] In a specific embodiment of the present invention, the anticancer efficacy was evaluated after treating immunodeficient mice with immune cells (CAR-BD2m T cells) expressing a chimeric antigen receptor comprising a bromo domain variant of the present invention. As a result, when the CAR-BD2m T cells were treated, tumor proliferation was inhibited at a level similar to when CAR-T cells expressing a CAR not comprising the bromo domain variant of the present invention were treated. This indicates that cells expressing a chimeric antigen receptor comprising a bromo domain variant have anticancer function.

[0171] Additionally, in another embodiment, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating an autoimmune disease.

[0172] Additionally, in another embodiment, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating a viral disease.

[0173] Additionally, in another embodiment, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating heart disease.

[0174] The above autoimmune disease refers to a pathological response to self-antigens. Autoimmune diseases occur when an abnormal immune response to normal body components causes inflammation, cell damage, and other factors. The components targeted by an autoimmune reaction are called autoantigens. Autoimmune reactions can be systemic or tissue- or organ-specific, and can be acute or chronic. Autoimmune diseases caused by such pathological reactions include rheumatic diseases such as systemic lupus erythematosus and vasculitis, as well as diseases such as autoimmune thyroiditis and multiple sclerosis.

[0175] The above viral disease refers to an infection caused by a virus, and a virus is composed of at least nucleic acid and protein, and can be distinguished as containing DNA as the nucleic acid and containing RNA. The nucleic acid has viral activity (infectivity), and the virus proliferates in specific cells suitable for the virus, and tissues having affinity for the virus cause pathological changes due to infection. When a cell is infected with a virus, cell degeneration, necrosis, inflammation, giant cell formation, and inclusion body formation occur. The virus can enter the nucleus and cause tumors. The pathogenic viruses of the above viral disease include, for example, Retroviridae (e.g., human immunodeficiency virus such as HIV-1 (also called HDTV-Ⅲ, LAVE or HTLV-Ⅲ / LAV or HIV-Ⅲ); and other isolates such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronoviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., Hantaan virus, bunga virus, phlebovirus, and Nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses);Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of hepatitis delta (thought to be a defective satellite of hepatitis B virus), agents of non-A, non-B hepatitis (class 1 = transmitted internally; class 2 = transmitted parenterally (i.e., hepatitis C)); Norwalk and related viruses, and astroviruses);

[0176] The above heart disease is a disease that occurs in the heart and major blood vessels (arteries). Just as the structure of the heart can be divided into the heart muscle, heart blood vessels, valves, and the conduction system that handles the heart's electrical signals, heart disease can also be classified into diseases that occur in each part. Examples of heart disease include, but are not limited to, hypertension, ischemic heart disease, coronary artery disease, heart failure due to cardiac fibrosis, angina pectoris, myocardial infarction, atherosclerosis (arteriosclerosis), cerebrovascular disease, stroke, and arrhythmia.

[0177] In a specific embodiment of the present invention, immune cells expressing a chimeric antigen receptor including a bromo domain variant of the present invention (CAR-BD2m T cells) were treated with an AGB-1 protac compound, and then the degradation of the CAR including the bromo domain variant was investigated. As a result, it was confirmed that when the CAR-BD2m T cells were treated with the protac compound, the CAR, which is a recombinant polypeptide including the bromo domain variant, could be degraded. Accordingly, it was found that in cell therapy technology using a recombinant polypeptide including the bromo domain variant of the present invention, the cell therapy effect can be controlled by utilizing the relationship between the bromo domain variant and the protac compound. Therefore, the pharmaceutical composition of the present invention can exhibit a preventive or therapeutic effect in the treatment of autoimmune diseases, viral diseases, or heart diseases using the CAR-T cells as described above.

[0178] The above “prevention” means any act of suppressing the onset of a disease such as cancer, autoimmune disease, viral disease, or heart disease, or delaying the progression to the onset of the disease, by administering the composition of the present invention, and includes suppressing, alleviating, or eliminating the initial symptoms of the disease or symptoms that appear before the onset of the disease.

[0179] The above “treatment” means any act of suppressing or delaying the progression of a disease such as cancer, autoimmune disease, viral disease, or heart disease by administering the composition of the present invention, and includes suppressing the development of the disease and alleviating or eliminating symptoms.

[0180] The pharmaceutical composition may additionally include pharmaceutically acceptable excipients. Examples of such excipients include, but are not limited to, surfactants, preferably nonionic surfactants of the polysorbate series; buffers such as neutral buffered saline, human salt buffered saline, etc.; sugars or sugar alcohols such as glucose, mannose, sucrose, dextran, mannitol, etc.; amino acids such as glycine, histidine, etc., or proteins or polypeptides; antioxidants; chelating agents such as EDTA or glutathione, etc.; penetrants; adjuvants; and preservatives.

[0181] The pharmaceutical composition may be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal other than a human. The formulation may be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder.

[0182]

[0183] In addition, another aspect of the present invention provides a composition for degrading a recombinant polypeptide or chimeric antigen receptor expressed by an immune cell of the present invention.

[0184] The composition for degrading a recombinant polypeptide or chimeric antigen receptor of an immune cell of the present invention contains a Proteolysis-targeting chimera (PROTAC) compound as an active ingredient.

[0185] The above protac compound is a compound that induces degradation (induced proteolysis) of a target protein by using the ubiquitin-proteasome system (UPS), an endogenous protein degradation mechanism that cells use to degrade proteins. The protac compound is characterized by the fact that it can "control by removing the target protein" even for various pathological proteins that were previously considered difficult to understand or difficult to treat with drugs by utilizing the properties of this degradation-inducing technology. The protac compound can be applied to the protein that is the target of degradation and all E3 ligases that have the ability to ubiquitine. The protac compound can be used to degrade the target protein. For example, the protac compound may include lenalidomide, CC885, RIPK degrader 2, ARV771, etc., and specifically, it may be the AGB-1 protac compound.

[0186] The above protac compound can be used without limitation as long as it binds to a variant of the bromo domain expressed by the immune cell of the present invention. By binding to the variant of the bromo domain of the immune cell of the present invention, the protac compound detects the recombinant polypeptide or chimeric antigen receptor containing the variant of the bromo domain, and the protac compound can degrade the recombinant polypeptide or chimeric antigen receptor using the recombinant polypeptide or chimeric antigen receptor as a target protein.

[0187] Therefore, the composition can degrade a recombinant polypeptide or chimeric antigen receptor expressed by the immune cell by contacting the protac compound with the immune cell of the present invention.

[0188] Additionally, the composition can suppress adverse effects caused by immune cells by regulating their activity. As the composition degrades the recombinant polypeptide or chimeric antigen receptor, the composition can remove the recombinant polypeptide or chimeric antigen receptor from immune cells expressing the recombinant polypeptide or chimeric antigen receptor, thereby suppressing the immune activity of the immune cells.

[0189] The immune activity of the above immune cells may be cytokine secretion activity or cytotoxic activity of the immune cells.

[0190] The above cytokine may refer to a protein (~5-20 kDa) that plays a role in cell signaling. The cytokines are released by cells and influence the behavior of the immune cells and / or other cells that release the cytokines. Non-limiting examples of cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor, monokines, and colony-stimulating factors. Cytokines can be produced by a wide range of cells, including, but not limited to, immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, and monocytes, endothelial cells, fibroblasts, and stromal cells. The cytokine may be an interferon, an interleukin, or a mutant thereof. Many interleukins are synthesized by NK cells, helper CD4 T lymphocytes, as well as monocytes, macrophages, and endothelial cells. The interleukins can promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Non-limiting examples of the interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36 or mutants thereof.

[0191] In a specific embodiment of the present invention, T cells expressing a chimeric antigen receptor comprising a variant of the bromo domain of the present invention (CAR-BD2m T) were treated with an AGB-1 protac compound, and the IL-2 secretion activity and cytotoxic activity of the CAR-BD2m T cells were measured. As a result, it was confirmed that when the protac compound was treated, the amount of IL-2 secreted by the CAR-BD2m T cells was reduced and the cytotoxic activity was lowered. This shows that the chimeric antigen receptor of the CAR-BD2m T cells was degraded by the protac treatment, thereby reducing the immune activity of the immune cells mediated by the chimeric antigen receptor.

[0192] Accordingly, the composition for degrading the recombinant polypeptide or chimeric antigen receptor of the immune cell may include the protac compound, and by contacting the protac compound with the chimeric antigen receptor expressed by the immune cell, the chimeric antigen receptor expressed by the immune cell is degraded, thereby reducing the activity of the immune cell.

[0193] The above decrease in cytotoxic activity may mean that the activity of the measured immune cells is decreased by 10% or more when the protac compound is treated compared to the control group that is not treated with the protac compound, and specifically, may mean that the activity is decreased by 15% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0194] The composition for degrading the recombinant polypeptide or chimeric antigen receptor of the above immune cells can suppress excessive expression or excessive activity of the recombinant polypeptide or chimeric antigen receptor.

[0195] Specifically, immunotherapeutic agents expressing recombinant polypeptides or chimeric antigen receptors may cause unexpected side effects, such as cytokine release syndrome, neurotoxicity, and off-target drug effects. Therefore, there is a need to increase the safety of immunotherapeutic agents expressing recombinant polypeptides or chimeric antigen receptors by modulating their activity by eliminating immune cells expressing the recombinant polypeptides or chimeric antigen receptors, if necessary.

[0196] Therefore, by using the composition for degrading the chimeric antigen receptor of the immune cell of the present invention, the chimeric antigen receptor can be removed or degraded as needed from the immune cell expressing the chimeric antigen receptor, thereby preventing side effects due to excessive efficacy or action of the immune cell expressing the chimeric antigen receptor. For example, when using the immune cell expressing the chimeric antigen receptor for anticancer treatment, the side effects of the anticancer treatment caused by overexpression or overactivity of the chimeric antigen receptor of the immune cell can be suppressed using the composition for degrading the chimeric antigen receptor of the immune cell of the present invention.

[0197] Additionally, the composition can enhance the anticancer effects of immune cells by regulating their activity. Accordingly, the composition can be used as an anticancer adjuvant.

[0198] The above anticancer adjuvant can regulate the immune activity of immune cells expressing the recombinant polypeptide or chimeric antigen receptor by appropriately controlling the expression level of the recombinant polypeptide or chimeric antigen receptor in immune cells expressing the recombinant polypeptide or chimeric antigen receptor, thereby controlling the anticancer treatment effect.

[0199] Specifically, in one aspect, side effects may occur due to overexpression or overactivation of a recombinant polypeptide or chimeric antigen receptor in an immune cell as described above, but when a composition for degrading a recombinant polypeptide or chimeric antigen receptor of an immune cell of the present invention is used to treat a protac compound at a specific concentration or dose or more to an immune cell expressing the chimeric antigen receptor, the chimeric antigen receptor is degraded, thereby suppressing the overexpression or overactivation of the recombinant polypeptide or chimeric antigen receptor. When the overexpression or overactivation of the recombinant polypeptide or chimeric antigen receptor is suppressed as described above, excessive immune activity of the immune cell is also reduced, thereby reducing the side effects of anticancer treatment caused by excessive immune activity of the immune cell as described above.

[0200] In another aspect, when a chimeric antigen receptor is overexpressed in the above-described immune cells, the overexpressed chimeric antigen receptor may accelerate the exhaustion of the immune cells, thereby accelerating the aging of the immune cells and lowering the immune activity of the immune cells. In this case, by appropriately treating the immune cells expressing the chimeric antigen receptor with a protac compound at a concentration or dose below a certain level using the composition for decomposing the chimeric antigen receptor of the immune cells of the present invention, and decomposing the chimeric antigen receptor to an appropriate degree, the exhaustion of the immune cells caused by the chimeric antigen receptor can be suppressed, and as a result, the immune activity of the immune cells can be maintained for a longer period of time.

[0201] In summary, when using the composition for degrading a recombinant polypeptide or chimeric antigen receptor of an immune cell of the present invention, the immune activity of an immune cell expressing the recombinant polypeptide or chimeric antigen receptor can be suppressed or increased by controlling the concentration or dose of the protac compound treated to the immune cell. Therefore, the composition for degrading a recombinant polypeptide or chimeric antigen receptor of an immune cell of the present invention has the property of regulating the immune activity of an immune cell of the present invention expressing the chimeric antigen receptor, and can be used as an anticancer adjuvant.

[0202] In a specific embodiment of the present invention, it was confirmed that when the CAR-BD2m T cells were treated with a protac compound, the tumor was completely remitted (complete remission), showing a significantly superior anticancer effect compared to when the CAR-BD2m T cells were treated alone. Therefore, it was found that the immune cells of the present invention and the protac compound can be used together to prevent or treat cancer.

[0203] The specific concentration or dose of the protac compound for regulating the immune activity of the immune cells may mean that the number of immune cells in which the recombinant polypeptide or chimeric antigen receptor is degraded as a result of the protac compound treatment is 10% or more compared to the number of immune cells initially expressing the recombinant polypeptide or chimeric antigen receptor, and specifically, it may be the concentration or dose of the treated protac compound when it is 15% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0204] In a specific embodiment of the present invention, in order to confirm the effect of improving anticancer efficacy when the CAR-BD2m T cells of the present invention and AGB-1 are used in combination, the anticancer activity of cases treated with PBS alone, cases treated with the CAR-BD2m T cells of the present invention alone, and cases treated with the CAR-BD2m T cells of the present invention and AGB-1 together was compared in an in vivo animal experiment. As a result, it was confirmed that the anticancer activity was significantly superior when the CAR-BD2m T cells of the present invention and AGB-1 were treated together.

[0205]

[0206] Another aspect of the present invention provides a kit for preventing or treating cancer, comprising the pharmaceutical composition and a PROTAC compound.

[0207] The description of the above immune cells, pharmaceutical compositions containing the immune cells, and protac compounds is the same as the description above, and therefore will not be described again.

[0208] The kit may further include an instruction manual describing that the cytotoxic activity of the immune cells can be controlled by degrading the recombinant polypeptide or chimeric antigen receptor expressed by the immune cells using the protac compound.

[0209] The above description may describe that the cytotoxic activity of the immune cells can be controlled depending on the treatment concentration of the protac compound.

[0210] The regulation of the cytotoxic activity of the immune cells according to the treatment concentration of the above protac compound can be understood as follows.

[0211] Specifically, in one aspect, side effects may occur due to overexpression or overactivation of a recombinant polypeptide or chimeric antigen receptor in immune cells as described above, but when the kit of the present invention is used to treat immune cells expressing the recombinant polypeptide or chimeric antigen receptor with a protac compound at a specific concentration or dose or higher, the recombinant polypeptide or chimeric antigen receptor is decomposed, thereby inhibiting the overexpression or overactivation of the recombinant polypeptide or chimeric antigen receptor. When the overexpression or overactivation of the recombinant polypeptide or chimeric antigen receptor is inhibited as described above, the excessive cytotoxic activity of the immune cells is also reduced, thereby reducing the side effects of anticancer treatment caused by the excessive cytotoxic activity of the immune cells as described above.

[0212] In another aspect, when a chimeric antigen receptor is overexpressed in the above-described immune cells, the overexpressed chimeric antigen receptor may accelerate the exhaustion of the immune cells, thereby accelerating the aging of the immune cells and lowering the cytotoxic activity of the immune cells. In this case, by appropriately treating the protac compound with a specific concentration or dose or less to the immune cells expressing the chimeric antigen receptor using the kit of the present invention to degrade the chimeric antigen receptor to an appropriate degree, the exhaustion of the immune cells due to the overexpression of the chimeric antigen receptor can be suppressed, and as a result, the immune activity of the immune cells can be sustained or increased for a longer period of time.

[0213] In a specific embodiment of the present invention, the anticancer effect of treating only the CAR-BD2m T cells alone and treating the CAR-BD2m T cells together with a protac compound was compared and analyzed in an in vivo animal experiment. As a result, it was confirmed that when the CAR-BD2m T cells and the protac compound were treated together and the CAR-BD2m was degraded to an appropriate degree, the tumor was completely remitted (complete remission). In addition, when the CAR-BD2m T cells and the protac compound were treated together, the anticancer effect was significantly superior to when the CAR-BD2m T cells were treated alone. Therefore, it was found that the kit of the present invention can regulate the activity of immune cells by using the immune cells of the pharmaceutical composition together with the protac compound, thereby preventing or treating cancer.

[0214] In summary, the instructions for the kit may include content that by controlling the concentration or dosage of the protac compound, the immune activity of immune cells expressing the recombinant polypeptide or chimeric antigen receptor can be suppressed or increased. The instructions may include content that by controlling the activity of the immune cells according to the treatment concentration of the protac compound, side effects caused by the immune cells can be suppressed or the anticancer effect of the immune cells can be enhanced.

[0215] The description of the appropriate concentration or dosage of the above protac compound can be understood in the same way as described above, and therefore will not be described again.

[0216] The kit may further include instructions describing how to inhibit overexpression of a recombinant polypeptide or chimeric antigen receptor expressed by the immune cells using the protac compound.

[0217]

[0218] Another aspect of the present invention provides a method for degrading a recombinant polypeptide or chimeric antigen receptor of an immune cell of the present invention, comprising the step of contacting the immune cell with the protac compound.

[0219] Another aspect of the present invention provides a method for inhibiting the activity of an immune cell, comprising the step of contacting the protac compound to the immune cell.

[0220] The above-mentioned immune cells, the protac compound, the decomposition of the recombinant polypeptide or chimeric antigen receptor of the immune cells, and the inhibition of the activity of the immune cells mean the same as the above-mentioned contents, and therefore, they are not described repeatedly.

[0221]

[0222] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0223] However, the following examples and experimental examples are only intended to illustrate the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

[0224]

[0225] [Example 1]

[0226] CAR disruption using bromodomain variants and protactic compounds

[0227] First, in order to select a safe protac compound that effectively degrades the target protein and has low cytotoxicity, experiments were conducted with lenalidomide, RIPK degrader 2, ARV771, and AGB-1 protac compounds. It is known that lenalidomide is a protac compound that targets CSNK1A1 (Casein Kinase 1 Alpha 1, CK1α), RIPK degrader 2 is a protac compound that targets RIPK2, and ARV771 and AGB-1 are protac compounds that target the wild-type bromo domain. Meanwhile, in the present invention, a mutant of the bromo domain was produced by introducing a mutation into the wild-type bromo domain, and AGB-1 was used as a protac compound that targets the bromo domain mutant.

[0228] CAR-Jurkat cells expressing a chimeric antigen receptor (CAR) including CK1α, RIPK2, a wild-type BD2 domain (BD2w), and a mutant BD2 domain (BD2m), which are proteins targeted by the above four types of protac compounds, were constructed (hereinafter, CAR-CK1α-Jurkat, CAR-RIPK2-Jurkat, CAR-BD2w-Jurkat, and CAR-BD2m-Jurkat cells, respectively). Vectors were constructed using a base sequence encoding CK1α, a base sequence encoding RIPK2, a base sequence encoding BD2w, a base sequence encoding BD2m, and a base sequence encoding CAR, and these recombinant vectors were transduced into Jurkat T cells to construct CAR-Jurkat cells expressing CARs including target domains of CK1α, RIPK2, BD2w, and BD2m, respectively (Fig. 1, Fig. 4). Specifically, the genetic construct of CAR including a bromo domain variant is described below. A variant (SEQ ID NO: 1) of the bromo domain targeted by the AGB-1 protac compound was produced, and specifically, in the wild-type bromo domain 2 (Bromodomain 2, BD2) having the amino acid sequence of SEQ ID NO: 26, the 387th leucine (L) was changed to valine (V), thereby producing an L387V variant of BD2 (SEQ ID NO: 1).Thereafter, in order to produce a gene construct of a CAR including a variant of the bromo domain, a base sequence encoding a signal peptide (SEQ ID NO: 13), a base sequence encoding CD19 scFv (SEQ ID NO: 9), a base sequence encoding linker 1 (SEQ ID NO: 14), a base sequence encoding CD28 (SEQ ID NO: 10), a base sequence encoding Arv2 (SEQ ID NO: 15), a base sequence encoding the variant of the bromo domain (SEQ ID NO: 12), a base sequence encoding Nsi1 (SEQ ID NO: 16), and a base sequence encoding CD3ζ (SEQ ID NO: 11) were sequentially linked to design a gene construct encoding a chimeric antigen receptor including a variant of the bromo domain (Fig. 2). The gene construct designed as described above was produced and cloned into a lentiviral vector to produce a recombinant vector for expressing the chimeric antigen receptor of the present invention. The recombinant vector was introduced into Jurkat cells and KHYG-1 NK cells to produce CD19 CAR-BD2m Jurkat cells and CD19 CAR-BD2m KHYG-1 cells expressing a CAR containing a variant of the bromo domain. In addition, CAR-Jurkat cells and CAR-KHYG-1 cells that do not contain the wild-type bromo domain or its variant were produced (Fig. 2). Thereafter, lenalidomide, a protac compound targeting CK1α, was treated to the CAR-CK1α-Jurkat cells at various concentrations, and the degradation of the CAR-CK1α in the Jurkat cells was analyzed by Western blotting. In addition, RIPK degrader 2, a protac compound targeting RIPK2, was treated to the CAR-RIPK2-Jurkat cells at various concentrations, and the degradation of CAR-RIPK2 was analyzed by Western blotting.In addition, the CAR-BD2w-Jurkat cells and the CAR-BD2m Jurkat cells were treated with ARV771 and AGB-1, which are protease compounds targeting the bromo domain or its variants, at various concentrations, respectively, and then it was confirmed through Western blot whether the CAR-BD2w and CAR-BD2m were degraded.

[0229] As a result, as shown in Fig. 1, lenalidomide and RIPK degrader 2 protease compounds were found to be unable to degrade the CAR. On the other hand, as shown in Fig. 4, ARV771 and AGB-1 were found to effectively degrade the CAR comprising the wild-type BD2 or its mutant. However, the degrading ability of AGB-1 for the CAR with the bromo domain mutant (CAR-BD2m) was confirmed to be approximately 10 times better than that of ARV771 for the CAR with the wild-type bromo domain (CAR-BD2w).

[0230]

[0231] [Example 2]

[0232] [2-1] Selective destruction of CAR containing bromo domain variants by AGB-1

[0233] The alpha screen enzyme assay was performed using purified proteins, BD2 wild type (hereinafter, BD2w) and BD2 L387V mutant (hereinafter, BD2m), to determine the binding affinity (IC) of ARV771 and AGB-1 to BD2w and BD2m polypeptides. 50 The value) was measured.

[0234] As a result, as shown in Fig. 3, it can be seen that AGB-1 has almost no binding activity toward BD2w, but has high selectivity toward BD2m. Thus, since AGB-1 shows high selectivity only toward BD2m, it was expected that it would be able to destroy only CARs containing BD2m without cytotoxicity toward general normal cells containing BD2w.

[0235] To demonstrate this, cytotoxicity experiments were conducted using normal cells. Specifically, peripheral blood mononuclear cells (PBMCs) and T cells were treated with various concentrations of the ARV771 and AGB-1 protac compounds, cultured for 72 hours, and then the viability of PBMCs and T cells was measured using a luminescence-based cell viability assay.

[0236] As a result, as shown in Fig. 3, unlike ARV771, AGB-1 was confirmed to have no cytotoxicity at all even at 2 uM. This suggests that the use of AGB-1 can modulate the activity of T cells expressing CARs containing the bromo domain variant without toxicity to normal cells.

[0237] Furthermore, in addition to the CD19 CAR-BD2m targeting CD19 produced in Example 1, it was investigated whether AGB-1 could degrade a CAR including a variant of the bromo domain of the present invention when a CAR targeting another type of antigen includes a variant of the bromo domain of the present invention. Specifically, the gene constructs of GD2-CAR-BD2m targeting GD2 as a target antigen and CD79b-CAR-BD2m targeting CD79b as a target antigen were produced. Specifically, in the gene construct of Example 1, instead of the base sequence encoding the CD19 scFv, the base sequence encoding the GD2 scFv (SEQ ID NO: 19) and the base sequence encoding the CD79b scFv (SEQ ID NO: 20) were respectively linked to produce a gene construct, which was transfected into 293T cells and then treated with AGB-1. After the above AGB-1 treatment, it was confirmed through Western blotting whether CAR was degraded in 293T cells transfected with the above GD2-CAR-BD2m or CD79b-CAR-BD2m.

[0238] As a result, as shown in Fig. 5, it was confirmed that both GD2-CAR-BD2m and CD79b-CAR-BD2m were destroyed by AGB1 in a concentration-dependent manner. That is, when a CAR protein includes a variant of the bromo domain of the present invention, the CAR can be destroyed or decomposed by AGB-1 regardless of the type of antigen, and thus it was found that the CAR destruction system utilizing the variant of the bromo domain of the present invention can be applied to all CARs.

[0239] [2-2] Confirmation of the mechanism of action of CAR-BD2m degradation by AGB-1

[0240] The effect of proteasome, a protein-degrading enzyme, on the degradation of CAR-BD2m by the above AGB-1 was investigated. Specifically, CAR-BD2m T cells were treated with AGB-1 at concentrations of 10 nM, 100 nM, and 1 uM, and then cultured with epoxomicin, a proteasome inhibitor, at concentrations of 10 nM and 50 nM. Western blotting was performed to confirm the degree of CAR degradation. The group not treated with epoxomicin served as a control.

[0241] As a result, as shown in Fig. 6, in the epoxomicin-untreated group, CAR was degraded by AGB-1 in CAR-BD2m T cells, but when the proteasome was inhibited by epoxomicin treatment, the CAR was confirmed to be expressed. Therefore, it was confirmed that the proteasome is related to the degradation of CAR by AGB-1.

[0242] [2-3] Inhibition of activity of immune cells of the present invention by AGB-1

[0243] In order to confirm whether the activity of CAR-BD2m of the present invention is regulated by the protac compound, the cytokine (IL-2) secretion activity and cytotoxic activity of the CAR-BD2m Jurkat cells were measured. First, 1×10 of the CAR-BD2m Jurkat cells were 5 3×10 Nalm6 cells positive for dogs and CD19 4 After treating dogs with the AGB-1 protease compound at various concentrations, co-culture was performed for 24 hours. The supernatant of the culture was obtained, and the content of IL-2 contained in the supernatant was measured.

[0244] As a result, as shown in Fig. 7, when Nalm6 cells and the CAR-BD2m Jurkat cells were co-cultured, IL-2 secretion increased, and it was confirmed that IL-2 secretion decreased as AGB-1 was treated.

[0245] In addition, the CAR-BD2m gene construct of Example 1 was cloned into a retroviral vector, and then the retrovirus was infected into human primary T cells to establish CD19 CAR-BD2m T cells. At this time, non-transduced T cells (NT), T cells expressing GFP (GFP-T), and T cells expressing only CAR (CAR-T) were used as controls. The NT, GFP-T, and CAR-BD2m T cells were treated with the AGB-1, and the change in the expression level of CAR on the T cell surface after the AGB-1 treatment was observed using a fluorescence activated cell sorter (FACS). Next, 3×10 of the NT, GFP-T, and CAR-BD2m T cells were cultured. 4 Nalm6 cancer cells expressing luciferase and dog were treated with 10 nM or 100 nM of AGB-1 and then co-cultured. The luciferase-expressing Nalm6 cells represent target cells (T), and the CAR-BD2m T cells or NT or GFP-T cells of the control group represent effector cells (E). After 4 and 24 hours of incubation, the cytotoxicity of T cells in the cultures was measured.

[0246] As a result, as shown in Fig. 8, CAR expression disappeared in CAR-BD2m T cells when treated with AGB-1, whereas there was no change in CAR-T cells (WB, FACS). This means that AGB-1 selectively destroys CARs containing BD2m. In addition, when treated with DMSO, the CAR-BD2m T cells showed excellent cytotoxicity, but it was confirmed that the cytotoxicity decreased in a concentration-dependent manner when treated with AGB-1. This means that the activity of CAR-BD2m T cells decreased as CARs containing BD2m were destroyed by AGB-1.

[0247] [2-4] Inhibition of cytotoxic activity of CAR-BD2m KHYG-1 cells (NK cell line) of the present invention by AGB-1

[0248] For NK cells expressing CAR containing the above BD2 variant (CAR-BD2m KHYG-1), it was confirmed whether AGB-1 regulates the activity of NK cells. Specifically, 1 × 10 Nalm6 cells expressing luciferase 4 2×10 CAR-BD2m KHYG-1 cells in dogs 4 Dog and 1×10 5 The cells were co-cultured for 4 hours in medium treated with various concentrations of AGB-1 or in medium treated only with DMSO. At this time, KHYG-1 NK cells expressing only CAR (CAR KHYG-1) were used as a control. The Nalm6 cells expressing luciferase represent target cells (T), and the CAR-BD2m KHYG-1 cells or the control CAR KHYG-1 cells represent effector cells (E). Thereafter, the cytotoxic activity of NK cells in the cultures was measured.

[0249] As a result, as shown in Fig. 9, the CAR-BD2m KHYG-1 cells exhibited cytotoxicity in DMSO, but the cytotoxicity was confirmed to decrease when treated with AGB-1. However, the control group, CAR KHYG-1, did not show a decrease in cytotoxicity even when treated with AGB-1.

[0250] Through the above experimental results, it was found that when a cell expresses a recombinant polypeptide or receptor including a bromo domain variant of the present invention, AGB-1 can bind to the bromo domain variant of the cell and decompose or destroy the recombinant polypeptide or receptor including the bromo domain variant, thereby regulating the activity of the cell using the recombinant polypeptide or receptor including the bromo domain variant. Accordingly, by utilizing the property of AGB-1 binding to the bromo domain variant of the present invention and destroying the recombinant polypeptide including the bromo domain variant, a system capable of regulating the activity of a cell expressing a recombinant polypeptide or receptor including the bromo domain variant can be constructed.

[0251]

[0252] [Example 3]

[0253] Comparison of immune cell activity according to the linking order of genes of variants of the bromo domain in the CAR of the present invention

[0254] In the gene construct used for expression of the variant of the bromo domain used in Example 1, the linking position of the sequence of the variant of the bromo domain was changed to produce a gene construct, and after expressing a vector including this gene construct in a T cell, the activity of the immune cell of the present invention according to the position of the variant sequence of the bromo domain was analyzed. Specifically, unlike the gene construct of Example 1, in this experiment, a gene construct encoding a signal peptide (SEQ ID NO: 13), a CD19 scFv encoding sequence (SEQ ID NO: 9), a linker 1 encoding sequence (SEQ ID NO: 14), a CD28 encoding sequence (SEQ ID NO: 10), a linker 2 encoding sequence (SEQ ID NO: 18), a CD3ζ encoding sequence (SEQ ID NO: 11), and a bromo domain variant encoding sequence (SEQ ID NO: 12) was designed and sequentially linked, and this was produced and cloned into a retro vector to produce a recombinant vector for expressing the chimeric antigen receptor of the present invention. The recombinant vector was introduced into T cells to produce CAR-T cells (hereinafter, CAR-BD2m-C) expressing a CAR including a bromo domain variant. Afterwards, the CAR-BD2m-C T cells and CAR-BD2m T cells were treated with AGB-1 at a concentration of 5 nM, 50 nM, or 500 nM, and Western blot and cytotoxicity analysis experiments were performed in the same manner as in Example 1 and Example 2.

[0255] As a result, as shown in Fig. 10, it was confirmed that the CAR-BD2m-C T cells showed CAR protein degradation as AGB-1 was treated, and cytotoxicity decreased in a concentration-dependent manner as AGB-1 was treated. Accordingly, it was found that in the genetic construct for producing an immune cell of the present invention expressing a CAR including a variant of the bromo domain, as long as the base sequence of the variant of the bromo domain is linked so that the immune cell can express a chimeric antigen receptor including a variant of the bromo domain, regardless of the position of the linkage with the base sequence of the CAR, it exhibits the effect of degradation of the immune cell and reduction of cytotoxicity by AGB-1.

[0256]

[0257] [Example 4]

[0258] Restoration of CAR expression in immune cells of the present invention following removal of protac compounds (reversible regulation of CAR by protac compounds)

[0259] When the protac compound was removed, it was confirmed whether the immune cells of the present invention were re-expressed and whether the cytotoxic activity of the immune cells was restored.

[0260] Specifically, the CAR-T cells and CAR-BD2m T cells of the above Examples 2-3 were treated with 100 nM of AGB-1, and then washed to remove AGB-1. Three days after removing AGB-1, Western blotting and flow cytometry were performed. In addition, the CAR-T cells and CAR-BD2m T cells that had been treated with AGB-1 and then removed as described above were co-cultured with Nalm6 cancer cells expressing luciferase. At this time, as a control, the CAR-T cells and CAR-BD2m T cells were used by co-culture with 293 T cells that do not express the CD19 receptor but express luciferase. The cytotoxic activity of the T cells in the above culture was measured.

[0261] As a result, as shown in Fig. 11, it was confirmed that the CAR protein was re-expressed in CAR-BD2m T cells after AGB-1 was removed. In addition, the cytotoxic activity of CAR-BD2m T cells decreased in the presence of AGB-1, but when AGB-1 was removed, the cytotoxic activity of CAR-BD2m T cells was confirmed to recover to the level of the AGB-1 untreated group. Therefore, it was confirmed that the degradation and activity of immune cells expressing a CAR including a bromo domain variant of the present invention can be controlled depending on whether AGB-1 is treated. In other words, by controlling the expression of CAR in the body through a protac compound that destroys the bromo domain variant, the activity of CAR-T cells in the body can be reversibly controlled.

[0262]

[0263] [Example 5]

[0264] Analysis of anticancer efficacy of CAR-BD2m T cells of the present invention by AGB-1 (in vivo)

[0265] Since the bromo domain variant used for activity regulation may interfere with the inherent function of CAR, we sought to confirm the in vivo anticancer efficacy of CAR-BD2m T cells produced in Examples 2-3. To this end, the CAR-BD2m T cells were administered to immunodeficient mice, and then tumor proliferation in vivo and mouse survival rates were measured.

[0266] Specifically, 1 × 10 CD19-positive NALM6 cancer cells, which are tumor cells expressing luciferase, were injected into immunodeficient NSG mice (Charles River). 6 The dog was injected intravenously into the tail. The next day, 7.5 × 10 CAR-BD2m T cells were administered. 6Dogs were injected intravenously. At this time, PBS, NT cells of Example 2, and CAR-T cells were used as controls. Afterwards, the anticancer activity was observed by checking the survival rate of the mice.

[0267] As a result, as illustrated in Fig. 12, when the CAR-BD2m T cells and CAR-T cells of the present invention were injected, the survival rate of mice was confirmed to be significantly higher than when PBS or NT cells were injected. Therefore, it was confirmed that the immune cells of the present invention have excellent anticancer activity even in vivo. In other words, it was confirmed that the bromo domain variant inserted for CAR regulation did not inhibit the inherent anticancer function of the CAR.

[0268] In addition, the anticancer efficacy modulation effect of the combination of CAR-BD2m T cells of the present invention and AGB-1 was investigated. First, 1x10 Nalm6 cancer cells were injected into the NSG mice. 6 The dogs were transplanted by subcutaneous administration (SC). Afterwards, the NSG mice transplanted with the cancer cells were treated with only PBS or CAR-BD2m T cells (1x10) of the present invention. 7 Dogs) alone or AGB-1 (100 mpk) and the above CAR-BD2m T cells (1x10 7 After co-treatment with the AGB-1 and CAR-BD2m T, tumor volume was measured in the mice for 122 days to compare the anticancer efficacy. In the AGB-1 and CAR-BD2m T treatment groups, AGB-1 was administered subcutaneously once daily from days 16 to 36 after the tumor transplantation, AGB-1 administration was discontinued from days 37 to 39, and AGB-1 was administered intraperitoneally (IP) once daily from days 40 to 52.

[0269] As a result, as shown in FIG. 13, unlike when only the CAR-BD2m T cells of the present invention were treated, when the CAR-BD2m T cells of the present invention and AGB-1 were treated together, the tumor volume increased when AGB-1 treatment was started, and the tumor size decreased again when AGB-1 administration was stopped. This means that the CAR of the CAR-BD2m T cells was degraded by the AGB-1, so that the anticancer activity of the immune cells was somewhat reduced, and when the AGB-1 drug administration was stopped, the CAR was expressed again, and the anticancer activity of CAR-BD2m was restored. This indicates that, as confirmed in FIG. 11, the expression of the CAR in the mouse is reversibly regulated by the AGB-1 compound. In particular, as illustrated in FIG. 14, when only the CAR-BD2m T cells of the present invention were treated, the tumor volume increased from about 44 days onwards, but when the CAR-BD2m T cells of the present invention and AGB-1 were treated together, complete remission was achieved where the tumor did not grow at all. Accordingly, it was confirmed that when the CAR-BD2m T cells of the present invention and AGB-1 were treated together, the anticancer activity was significantly superior compared to when only the CAR-BD2m T cells were treated. In other words, it was confirmed that when the CAR was appropriately destroyed by AGB-1, the exhaustion phenomenon of CAR-T was resolved, greatly enhancing the anticancer activity of CAR-T in the long term.

[0270]

[0271] Although representative embodiments of the present application have been described above as examples, the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the relevant field will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A recombinant polypeptide comprising a variant of a bromodomain.

2. In claim 1, A recombinant polypeptide, wherein the bromo domain is bromodomain 1 (BD1) or bromodomain 2 (BD2) of any one protein selected from the group consisting of bromodomain-containing protein 2 (BRD2), bromodomain-containing protein 3 (BRD3), bromodomain-containing protein 4 (BRD4), and bromodomain-containing protein T (BRDT).

3. In claim 2, A recombinant polypeptide in which a mutation is introduced into any one amino acid selected from the group consisting of the 63rd leucine of BD1 of the BRDT, the 70th leucine of BD1 of the BRD3, the 94th leucine of BD1 of the BRD4, the 110th leucine of BD1 of the BRD2, the 306th leucine of BD2 of the BRDT, the 345th leucine of BD2 of the BRD3, the 383rd leucine of BD2 of the BRD2, and the 387th leucine of BD2 of the BRD4, wherein the variant of the bromo domain is a recombinant polypeptide in which a mutation is introduced into any one amino acid selected from the group consisting of the 63rd leucine of BD1 of the BRDT, the 70th leucine of BD1 of the BRD3, the 94th leucine of BD1 of the BRD4, the 110th leucine of BD1 of the BRD2, the 306th leucine of BD2 of the BRDT, the 345th leucine of BD2 of the BRD3, the 383rd leucine of BD2 of the BRD2, and the 387th leucine of BD2 of the BRD4.

4. In claim 3, A recombinant polypeptide in which any one amino acid selected from the group consisting of the 63rd leucine of BD1 of the BRDT, the 70th leucine of BD1 of the BRD3, the 94th leucine of BD1 of the BRD4, the 110th leucine of BD1 of the BRD2, the 306th leucine of BD2 of the BRDT, the 345th leucine of BD2 of the BRD3, the 383rd leucine of BD2 of the BRD2, and the 387th leucine of BD2 of the BRD4 is substituted with any one amino acid selected from the group consisting of alanine (A), valine (V), isoleucine (I), and methionine (M).

5. In claim 1, A recombinant polypeptide, wherein the amino acid sequence of the variant of the above bromo domain comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

8.

6. Extracellular binding domain containing an antigen binding site; transmembrane domain; and An intracellular domain of a receptor comprising a recombinant polypeptide according to any one of claims 1 to 5; Chimeric antigen receptor (CAR).

7. In claim 6, The above antigen binding site is a chimeric antigen receptor that specifically binds to an antigen expressed in all cancer cells.

8. In claim 6, A chimeric antigen receptor, wherein the antigen binding site is at least one selected from the group consisting of an antibody or an antigen binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a linear antibody, a single-domain antibody (sdAb), a natural ligand, a designed ankyrin repeat protein (DARPin), a nanobody, an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, an anticalin, a synthelin, and a single chain variable fragment (scFv) of an antibody.

9. In claim 6, A chimeric antigen receptor, wherein the extracellular domain further comprises at least one selected from the group consisting of a hinge domain and a spacer domain.

10. In claim 9, A chimeric antigen receptor, wherein the hinge domain or spacer domain is at least one selected from the group consisting of a hinge domain of an alpha (α), beta (β) or zeta (ζ) chain of a T-cell receptor (TCR), a CD28 hinge domain, a CD3 epsilon (ε) hinge domain, a CD45 hinge domain, a CD4 hinge domain, a CD5 hinge domain, a CD8 hinge domain, a CD9 hinge domain, a CD16 hinge domain, a CD22 hinge domain, a CD33 hinge domain, a CD37 hinge domain, a CD64 hinge domain, a CD80 hinge domain, a CD86 hinge domain, a CD134 hinge domain, a CD137 hinge domain, a CD154 hinge domain and Fc.

11. In claim 6, A chimeric antigen receptor, wherein the transmembrane domain is any one selected from the group consisting of the alpha (α), beta (β) or zeta (ζ) chain of a T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

12. In claim 6, A chimeric antigen receptor, wherein the intracellular domain further comprises a signaling domain.

13. In claim 12, A chimeric antigen receptor, wherein the signaling domain is a polypeptide of at least one intracellular domain selected from the group consisting of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ, and Fc Receptorassociated γ chain.

14. A genetic construct comprising a base sequence encoding a recombinant polypeptide according to any one of claims 1 to 5; or a chimeric antigen receptor according to any one of claims 6 to 13.

15. A cell expressing a recombinant polypeptide according to any one of claims 1 to 5; or a chimeric antigen receptor according to any one of claims 6 to 13.

16. In claim 15, The above cells are immune cells.

17. In claim 16, The above immune cells are T cells or NK cells.

18. In claim 15, The above cell is a cell produced by introducing the genetic construct of claim 14 into the cell.

19. A pharmaceutical composition for preventing or treating cancer, comprising the cell of any one of claims 16 to 18 as an active ingredient.

20. A composition for degrading a recombinant polypeptide or chimeric antigen receptor of a cell according to any one of claims 16 to 18, comprising a proteolysis-targeting chimera (PROTAC) compound as an active ingredient.

21. In claim 20, A composition wherein the above protactic compound targets and destroys a variant of the bromo domain.

22. In claim 21, A composition wherein the above protac compound is AGB-1.

23. In claim 20, The composition above is a composition that can suppress side effects caused by immune cells by regulating the activity of immune cells or enhance the anticancer effect of immune cells.

24. In claim 20, The above composition is an anticancer adjuvant composition.

25. The pharmaceutical composition of claim 19; and A kit for preventing or treating cancer, comprising a proteolysis-targeting chimera (PROTAC) compound.

26. In claim 25, A kit for preventing or treating cancer, wherein the kit further includes an instruction manual describing that the activity of the immune cells can be controlled according to the treatment concentration of the protac compound.

27. In claim 26, A kit for preventing or treating cancer, wherein the above description describes that the activity of the immune cells can be controlled according to the treatment concentration of the protac compound, thereby suppressing side effects caused by the immune cells or enhancing the anticancer effect of the immune cells.

28. In claim 25, A kit for preventing or treating cancer, wherein the above protac compound can target and destroy a variant of the above bromo domain.

29. In claim 28, A kit for preventing or treating cancer, wherein the above protac compound is AGB-1.

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