Fusion protein comprising CD80 protein and il-2 protein, and composition for preventing or treating cancer comprising t cells expressing chimeric antigen receptors

Abstract

The present invention relates to: a fusion protein comprising an IL-2 protein and a CD80 protein; and a pharmaceutical composition for preventing or treating a cancer comprising chimeric antigen receptor (CAR)-expressing immune cells. When the fusion protein of the present invention is co-administered with the CAR-expressing immune cells, specifically CAR-T cells, immune activity of the CAR-T cells in tumors is enhanced and sustained. Moreover, when the fusion protein of the present invention is administered alone to a subject with recurrent cancer after the CAR-T cells have been administered, reactivation of the administered CAR-T cells may be induced to kill the recurrent cancer cells. Even when cancer cells are re-transplanted in an animal model in which cancer cells have been completely killed by co-administering the CAR-T cells and the fusion protein of the present invention, the immune activity of the CAR-T cells may be maintained for a long period of time by the fusion protein of the present invention, thereby continuously inhibiting the proliferation of the re-transplanted cancer cells. Accordingly, the fusion protein of the present invention not only enhances and prolongs the immune anticancer activity of the CAR-T cells, but also repeatedly reactivates the administered CAR-T cells, thereby maximizing the efficiency of the CAR-T cells as therapeutic cell resources to ultimately significantly improve the survival rate of patients.

Description

A composition for preventing or treating cancer comprising a fusion protein comprising CD80 protein and IL-2 protein and a T cell expressing a chimeric antigen receptor

[0001] The present invention relates to a pharmaceutical composition for treating cancer comprising a fusion protein comprising CD80 protein and IL-2 protein and a T cell expressing a chimeric antigen receptor.

[0002]

[0003] Interleukin-2 (IL-2) is a cytokine released by T cells that promotes T cell proliferation and differentiation. IL-2 and the IL-2R system are known to promote the proliferation and anticancer activity of chimeric antigen receptor T cells (CAR-T cells) (Zhang et al., Sci Transl Med., 13(625), 2021). However, the use of IL-2 has been reported to risk the proliferation of regulatory T cells, which can interfere with anticancer responses (Bellet et al., Function. Front Immunol. 12:684-642, 2021).

[0004] Meanwhile, the human CD19 molecule is a cell surface receptor expressed on the surface of human B cells, such as pre-B cells, immature B cells in the early stages of development, mature B cells, and malignant B cells. Most B cell lineage malignancies, including non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphocytic leukemia (ALL), express CD19. Despite recent active research on various anticancer agents, CD19-expressing tumors have a poor prognosis and low responsiveness to conventional treatments, so there is still a high demand for the development of improved treatment methods or therapeutic compositions.

[0005] Meanwhile, CAR-T therapy targeting the CD19 antigen demonstrates excellent antitumor efficacy against several B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL), significantly improving survival rates. However, approximately 60% of patients receiving CAR-T cell therapy experience disease progression or relapse. For this reason, strategies to improve the long-term therapeutic efficacy of CAR-T cells are urgently needed.

[0006] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0007]

[0008] The present inventors have conducted extensive research to develop an efficient combination therapy method that can significantly improve the anticancer activity and durability of immunotherapeutic agents. As a result, the inventors have completed the present invention by demonstrating that a fusion protein comprising CD80 and IL-2 significantly enhances the immune activity of immune cells expressing a chimeric antigen receptor (CAR), specifically CAR-T cells, and that this activity can be sustained and repeatedly reactivated over the long term.

[0009] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises a fusion protein comprising CD80 protein and IL-2 protein and a CAR-expressing immune cell.

[0010] Another object of the present invention is to provide an anticancer composition for co-administration with CAR-expressing immune cells, which comprises a fusion protein comprising CD80 protein and IL-2 protein.

[0011] Another object of the present invention is to provide a composition for enhancing the activity of CAR-expressing immune cells, comprising a fusion protein comprising CD80 protein and IL-2 protein.

[0012]

[0013] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0014]

[0015] To achieve the above purpose, one aspect of the present invention provides a composition for enhancing the activity of immune cells expressing a chimeric antigen receptor, comprising a fusion protein comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof as an active ingredient.

[0016] According to another aspect of the present invention, the present invention provides a method for enhancing the activity of an immune cell expressing a chimeric antigen receptor, comprising the step of administering to a subject a fusion protein comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof.

[0017] As used herein, the term “fusion protein” refers to a recombinant protein molecule in which an amino acid sequence derived from a specific protein or domain is fused with another amino acid sequence derived from a different protein or domain. The amino acid sequences of different origins may be directly linked within the fusion protein, or may be linked via a linker sequence, a tag sequence, a self-cleaving sequence, or a combination thereof.

[0018] As used herein, the term "IL-2 (Interleukin-2)" refers to wild-type IL-2 obtained from any vertebrate source, including primates and rodents. The IL-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-2. In addition, the IL-2 may be wild-type IL-2 or a variant thereof. The IL-2 may be in a mature form. Specifically, the mature IL-2 may not include a signal sequence and may have the amino acid sequence of SEQ ID NO: 10. In this case, the IL-2 may be used as a concept including a truncated fragment of the N-terminus or C-terminus of the wild-type IL-2.

[0019] As used herein, the term "IL-2 variant" refers to a form in which a portion of an amino acid is substituted in full-length IL-2 or a fragment of the IL-2 described above. However, the IL-2 variant may have activity equivalent to or similar to wild-type IL-2. Here, "IL-2 activity" may refer to, for example, specific binding to an IL-2 receptor, and the specific binding can be measured using a method known to those skilled in the art.

[0020] Specifically, the IL-2 variant may be a variant in which some of the amino acids of the wild-type IL-2 are substituted, and at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 may be substituted.

[0021] More specifically, the variant of the IL-2 protein may be a variant comprising a substitution of one or more of the 38th amino acid, the 42nd amino acid, and the 61st amino acid in the amino acid sequence of SEQ ID NO: 10.

[0022] In one specific example, the IL-2 variant may be a variant in which amino acid substitutions occur at two residue positions. Specifically, the IL-2 variant may be one in which the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be one in which the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be one in which the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be one in which the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be one in which the 42nd and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 61st and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10.

[0023] Furthermore, the IL-2 variant may be a variant in which amino acid substitutions occur at three residue positions. Specifically, the IL-2 variant may be one in which the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may have the 38th, 45th, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may have the 38th, 61st, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may have the 42nd, 45th, and 61st amino acids substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may have the 42nd, 45th, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may have the 45th, 61st, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10.

[0024] At this time, the "other amino acid" introduced by the residue position listed above may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, in the amino acid substitution of the IL-2 variant, the 38th position in the amino acid sequence of SEQ ID NO: 10 cannot be substituted with arginine, the 42nd position cannot be substituted with phenylalanine, the 45th position cannot be substituted with tyrosine, the 61st position cannot be substituted with glutamic acid, and the 72nd position cannot be substituted with leucine.

[0025] In the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than arginine. Preferably, in the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with alanine (R38A).

[0026] In the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than phenylalanine. Preferably, in the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (F42A).

[0027] In the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than tyrosine. Preferably, in the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (Y45A).

[0028] In the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than glutamic acid. Preferably, in the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with arginine (E61R).

[0029] In the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than leucine. Preferably, in the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with glycine (L72G).

[0030] Specifically, the IL-2 variant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10.

[0031] Specifically, the IL-2 variant may have two or three substitutions selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

[0032] In addition, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A and F42A are substituted. In addition, in one specific example, the IL-2 variant may be a form in which R38A and Y45A are substituted. In addition, in one specific example, the IL-2 variant may be a form in which R38A and E61R are substituted. In addition, in one specific example, the IL-2 variant may be a form in which R38A and L72G are substituted. In addition, in one specific example, the IL-2 variant may be a form in which F42A and Y45A are substituted. In addition, in one specific example, the IL-2 variant may be a form in which F42A and E61R are substituted. In addition, in one specific example, the IL-2 variant may be a form in which F42A and L72G are substituted. Additionally, as a specific example, the IL-2 variant may have substitutions of E61R and L72G.

[0033] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A, F42A, and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at Y45A, E61R, and L72G.

[0034] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and E61R. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, Y45A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at F42A, Y45A, E61R, and L72G.

[0035] Furthermore, the IL-2 variant may have substitutions such as R38A, F42A, Y45A, E61R, and L72G.

[0036] Specifically, the IL-2 variant may have one or more substitutions selected from the group consisting of R38A, F42A, and E61R in the amino acid sequence of SEQ ID NO: 10. More specifically, the IL-2 variant may have one or more substitutions selected from the group consisting of R38A, F42A, and E61R in the amino acid sequence of SEQ ID NO: 10.

[0037] The IL-2 variant described above used in the present invention has reduced in vivo toxicity caused by binding to the alpha form of the IL-2 receptor (IL-2Rα) compared to wild-type IL-2.

[0038]

[0039] In this specification, "CD80," also referred to as "B7-1," is a membrane protein present on dendritic cells, activated B cells, and monocytes that provides costimulatory signals essential for T cell activation and survival. CD80 consists of 288 amino acids and is known to be a ligand for two different proteins present on the surface of T cells: CD28 and CTLA-4.

[0040] As used herein, the term "CD80 fragment" refers to a fragment of the full-length CD80 protein in which some amino acid residues have been deleted, and thus is an analog of the full-length protein that retains its original biological activity and function. Specifically, it refers to a minimal fragment of CD80 capable of transmitting a costimulatory signal for T cell activation. For example, the functional fragment of CD80 may be the extracellular domain of CD80 or a fragment of the full-length CD80 protein comprising the same.

[0041] A specific example of a fragment of CD80 may be one in which all or part of the 1st to 34th amino acids from the N-terminus, which is the signal sequence of CD80, are removed. In addition, a specific example of the CD80 fragment may be a protein consisting of the 35th to 242nd amino acids of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of the 35th to 232nd amino acids of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of the 35th to 139th amino acids of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of the 142nd to 242nd amino acids of SEQ ID NO: 11.

[0042] More specifically, the fragment of the CD80 protein may be an extracellular domain of the CD80 protein. More specifically, the CD80 fragment may have the amino acid sequence of SEQ ID NO: 2.

[0043] According to a specific embodiment of the present invention, the chimeric antigen receptor expressed by the immune cell of the present invention comprises all or part of an intracellular signaling domain selected from the group consisting of CD3z, CD28, and 4-1BB.

[0044] The term "part" used herein to refer to a specific protein or domain refers to an analog of the full-length protein, with some amino acid residues deleted, that retains its original biological activity and function. Therefore, the term "part" has the same meaning as "functional portion."

[0045] According to a specific embodiment of the present invention, the chimeric antigen receptor comprises a transmembrane domain comprising all or part of CD28 or CD8.

[0046] According to a specific embodiment of the present invention, the chimeric antigen receptor comprises a hinge domain comprising all or part of a CD8 hinge or a CD28 hinge.

[0047] According to a specific embodiment of the present invention, the chimeric antigen receptor comprises an extracellular antigen binding domain that binds to CD19 or BCMA (B-cell maturation antigen).

[0048] More specifically, the chimeric antigen receptor of the present invention may have a CD8 hinge domain, a CD28 transmembrane domain, and a CD3z intracellular signaling domain sequentially positioned from the N-terminus to the C-terminus. Most specifically, the chimeric antigen receptor of the present invention may be ProMab ⓡ It may include a hinge domain, a transmembrane domain, and an intracellular signaling domain of an anti-CD19 CAR (Cat.# PM-CAR1001).

[0049] More specifically, the chimeric antigen receptor of the present invention may have a CD8 hinge domain - a CD8 transmembrane domain - a 4-1BB intracellular signaling (co-stimulatory) domain - a CD3z intracellular signaling domain sequentially positioned from the N-terminus to the C-terminus. Most specifically, the chimeric antigen receptor of the present invention may be Kymriah. ⓡ Anti-CD19 CAR or Abecma ⓡ It may include a hinge domain, a transmembrane domain, and an intracellular signaling domain of an anti-BCMA CAR.

[0050] More specifically, the chimeric antigen receptor of the present invention may have a CD8 hinge domain - a CD28 transmembrane domain - a CD28 intracellular signaling (co-stimulatory) domain - a CD3z intracellular signaling domain sequentially positioned from the N-terminus to the C-terminus. Most specifically, the chimeric antigen receptor of the present invention may be ProMab ⓡIt may include the hinge domain, transmembrane domain, and intracellular signaling domain of anti-BCMA CAR (Cat.# PM-CAR1031).

[0051] More specifically, the chimeric antigen receptor of the present invention may have a CD28 hinge domain - a CD28 transmembrane domain - a CD28 intracellular signaling (co-stimulatory) domain - a CD3z intracellular signaling domain sequentially positioned from the N-terminus to the C-terminus. Most specifically, the chimeric antigen receptor of the present invention may be Yescarta. ⓡ It may include a hinge domain, a transmembrane domain, and an intracellular signaling domain of an anti-CD19 CAR.

[0052] More specifically, the chimeric antigen receptor of the present invention may have a CD8 hinge domain - a CD28 transmembrane domain - a 41BB intracellular signaling (co-stimulatory) domain - a CD3z intracellular signaling domain sequentially positioned from the N-terminus to the C-terminus. Most specifically, the chimeric antigen receptor of the present invention may be ProMab ⓡ It may include a hinge domain, a transmembrane domain, and an intracellular signaling domain of an anti-BCMA CAR (Cat.# PM-CAR1037).

[0053] As used herein, the term "extracellular antigen binding domain" refers to a domain that, when a chimeric antigen receptor is expressed in a target cell (e.g., an immune cell), is located in the extracellular portion and specifically recognizes the target antigen, thereby activating the apoptotic activity of the immune cell specifically in the target cell (e.g., a cancer cell), and may be an antigen-binding fragment of an antibody, such as an Fc receptor or a single-chain variable fragment (ScFv). Therefore, the term "extracellular antigen binding domain" in the present invention is used with the same meaning as "extracellular domain", "antigen recognition fragment" or "antigen-binding fragment".

[0054] As used herein, the term "specifically binds" or "specifically recognizes" means that an antibody or antigen-binding fragment thereof forms a stable complex with an antigen with an affinity greater than a certain level under physiological conditions. Specific binding may be, for example, at least about 1 x 10 -6 It can be defined as an equilibrium dissociation constant less than M. Methods for determining whether two molecules bind specifically are well known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0055] As used herein, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity can generally be expressed as a dissociation constant (Kd) and can be measured by conventional methods known in the art.

[0056] According to the present invention, the extracellular antigen binding domain used in the chimeric antigen receptor of the present invention is an antigen binding fragment of an anti-CD19 antibody or an antigen binding fragment of an anti-BCMA antibody.

[0057] The term “antibody” as used herein means an antibody against CD19 protein or BCMA, which specifically recognizes and binds to a specific epitope thereof, and includes not only a complete full-length antibody form but also an antigen-binding fragment (antibody fragment) of the antibody molecule.

[0058] A complete antibody has two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The heavy chain constant regions are of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and are subclassed into gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant regions are of the kappa (κ) and lambda (λ) types.

[0059] The term “antigen-binding fragment of an antibody” as used herein means a fragment possessing antigen-antibody binding function within the entire antibody molecule, and specifically includes a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, and an Fv fragment.

[0060] Among antibody fragments, Fab has a structure with the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv is the smallest antibody fragment that has only the heavy chain variable region and the light chain variable region. Two-chain Fv (two-chain Fv) has the heavy chain variable region and the light chain variable region connected by a non-covalent bond, and single-chain Fv (single-chain Fv, ScFv) has the heavy chain variable region and the single chain variable region connected by a covalent bond, usually through a peptide linker, or directly at the C-terminus, so it can form a dimer-like structure like two-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction digestion of whole antibodies with papain yields Fab fragments, and digestion with pepsin yields F(ab')2 fragments), or they can be produced using genetic recombination techniques.

[0061] The antigen-binding fragment of the anti-CD19 antibody used in the present invention may be a scFv of the anti-CD19 antibody.

[0062] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD19 antibody comprises a heavy chain variable region comprising an HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 38, and 47, an HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 39, and 48, and an HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 40, and 49.

[0063] The term “heavy chain” as used herein refers to a variable region domain V comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H and three constant region domains C H1 , C H2 and C H3 It refers to both full-length heavy chains and fragments thereof.

[0064] As used herein, the term “CDR (complementarity determining region)” refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains. The heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, which provide key contact residues for antibody binding to an antigen or epitope.

[0065] The scope of the antibodies or antigen-binding fragments of the present invention includes variants having conservative amino acid substitutions in the CDR regions. In addition, the antigen-binding fragments of the present invention may include variants of the amino acid sequences set forth in the attached sequence listing, as long as they can specifically recognize CD19. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody, and are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be considered biologically functional equivalents.

[0066] More specifically, the heavy chain variable region of the antigen-binding fragment of the anti-CD19 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 41, and 50.

[0067] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD19 antibody additionally comprises a light chain variable region comprising LCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 42, and 51, LCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 43, and 52, and LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 44, and 53.

[0068] The term “light chain” in this specification encompasses both full-length light chains and fragments thereof, which comprise a variable region domain VL and a constant region domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen.

[0069] According to a specific embodiment of the present invention, the light chain variable region of the antigen-binding fragment of the anti-CD19 antibody comprises an amino acid sequence selected from the group consisting of sequences 36, 45, and 54.

[0070] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD19 antibody is any one selected from the following (a) to (c):

[0071] (a) an antigen-binding fragment comprising a heavy chain variable region comprising HCDR1 of SEQ ID NO: 29, HCDR2 of SEQ ID NO: 30, and HCDR3 of SEQ ID NO: 31; and a light chain variable region comprising LCDR1 of SEQ ID NO: 33, LCDR2 of SEQ ID NO: 34, and LCDR3 of SEQ ID NO: 35;

[0072] (b) an antigen-binding fragment comprising a heavy chain variable region comprising HCDR1 of SEQ ID NO: 38, HCDR2 of SEQ ID NO: 39, and HCDR3 of SEQ ID NO: 40; and a light chain variable region comprising LCDR1 of SEQ ID NO: 42, LCDR2 of SEQ ID NO: 43, and LCDR3 of SEQ ID NO: 44; and

[0073] (c) an antigen-binding fragment comprising a heavy chain variable region comprising HCDR1 of SEQ ID NO: 47, HCDR2 of SEQ ID NO: 48, and HCDR3 of SEQ ID NO: 49; and a light chain variable region comprising LCDR1 of SEQ ID NO: 51, LCDR2 of SEQ ID NO: 52, and LCDR3 of SEQ ID NO: 53.

[0074] More specifically, the above (a) includes a heavy chain variable region of sequence number 32 and a light chain variable region of sequence number 36. Most specifically, the above (a) is ProMab ⓡ It may be the anti-CD19 ScFv used in anti-CD19 CAR (Cat.# PM-CAR1001).

[0075] More specifically, the above (b) comprises a heavy chain variable region of sequence number 41 and a light chain variable region of sequence number 45. Most specifically, the above (b) comprises Kymriah ⓡ It may be an anti-CD19 ScFv used in an anti-CD19 CAR.

[0076] More specifically, the above (c) includes a heavy chain variable region of sequence number 50 and a light chain variable region of sequence number 54. Most specifically, the above (c) includes Yescarta ⓡ It may be an anti-CD19 ScFv used in an anti-CD19 CAR.

[0077]

[0078] The antigen-binding fragment of the anti-BCMA antibody used in the present invention may be a scFv of the anti-BCMA antibody.

[0079] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-BCMA antibody comprises a heavy chain variable region comprising an HCDR1 having an amino acid sequence of SEQ ID NO: 59, an HCDR2 having an amino acid sequence of SEQ ID NO: 60, and an HCDR3 having an amino acid sequence of SEQ ID NO: 61.

[0080] According to another specific embodiment of the present invention, the antigen-binding fragment of the anti-BCMA antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 62, and 68.

[0081] According to a specific embodiment of the present invention, the extracellular antigen binding domain additionally includes a light chain variable region including LCDR1 having an amino acid sequence of SEQ ID NO: 63, LCDR2 having an amino acid sequence of SEQ ID NO: 64, and LCDR3 having an amino acid sequence of SEQ ID NO: 65.

[0082] According to another specific embodiment of the present invention, the antigen-binding fragment of the anti-BCMA antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 66, and 69.

[0083] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-BCMA antibody is any one selected from the following (d) to (f):

[0084] (d) an antigen-binding fragment comprising a heavy chain variable region of sequence number 56; and a light chain variable region of sequence number 57;

[0085] (e) an antigen-binding fragment comprising a heavy chain variable region comprising HCDR1 of SEQ ID NO: 59, HCDR2 of SEQ ID NO: 60, and HCDR3 of SEQ ID NO: 61; and a light chain variable region comprising LCDR1 of SEQ ID NO: 63, LCDR2 of SEQ ID NO: 64, and LCDR3 of SEQ ID NO: 65; or

[0086] (f) an antigen-binding fragment comprising a heavy chain variable region of sequence number 68; and a light chain variable region of sequence number 69.

[0087] More specifically, the above (d) is ProMab ⓡ It may be the anti-BCMA ScFv used in anti-BCMA CAR (Cat.# PM-CAR1031).

[0088] More specifically, the above (e) comprises a heavy chain variable region of sequence number 62 and a light chain variable region of sequence number 66. Most specifically, the above (e) comprises Abecma ⓡ It may be an anti-BCMA ScFv used in anti-BCMA CAR.

[0089] More specifically, the above (f) is ProMab ⓡ It may be the anti-BCMA ScFv used in anti-BCMA CAR (Cat.# PM-CAR1037).

[0090]

[0091] According to the present invention, the immune cell of the present invention is introduced with a nucleic acid molecule encoding the chimeric antigen receptor of the present invention described above and expresses it.

[0092] The term “nucleic acid molecule” in this specification encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base portion is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)). The nucleic acid molecule of the present invention can be introduced into a target cell by being inserted into a gene delivery vehicle in order to express the chimeric antigen receptor of the present invention described above in a target cell, for example, an immune cell.

[0093] The term “express” as used herein means to artificially introduce a gene using a gene vector to cause a target cell to express an exogenous gene or to increase the natural expression level of an endogenous gene, thereby making the gene replicable as an extrachromosomal element or through chromosomal integration within the target cell. Therefore, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.”

[0094] As used herein, the term “gene delivery vehicle” or “gene delivery system” means any means of transporting a gene into a cell, and gene delivery has the same meaning as transduction of a gene into a cell. At the cell or tissue level, gene delivery has the same meaning as spread of a gene. Therefore, the gene delivery system of the present invention may be described as a gene transduction system and a gene spread system.

[0095] To prepare the gene delivery system of the present invention, the nucleotide sequence of the present invention is preferably operably linked to a suitable expression control sequence within a suitable expression construct. As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the control sequence regulates the transcription and / or translation of the other nucleic acid sequence. The promoter linked to the target gene of the present invention is capable of operating in animal cells, more specifically mammalian cells, and most specifically immune cells to control transcription of the target gene, and includes promoters derived from mammalian viruses and promoters derived from the genome of mammalian cells, such as, but not limited to, the CMV (mammalian cytomegalovirus) promoter, the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the tk promoter of HSV, the RSV promoter, the EF1 alpha promoter, the metallothionine promoter, the beta-actin promoter, the promoter of the human IL-2 gene, the promoter of the human IFN gene, the promoter of the human IL-4 gene, the promoter of the human lymphotoxin gene, and the promoter of the human GM-CSF gene.

[0096] The nucleotide sequence of the target gene can be applied to any gene delivery system conventionally used for gene introduction, for example, plasmids, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, liposomes or niosomes.

[0097] As used herein, the term "immune cell" refers to any cell involved in the initiation or promotion of an immune response, and more specifically, refers to an immune effector cell. Immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, and mast cells. More specifically, the immune cells used in the present invention are selected from the group consisting of T cells, natural killer cells, and natural killer T (NKT) cells, and most specifically, T cells.

[0098] As used herein, the term “treatment” means (a) suppressing the development of a disease, condition, or symptom; (b) alleviating the disease, condition, or symptom; or (c) eliminating the disease, condition, or symptom. When the fusion protein and CAR-expressing immune cells of the present invention are administered to a subject, they induce the death of CD19-positive cancer cells, thereby suppressing the development of, eliminating, or alleviating symptoms caused by various CD19-positive tumors, including lymphoma. Therefore, the composition of the present invention may be a cell therapy composition on its own for a disease, or may be administered together with other pharmacological ingredients and used as an adjuvant treatment for the disease. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjuvant treatment” or “adjuvant treatment agent.”

[0099] As used herein, the term “administration” or “administer” refers to directly injecting a therapeutically effective amount of the composition of the present invention into a subject so that the same amount is formed in the body of the subject.

[0100] In the present invention, the term “therapeutically effective amount” means the content of the composition contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the composition of the present invention is to be administered, and includes “prophylactically effective amount”.

[0101] The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0102] According to a specific embodiment of the present invention, the cancer that can be prevented or treated with the composition of the present invention is CD19-positive cancer. "CD19-positive cancer" refers to a solid cancer or hematological cancer in which CD19 is measurably overexpressed compared to normal cells or CD19-negative tumors.

[0103] CD19 is a cell surface receptor expressed on the surface of B cells, such as pre-B cells, immature B cells in the early stages of development, mature B cells, and malignant B cells. Most malignant tumors of the B cell lineage express it, and active research is being conducted as a therapeutic target.

[0104] More specifically, the CD19 positive cancer is a B cell malignancy.

[0105] The above B-cell malignancies include, for example, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia (CALLA), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, splenic lymphoma, marginal zone lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, and Hodgkin's lymphoma.

[0106] According to a specific embodiment of the present invention, the cancer that can be prevented or treated with the composition of the present invention is a BCMA-positive cancer. "BCMA-positive cancer" refers to a solid cancer or blood cancer in which BCMA is measurably overexpressed compared to normal cells or BCMA-negative tumors.

[0107] BCMA, encoded by the TNFRSF17 gene, is a cell-surface receptor of the TNF receptor superfamily, primarily expressed on the surface of mature B cells and recognizing B-cell activating factors. It is particularly highly expressed in multiple myeloma and is widely studied as a diagnostic and therapeutic target.

[0108] More specifically, the BCMA positive cancer is multiple myeloma.

[0109] According to another aspect of the present invention, the present invention provides an anticancer composition for co-administration with an immune cell expressing a chimeric antigen receptor, the composition comprising a fusion protein comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof as an active ingredient.

[0110] Since the fusion protein and CAR-expressing immune cells used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0111] The fusion protein of the present invention, when administered in combination with CAR-expressing immune cells, maximizes the anticancer effect and improves patient survival by enhancing and sustaining the cancer cell killing activity of the immune cells. The combination may be administered as a single formulation containing both the fusion protein of the present invention and CAR-expressing immune cells, or as separate formulations containing each component individually, administered simultaneously or sequentially in any order with an appropriate time gap.

[0112]

[0113] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as active ingredients a fusion protein comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof; and an immune cell expressing a chimeric antigen receptor.

[0114] According to another aspect of the present invention, there is provided a method for preventing or treating cancer, comprising administering to a subject a fusion protein comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof; and an immune cell expressing a chimeric antigen receptor.

[0115] Since the fusion protein and CAR-expressing immune cells used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0116]

[0117] The features and advantages of the present invention are summarized as follows:

[0118] (a) The present invention provides a composition for enhancing the activity of immune cells expressing a chimeric antigen receptor, which comprises a fusion protein comprising CD80 protein and IL-2 protein as an active ingredient.

[0119] (b) When the fusion protein of the present invention is administered in combination with a chimeric antigen receptor (CAR)-expressing immune cell, specifically, a CAR-T cell, not only is the immune activity of the CAR-T cell strengthened and sustained in the tumor, but when the fusion protein of the present invention is administered alone to an individual whose cancer has relapsed after CAR-T cell administration, the reactivation of the previously administered CAR-T cell can be induced, thereby killing the relapsed cancer cells. In addition, when the fusion protein of the present invention and CAR-T cells are administered in combination and the cancer cells are re-transplanted into an animal model in which the cancer cells have been completely killed, the immune activity of the CAR-T cell is maintained for a long period of time by the fusion protein of the present invention, thereby continuously suppressing the proliferation of the re-transplanted cancer cells.

[0120] (c) Accordingly, the fusion protein of the present invention not only enhances and prolongs the immune anticancer activity of CAR-T cells, but also repeatedly reactivates previously administered CAR-T cells, thereby maximizing the efficiency of CAR-T cells as a therapeutic cell resource, ultimately significantly improving the survival rate of patients.

[0121]

[0122] Figure 1 is a schematic diagram of the structure of one example of a fusion protein (GI-101A).

[0123] Figure 2 is a schematic diagram of the structure of one example of a fusion protein (GI-102).

[0124] Figure 3 is a graph showing the results of measuring the number of dead Daudi cancer cells after co-culturing Daudi cancer cells, a B cell lymphoma, with non-transduced T cells or CAR-T cells, and treating them with culture medium (Non-treat), IgG4-Fc, and GI-101A, respectively, for 16 hours.

[0125] Figure 4 is a graph showing the results of measuring PD-1 expression in non-transduced T cells and CAR-T cells after co-culturing Daudi cancer cells, a B-cell lymphoma, with non-transduced T cells or CAR-T cells, and culturing them for 48 hours by treating them with culture medium (Non-treat), IgG4-Fc, and GI-101A, respectively.

[0126] Figure 5 is a graph showing the results of measuring the number of dead cancer cells after co-culturing multiple myeloma U266 cells with non-transduced T cells or CAR-T cells for 16 hours by treating them with culture medium (Non-treat), IgG4-Fc, GI-101A, and GI-102, respectively.

[0127] Figure 6 is a graph showing the results of measuring the number of dead cancer cells after co-culturing multiple myeloma MM.1S cells and non-transduced T cells or CAR-T cells for 16 hours by treating them with culture medium (Non-treat), IgG4-Fc, GI-101A, and GI-102, respectively.

[0128] Figure 7 is a schematic diagram of an experimental plan to confirm the combined effect of GI-101A and CAR-T cells using a lymphoma xenograft mouse model.

[0129] Figure 8 shows the results of measuring the body weight of the mouse model during Part 1 of Example 4.2.

[0130] Figure 9 shows the effect of IgG4-Fc alone (Vehicle, G1) and low dose (3 × 10) in a lymphoma xenograft mouse model. 5CAR-T cells and IgG4-Fc combination [CAR-T(low), G2], high dose (3 × 10 6 These are photographs taken using IVIS (In-vivo Imaging System) equipment on days 0, 7, 20, and 32 after administration of CAR-T cells and IgG4-Fc combination [CAR-T(high), G3], low-dose CAR-T cells and GI-101A combination (G4), and high-dose CAR-T cells and GI-101A combination (G5).

[0131] Figure 10 is a graph showing the results taken in Figure 9 as a total amount of light emission.

[0132] Figure 11 shows the results of measuring the body weight of the mouse model during Part 2 of Example 4.3.

[0133] Figure 12 shows a low-dose (3 × 10 5 CAR-T cells and IgG4-Fc combination [CAR-T(low), G2], high dose (3 × 10 6 The group that received CAR-T cells and IgG4-Fc combination [CAR-T (high), G3] was divided into groups and administered IgG4-Fc and GI-101A, respectively. The images were taken using IVIS equipment on days 42, 53, 62, 74, 84, 95, and 104 to show the extent of tumor formation.

[0134] Figure 13 is a graph showing the results taken in Figure 12 as a total amount of light emission.

[0135] Figure 14 shows the results of measuring the body weight of the mouse model during Part 3 of Example 4.4.

[0136] Figure 15 shows a low-dose (3 × 10 5 CAR-T cells and GI-101A in combination (G4) and high dose (3 × 10 6The group that received CAR-T cells and GI-101A combination (G5) was divided into groups, and after administering IgG4-Fc and GI-101A respectively, the tumor formation degree on days 42 and 53, and the cancer cells were re-transplanted on day 56, and the tumor formation degree on days 62, 74, 84, 95, and 104 were photographed using IVIS equipment.

[0137] Figure 16 is a graph showing the results taken in Figure 15 as a total amount of light emission.

[0138] Figure 17 shows FMC63 on the 1st, 11th, 21st and 33rd days in the Part 1 test of Example 4.2. + CD3 + T cell count, CD3 + CD4 + T cell count and CD3 + CD8 + This is a graph showing the results of measuring T cell counts.

[0139] Figure 18 shows FMC63 on days 33, 44, 54, 63, 75, 85, 95 and 105 in the Part 2 test of Example 4.3. + CD3 + T cell count, CD3 + CD4 + T cell count and CD3 + CD8 + This is a graph showing the results of measuring T cell counts.

[0140] Figure 19 shows FMC63 on days 33, 44, 54, 63, 75, 85, 95 and 105 in Part 3 of Example 4.4. + CD3 + T cell count, CD3 + CD4 + T cell count and CD3 + CD8 + This is a graph showing the results of measuring T cell counts.

[0141] Figure 20 is a schematic diagram of an experimental plan to confirm the combined effect of GI-102 and CAR-T cells using a lymphoma xenograft mouse model.

[0142] Figure 21 shows the effect of IgG4-Fc alone (Vehicle, G1) and low dose (3 × 10) in a lymphoma xenograft mouse model. 5 CAR-T cells and IgG4-Fc combination [CAR-T(low), G2], high dose (3 × 10 6 These are photographs taken with IVIS equipment on days 0, 10, 20, and 30 after administration of CAR-T cells and IgG4-Fc combination [CAR-T(high), G3], low-dose CAR-T cells and GI-102 combination (G4), and high-dose CAR-T cells and GI-102 combination (G5).

[0143] Figure 22 shows a low-dose (3 × 10 5 CAR-T cells and IgG4-Fc combination [CAR-T(low), G2], high dose (3 × 10 6 The group that received CAR-T cells and IgG4-Fc combination [CAR-T (high), G3] was divided into groups and administered IgG4-Fc and GI-102, respectively. The images were taken using IVIS equipment on days 40, 50, 66, 77, 87, and 95 to show the extent of tumor formation.

[0144] Figure 23 shows a low-dose (3 × 10 5 CAR-T cells and GI-101A in combination (G4) and high dose (3 × 10 6 The group that received CAR-T cells and GI-102 in combination (G5) was divided into groups, and after administering IgG4-Fc and GI-102 respectively, the tumor formation degree was taken on the 40th and 53rd days, and the cancer cells were re-transplanted on the 59th day, and the tumor formation degree was taken on the 66th, 77th, 87th, and 95th days using IVIS equipment.

[0145] Figure 24 is CD3 on the 11th day of Part 1 test of Example 6.2. + CD4 + T cell count and CD3 + CD8 + This is a graph showing the results of measuring T cell counts.

[0146] Figure 25 is CD3 on the 52nd day in Part 2 of Example 6.3. + CD4 + T cell count and CD3 + CD8 + This is a graph showing the results of measuring T cell counts.

[0147] Figure 26 is CD3 on the 52nd day in the Part 3 test of Example 6.4. + CD4 + T cell count and CD3 + CD8 + This is a graph showing the results of measuring T cell counts.

[0148] Figure 27 is a graph showing the degree of tumor formation measured on day 23 after administration of BCMA-targeting CAR-T cells and IgG4 and BCMA-targeting CAR-T cells and GI-102 to a multiple myeloma mouse model, expressed as total flux.

[0149] Figure 28 shows the CD3 expression in blood samples 24 days after administration of BCMA-targeting CAR-T cells and IgG4 and BCMA-targeting CAR-T cells and GI-102 in a multiple myeloma mouse model. + CD4 + T cell count and CD3 + CD8 + This is a graph showing the results of measuring T cell counts.

[0150]

[0151] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0152] Example

[0153] I. Production of fusion proteins

[0154] Manufacturing Example 1. Manufacturing of GI-101A

[0155] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide comprising a base sequence (SEQ ID NO: 8) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of Thermo Fisher Scientific and loaded into the pcDNA3.4 vector (Table 1).

[0156] 구분서열정보서열번호signal peptide (TPA)MDAMLRGLCCVLLLCGAVFVSPSHA1CD80(hB7-1:35-242)VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN2hingeGSGGGGSGGGGSGGGGSAESKYGPPCPPCP3Immunoglobulin FcAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLG4linkerGGGGS5hIL2v2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT6CD80-IgG4Fc-IL2v2TGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCTAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGACATTGCCGTGGAATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGTCTAGATGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGCTGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCTCTTGGAGGTGGTGGCGGTTCTGCCCCTACCAGCTCCTCTACCAAGAAAACCCAGCTCCAGTTGGAGCATCTGCTGCTGGACCTCCAGATGATTCTGAACGGGATCAACAACTATAAGAACCCCAAGCTGACCGCCATGCTGACCGCTAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCACCTCCAGTGCCTGGAAGAAGAACTGAAGCCCCTGGAAGAGGTGCTGAATCTGGCCCAGTCCAAGAACTTCCACCTGAGGCCACGGGACCTGATCAGCAACATCAACGTGATCGTGCTGGAACTGAAGGGCTCCGAGACAACCTTTATGTGCGAGTACGCCGACGAGACAGCCACCATCGTGGAATTTCTGAACCGGTGGATCACCTTCTGCCAGAGCATCATCTCCACACTGACCTGATGA8CD80-IgG4Fc-IL2v2VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWL ENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNGSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHN HYTQKSLSLSLGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT9

[0157] In addition, the vector was introduced into CHO cells (Expi-CHO) to express the fusion protein of sequence number 9. The stable cell line expressing the fusion protein of sequence number 9 was supplied with feed medium at a ratio of 7.0% (v / v) every other day from the 3rd to the 7th day of culture in a fed-batch culture manner, and the culture medium was collected on the 7th day. The fusion protein (GI-101A) was purified from the collected culture medium through chromatography.

[0158]

[0159] Manufacturing Example 2. Manufacturing of GI-102

[0160] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, E61R) (GI101-M61), a polynucleotide comprising the following sequence (SEQ ID NO: 27) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 23) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of Thermo Fisher Scientific and loaded into the pcDNA3.4 vector (Table 2).

[0161] 구분서열정보서열번호signal peptide (TPA)MDAMLRGLCCVLLLCGAVFVSPSHA1CD80(hB7-1:35-242)VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN2hingeGSGGGGSGGGGSGGGGSAESKYGPPCPPCP3Immunoglobulin FcAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLG4linkerGGGGS5hIL2v3APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT23CD80-IgG4Fc-IL2v3TGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCTAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGACATTGCCGTGGAATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCGTGGACAAGTCTAGATGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGCTGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCTCTTGGAGGTGGTGGCGGTTCTGCCCCTACCAGCTCCTCTACCAAGAAAACCCAGCTCCAGTTGGAGCATCTGCTGCTGGACCTCCAGATGATTCTGAACGGGATCAACAACTATAAGAACCCCAAGCTGACCGCCATGCTGACCGCTAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCACCTCCAGTGCCTGGAAAGGGAACTGAAGCCCCTGGAAGAGGTGCTGAATCTGGCCCAGTCCAAGAACTTCCACCTGAGGCCACGGGACCTGATCAGCAACATCAACGTGATCGTGCTGGAACTGAAGGGCTCCGAGACAACCTTTATGTGCGAGTACGCCGACGAGACAGCCACCATCGTGGAATTTCTGAACCGGTGGATCACCTTCTGCCAGAGCATCATCTCCACACTGACC27CD80-IgG4Fc-IL2v3VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWL ENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNGSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNH YTQKSLSLSLGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT28

[0162]

[0163] In addition, the vector was introduced into CHO cells (Expi-CHO) to express the fusion protein of sequence number 28. The stable cell line expressing the fusion protein of sequence number 28 was supplied with feed medium at a ratio of 7.0% (v / v) every other day from the 3rd to the 11th day of culture in a fed-batch manner, and the culture medium was collected on the 12th day. The fusion protein (GI-102) was purified from the collected culture medium through chromatography.

[0164]

[0165] II. Confirmation of the effect of fusion proteins on enhancing CAR-T cell immune activity: in vitro

[0166] Example 1. Enhancement of cell-mediated cytotoxicity of CD19-targeted CAR-T cells by GI-101A

[0167] To distinguish between target Daudi cancer cells (Biocytogen Pharmaceuticals, Cat no. 310728) and effector T cells, Daudi cancer cells were labeled with CTV (CellTrace Violet, Invitrogen) dye. Specifically, Daudi cancer cells inserted with luciferase cDNA were labeled by reacting with 1 μM CTV dye at 37°C for 20 minutes. After the reaction was completed, culture medium 5 times the volume of the staining solution was added and reacted for 5 minutes. Then, the CTV dye not bound to the cells was removed by centrifugation at 1,300 rpm for 5 minutes. CTV-labeled Daudi cancer cells were resuspended in culture medium [RPMI1640 medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin / streptomycin, and 2 mM L-glutamine] and seeded in 96-well microplates at a density of 5 × 10 per well. 4 Each dog was busy.

[0168] T cells (ProMab Biotechnologies, Cat no. PM-CAR1001) transduced with chimeric antigen receptor (CAR) gene (CD19scFv-CD28-CD3z) and non-transduced T cells (ProMab Biotechnologies, Cat no. PM-CAR2003) without CAR gene as a control were prepared and seeded in 96-well microplates containing Daudi cancer cells labeled with CTV dye at a density of 5 × 10 per well. 4 Each well was divided into 1:1 (ET ratio). The same amount of culture medium was added to the wells containing only target cells (control group).

[0169] Each well of a 96-well microplate containing target and effector cells was treated with IgG4-Fc (50 nM) or GI-101A (50 nM) and cultured in an incubator at 37°C and 5% CO2 for 16 h. The cells co-cultured for 16 h were centrifuged, the supernatant was removed, and the cells were resuspended in FACS buffer [DPBS buffer containing 2% fetal bovine serum (FBS)] and centrifuged again. Then, Annexin V binding buffer (Biolegend, Cat no. 422201), 7-AAD (Biolegend, Cat no. 420403), and FITC Annexin V (Biolegend, Cat no. 640906) were treated and reacted at 4°C for 20 min. After the reaction was completed, analysis was performed using a Cytek Aurora (Cytek Biosciences) flow cytometer and FlowJo Software (BD Biosciences) analysis program.

[0170] As a result, non-transduced T cells showed minimal tumor-killing activity against Daudi cancer cells, and the change due to GI-101A treatment was not significant. On the other hand, CAR-T cells showed tumor-killing activity against Daudi cancer cells, and the tumor-killing activity of CAR-T cells increased by GI-101A treatment (Fig. 3).

[0171]

[0172] Example 2. Confirmation of decreased PD-1 expression in CAR-T cells by GI-101A

[0173] The target cells, Daudi cancer cells, were labeled with CTV dye in the same manner as in Example 1. The labeled Daudi cancer cells were resuspended in culture medium [RPMI1640 medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin / streptomycin, and 2 mM L-glutamine], and seeded in a 96-well microplate at a density of 5 × 10 per well. 4Each dog was busy.

[0174] T cells transduced with CAR genes and non-transduced T cells as a control were prepared, and 5 × 10 per well were plated in a 96-well microplate containing Daudi cancer cells labeled with CTV dye. 4 Each well was divided into 1:1 (ET ratio). The same amount of culture medium was added to the wells containing only target cells (control group).

[0175] Each well of a 96-well microplate containing target and effector cells was treated with IgG4-Fc (50 nM) or GI-101A (50 nM) and cultured in an incubator at 37°C and 5% CO2 for 48 h. The cultured cells were centrifuged to remove the supernatant, resuspended in FACS buffer [DPBS buffer containing 2% fetal bovine serum (FBS)], and centrifuged again. After centrifugation, the cells were resuspended in FACS buffer and treated with Fc blocker (BD Biosciences, Cat no. 564219) and incubated at 4°C for 5 minutes. After completion of the reaction, the cells were treated with APC anti-FMC63 (ACRO Biosystems, FM3-AY54A1-25tests), Fixable Viability Dye eFluor™ 780 (eBioscience, Cat no. 65-0865-14), PE-Cy7 anti-CD3 (Biolegend, Cat no. 300420), and BV510 anti-PD-1 (BD Biosciences, Cat no. 563076), incubated at 4°C for 20 minutes, and washed with FACS buffer. The cell pellet was resuspended in FACS buffer and analyzed using a Cytek Aurora flow cytometer and FlowJo Software analysis program.

[0176] As a result, in non-transduced T cells, there was little change in PD-1 expression before and after co-culture with Daudi cancer cells, whereas in CAR-T cells, PD-1 expression increased after co-culture with Daudi cancer cells. In addition, in CAR-T cells treated with IgG4-Fc, the change in PD-1 expression due to co-culture was not significant, but in CAR-T cells treated with GI-101A, PD-1 expression was significantly reduced due to co-culture (Fig. 4).

[0177] Decreased expression of PD-1 in CAR-T cells is known to enhance their ability to attack tumor cells. In other words, decreased PD-1 expression increases CAR-T cell infiltration into tumor cells, thereby suppressing tumor formation and enhancing therapeutic responses.

[0178] Through Figure 4, it was confirmed that the effector function of CAR-T cells to destroy tumor cells and induce an immune response was increased by GI-101A.

[0179]

[0180] Example 3. Enhancement of cell-mediated cytotoxicity of BCMA-targeted CAR-T cells by GI-101A or GI-102

[0181] To distinguish between target cells, U266 (human multiple myeloma cells) or MM.1S (multiple myeloma human B lymphoblasts), and effector cells, T cells, multiple myeloma cells were labeled with CTV (CellTrace™ Violet, Invitrogen™) dye. Specifically, Daudi cancer cells inserted with luciferase cDNA were labeled by reacting with 1 μM CTV dye at 37°C for 20 minutes. After the reaction was completed, a culture medium volume five times the volume of the staining solution was added, reacted for 5 minutes, and centrifuged at 1,300 rpm for 5 minutes to remove CTV dye not bound to the cells. CTV-labeled multiple myeloma cells were resuspended in culture medium [RPMI1640 medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin / streptomycin, and 2 mM L-glutamine] and seeded in 96-well microplates at a density of 5 × 10 per well. 4 Each dog was busy.

[0182] T cells (ProMab Biotechnologies, Cat no. PM-CAR1031) transduced with CAR gene (BCMAscFv-CD28-CD3z) and non-transduced T cells (ProMab Biotechnologies, Cat no. PM-CAR2003) without CAR gene as a control were prepared and seeded in 96-well microplates containing Daudi cancer cells labeled with CTV dye at a density of 5 × 10 per well. 4 Each well was divided into 1:1 (ET ratio). The same amount of culture medium was added to the wells containing only target cells (control group).

[0183] Each well of a 96-well microplate containing target and effector cells was treated with IgG4-Fc (50 nM), GI-101A (50 nM), or GI-102 (50 nM) and cultured in an incubator at 37°C and 5% CO2 for 16 h. The cells co-cultured for 16 h were centrifuged, the supernatant was removed, and the cells were resuspended in FACS buffer [DPBS buffer containing 2% fetal bovine serum (FBS)] and centrifuged again. Then, Annexin V binding buffer (Biolegend, Cat no. 422201), 7-AAD (Biolegend, Cat no. 420403), and FITC Annexin V (Biolegend, Cat no. 640906) were treated and reacted at 4°C for 20 min. After the reaction was completed, analysis was performed using a Cytek Aurora (Cytek Biosciences) flow cytometer and FlowJo Software (BD Biosciences) analysis program.

[0184] As a result, non-transduced T cells showed minimal killing capacity against U266 and MM.S1 cancer cells, and the effect of GI-101A or GI-102 treatment was not significant. In contrast, CAR-T showed killing capacity against U266 and MM.S1 cancer cells, and the killing capacity was increased by GI-101A and GI-102 (Figs. 5 and 6).

[0185]

[0186] III. Confirmation of the effect of fusion proteins on enhancing CAR-T cell immune activity: in vivo

[0187] Example 4. Confirmation of the CD19-targeted CAR-T cell reactivation and cancer cell killing effect of GI-101A in a lymphoma xenograft mouse model.

[0188] Example 4.1. Creation of a lymphoma NSG mouse model and separation of test groups

[0189] To establish a lymphoma xenograft mouse animal model, 5 × 10 luciferase-expressing B-Luc Daudi cancer cells were injected into the tail vein of 4-8 week old NSG mice (NOD scid gamma mice, Jackson Labs). 5 Each animal was injected with 200 μl / animal. In vivo imaging was performed 7 days before CAR-T cell infusion and after B-Luc Daudi cell administration. Comparable bioluminescence intensities were confirmed between groups using IVIS Lumina III (PerkinElmer, Inc.), and then the groups were classified. To measure bioluminescence, luciferin was injected intraperitoneally 10 minutes before in vivo imaging. Animals were maintained under inhaled isoflurane anesthesia during imaging. After in vivo imaging, a region of interest (ROI) was designated for each subject, and the photon values ​​in that region were recorded. To monitor the progress of the experiment, bioluminescence measurements were performed 11 times in total: on days 0, 7, 20, 32, 42, 53, 62, 74, 84, 95, and 104.

[0190] Thirty healthy mice were randomly selected based on physiological status, body weight changes, and tumor growth rates. The selected animals were divided into groups of six, with tumor formation and body weight being as even as possible.

[0191]

[0192] Example 4.2. Administration of Test Substances and Evaluation of Tumor Growth (Part 1)

[0193] The test groups were organized and the test substances were administered as shown in Table 3 below. The test from the day of CAR-T administration (Day 0) and the day of test substance administration (Day 1) to Day 33 was defined as Part 1.

[0194] Group Sex Individual Number Test Substance Administration Dosage Volume (mL / kg) Route Cycle G1F6IgG4 1.1 mg / kg 5 S.C. Once every 10 days (Q10D) x 3 G2F6CAR-T (low) 3 x 10 5 cells / head5I.V.1 timeIgG41.1 mg / kg5S.C.Q10D x 3G3F6CAR-T (high)3 x 10 6 cells / head5I.V.1 timeIgG41.1 mg / kg5S.C.Q10D x 3G4F6CAR-T (low)3 x 10 5 cells / head5I.V.1 timeGI-101A3.0 mg / kg5S.C.Q10D x 3G5F6CAR-T (high)3 x 10 6 cells / head5I.V.1 timeGI-101A3.0 mg / kg5S.C.Q10D x 3

[0195] At this time, T cells (ProMab Biotechnologies, Cat no. PM-CAR1001) introduced with the CAR gene (CD19scFv-CD28-CD3z) were administered to a lymphoma xenograft mouse model, and then IgG4-Fc and GI-101A were administered at 10-day intervals from the next day until day 33. Body weight was measured regularly during the test period.

[0196] As a result of measuring body weight during the test period (Fig. 8), the body weight of the control group G1 Vehicle (IgG4 1.1 mg / kg) gradually increased but began to decrease from day 16. As a result, a statistically significant difference in body weight was observed between the groups administered CAR-T and the test substance and G1 from day 27. In terms of body weight change, the groups administered CAR-T showed a tendency for greater weight gain than the G1 group that did not receive administration, and this was observed regardless of the number of CAR-T cells administered.

[0197] The results of bioluminescence total flux, which measured the extent of tumor growth on days 0 to 33, showed that the total flux values ​​were significantly lower in all groups administered CAR-T and test substances from day 7 to day 32 compared to the control group G1.

[0198] Bioluminescence imaging results showed that high doses (3 × 10 6 Groups (G3 and G5) administered CAR-T cells at low doses (3 × 10 5 In the vehicle group (G1), GI-101A showed a better antitumor effect than the groups (G2 and G4) that received CAR-T cells (Figs. 9 and 10). Regarding clinical symptoms, crawling posture, hypothermia, and weakness symptoms appeared from day 21 in the vehicle group, and all subjects showed these symptoms on day 33. These symptoms were not observed in the CAR-T treatment groups (G2, G3, G4, G5), suggesting a protective or therapeutic effect of CAR-T treatment. In addition, bioluminescence imaging showed that GI-101A showed a superior antitumor effect compared to the vehicle (IgG4), and some subjects in the groups (G4 and G5) that received GI-101A showed complete tumor disappearance. Therefore, from day 33, each group was divided into two subgroups of three subjects each, and the trial continued.

[0199]

[0200] Example 4.3. Administration of Test Substances and Evaluation of Tumor Growth (Part 2)

[0201] In Part 1 of the trial, the G2 and G3 groups, which received IgG4 together with CAR-T, were divided into two subgroups and administered GI-101A or IgG4 under the conditions listed in Table 4 and monitored until Day 105. This phase was defined as Part 2 of the trial.

[0202] Group Sex Number of individuals Test substance Administration Dosage Volume (mL / kg) Route Cycle G1F6IgG4 1.15S.C-G2-1F3IgG4 1.15S.CQ10D x 7 G2-2F3GI-10 1A3.05S.CQ10D x 7 G3-1F3IgG4 1.15S.CQ10D x 7 G3-2F3GI-101A3.05S.CQ10D x 7

[0203] Body weight was measured during the trial period, and significant differences in weight change results were observed between the groups (Fig. 11). In particular, the groups receiving GI-101A (G2-2 and G3-2) showed sustained weight gain compared to the groups receiving IgG4 (G2-1 and G3-1), and this effect was more pronounced in the group receiving a large number of CAR-T cells in Part 1 (G3-2).

[0204] Tumor growth confirmed by bioluminescence imaging showed that the groups receiving GI-101A (G2-2 and G3-2) showed increased tumor suppression over time (Figs. 12 and 13), while the groups receiving IgG4 (G2-1 and G3-1) showed hypothermia and weakness before dying. Similar to the weight gain results, this phenomenon was observed in part 1 at a high dose (3 × 10 6 The group (G3-2) that received CAR-T cells (cells) showed a superior antitumor effect. The results from the Part 2 trial highlight that combination therapy plays an important role in achieving better cancer treatment outcomes.

[0205]

[0206] Example 4.4. Administration of Test Substances and Evaluation of Tumor Growth (Part 3)

[0207] In Part 1 of the trial, the G4 and G5 groups, which received GI-101A together with CAR-T, were each divided into two subgroups and administered GI-101A or IgG4 under the conditions in Table 5 until day 55, followed by Daudi-Luc cell re-transplantation on day 56, and then monitored until day 105.

[0208] Group Sex Number of individuals Days 33-55 Day 56 Test substance administration Tumor reimplantation (Daudi-Luc) Dosage Volume (mL / kg) Route Cycle G4-1F3IgG41.15S.C.Q10D x 35 x 10 5 cells / headG4-2F3GI-101A3.05S.C.Q10D x 35 x 10 5 cells / headG5-1F3IgG41.15S.C.Q10D x 35 x 10 5 cells / headG5-2F3GI-101A3.05S.C.Q10D x 35 x 10 5 cells / head

[0209] As a result of measuring body weight during the test period, no significant difference was found between G4-2 and G4-1, G5-2 and G5-1 (Fig. 14).

[0210] According to the bioluminescence imaging results, some animals with residual tumors at the beginning of Part 3 showed complete tumor resolution by Day 53, with no detectable tumors remaining in the body (Figs. 15 and 16). Therefore, tumor rechallenge was performed on Day 56 for all groups in Part 3, and no additional administration was performed thereafter. No weight change was observed due to the substances administered at the beginning of Part 3, and no notable clinical symptoms were observed. However, even after tumor rechallenge, no tumors were detected in the G4-2 and G5-2 groups until the end of the trial. On the other hand, tumors recurred over time in the G4-1 and G5-1 groups. These observations indicate that the effect of GI-101A administered to G4-2 and G5-2 in the early phase of Part 3 persisted. Therefore, this tumor recurrence phenomenon was observed at a low dose (3 × 10 5 The results of Part 3 confirmed the sustained antitumor effect of GI-101A.

[0211] Across all three trials, combination therapy with GI-101A and CAR-T cells demonstrated improved health status and suppressed tumor growth in the subjects. In addition, high doses (3 × 10 6 When CAR-T cells (CAR-T cells) were co-administered with GI-101A, no clinical symptoms appeared, indicating the safety of this combination administration method. Furthermore, GI-101A showed potential as a maintenance therapy after CAR-T treatment.

[0212]

[0213] Example 5. Analysis of the effect of enhancing immune activity by combining GI-101A and CD19-targeting CAR-T.

[0214] Example 5.1. Sample Preparation

[0215] Blood samples were prepared by collecting blood from mice in the test group on days 1, 11, 21, and 34 after the tumor formation analysis in Example 3.2 and on days 34, 44, 54, 63, 75, 85, 95, and 105 after the tumor formation analysis in Examples 3.3 and 3.4. Thereafter, FACS analysis was performed on CD4+ T cells and CD8+ T cells using the blood samples.

[0216] Specifically, 200 μl of blood sample was treated with 5 ml RBC Lysis buffer (eBioscience, Cat no. 00-4300-54) and reacted at room temperature for 6 minutes. The sample was filled with serum-free RPMI medium (Gibco, Cat no. A1049101) and centrifuged at 500 × g for 5 minutes. The supernatant was removed, treated with 5 ml RBC Lysis buffer again, reacted for 6 minutes, and then the sample was filled with serum-free RPMI medium and centrifuged at 500 × g for 5 minutes. Afterwards, it was resuspended in 100-200 μl of FACS buffer [PBS buffer containing 2% HI-FBS (Heat-inactivated FBS)]. The cell number was measured using an ADAM cell counter system (NanoEntech).

[0217] Add 50 μL of human Fc blocker to the FACS tube and incubate at room temperature (RT) for 15 minutes, then add antibody cocktail for cell surface staining (Alexa Flour 700 anti-mouse CD45 antibody (Biolegend, Cat. No. 103128), BV510 anti-human CD45 antibody (Biolegend, Cat. No. 368526), ​​PerCP anti-human CD19 antibody (BD, Cat. No. 552851), PerCP-Cy5.5 anti-human CD3 antibody (BD, Cat. No. 560835), BV785 anti-human CD8a antibody (Biolegend, Cat. No. 301046), PE-Cy7 anti-human CD4 antibody (Biolegend, Cat. No. 317414), Fixable viability dye eFluor780 (Invitrogen, Cat. No. 65-0865-14), The cells were reacted with FITC anti-FMC63 antibody (Acrobiosystems, Cat. No. FM3-FY45P1) at 4°C for 30 minutes. Then, the cells were centrifuged for 5 minutes, the supernatant was removed, and the cells were washed twice with 200 μL of FACS buffer.

[0218] Afterwards, the cell pellet was resuspended in FACS buffer and measured using a Cytek Aurora flow cytometer and analyzed using the FlowJo Software analysis program.

[0219]

[0220] Example 5.2. Analysis of the immune activity enhancement effect in the test group in Part 1.

[0221] Analysis of the Part 1 trial revealed that in the groups administered only CAR-T cells (G2 and G3), a decreasing trend in the proportion of FMC63+ CD3+ T cells was observed from day 11 to day 34 after administration (Fig. 17). In the groups administered CAR-T cells together with GI-101A (G4 and G5), the proportion of FMC63+ CD3+ T cells among total cells was higher than that in the CAR-T monotherapy groups (G2 and G3). Although the proportion of FMC63+ CD3+ T cells in the G4 group decreased on day 21, the G4 and G5 groups maintained higher FMC63+ CD3+ T cell proportions than the G2 and G3 groups until day 34. The numbers of CD4+ T cells and CD8+ T cells were confirmed to be higher in the group administered with CAR-T and GI-101A in combination than in the group administered only CAR-T and the control group (G2 vs. G4, G3 vs. G5).

[0222]

[0223] Example 5.3. Confirmation of CAR-T cell reactivation following GI-101A administration in mice with cancer relapse after CAR-T cell administration (Analysis of the immune activity enhancement effect for the Part 2 test group)

[0224] Analysis of the Part 2 trial showed that the group receiving GI-101A had an increased proportion of CAR-T cells, and after day 63, when complete remission was observed, the proportion of CAR-T cells decreased (Fig. 18). In the G2-2 and G3-2 groups receiving GI-101A, the number of CD4+ T cells and CD8+ T cells tended to increase until the end of the trial compared to the control group (G2-1 and G3-1). In particular, there was a difference in the number of CD4+ T cells and CD8+ T cells between the GI-101A and IgG4 groups from day 54. In addition, in the G2-2 and G3-2 groups that continued to receive GI-101A, it was confirmed that the number of CD4+ and CD8+ T cells was maintained until day 105, the end of the experiment. Tumor recovery and an increase in the number of CD4+ T cells were observed in one mouse in the G2-1 group.

[0225]

[0226] Example 5.4. Confirmation of the maintenance of CAR-T cell efficacy following cancer cell reimplantation in mice with cancer cell abolition after administration of CAR-T cells and GI-101A (Analysis of the immune activity enhancement effect for the Part 3 test group)

[0227] Analysis of Part 3 of the trial revealed that the proportion of FMC63+ CD3+ T cells in the G4-2 and G5-2 groups increased rapidly on day 63 after Luc-daudi retransplantation (Figure 19). Consistent with this result, CD4+ and CD8+ T cell counts also increased. The increased CD4+ and CD8+ T cell counts gradually decreased until the end of the trial, which correlated with the maintenance of complete remission (CR) in the tumor.

[0228] Combining CAR-T cell therapy with GI-101A, and administering GI-101A additionally at the time of relapse, significantly enhanced CAR-T cell expansion and persistence. These results suggest that GI-101A can enhance the anti-tumor efficacy of CAR-T cell therapy and may be a promising strategy for improving clinical outcomes in patients with lymphoid tissue malignancies. Furthermore, GI-101A shows promise as a maintenance therapy following CAR-T therapy.

[0229]

[0230] Example 6. Confirmation of the effect of GI-102 on CD19-targeted CAR-T cell reactivation and cancer cell killing in a lymphoma xenograft mouse model.

[0231] Example 6.1. Creation of a lymphoma NSG mouse model and isolation of test groups

[0232] To establish a lymphoma xenograft mouse animal model, 5 × 10 luciferase-expressing B-Luc Daudi cancer cells were injected into the tail vein of 4-8 week old NSG mice (NOD scid gamma mice, Jackson Labs). 5 Each animal was injected with 200 μl of B-Luc Daudi cells. In vivo imaging was performed 7 days before CAR-T cell infusion, and comparable bioluminescence intensities were confirmed between groups using IVIS Lumina III (PerkinElmer, Inc.), and then the groups were classified. To measure bioluminescence, luciferin was injected intraperitoneally 10 minutes before in vivo imaging. Animals were maintained under inhaled isoflurane anesthesia during imaging. After in vivo imaging, a region of interest (ROI) was designated for each subject, and the photon values ​​in that region were recorded. To monitor the progress of the experiment, bioluminescence was measured on days 0, 7, 20, 30, 40, 50, 66, 77, 87, and 95.

[0233] Mice in good health were randomly selected based on physiological status, body weight changes, and tumor growth rates. The selected animals were divided into groups of seven, with tumor formation and body weight being as even as possible.

[0234]

[0235] Example 6.2. Administration of Test Substances and Evaluation of Tumor Growth (Part 1)

[0236] The test group was composed and the test substance was administered as shown in Table 6 below. The period from the day of CAR-T administration (Day 0) and the day of test substance administration (Day 1) to 31 days was defined as Part 1.

[0237] Group Sex Individual Number Test Substance Administration Dosage Volume (mL / kg) Route Cycle G1F7IgG4 1.1 mg / kg 5 S.C. Once every 10 days (Q10D) x 3 G2F7CAR-T (low) 3 x 10 5 cells / head5I.V.1 timeIgG41.1 mg / kg5S.C.Q10D x 3G3F7CAR-T (high)3 x 10 6 cells / head5I.V.1 timeIgG41.1 mg / kg5S.C.Q10D x 3G4F7CAR-T (low)3 x 10 5 cells / head5I.V.1 timeGI-1023.0 mg / kg5S.C.Q10D x 3G5F7CAR-T (high)3 x 10 6 cells / head5I.V.1 timeGI-1023.0 mg / kg5S.C.Q10D x 3

[0238] At this time, T cells (ProMab Biotechnologies, Cat no. PM-CAR1001) into which CAR genes (CD19scFv-CD28-CD3z) were introduced were administered to the lymphoma xenograft mouse model, and then IgG4-Fc and GI-102 were administered at 10-day intervals from the next day until day 31.

[0239] Total bioluminescence flux, which measures the extent of tumor growth on days 0 to 31, was significantly lower in all groups administered CAR-T and test substances from day 10 to day 30 compared to the control group G1 (Fig. 21).

[0240] Bioluminescence imaging results showed that high doses (3 × 10 6 Groups (G3 and G5) administered CAR-T cells at low doses (3 × 10 5 The groups (G2 and G4) that received CAR-T cells showed better anti-tumor effects than the groups that received CAR-T cells (G2 and G4). In addition, bioluminescence imaging showed that GI-102 showed a superior anti-tumor effect compared to IgG4 Fc, and some individuals in the group (G4) that received a small number of CAR-T and GI-102 had complete tumor disappearance after day 20, while all individuals in the group (G5) that received a large number of CAR-T and GI-102 had complete tumor disappearance after day 20.

[0241]

[0242] Example 6.3. Administration of Test Substances and Evaluation of Tumor Growth (Part 2)

[0243] In Part 1 of the trial, groups G2 and G3, who received IgG4 together with CAR-T, and groups G4 and G5, who received GI-102 together with CAR-T, were divided into two subgroups and administered GI-102 or IgG4 under the conditions listed in Table 7 and monitored. This phase was defined as Part 2 of the trial.

[0244] Group Sex Number of individuals Test substance Administration Dosage Volume (mL / kg) Route Cycle G2-1F3IgG4 1.15S.C.Q10D x 6 G2-2F4GI-1023.05SCQ10D x 6 G3-1F3IgG4 1.15S.C.Q10D x 6 G3-2F4GI-1023.05S.CQ10D x 6 G4-1F3IgG4 1.15S.C.Q10D x 3 G4-2F4GI-1023.05SCQ10D x 3 G5-1F3IgG4 1.15S.C.Q10D x 3 G5-2F4GI-1023.05S.CQ10D x 3

[0245] Tumor growth confirmed by bioluminescence imaging showed that the tumor suppression effect increased over time in the group receiving GI-102 (G3-2) (Fig. 22). The group receiving a higher number of CAR-T cells in Part 1 (G3-2) exhibited even greater antitumor effects.

[0246]

[0247] Example 6.4. Administration of Test Substances and Evaluation of Tumor Growth (Part 3)

[0248] In Part 1 of the trial, the G4 and G5 groups, which received GI-102 together with CAR-T, were each divided into two subgroups and administered GI-102 or IgG4 under the conditions in Table 8 until Day 58, followed by Daudi-Luc cell re-transplantation on Day 59, and monitored until Day 87.

[0249] Group Sex Number of individuals Days 31-58 Day 59 Test substance administration Tumor reimplantation (Daudi-Luc) Dose (mg / kg) Volume (mL / kg) Route Cycle G4-1F3IgG41.15S.C.Q10D x 35 x 10 5 cells / headG4-2F4GI-1023.05SCQ10D x 35 x 10 5 cells / headG5-1F3IgG41.15S.C.Q10D x 35 x 10 5 cells / headG5-2F4GI-1023.05SCQ10D x 35 x 10 5 cells / head

[0250]

[0251] Bioluminescence imaging results showed that no detectable tumors remained in the body in any subject from the start of Part 3 to Day 50 (Figure 23). Tumor reimplantation was performed on Day 59 for all groups in Part 3. Even after tumor reimplantation, no tumors were detected in any group until Day 66. In contrast, tumors recurred over time in the G4-1 and G5-1 groups. These observations indicate that the effects of GI-102 administered to G4-2 and G5-2 in the early phase of Part 3 were sustained. The results from Part 3 confirmed the sustained antitumor effect of GI-102.

[0252]

[0253] Example 6.5. Administration of Test Substances and Evaluation of Tumor Growth (Part 4)

[0254] In Part 3 of the trial, the G4-2 and G5-2 groups remained tumor-free until Day 87, despite tumor reimplantation on Day 59. A second tumor reimplantation was performed on Day 88, and monitoring continued until Day 95, but no tumors were detected until the end of the trial (Figure 23). The results of Part 4 confirmed the sustained antitumor effect of GI-102.

[0255] Group sex, number of individuals, 88 days, tumor reimplantation (Daudi-Luc), G4-1F35 x 10 5 cells / headG4-2F45 x 10 5 cells / headG5-1F35 x 10 5 cells / headG5-2F45 x 10 5 cells / head

[0256]

[0257] Throughout the trial, up to Part 4, the combination of GI-102 and CAR-T cells demonstrated improved health status and suppressed tumor growth in the subjects. Furthermore, when CAR-T cells and GI-102 were combined in the initial treatment phase, they demonstrated excellent tumor growth inhibition, and the anti-tumor efficacy was maintained even without additional GI-102 administration, demonstrating the potential of GI-102 as a maintenance therapy following CAR-T therapy.

[0258]

[0259] Example 7. Analysis of the effect of enhancing immune activity by combining GI-102 and CD19-targeting CAR-T.

[0260] Example 7.1. Sample Preparation

[0261] Blood samples were prepared by collecting blood from mice in the test group on day 11 after the tumor formation assay in Example 6.2 and on day 52 after the tumor formation assay in Examples 6.3 and 6.4. Thereafter, FACS analysis was performed on CD4+ T cells and CD8+ T cells using the blood samples.

[0262] The specific experimental method is the same as Example 5.1.

[0263] FACS analysis results showed that only the group (G5) that received a large number of CAR-T cells and GI-102 in the Part 1 trial showed an increase in the proportion of CD4+ T cells and CD8+ T cells on day 11 after administration (Fig. 24). In addition, in the Part 2 trial, only the group (G3-2) that received a large number of CAR-T cells and GI-102 in the Part 1 trial showed an increase in the proportion of CD4+ T cells and CD8+ T cells on day 52 (Fig. 25). In the Part 3 trial, only the group (G5-2) that received a large number of CAR-T cells and GI-102 in the Part 1 trial and additionally received GI-102 showed an increase in the proportion of CD4+ T cells and CD8+ T cells on day 52 (Fig. 26).

[0264] Combining CAR-T cell therapy with GI-102, and administering GI-102 additionally at the time of relapse, significantly enhanced CAR-T cell expansion and persistence. These results suggest that GI-102 may be a promising strategy for enhancing the anti-tumor efficacy of CAR-T cell therapy and improving clinical outcomes in patients with lymphoid tissue malignancies. Furthermore, GI-102 shows promise as a maintenance therapy following CAR-T therapy.

[0265]

[0266] Example 8. Confirmation of the effect of GI-102 on BCMA-targeted CAR-T cell reactivation and cancer cell killing in a multiple myeloma xenograft mouse model.

[0267] Example 8.1. Creation of a multiple myeloma mouse model and separation of test groups

[0268] 5×10 Luciferase-expressing U266 (human multiple myeloma cell) cells were injected into the tail vein of NSG mice at 4–8 weeks of age. 6 A multiple myeloma xenograft mouse model was created by intravenous injection of 200 μl / animal. After intraperitoneal injection, D-luciferin dissolved in phosphate-buffered saline (PBS) was injected intraperitoneally, and the fluorescence intensity was measured using an IVIS (in vivo fluorescence spectrometer) 15–20 minutes later to confirm the presence of transplantation. The animals were maintained under inhaled isoflurane anesthesia during imaging. After in vivo imaging, the region of interest (ROI) of each subject was designated, and the photon value in the region was recorded. After tumor formation was confirmed by biofluorescence analysis after 14 days, T cells introduced with the CAR gene (BCMAscFv-41BB-CD3z) (ProMab Biotechnologies, Cat no. PM-CAR1037, 1 X 10 6 / head) was administered intravenously (day 0). GI-102 or IgG4 was administered the following day (day 1), and tumor formation was observed by measuring the flux value through biofluorescence analysis. Bioluminescence was measured on days 0, 7, 16, and 23 to monitor the progress of the experiment.

[0269] In the established multiple myeloma model, individual body weights and adverse reactions were observed twice a week.

[0270]

[0271] Example 8.2. Administration of test substances and evaluation of tumor growth

[0272] The test group was formed as shown in Table 10 below and the test substance was administered.

[0273] Group Test substance Administration Dosage Volume (mL / kg) Route Cycle G1IgG41.1 mg / kg5S.C. Once each on Day 8 and Day 17 G2CAR-T1 x 10 6 cells / head 5I.V. 1 time on day 0IgG 4 1.1 mg / kg 5S.C. 1 time each on days 8 and 17G3CAR-T1 x 10 6 cells / head 5I.V. 1 time on day 0GI-1023.0 mg / kg5S.C. 1 time each on day 8 and 17

[0274]

[0275] After CAR-T cells were administered to a multiple myeloma xenograft mouse model, IgG4-Fc and GI-102 were administered on days 7 and 16, respectively.

[0276] The total bioluminescence flux, which measures the extent of tumor growth, was significantly reduced in the group administered CAR-T and GI-102 in combination (G3) compared to the group administered CAR-T alone (CAR-T + hIgG4, G2) (Fig. 27).

[0277] No weight loss or special toxicity symptoms were observed following substance administration.

[0278]

[0279] Example 9. Analysis of the effect of enhancing immune activity by combining GI-102 and BCMA-targeted CAR-T.

[0280] On day 24 of T cell administration in Example 8.2, blood samples were collected from mice in the test group to prepare blood samples. Subsequently, FACS analysis was performed on CD4+ T cells and CD8+ T cells using the blood samples.

[0281] The specific experimental method is the same as Example 5.1.

[0282] FACS analysis results showed that the proportion of CD4+ T cells and CD8+ T cells increased after administration only in the group that received CAR-T cells and GI-102 (Fig. 28).

[0283]

[0284] ScFv도메인아미노산 서열(서열번호)가변 영역CDR1CDR2CDR3Anti-CD19ScFv(Promab)VHEVQLVESGGGLVQPGGSLRLSCAASGVSLPDYGVSWVRQAPGKGLEWVSVIWGSETTYYNSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(32)GVSLPDYGVS(29)VIWGSETTYYNSALK(30)KHYYYGGSYAMDY(31)VLDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPYTFGGGTKVEIK(36)RASQDISKYLN(33)HTSRLH(34)QQGNTLPY(35)링커GSTSGSGKPGSGEGSTKG(37)Anti-CD19ScFv(Kymriah)VHEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(41)GVSLPDYGVS(38)VIWGSETTYYNSALK(39)KHYYYGGSYAMDY(40)VLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT(45)RASQDISKYLN(42)HTSRLH(43)QQGNTLPY(44)링커(46)GGGGSGGGGSGGGGSAnti-CD19ScFv(Yescarta)VHEVQLVESGGGLVQPGRSLRLSCTASGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(50)GVSLPDY(47)VIWGSETTYYNSALKS(48)HYYYGGSYAMDY(49)VLDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPDQAPKLLIKHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGQGTKLEIK(54)RASQDISKYLN (51)HTSRLHS(52)QQGNTLPYT(53)링커GSTSGSGKPGSGEGSTKG(55)Anti-BCMAScFv(Promab)VHDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIK(56)---VLVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDATKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARYNYDGYFDVWGAGTTVTVSS(57)---링커GGGGSGGGGSGGGGS(58)Anti-BCMAScFv(Abecma)VHQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS(62)DYSIN(59)WINTETREPAYAYDFRG(60)DYSYAMDY(61)VLMALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK(66)RASESVTILGSHLIH(63)LASNVQT(64)LQSRTIPRT(65)링커GSTSGSGKPGSGEGSTKG(67)Anti-BCMAScFv(Promab_CAR1037)VHEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYASQSITGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPTFGGGTKVEIKLE(68)---VLMALPVTALLLPLALLLHAAR PASQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYNDATKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARYNYDGYFDVWGQGTLVTVSS(69)---LinkerGGGGSGGGGSGGGGS(70)

[0285]

[0286] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for enhancing the activity of immune cells expressing a chimeric antigen receptor, comprising a fusion protein comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof as an active ingredient.

2. A composition according to claim 1, characterized in that the fragment of the CD80 protein is an extracellular domain of the CD80 protein.

3. A composition according to claim 1, characterized in that the IL-2 protein variant is a variant comprising a substitution of at least one of the 38th amino acid, the 42nd amino acid, and the 61st amino acid in the amino acid sequence of SEQ ID NO:

10.

4. A composition according to claim 1, characterized in that the chimeric antigen receptor comprises all or part of an intracellular signaling domain selected from the group consisting of CD3z, CD28, and 4-1BB.

5. A composition according to claim 1, wherein the chimeric antigen receptor comprises a transmembrane domain comprising all or part of CD28 or CD8.

6. A composition according to claim 1, wherein the chimeric antigen receptor comprises a hinge domain comprising all or part of a CD8 hinge.

7. A composition according to claim 1, characterized in that the chimeric antigen receptor comprises an extracellular antigen binding domain that binds to CD19 or BCMA.

8. A composition according to claim 7, wherein the extracellular antigen binding domain specifically binds to CD19 and comprises a heavy chain variable region comprising an HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 38, and 47, an HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 39, and 48, and an HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 40, and 49.

9. A composition according to claim 8, characterized in that the heavy chain variable region comprises an amino acid sequence selected from the group consisting of sequence numbers 32, 41, and 50.

10. A composition according to claim 8, characterized in that the extracellular antigen binding domain further comprises a light chain variable region comprising LCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 42, and 51, LCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 43, and 52, and LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 44, and 53.

11. A composition according to claim 10, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of sequence numbers 36, 45, and 54.

12. A composition according to claim 7, wherein the extracellular antigen binding domain specifically binds to BCMA and comprises a heavy chain variable region comprising an HCDR1 having an amino acid sequence of SEQ ID NO: 59, an HCDR2 having an amino acid sequence of SEQ ID NO: 60, and an HCDR3 having an amino acid sequence of SEQ ID NO:

61.

13. A composition according to claim 7, wherein the extracellular antigen binding domain specifically binds to BCMA and comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of sequences 56, 62, and 68.

14. A composition according to claim 12, characterized in that the extracellular antigen binding domain additionally comprises a light chain variable region comprising LCDR1 having an amino acid sequence of SEQ ID NO: 63, LCDR2 having an amino acid sequence of SEQ ID NO: 64, and LCDR3 having an amino acid sequence of SEQ ID NO:

65.

15. A composition according to claim 7, wherein the extracellular antigen binding domain specifically binds to BCMA and includes a light chain variable region comprising an amino acid sequence selected from the group consisting of sequence numbers 57, 66, and 69.

16. A composition according to claim 1, characterized in that the immune cells are selected from the group consisting of T cells, natural killer cells, and natural killer T (NKT) cells.

17. An anticancer composition for co-administration with an immune cell expressing a chimeric antigen receptor, comprising a fusion protein comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof as an active ingredient.

18. A composition according to claim 17, characterized in that the anticancer composition is a composition for preventing or treating B cell malignancy.

19. A pharmaceutical composition for preventing or treating cancer, comprising a fusion protein comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof; and an immune cell expressing a chimeric antigen receptor as an active ingredient.

20. A composition according to claim 19, wherein the cancer is a B cell malignancy.

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