Recombinant immune cell-based cell therapeutic agent for treating cancer via polyclonal expansion of tumor-infiltrating t cell in tumor

Abstract

The invention relates to a recombinant immune cell into which (i) a first nucleic acid sequence encoding a single chain variable fragment (scFv) of an anti-CD3 antibody and (ii) a second nucleic acid sequence encoding a membrane protein are introduced. When the recombinant immune cells of the present invention are injected into tumors, the expression of the anti-CD3 epsilon chain scFv gene is induced on the cell membrane surface through membrane proteins, and intratumoral polyclonal proliferation of tumor-infiltrating T cells is induced through various costimulatory molecules of immune cells, thereby delaying or inhibiting tumor growth. In addition, the present invention relates to a composition comprising the recombinant immune cells as an active ingredient for treating cancer, wherein the recombinant immune cells are directly injected into a tumor.

Description

Cell therapy for cancer treatment based on recombinant immune cells through intratumoral polyclonal expansion of tumor-infiltrating T cells

[0001] The present invention relates to a recombinant immune cell into which an anti-CD3 gene can be expressed on the surface of a cell membrane, and a composition for treating cancer comprising the same.

[0002]

[0003] Worldwide, countless people are dying from various cancers. Despite the use of various cancer treatments, including radiation and chemotherapy, over the past several years, the number of cancer patients continues to increase. Furthermore, adverse effects of radiation and chemotherapy, including illness and even death in some patients, are widely reported. Furthermore, for certain cancers, antigenic heterogeneity makes it difficult to identify target antigens, and in many cases, these target antigens are not widely known.

[0004] Solid tumors, specifically those formed within tissues or organs, can develop in a variety of organs, including the lungs, breasts, colon, prostate, and brain. Due to antigenic heterogeneity, identifying target antigens for solid tumors is challenging. Furthermore, the types of target antigens for solid tumors are often not widely known. Furthermore, because tumors are often composed of diverse cell types, responses to treatment can vary significantly due to differences in the genetic and molecular characteristics of the cells, making it difficult to predict treatment outcomes.

[0005] In this regard, tumor-infiltrating T cell therapy is currently under development. This therapy involves isolating tumor-infiltrating T cells ex vivo, expanding them, and using them as a therapeutic agent, even if the cancer cell target antigen is unknown. This tumor-infiltrating T cell therapy has demonstrated promising therapeutic effects in clinical trials targeting patients with metastatic melanoma, a condition difficult to treat with existing anticancer treatments. However, the method for mass-proliferating tumor-infiltrating T cells presents challenges: the ex vivo expansion process is complex and time-consuming.

[0006] Therefore, with regard to tumor-infiltrating T cell therapy for treating cancer, there is a need and demand for recombinant immune cells that can simply and rapidly proliferate tumor-infiltrating T cells within a tumor, thereby improving or enhancing the effectiveness of existing anticancer drugs and inhibiting or delaying tumor growth.

[0007] Accordingly, while researching a method to overcome the limitations of existing tumor-infiltrating T cell therapeutics, the inventors of the present invention discovered a recombinant immune cell that allows tumor-infiltrating T cells isolated from a tumor to polyclonally proliferate directly within the tumor rather than in vitro, thereby exhibiting an excellent anticancer therapeutic effect, and completed the present invention.

[0008]

[0009] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0010]

[0011] Summary of the invention

[0012] The purpose of the present invention is to provide a recombinant immune cell that enables polyclonal proliferation of tumor-infiltrating T cells directly within a tumor for the treatment of cancer, particularly solid cancer, and a composition for preventing or treating cancer comprising the same.

[0013] Another object of the present invention is to provide a cancer prevention or treatment method comprising the recombinant immune cell.

[0014] Another object of the present invention is to provide a method for producing a composition for preventing or treating cancer using the recombinant immune cells.

[0015] Another object of the present invention is to provide a method for preventing or treating cancer using the recombinant immune cells.

[0016]

[0017] To achieve the above purpose, the present invention provides a recombinant immune cell, into which (i) a first nucleic acid sequence encoding a single chain variable fragment (scFv) of an anti-CD3 antibody; and (ii) a second nucleic acid sequence encoding a membrane protein are introduced.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the recombinant immune cells and a pharmaceutically acceptable carrier.

[0019] The present invention also provides a use of the recombinant immune cells for preventing or treating cancer.

[0020] The present invention also provides a use for preparing a composition for preventing or treating cancer using the recombinant immune cells.

[0021] The present invention also provides a method for preventing or treating cancer, comprising a step of administering the recombinant immune cells.

[0022]

[0023] Figure 1 is a schematic diagram of a synthetic gene (M-aCD3εscFv; top of Figure 1) in which the variable region sequence of an anti-CD3ε antibody (145-2C11) and the GPI-anchoring sequence of a GPI-anchoring protein (LFA-3) are grafted, and a synthetic gene (M-EGFP; bottom of Figure 1) in which the sequence encoding a fluorescent protein (EGFP) and the GPI-anchoring sequence of a GPI-anchoring protein (LFA-3) are grafted.

[0024] Figure 2 is a diagram showing the expression of M-aCD3εscFv cell membrane over time by flow cytometry after transfection of M-aCD3εscFv mRNA into proinflammatory bone marrow-derived macrophages (M1 BMDM).

[0025] Figures 3a to 3d are diagrams showing the results of interaction between TIL-ex MΦ and spleen T cells (Figure 3a) and the results of spleen T cell proliferation (Figures 3b to 3d) after co-culture of M-EGFP-M1 or TIL-ex MΦ, which were produced by transfecting M1 BMDM with M-EGFP or M-aCD3εscFv mRNA, with T cells isolated from mouse spleen.

[0026] Figures 4a to 4h are diagrams showing the results of the interaction between intratumoral T cells (TILs) and macrophages (Figure 4a), the number of CD8+ T cells and CD4+ T cells (Figures 4b and 4c), the proportion of Ki-67+ cells (Figures 4d and 4e), the results of the proliferation of Foxp3+ Treg cells (Figures 4f and 4g), and the results of the tumor antigen-specific tumor cell killing ability of TILs (Figure 4h) after intratumoral injection of TIL-ex MΦ or M-EGFP-M1 into a B16F10 mouse melanoma model.

[0027] Figures 5a to 5d are diagrams showing the tumor suppression effect and the presence or absence of changes in body weight after intratumoral injection of TIL-ex MΦ or M-EGFP-M1 into a B16F10 mouse melanoma model (Figures 5a and 5b), and the tumor suppression effect and the presence or absence of changes in body weight after intratumoral injection of TIL-ex MΦ or M-EGFP-M1 into a MC38 mouse colon cancer model (Figures 5c and 5d).

[0028]

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0030]

[0031] In the present invention, with respect to a tumor-infiltrating T cell therapeutic agent for treating cancer, a recombinant immune cell capable of simply and rapidly proliferating tumor-infiltrating T cells within a tumor and improving or enhancing the effect of existing anticancer drugs to suppress or delay tumor growth was developed, and it was confirmed that when the immune cell of the present invention is injected into a tumor, tumor-infiltrating T cells are directly polyclonal proliferated in vitro and within the tumor through scFv containing the epsilon chain of an anti-CD3 antibody expressed on the cell membrane, thereby delaying or suppressing tumor growth and exhibiting an excellent anticancer therapeutic effect.

[0032] The "T cell" according to the present invention refers to one of the lymphocytes that conduct antigen-specific adaptive immunity, and the T cell is classified into a naive T cell that has not yet encountered an antigen, a mature T cell that has encountered an antigen, and a memory T cell. At this time, the mature T cell includes a cytotoxic T cell, an αβT cell, and a γδT cell, and is called a "memory T cell (Central memory T cell, T CM )" includes T cells having TCRαβ and TCR γδ.

[0033] Accordingly, the present invention relates, in one aspect, to a recombinant immune cell into which (i) a first nucleic acid sequence encoding a single chain variable fragment (scFv) of an anti-CD3 antibody; and (ii) a second nucleic acid sequence encoding a membrane protein are introduced.

[0034]

[0035] In the present invention, the term "anti-CD3 antibody" encompasses both polyclonal antibodies and monoclonal antibodies, and may be in the form of a complete whole antibody. The whole antibody has a structure comprising two full-length light chains and two full-length heavy chains, including a constant region, and each light chain is linked to a heavy chain by a disulfide bond.

[0036] In the present invention, the entire antibody of the anti-CD3 antibody is a concept including IgA, IgD, IgE, IgM and IgG forms, and IgG is a subtype including IgG1, IgG2, IgG3 and IgG4.

[0037] In the present invention, the anti-CD3 antibody may be any known anti-CD3 antibody and may bind to human and mouse CD3.

[0038] In the present invention, the scFv of the anti-CD3 antibody may be characterized by including a heavy chain variable region amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5; and a light chain variable region amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7, but is not limited thereto.

[0039] In addition, in the present invention, the first nucleic acid sequence encoding the scFv of the anti-CD3 antibody may be characterized by including, but not limited to, the base sequence of SEQ ID NO: 2 or SEQ ID NO: 6; and the base sequence of SEQ ID NO: 4 or SEQ ID NO: 8.

[0040] In the present invention, the scFv of the anti-CD3 antibody may be characterized by including the amino acid sequence of SEQ ID NO: 9, but is not limited thereto.

[0041] In addition, in the present invention, the first nucleic acid sequence may be characterized by including the base sequence of sequence number 10, but is not limited thereto.

[0042] Meanwhile, a substituted antibody or scFv of an antibody comprising a heavy chain variable region and a light chain variable region having a sequence having 80% or more sequence homology, preferably 90% or more sequence homology, and more preferably 99% sequence homology with the heavy chain variable region of SEQ ID NO: 1 or 5 and the light chain variable region of SEQ ID NO: 3 or 7, respectively, and having the same characteristics as the scFv of the anti-CD3 antibody or antibody according to the present invention is also included in the scope of the scFv of the anti-CD3 antibody according to the present invention.

[0043] In the present invention, the anti-CD3 antibody or scFv of the anti-CD3 antibody according to the present invention also includes an antibody or scFv of the antibody in which a part of the amino acid sequence is substituted through conservative substitution in the anti-CD3 antibody or scFv of the anti-CD3 antibody according to the present invention.

[0044] In the present invention, a "conservative substitution" refers to a modification of a polypeptide that involves replacing one or more amino acids with amino acids having similar biochemical properties without causing a loss of the biological or biochemical function of the polypeptide. A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are well known and defined in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is anticipated that antibodies of the present invention may have conservative amino acid substitutions and still retain activity.

[0045] In the present invention, the anti-CD3 antibody sequence may differ from the sequence provided in the present application. For example, the amino acid sequence may (a) separate the variable region from the constant domain of the light chain, (b) differ from the amino acid sequence according to the present invention but may not significantly affect the chemical properties of the residues (so-called conservative substitutions), and (c) exhibit, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence according to the present invention. Alternatively, the nucleic acid encoding the antibody may (a) be separated from the constant domain of the light chain, (b) differ from the nucleic acid sequence according to the invention without changing the encoded residues, and (c) exhibit 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleic acid sequence according to the invention.

[0046] In the present invention, when making conservative changes in an amino acid sequence, the hydropathic index of an amino acid may be considered. The importance of the hydropathic amino acid index in conferring mutual biological functions to a protein is generally understood in the art. The relative hydropathic properties of amino acids contribute to the secondary structure of a resulting protein, which determines its interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens.

[0047] It is also well known in the art that similar amino acid substitutions can be efficiently performed based on hydrophilicity. For example, the highest local average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, is related to the biological properties of the protein. It is understood that amino acids can be substituted with other amino acids having similar hydrophilicity, resulting in biologically or immunologically modified proteins. In such modifications, substitution with amino acids having a hydrophilicity value within ±2 is preferred, with amino acids within ±1 being particularly preferred, and amino acids within ±0.5 being even more particularly preferred.

[0048] As described above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various properties described above are well known to those skilled in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; valine, leucine, and isoleucine.

[0049] In the present invention, the anti-CD3 antibody is a concept including an antigen-binding fragment thereof, which is a fragment having the function of binding to the antigen of the anti-CD3 antibody, i.e., CD3, and also includes Fab, Fab', F(ab')2, scFv, (scFv)2, scFv-Fc, and Fv, etc.

[0050] The Fab has a structure with variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) 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 heavy chain CH1 domain. F(ab')2 antibodies are produced when cysteine ​​residues in the hinge region of Fab' form a disulfide bond. The Fv (variable fragment) refers to the minimum antibody fragment that has only the heavy chain variable region and the light chain variable region. In the double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are connected by a disulfide bond, and in the single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally covalently connected via a peptide linker. These antibody fragments can be obtained using protein hydrolases (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and can be produced by genetic recombination technology (for example, amplification by PCR (Polymerase Chain Reaction) using DNA encoding the heavy chain or its variable region and DNA encoding the light chain or its variable region as templates and a primer pair, and amplification by combining DNA encoding a peptide linker and a primer pair such that both ends are linked to the heavy chain or its variable region and the light chain or its variable region, respectively).

[0051] In the present invention, the first nucleic acid sequence may be characterized by including an epsilon chain of an anti-CD3 antibody.

[0052] Although numerous CD3 antibodies known in the art have been reported to recognize the CD3 epsilon subunit of the CD3 complex, in fact, most of them bind to conformational epitopes and thus recognize only CD3 epsilon in the natural context of the T-cell receptor (TCR). A conformational epitope is characterized by the presence of two or more non-consecutive amino acid residues that are separated in the primary sequence but come together on the molecular surface when the polypeptide folds into a native protein / antigen ([Sela, (1969) Science 166, 1365] and [Laver, (1990) Cell 61, 553-6]). The conformational epitopes bound by CD3 epsilon antibodies known in the art can be divided into two groups. In an important group, the epitopes are formed by two CD3 subunits, e.g., the CD3 epsilon chain and the CD3 gamma or CD3 delta chain. For example, some studies have shown that the most widely used CD3 epsilon monoclonal antibodies, OKT3, WT31, UCHT1, 7D6, and Leu-4, do not bind to cells transfected with the CD3 epsilon chain alone. However, these antibodies stained cells doubly transfected with CD3 epsilon plus either CD3 gamma or CD3 delta (Tunnacliffe, supra; Law, Int. Immunol. 14 (2002), 389-400; Salmeron, J. Immunol. 147 (1991), 3047-52; Coulie, Eur. J. Immunol. 21 (1991), 1703-9). In a second subgroup, the conformational epitopes are formed within the CD3 epsilon subunit itself. A member of this group is, for example, the mAb APA 1 / 1, which is raised against the mutated CD3 epsilon ([Risueno, Blood 106 (2005), 601-8]).Taken together, most of the CD3 epsilon antibodies disclosed in the art recognize conformational epitopes located on two or more subunits of CD3. The discrete amino acid residues forming the three-dimensional structure of these epitopes may be located on the CD3 epsilon subunit itself, or on the CD3 epsilon subunit and other CD3 subunits, such as CD3 gamma or CD3 delta.

[0053] Anti-CD3 monoclonal antibodies work by highly specific recognition of their target molecules. They recognize only a single site, or epitope, on the CD3 molecule. For example, one of the most widely used and best characterized monoclonal antibodies specific for the CD3 complex is OKT-3. This antibody reacts with chimpanzee CD3, but not with CD3 homologs from other primates, such as macaques, or with dog CD3 (Sandusky et al., J. Med. Primatol. 15 (1986), 441-451). Similarly, WO2005 / 118635 or WO2007 / 033230 disclose human monoclonal CD3 epsilon antibodies that react with human CD3 epsilon, but not with CD3 epsilon of mouse, rat, rabbit or non-chimpanzee primate, e.g., rhesus macaque, cynomolgus macaque or baboon.

[0054]

[0055] In the present invention, the membrane protein may be characterized by including a GPI-anchoring domain of a GPI-anchoring protein or a transmembrane domain of a transmembrane protein.

[0056] In the present invention, “membrane protein” means a protein inserted into or attached to the surface of a membrane formed of a lipid bilayer, and includes “intrinsic membrane protein” which is a protein inserted into a lipid bilayer, “extrinsic membrane protein” which is a protein attached to the surface of a lipid bilayer, etc., and “intrinsic membrane protein” belongs to one type of “transmembrane protein” which crosses the membrane.

[0057] In the present invention, the "extrinsic membrane protein" is linked to the cell membrane through covalent or non-covalent bonds between the membrane protein and the lipid bilayer or the transmembrane protein. A lipoprotein is formed by covalent bonding between a protein molecule and a lipid molecule, and as a result of hydrophobic bonding between the lipid molecule of the lipoprotein and the lipid monolayer of the cell membrane, the protein is fixed to the surface of the cell membrane, such as an example of a Glycosylphosphatidylinositol (GPI)-anchored protein. The fatty acid of GPI is inserted into the lipid monolayer, and the phosphate group on the other side is covalently linked to the protein, thereby fixing the protein to the surface of the lipid bilayer.

[0058] In the present invention, "GPI-anchored protein" means a glycosylphosphatidylinositol (GPI)-anchored protein, which exhibits GPI-anchored protein activity by being covalently linked to the outer cell membrane layer.

[0059] In the present invention, the GPI-anchored protein may be any known GPI-anchored protein. In some embodiments, the GPI-anchored protein includes, but is not limited to, LFA-3.

[0060] GPI-anchored proteins are known to be attached to the outer surface of the plasma membrane via GPI-anchor. In various organisms, including protozoa and vertebrates, the GPI-anchor attachment process is carried out in the ER lumen by cleavage of the C-terminal carboxyl group of the protein and secondary addition of a core structure composed of ethanolamine phosphate, trimannosides, glucosamine, and inositolphospholipids (Udenfriend S, Kodukula K (1995) Annu Rev Biochem 64: 563591). GPI-anchor has been implicated in plasma membrane targeting, polarized targeting of apical plasma membrane, lipid raft assembly, plasma membrane recycling, and signal sensing and transduction (Mayor S, Riezman H (2004) Nat Rev Mol Cell Biol 5: 110-120).

[0061] In the present invention, an “intrinsic membrane protein” is a protein that penetrates the cell membrane and is mainly composed of three domains: a “transmembrane domain” that penetrates the membrane, a “cytoplasmic domain” located in the cytoplasm, and an “extracellular domain” exposed to the outside of the cell.

[0062] In the present invention, the number of transmembrane domains varies depending on the membrane protein. Since the lipid bilayer is composed of highly hydrophobic lipids, the transmembrane domains passing through this region are mostly composed of hydrophobic amino acids. Almost all transmembrane domains are composed of an α-helix structure. In membrane proteins with multiple transmembrane domains, the transmembrane domains are arranged in a circle to form a cylindrical structure. This cylindrical structure opens and closes in response to specific signals, and is used as a passage for transporting specific ions or biological substances. The transmembrane domains of an extremely limited number of membrane proteins are in a β-sheet structure. A cylindrical structure formed by arranging multiple domains in a β-sheet structure in a circle is called a 'β-cylinder (β). A representative example of an integral membrane protein composed of a β-cylinder is a porin in the outer membrane of mitochondria.

[0063] In the present invention, the transmembrane protein may be characterized by being selected from the group consisting of CD28, CD3 zeta, CD8 alpha, and CD4, but is not limited thereto.

[0064] In the present invention, the GPI-anchored domain may be characterized by including an amino acid sequence of SEQ ID NO: 13, and the transmembrane domain may be characterized by including an amino acid sequence of SEQ ID NO: 17, but is not limited thereto.

[0065] In the present invention, the second nucleic acid sequence encoding the GPI-anchored domain may be characterized by including a base sequence of SEQ ID NO: 14, and the second nucleic acid sequence encoding the transmembrane domain may be characterized by including a base sequence of SEQ ID NO: 18, but is not limited thereto.

[0066] Meanwhile, a protein having the same characteristics as the membrane protein according to the present invention, which includes an amino acid sequence having a sequence having 80% or more sequence homology, preferably 90% or more sequence homology, and more preferably 99% sequence homology with the amino acid sequence of SEQ ID NO: 13 or 17, is also included in the scope of the membrane protein according to the present invention.

[0067] In the present invention, the scFv of the anti-CD3 antibody may be characterized in that it is expressed on the cell membrane of an immune cell by a GPI-anchored domain or a transmembrane domain.

[0068] In the present invention, it may be characterized in that the first nucleic acid sequence encoding the scFv of the anti-CD3 antibody and the second nucleic acid sequence encoding the membrane protein are connected directly or through a linker.

[0069] In the present invention, the linker may be characterized as being an IgG4 hinge (S228P), but is not limited thereto.

[0070] In the present invention, the IgG4 hinge (S228P) may be characterized by including an amino acid sequence of SEQ ID NO: 19, and the sequence encoding the IgG4 hinge (S228P) may be characterized by including a base sequence of SEQ ID NO: 20.

[0071]

[0072] The sequence according to the present invention is as described in Table 1 below.

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] In the present invention, it may be characterized in that the first nucleic acid sequence encoding the scFv of the anti-CD3 antibody and the second nucleic acid sequence encoding the membrane protein are introduced by transfection.

[0079] In the present invention, the transfection may be performed using, but is not limited to, methods such as cationic liposome method, lithium acetate-DMSO, lipid-mediated transfection, calcium phosphate precipitation method, electroporation, lipofcetion, PEI (Polyethyleneimine)-mediated transfection, DEAE-dextran-mediated transfection, nanoparticle-mediated nucleic acid delivery, etc.

[0080] In the present invention, a reagent capable of liposomizing a DNA plasmid containing a genetic construct including the first nucleic acid sequence and the second nucleic acid sequence and delivering it into a cell may be used for the transfection, and a transfection reagent generally used in the art may be used, for example, Lipofectamine MessengerMAX™ reagent (Thermo Fisher), Lipofectamine 2000 reagent (Invitrogen), and Lipofectamine 3000 reagent (Invitrogen).

[0081] In the present invention, it may be characterized in that the first nucleic acid sequence and the second nucleic acid sequence are introduced by a vector including a first nucleic acid sequence encoding the scFv of the anti-CD3 antibody and a second nucleic acid sequence encoding the membrane protein.

[0082] In the present invention, the vector may be a known vector such as a plasmid vector, a cosmid vector, a bacteriophage vector, etc., and the vector may be easily manufactured by a person skilled in the art according to any known method using DNA recombination technology.

[0083]

[0084] In the present invention, the immune cell may be characterized as being a macrophage.

[0085] In the present invention, "immune cell" means a cell involved in an immune response in the body, and is not particularly limited in type as long as it is known as an immune cell in the art, and in particular, is known as an immune cell existing in the human body, but includes monocytes, macrophages, neutrophils, eosinophils, basophils, dendritic cells, natural killer cells, megakaryocytes, T cells, and B cells. Preferably, it may mean monocytes, macrophages, or neutrophils. More preferably, it may mean macrophages.

[0086] In the present invention, "immune response" as understood in the art generally refers to the biological response of a vertebrate to a foreign agent or abnormality (e.g., cancer cells), which response protects the organism against these agents and diseases caused by them. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by any of these cells, or by the action of the liver (including antibodies, cytokines, and complement) to selectively target, bind, damage, destroy, and / or remove from the body of the vertebrate an invading pathogen, a cell or tissue infected with a pathogen, a cancerous or other abnormal cell, or, in the case of autoimmune or pathological inflammation, a normal human cell or tissue. An immune response includes, for example, activation or inhibition of T cells, such as effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or activation or inhibition of any other cells of the immune system, such as NK cells.

[0087]

[0088] From another aspect, the present invention relates to a pharmaceutical composition for treating cancer comprising the recombinant immune cells and a pharmaceutically acceptable carrier.

[0089] In the present invention, the recombinant immune cells may be characterized in that they are directly injected into a tumor.

[0090] In the present invention, “cancer” is used with the same meaning as “tumor” and refers to or means a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0091] The cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited and includes both solid cancers and blood cancers. Examples of such cancers may be selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, large intestine cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, etc., but are not limited thereto.

[0092] In the present invention, a “pharmaceutically acceptable carrier” is a substance that can be added to an active ingredient to help formulate or stabilize the preparation, and does not cause significant harmful toxic effects to a patient.

[0093] The above carrier refers to a carrier or diluent that does not irritate the patient and does not inhibit the biological activity and properties of the administered compound. For compositions formulated as liquid solutions, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Other carriers are described, for example, in the literature [Remington's Pharmaceutical Sciences (EW Martin)]. Such compositions may contain a therapeutically effective amount of one or more recombinant immune cells according to the present invention.

[0094] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of extemporaneous sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions are preferably formulated for parenteral injection. The compositions may be formulated as solutions, microemulsions, liposomes, or other customized structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In some cases, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, may be included in the composition. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients from those described above. For sterile powders for the preparation of sterile injectable solutions, some preparation methods include vacuum drying and freeze-drying (lyophilization), which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0095] The dosage of the pharmaceutical composition according to the present invention is not particularly limited, but may vary depending on various factors including the patient's health condition and weight, the severity of the disease, the type of drug, the route of administration, and the time of administration. The pharmaceutical composition according to the present invention may be administered once or multiple times a day to mammals including rats, mice, livestock, and humans via a typically accepted route, such as oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration, but is not limited thereto.

[0096] In the present invention, a "therapeutically effective amount" refers to the amount of recombinant immune cells required to produce a measurable benefit in vivo in a patient requiring treatment. The precise amount will vary depending on numerous factors, including but not limited to the components and physical characteristics of the therapeutic composition, the intended patient population, and individual patient considerations, and can be readily determined by those skilled in the art. Taking these factors into full consideration, it is important to administer the minimum amount sufficient to achieve maximum effect without adverse effects, and this dosage can be readily determined by a skilled practitioner.

[0097] In the present invention, it relates to a method for treating cancer and inhibiting cancer growth, which comprises administering to a patient a pharmaceutical composition containing the recombinant immune cell as an active ingredient.

[0098] When a pharmaceutical composition comprising a recombinant immune cell according to the present invention as an active ingredient is administered, a first nucleic acid sequence encoding the scFv of the anti-CD3 antibody introduced into the recombinant immune cell is expressed on the cell membrane by the expression of a second nucleic acid sequence encoding a membrane protein introduced into the recombinant immune cell. At this time, polyclonal proliferation of tumor-infiltrating T cells within the tumor is induced through the scFv of the anti-CD3 antibody expressed on the cell membrane and various co-stimulatory molecules of macrophages, and tumor growth is inhibited or delayed.

[0099]

[0100] 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.

[0101]

[0102] Manufacturing Example 1. Manufacturing of a synthetic gene for cell membrane-expressed anti-CD3 epsilon scFv (M-aCD3εscFv)

[0103] The sequence encoding the variable region of the anti-CD3 epsilon antibody 145-2C11 and the GPI-anchoring coding sequence of the GPI-anchoring protein LFA-3 were linked, and a sequence encoding a G4S linker and a sequence encoding an IgG4 hinge (S228P) were linked between them. In addition, a sequence encoding an HA-tag (Human influenza hemagglutinin-tag) for detection, separation, and purification of the target protein was linked to produce an anti-CD3 epsilon scFv (M-aCD3εscFv) gene that can be expressed on the cell membrane surface through GPI-anchoring (Fig. 1, top). The DNA fragment encoding the synthetic gene was synthesized (Macrogen, Korea) and subcloned into the pMRNAxp vector (System Biosciences) for mRNA in vitro transcription.

[0104]

[0105] Manufacturing Example 2. Manufacturing of a synthetic gene for cell membrane-expressing EGFP (M-EGFP)

[0106] For expression of control mRNA without the target gene, the sequence encoding EGFP, a green tubular protein, and the GPI-anchoring coding sequence of LFA-3, a GPI-anchoring protein, were linked, and a sequence encoding a G4S linker and a sequence encoding an IgG4 hinge (S228P) were linked between them. Then, a sequence encoding an HA-tag (Human influenza hemagglutinin-tag) for detection, separation, and purification of the target protein was linked, thereby producing an EGFP (M-EGFP) gene in a form that can be expressed on the cell membrane surface through GPI-anchoring (Fig. 1, bottom). After synthesizing a DNA fragment encoding the synthetic gene (Macrogen, Korea), pMRNA was constructed. xp It was subcloned into a vector and used for mRNA in vitro transcription.

[0107]

[0108] Example 1. Cell membrane expression of M-aCD3εscFv in proinflammatory bone marrow-derived macrophages

[0109] 1.1. Production of proinflammatory bone marrow-derived macrophages (M1 BMDM)

[0110] Bone marrow cells isolated from the femurs and tibias of 7-week-old female C57BL / 6N mice were cultured in medium containing 20 ng / ml murine M-CSF (Peprotech). After 6 days of culture, differentiated macrophages (bone marrow-derived macrophages, BMDM) were obtained from the mouse bone marrow cells. These differentiated macrophages were treated with 40 ng / ml murine interferon-γ (IFN-γ), and after 24 hours, BMDM were polarized into proinflammatory bone marrow-derived macrophages (M1 BMDM).

[0111]

[0112] 1.2. Confirmation of cell membrane expression of M-aCD3εscFv

[0113] M-aCD3εscFv mRNA was prepared by in vitro transcription from the pMRNAxp vector containing the M-aCD3εscFv gene DNA fragment prepared in Manufacturing Example 1. The mRNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB), and the UTP of the mRNA was N 1 -Methylpseudo-UTP (Jena Bioscience) was completely replaced by the synthesized mRNA. In addition, the synthesized mRNA was used in the experiment after capping using the Vaccinia capping system (NEB) and mRNA Cap 2'-O-Methyltransferase (NEB) and tailing using E. colipoly (A) Polymerase (NEB).

[0114] 0.5 μg of the manufactured M-aCD3εscFv mRNA was treated with 1 μg of Lipofectamine™ MessengerMAX™ reagent, a transfection reagent, to inoculate 1X10 5 was transfected into M1 BMDM. After transfection, M-aCD3εscFv cell membrane expression over time was confirmed using flow cytometry.

[0115] As a result, it was confirmed that M-aCD3εscFv was expressed on the cell membrane for approximately 48 hours (Fig. 2).

[0116]

[0117] Example 2. Induction of polyclonal proliferation of T cells in vitro by TIL-ex MΦ

[0118] 2.1. Production of macrophages (TIL-ex MΦ and M-EGFP-M1) and their interaction with splenic T cells

[0119] The M-aCD3εscFv gene DNA fragment prepared in Manufacturing Example 1 was prepared into M-aCD3εscFv mRNA using the same method as in Example 1, and then transfected into M1 BMDM to prepare macrophages (TIL-ex MΦ).

[0120] Meanwhile, to produce control macrophages that do not contain the target gene, the M-EGFP gene produced in Manufacturing Example 2 was produced as M-EGFP mRNA using the same method as in Example 1, and then transfected into M1 BMDM to produce macrophages (M-EGFP-M1).

[0121] Splenic T cells were isolated from the spleen of 7-week-old female C57BL / 6N mice using the mouse Pan T Cell Isolation Kit II (Miltenyi Biotec). The isolated 5X104 splenic T cells were divided into 5X10 4TIL-ex MΦs were co-cultured with splenic T cells. After 24 hours of co-culture, the interaction between TIL-ex MΦs and splenic T cells was confirmed through immunocytochemistry and confocal imaging (Fig. 3a).

[0122]

[0123] 2.2. Induction of polyclonal expansion of T cells in vitro by TIL-ex MΦ and M-EGFP-M1

[0124] After staining splenic T cells with CellTrace™Violet (CTV), 1x10 5 2x10 CTV-T cells from dogs 4 M-EGFP-M1 or TIL-ex MΦ and 50 μM β-mercaptoethanol (Sigma) were co-cultured in 10% FBS RPMI medium using round bottom 96-well plates (SPL), respectively. After 3 days of co-culture, the degree of T cell proliferation was confirmed by microscopy and flow cytometry. As a result, it was confirmed that TIL-ex MΦ expressing M-aCD3εscFv induced polyclonal proliferation of T cells more than M-EGFP-M1 not expressing M-aCD3εscFv (Fig. 3b to 3d).

[0125]

[0126] Example 3. Confirmation of induction of proliferation of tumor-infiltrating T cells in vivo by TIL-ex MΦ

[0127] 3.1. Preparation of B16F10 mouse melanoma model and interaction between TILs and macrophages

[0128] 5x10 on the right flank of 7-week-old female C57BL / 6N mice 5 A B16F10 mouse melanoma model was prepared by subcutaneously injecting B16F10 tumor cells into the mouse. Eight days after injection of B16F10 tumor cells, 2x10 6TIL-ex MΦ or M-EGFP-M1 were injected intratumorally. One day after intratumoral injection, tumors were harvested, and the interaction between tumor-infiltrating T cells (TILs) and macrophages was confirmed through immunohistochemistry and confocal imaging (Fig. 4a).

[0129]

[0130] 3.2. In vivo induction of tumor-infiltrating T cell proliferation by TIL-ex MΦ and M-EGFP-M1

[0131] Three days after intratumoral injection, tumors were harvested and the number of TILs was measured by flow cytometry to confirm TIL proliferation. As a result, CD8 was significantly higher in the TIL-ex MΦ-administered group compared to the PBS control group and M-EGFP-M1-administered group. + T cells and CD4 + The number of T cells significantly increased, and in particular, the number of CD8+ T cells increased significantly (Figures 4b and 4c).

[0132]

[0133] 3.3. Ki-67 of TIL-ex MΦ and M-EGFP-M1 + Cells and Foxp3 + T reg The ratio of

[0134] The proportion of Ki-67+ cells, a proliferating cell marker, was confirmed. As a result, Ki-67 + CD8 + In the case of T cells, Ki-67 was significantly increased in the TIL-ex MΦ administration group compared to the PBS control group and M-EGFP-M1 administration group. + CD4 + In the case of T cells, there was a significant increase in the TIL-ex MΦ administration group compared to the PBS control group (Fig. 4d and Fig. 4e).

[0135] Meanwhile, anti-inflammatory T by TIL-ex MΦ administration reg As a result of confirming that proliferation of Foxp3 is induced + Treg In this case, it was rather decreased in the TIL-ex MΦ administration group compared to the PBS control group and M-EGFP-M1 administration group (Fig. 4f and Fig. 4h).

[0136] That is, TIL-ex MΦ, when injected intratumorally, + Not only does it induce the proliferation of TILs, but it also reg By reducing the ratio of , it can be confirmed that the anti-inflammatory tumor microenvironment is regulated to an inflammatory one.

[0137]

[0138] 3.4. Antigen-specific tumor cell killing ability of intratumoral proliferated TILs

[0139] 5x10 on the right flank of 7-week-old female C57BL / 6N mice 5 A B16F10-OVA mouse melanoma model was constructed by subcutaneously injecting B16F10-OVA tumor cells. 2 × 106 TIL-ex MΦ or M-EGFP-M1 were injected into B16F10-OVA tumors. Three days after injection, intratumoral TILs were isolated by magnetic associated cell separation (MACS) using CD90.2 microbeads (Miltenyi Biotec), and the isolated TILs were co-cultured with EL4 or E.G7-OVA cancer cell lines treated with 100 ng / ml of IFN-γ for 48 hours. After 12 hours of co-culture, viable tumor cells were quantified by flow cytometry to confirm OVA-specific tumor cell death. As a result, it was confirmed that both TILs isolated from tumors of the TIL-ex MΦ administration group and the M-EGFP-M1 administration group normally exhibited OVA-specific cytotoxicity (Fig. 4h).

[0140]

[0141] Example 4. Anti-tumor effect of TIL-ex MΦ

[0142] 4.1. Antitumor effect of TIL-ex MΦ in the B16F10 mouse melanoma model

[0143] 2x10 into the right flank of 7-week-old female C57BL / 6N mice 5 A B16F10 mouse melanoma model was prepared by subcutaneously injecting B16F10 tumor cells into the mouse. Nine days after tumor cell injection, 3x10 6 TIL-ex MΦ or M-EGFP-M1 were injected intratumorally, and tumor volumes were measured at 2-day intervals. As a result, it was confirmed that tumor growth was significantly inhibited or delayed in the TIL-ex MΦ administration group compared to the M-EGFP-M1 administration group without any change in body weight (Figs. 5a and 5b).

[0144]

[0145] 4.2. Anti-tumor effect of TIL-ex MΦ in the MC38 mouse colon cancer model

[0146] 5x10 on the right flank of 7-week-old female C57BL / 6N mice 5 A MC38 mouse colon cancer model was prepared by subcutaneously injecting MC38 tumor cells. Eight days after tumor cell injection, 2x10 6 TIL-ex MΦ or M-EGFP-M1 were injected intratumorally, and tumor volumes were measured at 2-day intervals. As a result, it was confirmed that tumor growth was significantly inhibited or delayed in the TIL-ex MΦ-administered group compared to the M-EGFP-M1-administered group without any change in body weight (Figs. 5c and 5d).

[0147]

[0148] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0149]

[0150] The immune cells of the present invention, when injected into a tumor, induce the expression of an anti-CD3 single-chain variable fragment (scFv) gene containing the epsilon chain of an anti-CD3 antibody on the cell membrane surface through the action of the GPI-anchoring domain of a GPI-anchoring protein or the transmembrane domain of a transmembrane protein. In addition, the immune cells of the present invention induce polyclonal proliferation of tumor-infiltrating T cells in vitro. In addition, the immune cells of the present invention induce polyclonal proliferation of tumor-infiltrating T cells within a tumor. In addition, the immune cells of the present invention exhibit excellent anticancer therapeutic effects by delaying or inhibiting tumor growth.

[0151] Furthermore, the immune cells of the present invention can achieve effective anticancer immunotherapy when injected into a tumor even in situations where information about cancer cell-specific antigens, which is a major limitation in the treatment of cancer, especially solid cancer, is not known in advance.

[0152]

[0153] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0154]

[0155] Electronic file attached.

Claims

1. A recombinant immune cell, comprising: (i) a first nucleic acid sequence encoding a single chain variable fragment (scFv) of an anti-CD3 antibody; and (ii) a second nucleic acid sequence encoding a membrane protein.

2. A recombinant immune cell according to claim 1, wherein the scFv of the anti-CD3 antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 5; and a light chain variable region amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

7.

3. A recombinant immune cell according to claim 1, characterized in that the first nucleic acid sequence comprises a base sequence of SEQ ID NO: 2 or SEQ ID NO: 6; and a base sequence of SEQ ID NO: 4 or SEQ ID NO:

8.

4. A recombinant immune cell according to claim 1, characterized in that the scFv of the anti-CD3 antibody comprises the amino acid sequence of SEQ ID NO:

9.

5. A recombinant immune cell according to claim 1, characterized in that the first nucleic acid sequence comprises the base sequence of sequence number 10.

6. A recombinant immune cell according to claim 1, characterized in that the first nucleic acid sequence comprises an epsilon chain of an anti-CD3 antibody.

7. A recombinant immune cell according to claim 1, characterized in that the membrane protein comprises a GPI-anchoring domain of a GPI-anchoring protein or a transmembrane domain of a transmembrane protein.

8. A recombinant immune cell according to claim 7, wherein the GPI-anchored domain comprises an amino acid sequence of SEQ ID NO:

13.

9. A recombinant immune cell according to claim 7, characterized in that the transmembrane protein is selected from the group consisting of CD28, CD3 zeta, CD8 alpha, and CD4.

10. A recombinant immune cell according to claim 7, characterized in that the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

17.

11. A recombinant immune cell according to claim 1, characterized in that the scFv of the anti-CD3 antibody is expressed on the cell membrane of the immune cell by a GPI-anchored domain or a transmembrane domain.

12. A recombinant immune cell according to claim 1, characterized in that the first nucleic acid sequence and the second nucleic acid sequence are introduced by transfection.

13. A recombinant immune cell characterized in that the first nucleic acid sequence and the second nucleic acid sequence are introduced by a vector comprising the first nucleic acid sequence and the second nucleic acid sequence in the first paragraph.

14. A recombinant immune cell according to claim 1, characterized in that the immune cell is a macrophage.

15. A pharmaceutical composition for treating cancer comprising a recombinant immune cell according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition for treating cancer, characterized in that the recombinant immune cells in claim 15 are directly injected into a tumor.

17. A pharmaceutical composition according to claim 15, characterized in that the cancer is a solid cancer.

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