KR20220138790A - Transformed professional antigen presenting cells specifically binding to antigen containing chimeric antigen receptor(CAR) and uses thereof - Google Patents

Abstract

The present invention relates to a transformed professional antigen-expressing cell specifically binding to an antigen containing a chimeric antigen receptor (CAR) and uses thereof. In the professional antigen-expressing cell according to the present invention, the CAR comprises an antigen binding domain specifically binding to CD47, a transmembrane domain, and an intracellular signaling domain. Therefore, the professional antigen-expressing cell according to the present invention can be used as an immune cell therapeutic agent for the treatment of carcinoma expressing CD47.

Description

Transformed professional antigen presenting cells specifically binding to antigen containing chimeric antigen receptor (CAR) and uses thereof

The present invention is a transformed antigen-specific professional antigen-presenting cell comprising a chimeric antigen receptor (CAR), specifically, the chimeric antigen receptor comprises an antigen-binding domain that specifically binds to CD47, the cell comprising: It relates to a cell therapeutic agent or a pharmaceutical composition for the treatment of cancer comprising as an active ingredient.

Professional antigen presenting cells include macrophages, dendritic cells, and naive B cells (Makala et al., 2004). When macrophages and dendritic cells recognize antigens, they break down antigens by phagocytosis, and major histocompatibility complex (MHC) antigens expressed in macrophages and dendritic cells transfer the antigenic determinant (epitope) to T cells. (T cell) (Kambayashi and Laufer, 2014). In the case of naive B cells, the antigen is recognized by the B cell receptor (membrane bound IgM), and then the antigen is crushed and expressed in the naïve B cells (major histocompatibility). complex, MHC) antigen delivers epitope to T cells (Kambayashi and Laufer, 2014). Professional antigen-presenting cells also induce naive T cells by transduction of antigenic determinants by major histocompatibility complex (MHC) antigens and co-stimulatory signal transduction into effector T cells ( effector T cells) (Kambayashi and Laufer, 2014). In addition, the specialized antigen-expressing cells secrete various cytokines and chemokines to induce an inflammatory response or activate other immune cells (Kambayashi and Laufer, 2014). Therefore, an effective immune response is initiated by activation of professional antigen-presenting cells, and an effective immune response cannot be induced without activation of professional antigen-presenting cells (Makala et al., 2004).

Recently, a method for treating tumors by inducing the activation of specialized antigen-expressing cells has been devised. That is, it is a method in which tumor-specific antigens are treated with cancer patient-derived specialized antigen-expressing cells, and then, professional antigen-expressing cells activated by tumor-specific antigens are put into the cancer patient's body (van Willigen et al., 2018). . Activated specialized antigen-expressing cells implanted in cancer patients induce the activation of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) that directly target tumor cells, thereby causing tumor cells (Galluzzi et al., 2018; Lu et al., 2020).

However, the biggest challenge for such immunotherapy is that tumor-specific antigens have not been discovered so far. Therefore, recently, a single-chain variable fragment (scFv) portion of an antibody that recognizes a protein expressed on the cell surface of cancer has been replaced with CD3 zeta or the cytoplasmic signaling domain of another protein. ) grafted onto the chimeric antigen receptor (CAR) is expressed in cytotoxic T cells and natural killer cells to recognize proteins expressed on the surface of cancer cells and to communicate with cytotoxic T cells by signal transduction. A new anticancer treatment method that induces cancer-specific activity of natural killer cells has been introduced. That is, when the chimeric antigen receptor is grafted onto T cells or natural killer cells, the anticancer action of T cells or natural killer cells only by recognizing specific antigens of scFv regardless of signal transduction by specialized antigen-expressing cells that recognize cancer-specific antigens. In addition, since it is not limited to HLA type, it can be used as a more efficient treatment method that can be used universally by many people. Indeed, these chimeric antigen receptor-expressing T cells or natural killer cells have shown efficacy in several cancers (Holzinger et al., 2016; Pettitt et al., 2018; Wang et al., 2020).

On the other hand, CD47 is a 50 kDa integral membrane protein belonging to the immunoglobulin (Ig) superfamily, and is observed in various carcinomas (malignant cells) and immune cells (Brown and Frazier, 2001; Sick et al., 2012). In particular, cancer cells overexpress CD47 and bind to the signaling regulatory protein alpha (SIRP-α) expressed in macrophages, and avoid phagocytosis by inhibiting macrophage activity according to the signaling mechanism of SIRP-α. (Weissman, 2016; Brightwell et al., 2016). Accordingly, a neutralizing antibody capable of inhibiting CD47 / SIRP-α signaling was developed, and it was concluded that the neutralizing antibody could more effectively remove cancer (Weiskopf et al., 2016). Carcinomas expressing CD47 so far have been ovarian cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, glioblastoma, and hepatocellular carcinoma. ), squamous cell carcinoma, leukemia, cancer stem cells, and non-Hodgkin's lymphoma (Campbell et al., 1992; Jaiswal et al., 2009; Li et al., 2018; Majeti et al., 2009; Michaels et al., 2018; pai et al., 2019; Theocharides et al., 2012; Willingham et al., 2012; Zhang et al., 2015).

In addition, signaling regulatory protein alpha (SIRP-α) is an integral membrane protein belonging to the immunoglobulin (Ig) superfamily, and has an extracellular region (domains 1 to 3). have (Barclay and Van den Berg, 2014). SIRP-α has an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, and is known to block phagocytosis by inhibiting macrophage activity when it binds to CD47 (Okazawa et al., 2005).

Currently, CAR-T cells that recognize CD47 as a tumor antigen (US Patent Publication No. 2019-0338028) or SIRP-α polypeptide compositions and methods of use (US Patent Publication No. 2020-0270324) are disclosed, but intracellular There has been no description of immuno-cancer therapeutics using specialized antigen-expressing cells that enhance the signal transduction domain and express a chimeric antigen receptor including the extracellular domain of SIRP-α.

Under this background, the present inventors devised a new treatment method that grafts the function of the professional antigen-presenting cell and the function of the chimeric antigen receptor. That is, after recognizing a receptor and a ligand that can recognize a tumor cell surface protein as a ligand, a novel chimeric signaling domain that can induce the activity of a specialized antigen-presenting cell by the chimeric antigen receptor was designed, It was demonstrated that professional antigen-presenting cells activated by such a chimeric signaling domain can have cytotoxicity against cancer. That is, using the fact that human CD47 and SIRP-α are capable of binding, the extracellular domain of SIRP-α, the transmembrane domain of toll-like receptor 2 (TLR-2), and TLR-3 Intracellular domain of TLR-4, IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ) or IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I) Beta chain, FcεRIβ) immunoreceptor tyrosine-based activation motif (ITAM) as a subunit (subunit), three ITAMs are linked with a new synthetic intracellular signal transduction domain linked by a linker (linker) Chimeric antigen receptor-expressing macrophages expressing recombinant proteins were constructed. At this time, the extracellular domain of SIRP-α expressed in macrophages recognizes CD47 and, when combined with a specific cancer cell expressing CD47, can deliver an activation signal to the inside of these macrophages, which in turn activates the macrophages. It makes it possible to have cytotoxicity against cancers expressing CD47. In fact, it was confirmed through an in vitro experiment that the macrophages have specific cytotoxicity to CD47-expressing cells.

US Patent Publication No. 2019-0338028 (2019. 11. 07) US Patent Publication No. 2020-0270324 (2020.08.27)

Accordingly, an object of the present invention is a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain that specifically binds to CD47, a transmembrane domain and an intracellular signaling domain. And, the cell is to provide a transformed cell, including a macrophage, a dendritic cell or a naive B cell (naive B cell) as an antigen-specific professional antigen-expressing cells having a targeted effector activity.

Another object of the present invention is to provide a cell therapeutic agent comprising the transformed cell as an active ingredient or a pharmaceutical composition for the treatment of cancer that kills cancer cells expressing CD47.

In order to achieve the object of the present invention as described above, as a transformed cell comprising the present chimeric antigen receptor (CAR), the chimeric antigen receptor has an antigen binding domain that specifically binds to CD47, a transmembrane domain and A transformed cell comprising an intracellular signaling domain, wherein the cell comprises a macrophage, dendritic cell or naive B cell as an antigen-specific professional antigen-presenting cell having a targeted effector activity to provide.

In addition, the present invention provides a pharmaceutical composition for the treatment of cancer that kills cancer cells expressing a cell therapeutic agent or CD47, including the transformed cells.

In one embodiment of the present invention, the antigen-binding domain of the chimeric antigen receptor (CAR) may be an extracellular domain of signaling regulatory protein alpha (SIRP-α) that specifically binds to the CD47.

In one embodiment of the present invention, the transmembrane domain may be TLR-2.

In one embodiment of the present invention, the intracellular signaling domain is TLR-3, TLR-4, IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα), an immunoreceptor tyrosine-based activation motif (ITAM) of the IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ), or a combination thereof.

The CD47-expressing carcinoma is ovarian cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, glioblastoma, liver cancer (hepatocellular carcinoma) , squamous cell carcinoma, leukemia, cancer stem cell and non-Hodgkin's lymphoma.

The transformed cell according to the present invention provides an enhanced chimeric signaling domain that induces the activity of the professional antigen-presenting cell when the professional antigen-presenting cell specifically binds to an antigen. As an example, the extracellular domain of SIRP-α and TLR-3, TLR-4 and IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ) or IgG receptor 2A that play an important role in signal transduction of professional antigen-presenting cells Using the immunoreceptor tyrosine-based activation motif (ITAM) of the alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or the IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) as a subunit, three ITAMs are linkers ), a recombinant protein comprising a novel synthetic intracellular signal transduction domain linked to, and has specific cytotoxicity to CD47-expressing carcinoma in the case of professional antigen-expressing cells transformed with a vector capable of overexpressing it. Therefore, the professional antigen-expressing cell overexpressing the chimeric antigen receptor according to the present invention can be usefully used as a therapeutic agent for immune cells for the treatment of CD47-expressing carcinoma.

1 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
2 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
3 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
4 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
5 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
6 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
7 is a schematic diagram showing the form of one of the lentiviral vectors according to an embodiment of the present invention.
8 is a schematic diagram illustrating a form of one of lentiviral vectors according to an embodiment of the present invention.
9 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
10 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
11 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
12 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of a professional antigen-expressing cell according to an embodiment of the present invention.
13 is a diagram showing the results of measuring the expression level of CD47 in cancer cells (CD47 positive cells) expressing CD47, the target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
14 is a diagram showing the results of comparing the expression ratio of GFP in professional antigen-presenting cells (SIRP-α CAR-pAPC) transduced with the expression vector according to an embodiment of the present invention using a lentiviral system.
15 is a diagram showing whether CD47 is recognized by professional antigen-presenting cells (SIRP-α CAR-pAPC) transduced with an expression vector according to an embodiment of the present invention using a lentiviral system using flow cytometry.
16 is a CD47-expressing cancer cell (CD47) using professional antigen-presenting cells (SIRP-α CAR-pAPC) transduced with a chimeric antigen receptor or empty vector according to an embodiment of the present invention as effector cells. It is a diagram showing the result of measuring the cytotoxicity to positive cells) after 24 hours.
17 is a CD47-expressing cancer cell (CD47) using professional antigen-presenting cells (SIRP-α CAR-pAPC) transduced with a chimeric antigen receptor or empty vector according to an embodiment of the present invention as effector cells. A diagram showing the results of measuring cytotoxicity to positive cells) after 48 hours.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

In one aspect, the present invention provides a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor has an antigen binding domain that specifically binds to CD47, a transmembrane domain and an intracellular signaling domain. Including, wherein the cell provides a transformed cell, including a macrophage, a dendritic cell or a naive B cell (naive B cell) as an antigen-specific professional antigen-presenting cell having a targeted effector activity.

The chimeric antigen receptor protein according to an embodiment of the present invention may include a functional equivalent. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably the amino acid sequence of the chimeric antigen receptor protein as a result of the addition, substitution, or deletion of amino acids. refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity. 'Substantially homogenous physiological activity' means having an activity capable of specifically binding to CD47.

The present invention also includes fragments, derivatives and analogues of chimeric antigen receptors. As used herein, the terms 'fragment', 'derivative' and 'analog' refer to a polypeptide that retains substantially the same biological function or activity as the chimeric antigen receptor protein of the present invention. Fragments, derivatives and analogs of the present invention may comprise (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, wherein the substituted amino acid residues are encoded by the genetic code. may or may not be) or (ii) a polypeptide having substituent(s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol); a polypeptide derived from the associated mature polypeptide, or (iv) an additional amino acid sequence (eg, a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence, or a fusion protein); It may be a polypeptide derived from said polypeptide to which it is bound. The fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

In the present invention, CD47 is found in various malignant cells. Carcinomas expressing CD47 so far have been ovarian cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, glioblastoma, and hepatocellular carcinoma. ), squamous cell carcinoma, leukemia, cancer stem cell or non-Hodgkin's lymphoma.

In the present invention, "chimeric signaling domain" binds to a desired ligand, induces activation of professional antigen-presenting cells through ligand-receptor reaction, and is expressed in specialized antigen-expressing cells to attack cells expressing the ligand It may mean a fusion protein for In other words, when expressed in specialized antigen-expressing cells, it can be seen as a protein that binds to a ligand and induces activation of these cells. Therefore, the novel chimeric signaling domain provided by the present invention is a novel chimeric signaling domain in a general sense that can induce the activation of professional antigen-presenting cells using all ligand-receptor reactions including all antigen-antibody reactions. to provide.

That is, the chimeric antigen receptor can be regarded as a protein that binds to an antigen and induces activation of these cells when expressed in professional antigen-expressing cells. Through this, it may be a protein recognizing an antigen specific to a cell to cause an immune response, and the cell to cause an immune response may refer to a cell existing in a specific tissue or constituting a tissue causing a lesion.

The antigen binding domain of the chimeric antigen receptor (CAR) may be an extracellular domain of SIRP-α that specifically binds to the CD47. The SIRP-α protein is an integral membrane protein belonging to the immunoglobulin (Ig) superfamily, and has three extracellular domains. The extracellular domain of SIRP-α may be any one of domains 1 to 3 or a combination thereof, and preferably domain 1, but is not limited thereto.

The extracellular domain 1 (domain 1) of the SIRP-α may consist of SEQ ID NO: 1 or an amino acid sequence having 95% or more homology thereto, but is not limited thereto.

The antigen binding domain may be in the form of a dimer in which a pair of extracellular domains of SIRP _- α are linked to a human immunoglobulin G ₁ heavy chain constant region to amplify signal transduction, respectively. , but is not limited thereto.

The human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) may consist of SEQ ID NO: 2 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

The intracellular signaling domain, which is one component of the chimeric antigen receptor of the present invention, refers to a site that activates an immune response of an immune cell by binding to an antigen-binding domain. The signaling domain may be TLR-3, TLR-4, tyrosine-based activation motif (ITAM), or a combination thereof. In a preferred embodiment, the signaling domain may be in the form of connecting ITAM as a subunit to the structure of TLR-3-TLR-4 with a linker, wherein one or more ITAMs are linked may be, and preferably may be in the form of three connected, but is not limited thereto.

The ITAM is based on immunoreceptor tyrosine of IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) It may be an activation motif, but is not limited thereto.

The TLR-3 is SEQ ID NO: 3 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid represented by SEQ ID NO: 3 may consist of an amino acid sequence exhibiting a function substantially equivalent to the sequence; The TLR-4 is SEQ ID NO: 4 or more than 70%, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid represented by SEQ ID NO: 4 may consist of an amino acid sequence exhibiting a function substantially equivalent to the sequence; The ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Alternatively, it may consist of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 7.

The linker may consist of an amino acid sequence having a function substantially equivalent to that of the amino acid sequence represented by SEQ ID NO: 8.

The transmembrane domain of the present invention is a site that connects the extracellular domain of SIRP-α with the co-stimulatory and essential signaling domains between the cell membrane, and the intracellular signaling domain is the domain of immune cells by binding of the antigen-binding domain. It refers to a site that activates an immune response.

One component of the transmembrane domain of the chimeric antigen receptor of the present invention is CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and a transmembrane domain of a protein selected from the group consisting of TLR-2. Preferably, the transmembrane domain may be TLR-2, which may consist of SEQ ID NO: 9 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.

The antigen-binding domain may include a signal peptide, and the signal peptide may be a TLR-4 signal peptide, and may consist of SEQ ID NO: 10 or an amino acid sequence showing 95% or more homology thereto, but is limited thereto not.

As the vector used in the present invention, a variety of vectors known in the art may be used, and depending on the type of host cell to produce the antigen receptor, such as a promoter, terminator, enhancer, etc. Expression control sequences, sequences for membrane targeting or secretion, etc. can be appropriately selected and variously combined according to the purpose. The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose.

In the present invention, as a preferred example, a lenti-virus vector can be used, and in the following examples of the present invention, pCDH-CMV-MCS-EF1-copGFP vector (lenti-virus vector) was used ( FIGS. 5 to 5 ). 8).

In addition, the cell can be transformed by introducing the chimeric antigen receptor that specifically binds to CD47 of the present invention into the cell through the vector. The cells may be cytotoxic T cells and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NKT cells. It may be a naive B cell. In some embodiments, the cells may be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells or umbilical cord blood. In some embodiments, the cell is a human cell.

As an embodiment of the present invention, a chimeric antigen receptor that specifically binds to CD47 can be transformed into specialized antigen-expressing cells using the vector described above.

As described above, the cells transformed by introducing the chimeric antigen receptor of the present invention recognize CD47 as an antigen and have a characteristic of strongly binding thereto.

In the present invention, "chimeric antigen receptor professional antigen presenting cell (hereinafter abbreviated as 'CAR-pAPC cell')" is a method such as transduction of normal professional antigen presenting cells It refers to a specialized antigen-presenting cell expressing a chimeric antigen receptor that specifically responds to cancer cells rather than the original specialized antigen-expressing cell surface receptor. Professional antigen-expressing cells with this receptor induce apoptosis of target cells and exhibit cytotoxicity.

In the present invention, in particular, the CAR-pAPC cell may be a cell in which the chimeric antigen receptor of the present invention is introduced into a professional antigen-expressing cell. The cells have the advantage of anticancer-specific targeted therapy, which is the existing advantage of CAR-T therapeutics. In particular, the specialized antigen-expressing cells equipped with the chimeric antigen receptor of the present invention can recognize and effectively destroy CD47-expressing cancer cells. .

Therefore, another aspect of the present invention is a cell therapy agent comprising the cell; A pharmaceutical composition for the treatment of cancer expressing CD47 comprising the same as an active ingredient; and administering the cells to a subject.

In the present invention, the cell, the cell may be a cytotoxic T cell (cytotoxic T cell) and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, preferably, specialized antigen expression The cells may be macrophages, dendritic cells, or naive B cells. Or progenitor cells, for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and may be embryonic stem cells, which may be used in cell therapy such as anticancer therapy. The cells may come from a donor, or they may be cells obtained from a patient. Cells can be used, for example, in regeneration, replacing the function of diseased cells. Cells can also be modified to express heterologous genes so that biological agents can be delivered to specific microenvironments, such as, for example, diseased bone marrow or metastatic deposits.

In the present invention, "cell therapeutic" refers to cells and tissues manufactured through isolation, culture, and special manipulation from an individual, and is a drug (US FDA regulations) used for the purpose of treatment, diagnosis, and prevention, restoring the function of cells or tissues. It refers to a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological properties of cells in other ways.

As used herein, the term “prevention” refers to any act of inhibiting or delaying the development of CD47-expressing cancer by administration of the composition.

As used herein, the term "treatment" refers to any action in which symptoms caused by cancer expressing CD47 are improved or beneficially changed by administration of the composition.

The composition may include a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not inhibit the biological activity and properties of the injected compound without irritating the organism. The type of carrier usable in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sucrose, lactose. , gelatin, etc. may be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used.

The composition may be administered in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, infected virus type, and drug. Activity, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field.

The administration means introducing the composition of the present invention to the patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered intranasally, but is not limited thereto.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into two to three times. When the two active ingredients are single drugs, the number of administrations may be the same or different. In addition, the composition of the present invention may be used alone or in combination with other drug treatments for the prevention or treatment of hematologic cancer. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

The subject includes all humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that have or can develop cancer expressing CD47. means animals. If the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject, the type of subject is included without limitation.

In the present invention, the types of CD47-expressing cancers to be treated include ovarian cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, and glial cancer. It may be, but is not limited to, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma, leukemia, cancer stem cell or non-Hodgkin's lymphoma. .

Hereinafter, the present invention will be described in more detail through examples. These Examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these Examples.

Example 1. Gene by gene synthesis method cloning

In order to prepare the chimeric antigen receptor of the present invention, it has the sequences of domain 1 and the transmembrane domain of TLR-2 among the extracellular regions (domains 1 to 3) of SIRP-α, and different Four types of chimeric antigen receptor protein coding sequences having sequences coding for intracellular signal transduction domains were synthesized. The chimeric antigen receptor is an antigen recognition site that connects a pair of domain 1 (domain ₁ ) of SIRP-α to the human immunoglobulin G ₁ heavy chain constant region, respectively, and the transmembrane domain of TLR-2 , commonly comprising a TLR-4 signal peptide. Each of the signaling domains was constructed in four different configurations. Table 1 below describes the configuration of the four types of signaling domains.

division Signaling domain configuration One TLR-3 / TLR-4 / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif / linker / FcεR1 γ chain ITAM motif 2 TLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif / linker / FcγR2A α chain ITAM motif 3 TLR-3 / TLR-4 / linker/ FcεR1 β chain ITAM motif/ linker / FcεR1 β chain ITAM motif / linker / FcεR1 β chain ITAM motif 4 TLR-3 / TLR-4 / linker / FcγR2A α chain ITAM motif / linker / FcεR1 β chain ITAM motif / linker / FcεR1 γ chain ITAM motif

Schematic diagrams of cDNAs of each domain expressing the chimeric antigen receptor constructed above are shown in FIGS. 1 to 4 . The protein coding sequence of each domain part was confirmed in a gene database, and the accuracy of gene synthesis was confirmed through sequencing after a protein expression plasmid was prepared.

Example 2. Preparation of protein expression plasmids

In order to express in macrophages the recombinant protein obtained by fusion of the extracellular domain 1 of SIRP-α of the four chimeric antigen receptors prepared in Example 1 and a macrophage signaling protein, the gene was expressed in a lentivirus-derived expression vector It was cloned into pCDH-CMV-MCS-EF1-copGFP (System Biosciences) using restriction enzymes Xba I and Not I cleavage sites.

As a result, a recombinant vector expressing four proteins in which the extracellular domain of SIRP-α having the structure of FIGS. 9 to 12 and a novel signal transduction protein was fused was completed (see FIGS. 5 to 8 ).

Example 3. Screening of human cell lines expressing CD47

In order to screen a human cell line expressing CD47 on the cell surface, the expression of CD47 was confirmed in the Raji (ATCC) cell line, a cell line derived from B cell lymphoma. In order to confirm the expression, the cell line was treated with an antibody (Invitrogen) capable of binding to CD47 to which the fluorescent protein was attached, and then flow cytometry (fluorescence-activated cell sorting) was used. As a result of the analysis, it was confirmed that CD47 was expressed in the Raji cell line (see FIG. 13). Based on the above results, a Raji cell line expressing CD47 was used as a target cell.

Example 4. chimeric Antigen receptor expression specialized antigen-expressing cell production

Lentivirus using 293FT cells (Thermo Fisher Scientific) to introduce the four recombinant chimeric antigen receptor expression vectors prepared in Example 2 into THP-1 cells (human macrophage cell line), a type of specialized antigen-expressing cells. system was used.

First, 293FT cells were inoculated to become 2.5Х10 ⁶ cells in a 100π cell culture dish, and then cultured in DMEM medium containing 10% calf serum. After 24 hours of incubation, when the cells have grown to cover about 60-70% of the dish, 20 µg of each of the four vector DNAs of Example 2 is aliquoted, 10 µg of psPAX2 (Addgene) and 3 µg of pMD2.G ( Addgene) vector and calcium phosphate were crystallized using calcium phosphate and Hepes-buffered solution, and then introduced into 293FT cells. Then, the culture supernatant containing the virus (lentivirus) was collected at intervals of 24 hours after 48 hours of the 293FT cells introduced with the expression vector. The collected supernatant was centrifuged at 21,000 rpm in an ultra-high speed centrifuge for 2 hours to concentrate the virus. The concentrated virus was mixed with 4 μg/ml of polybrene and added to the culture medium of THP-1 cells, a macrophage cell line, for transduction. The efficiency of transduction was increased by changing the culture medium of THP-1 cells to the culture medium in which the concentrated virus was added twice at 24 hour intervals. 24 hours after the last culture medium change, a portion of the transduced THP-1 cells was used to measure the transduction efficiency. Transduction efficiency was measured using flow cytometry for the expression level of GFP inside the cell, and THP-1 cells (SIRP-α CAR) transduced with each of the four recombinant chimeric antigen receptor expression vectors were used in empty vector (Mock). The results of comparison with the transduction ratio of THP-1 cells transduced with is shown in FIG. 14 .

Example 5. chimeric Affinity determination of antigen receptors and human cell lines expressing CD47

In order to confirm that the four kinds of recombinant chimeric antigen receptors prepared in Example 4 have affinity with human cell lines expressing CD47, water-soluble recombinant human CD47 IgG (R&D system) was processed and confirmed through flow cytometry. As a result, the soluble recombinant human CD47 IgG did not bind to Mock CAR-pAPC (Mock), but compared with THP-1 cells (SIRP-α CAR) transduced with each of the four recombinant SIRP-α chimeric antigen receptor expression vectors. bound (see FIG. 15). Based on the above results, the four types of SIRP-α chimeric antigen receptor-expressing chimeric antigen receptor-expressing specialized antigen-expressing cells were used as effector cells, and the Raji cell line expressing CD47 was used as the target cell, CD47-specific cytotoxicity of α CAR-pAPC was verified.

Example 6. Cells expressing CD47 (CD47 positive cells) co-culture CD47-specific cytotoxicity verification through

Raji cells, the target cells selected in Example 3, were selected in Example 3 to determine whether the four prepared chimeric antigen receptor-expressing THP-1 cells selectively recognized CD47-expressing cells (CD47 positive cells, CD47 ⁺ cells) and showed toxicity. was co-cultured with 4 types of chimeric antigen receptor-expressing THP-1 cells or THP-1 cells (Mock) introduced with an empty vector. After co-culture, treatment with 7-Aminoactinomycin D (7-AAD) was performed 24 or 48 hours later, and the cytotoxicity of each chimeric antigen receptor-expressing THP-1 cell was measured by comparing the fluorescence expression level of 7-AAD by flow cytometry. did. 7-Aminoactinomycin D (7-AAD) binds to the DNA of the cell in which apoptosis is induced and becomes fluorescent (Zembruski et al., 2012).

First, each of the 4 types of chimeric antigen receptor-expressing THP-1 cells was dispensed at 5Х10 ⁵ into the wells of a 24-well cell culture dish, and the target cell CD47 ⁺ cells (Raji cells) were aliquoted by 1Х10 ⁵ to determine the effector: target ratio. Co-culture was carried out at a ratio of 5:1. The volume per well of the co-culture was proceeded to be 1 ml, and centrifuged at 250 g for 4 minutes using a centrifuge to make the intercellular space close. Then, after co-culture for 24 hours or 48 hours, the cells in each well were centrifuged at 300 g for 3 minutes using a centrifuge to remove the supernatant, and then the cells were stained with 7-AAD (Biolegend). Thereafter, the cytotoxicity of the four chimeric antigen receptor-expressing THP-1 cells prepared by measuring the proportion of cells exhibiting fluorescence by 7-AAD using flow cytometry was quantified.

As a result, as shown in FIG. 16 , when the prepared chimeric antigen receptor-expressing THP-1 cells were co-cultured with CD47 ⁺ cells (Raji cells), 24 hours passed, THP-1 not expressing the chimeric antigen receptor Cells (Mock) showed cytotoxicity of about 15.5%, whereas 3-4-3 THP-1 cells (TLR-3+TLR-4+FcεRIγ ITAM trimer), which are THP-1 cells expressing 4 types of chimeric antigen receptors , 3-4-2A THP-1 cells (TLR-3+TLR4+FcγR2Aα ITAM trimer), 3-4-1B THP-1 cells (TLR-3+TLR4+FcεRIβ ITAM trimer), 3-4-ABC THP- 1 cell (TLR-3+TLR4+FcγR2A ITAM monomer+FcεRIβ ITAM monomer+FcγR2Aα ITAM monomer) showed very high cytotoxicity of 24.7%, 20.7%, 26.7%, and 22.9%, respectively.

In addition, as shown in FIG. 17 , when the cytotoxicity of the co-cultured Raji cells was confirmed 48 hours after the co-culture, the Mock showed about 35% cytotoxicity, while the 4 types of chimeric antigens Receptor expressing THP-1 cells, 3-4-3 THP-1 cells (TLR-3+TLR-4+FcεRIγ ITAM trimer), 3-4-2A THP-1 cells (TLR-3+TLR4+FcγR2Aα ITAM trimer) , 3-4-1B THP-1 cells (TLR-3+TLR4+FcεRIβ ITAM trimer), 3-4-ABC THP-1 cells (TLR-3+TLR4+FcγR2A ITAM monomer+FcεRIβ ITAM monomer+FcγR2Aα ITAM monomer) showed very high cytotoxicity of 53.3%, 54.9%, 55.2%, and 54.7%, respectively. Through this, it was confirmed that the produced chimeric antigen recognition receptor-expressing THP-1 cells showed specific cytotoxicity to the CD47 protein.

According to the above results, the transformed THP-1 cells containing the chimeric antigen receptor according to the present invention were CD47 By showing the protein-specific cytotoxicity, it was confirmed that the treatment of related cancer cells was possible using the transformed antigen-specific specialized antigen-expressing cells.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> IMMUNOLOGICAL DESIGNINGLAB CO., LTD <120> SIRPa.CAR-MAC <130> KP21-0032-ILDL <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> SIRP-alpha Extracellular domain 1 <400> 1 Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala 1 5 10 15 Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 2 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 2 Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 3 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> hTLR-3 TIR domain <400> 3 Phe Glu Tyr Ala Ala Tyr Ile Ile His Ala Tyr Lys Asp Lys Asp Trp 1 5 10 15 Val Trp Glu His Phe Ser Ser Met Glu Lys Glu Asp Gln Ser Leu Lys 20 25 30 Phe Cys Leu Glu Glu Arg Asp Phe Glu Ala Gly Val Phe Glu Leu Glu 35 40 45 Ala Ile Val Asn Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val Ile 50 55 60 Thr His His Leu Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val His 65 70 75 80 His Ala Val Gln Gln Ala Ile Glu Gln Asn Leu Asp Ser Ile Ile Leu 85 90 95 Val Phe Leu Glu Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Leu Cys 100 105 110 Leu Arg Arg Gly Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro Val 115 120 125 Gln Lys Glu Arg Ile Gly Ala Phe Arg His Lys Leu Gln Val Ala 130 135 140 <210> 4 <211> 147 <212> PRT <213> Artificial Sequence <220> <223> hTLR-4 TIR domain <400> 4 Asn Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp 1 5 10 15 Val Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe 20 25 30 Gln Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala 35 40 45 Ala Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val 50 55 60 Val Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr 65 70 75 80 Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile 85 90 95 Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val 100 105 110 Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp 115 120 125 Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu 130 135 140 Leu Asp Gly 145 <210> 5 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> ITAM of IgE receptor gamma chain <400> 5 Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser 1 5 10 15 Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 20 25 <210> 6 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> ITAM of IgG receptor 2A alpha chain <400> 6 Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp 1 5 10 15 Lys Asn Ile Tyr Leu Thr Leu 20 <210> 7 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> ITAM of IgE receptor beta chain <400> 7 Lys Val Pro Glu Asp Arg Val Tyr Glu Glu Leu Asn Ile Tyr Ser Ala 1 5 10 15 Thr Tyr Ser Glu Leu Glu Asp Pro Gly Glu Met Ser Pro 20 25 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker <400> 8 Gly Gly Gly Gly Ser 1 5 <210> 9 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> hTLR-2 transmembrane domain <400> 9 Ala Leu Val Ser Gly Met Cys Cys Ala Leu Phe Leu Leu Ile Leu Leu 1 5 10 15 Thr Gly Val Leu Cys 20 <210> 10 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> hTLR-4 Signal peptide <400> 10 Met Met Ser Ala Ser Arg Leu Ala Gly Thr Leu Ile Pro Ala Met Ala 1 5 10 15 Phe Leu Ser Cys Val Arg Pro 20 <210> 11 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> SIRP-alpha Extracellular domain 1 <400> 11 gaggaggagc tgcaggtgat tcagcctgac aagtccgtgt tggttgcagc tggagagaca 60 gccactctgc gctgcactgc gacctctctg atccctgtgg ggcccatcca gtggttcaga 120 ggagctggac caggccggga attaatctac aatcaaaaag aaggccactt cccccgggta 180 acaactgttt cagacctcac aaagagaaac aacatggact tttccatccg catcggtaac 240 atcaccccag cagatgccgg cacctactac tgtgtgaagt tccggaaagg gagccccgat 300 gacgtggagt ttaagtctgg agcaggcact gagctgtctg tgcgcgccaa accctct 357 <210> 12 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 12 ggatccgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420 tcacgagatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660 aaccactaca cgcagaagag cctctccctg tctccgggta aa 702 <210> 13 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> hTLR-3 TIR domain <400> 13 tttgaatatg cagcatatat aattcatgcc tataaagata aggattgggt ctgggaacat 60 ttctcttcaa tggaaaagga agaccaatct ctcaaatttt gtctggaaga aagggacttt 120 gaggcgggtg tttttgaact agaagcaatt gttaacagca tcaaaagaag cagaaaaatt 180 atttttgtta taacacacca tctattaaaa gacccattat gcaaaagatt caaggtacat 240 catgcagttc aacaagctat tgaacaaaat ctggattcca ttatattggt tttccttgag 300 gagatccag attataaact gaaccatgca ctctgtttgc gaagaggaat gtttaaatct 360 cactgcatct tgaactggcc agttcagaaa gaacggatag gtgcctttcg tcataaattg 420 caagtagca 429 <210> 14 <211> 441 <212> DNA <213> Artificial Sequence <220> <223> hTLR-4 TIR domain <400> 14 aacatctatg atgcctttgt tatctactca agccaggatg aggactgggt aaggaatgag 60 ctagtaaaga atttagaaga aggggtgcct ccatttcagc tctgccttca ctacagagac 120 tttattcccg gtgtggccat tgctgccaac atcatccatg aaggtttcca taaaagccga 180 aaggtgattg ttgtggtgtc ccagcacttc atccagagcc gctggtgtat ctttgaatat 240 gagatgctc agacctggca gtttctgagc agtcgtgctg gtatcatctt cattgtcctg 300 cagaaggtgg agaagaccct gctcaggcag caggtggagc tgtaccgcct tctcagcagg 360 aacacttacc tggagtggga ggacagtgtc ctggggcggc acatcttctg gagacgactc 420 agaaaagccc tgctggatgg t 441 <210> 15 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> ITAM of IgE receptor gamma chain <400> 15 gctataacca gctatgagaa atcagatggt gtttacacgg gcctgagcac caggaaccag 60 gagacttacg agactctgaa gcatgag 87 <210> 16 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> ITAM of IgG receptor 2A alpha chain <400> 16 gacggcggct acatgactct gaaccccagg gcacctactg acgatgataa aaacatctac 60 ctgactctt 69 <210> 17 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> ITAM of IgE receptor beta chain <400> 17 aaggttccag aggatcgtgt ttatgaagaa ttaaacatat attcagctac ttacagtgag 60 ttggaagacc caggggaaat gtctcct 87 <210> 18 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Flexible linker <400> 18 ggtggcggtg gctcg 15 <210> 19 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> hTLR-2 transmembrane domain <400> 19 gcactggtgt ctggcatgtg ctgtgctctg ttcctgctga tcctgctcac cggtgtcctg 60 tgc 63 <210> 20 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> hTLR-4 Signal peptide <400> 20 atgatgtctg cctcgcgcct ggctgggact ctgatcccag ccatggcctt cctctcctgc 60 gtgagacca 69

Claims (16)

A transformed cell comprising a chimeric antigen receptor (CAR), comprising:
The chimeric antigen receptor comprises an antigen binding domain that specifically binds to CD47, a transmembrane domain and an intracellular signaling domain, wherein the cell is a macrophage as an antigen-specific professional antigen-presenting cell having a targeted effector activity; Transformed cells, including dendritic cells or naive B cells. According to claim 1,
The antigen-binding domain is the extracellular domain 1 (domain 1) of SIRP-α (signaling regulatory protein alpha) that specifically binds to CD47, the transformed cell. 3. The method of claim 2,
The extracellular domain 1 (domain 1) of the SIRP-α will include the amino acid sequence shown in SEQ ID NO: 1, the transformed cell. 3. The method of claim 2,
The antigen binding domain is a pair of extracellular domain 1 (domain 1) of SIRP-α in the form of a dimer, respectively, human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) characterized in that it is connected to transformed cells. 5. The method of claim 4,
Human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) is a transformed cell comprising the amino acid sequence represented by SEQ ID NO: 2. According to claim 1,
The intracellular signaling domain is linked to the structure of TLR-3-TLR-4 with an immunoreceptor tyrosine-based activation motif (ITAM) as a subunit and linked by a linker. transformed cells. 7. The method of claim 6,
The ITAM is based on immunoreceptor tyrosine of IgE receptor gamma chain (Fcε receptor I gamma chain, FcεRIγ), IgG receptor 2A alpha chain (Fcγ receptor 2A alpha chain, FcγR2Aα) or IgE receptor beta chain (Fcε receptor I beta chain, FcεRIβ) An activation motif, the transformed cell. 7. The method of claim 6,
The ITAM is a transformed cell that connects three with a linker. 7. The method of claim 6,
The TLR-3 is SEQ ID NO: 3; And TLR-4 is a transformed cell comprising the amino acid sequence shown in SEQ ID NO: 4. 7. The method of claim 6,
The ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Or comprising the amino acid sequence represented by SEQ ID NO: 7, the transformed cell. 7. The method of claim 6,
Wherein the linker comprises the amino acid sequence represented by SEQ ID NO: 8, the transformed cell. According to claim 1,
The transmembrane domain is TLR-2, and the transformed cell comprising the amino acid sequence shown in SEQ ID NO: 9. According to claim 1,
The chimeric antigen receptor comprises a signal peptide and will comprise the amino acid sequence shown in SEQ ID NO: 10, the transformed cell. A pharmaceutical composition for the treatment of cancer expressing CD47 comprising the cell of any one of claims 1 to 13 as an active ingredient. 15. The method of claim 14,
The cancer is ovarian cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma ( A composition selected from the group consisting of squamous cell carcinoma), leukemia, cancer stem cells and non-Hodgkin's lymphoma. A cell therapeutic agent comprising the cell of any one of claims 1 to 13 as an active ingredient.

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