WO2022215919A1 - Chimeric antigen receptor specifically binding to cd47 and use thereof - Google Patents

Abstract

The present invention relates to: a chimeric antigen receptor that specifically binds to CD47; a polynucleotide encoding the chimeric antigen receptor protein; a vector comprising the polynucleotide; a cell transformed with the vector; and a cellular therapeutic agent or a pharmaceutical composition for treating cancer, comprising the cell as an active ingredient. The chimeric antigen receptor according to the present invention comprises an extracellular domain of SIRP-α that specifically binds to CD47. Therefore, cytotoxic T cells or natural killer cells transformed with a vector capable of overexpressing the chimeric antigen receptor have cytotoxicity specifically to CD47-expressing carcinomas, and thus can be effectively used as an immune cell therapeutic agent for cancer treatment.

Description

Chimeric antigen receptor specifically binding to CD47 and uses thereof

The present invention relates to a chimeric antigen receptor (CAR) that specifically binds to CD47; a polynucleotide encoding the chimeric antigen receptor protein; a vector comprising the polynucleotide; T cells or natural killer cells transformed with the vector (Natural killer cell, NK cell); It relates to a cell therapeutic agent or a pharmaceutical composition for the treatment of cancer comprising the cell as an active ingredient.

CD47 is a 50 kDa integral membrane protein belonging to the immunoglobulin (Ig) superfamily, and is observed in various malignant cells and immune cells (Sick et al., 2012; Brown). and Frazier, 2001). 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).

It has been reported that cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) that directly target cancer cells are important for effective cancer treatment. Until now, studies on anticancer treatment using cytotoxic T cells or natural killer cells have either delivered specific cancer antigens derived from a patient to the patient's cytotoxic T cells to induce activity, or induce antibody-dependent cellular cytotoxicity. (Galluzzi et al., 2018; Lu et al., 2020). However, recently, a new anticancer treatment method using cytotoxic T cells and natural killer cells has been introduced. That is, a single-chain variable fragment (scFv) portion of an antigen-recognizing antibody is grafted onto a domain grafted to CD3 zeta or the cytoplasmic signaling domain of another protein. It was attempted as a method of delivering chimeric antigen receptor (CAR). When the chimeric antigen receptor is grafted onto T cells or natural killer cells, the anticancer action of T cells or natural killer cells is enhanced by the specific antigen recognition of single-chain Fv fragments regardless of signal transduction by antigen presenting cells (APCs). It can be activated, and it is not limited to the HLA type, so 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, signaling regulatory protein alpha (SIRP-α) is an integral membrane protein belonging to the immunoglobulin (Ig) superfamily, and the extracellular region (domains 1 to 3) is 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 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) have been disclosed, but CD47 The invention of using the SIRP-α extracellular domain having binding specificity for the expressed cancer cells as an antigen-binding domain as an immunocancer therapeutic agent has not been described.

Under this background, the present inventors took advantage of the fact that CD47 expression in various cancer cells is confirmed or induced and that CD47 and SIRP-α are capable of binding to CD47-expressing cancer using the extracellular domain of SIRP-α. Specific chimeric antigen receptor-expressing T cells and natural killer cells were prepared, and it was confirmed through experiments that the T cells or natural killer cells have specific cytotoxicity to CD47-expressing cells. At this time, the signal transduction strength was amplified by making a fusion protein linking each domain 1 of the extracellular domain of SIRP-α with the human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region). That is, while the existing chimeric antigen receptor has one site for recognizing an antigen by a single chain Fv fragment, the chimeric antigen receptor designed by the present inventors converts each of the extracellular domains of SIRP-α into human human immunoglobulin. By connecting with the G ₁ heavy chain constant region (IgG ₁ heavy chain constant region), the extracellular domain of two SIRP-α per chimeric antigen receptor recognizes the antigen, to amplify the signal transduction strength.

Accordingly, it is an object of the present invention to provide a chimeric antigen receptor that specifically binds to CD47.

Another object of the present invention is to provide a polynucleotide encoding the chimeric antigen receptor protein, a vector containing the polynucleotide, and T cells or natural killer cells transformed with the vector.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of cancer that kills cancer cells expressing cell therapeutics or CD47, including the transformed T cells or natural killer cells.

In order to achieve the object of the present invention as described above, the present invention provides an antigen-binding site, an extracellular domain; transmembrane domain; and an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain of SIRP-α that specifically binds to CD47. .

In addition, the present invention provides a polynucleotide encoding the chimeric antigen receptor protein, a vector containing the polynucleotide, and T cells or natural killer cells transformed with the vector.

In addition, the present invention provides a pharmaceutical composition for the treatment of cancer comprising the transformed T cells or natural killer cells, which kills cancer cells expressing a cell therapeutic agent or CD47.

The chimeric antigen receptor according to the present invention comprises an extracellular domain of SIRP-α that specifically binds to CD47. Therefore, in the case of cytotoxic T cells or natural killer cells transformed with a vector capable of overexpressing the chimeric antigen receptor, they have specific cytotoxicity to CD47-expressing carcinoma, so they are useful as immune cell therapeutics for cancer treatment. can be used

1 is a schematic diagram showing the cDNA region of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.

2A is a schematic diagram of a lentiviral vector according to an embodiment of the present invention.

2B is a schematic diagram of a retroviral vector according to an embodiment of the present invention.

3 is a schematic diagram of a chimeric antigen receptor expressed on the surface of cytotoxic T cells or natural killer cells according to an embodiment of the present invention.

4 is a diagram showing the results of measuring the expression level of CD47 in cancer cells expressing CD47, a target of the chimeric antigen receptor provided in the present invention, using flow cytometry.

Figure 5a is a diagram showing the result of comparing the expression rate of GFP in cytotoxic T cells (SIRP-α CAR-T cell) transduced with the expression vector according to an embodiment of the present invention using a lentiviral system. .

Figure 5b is a diagram showing the result of comparing the expression ratio of SIRP-α in natural killer cells (SIRP-α CAR-NK cells) transduced with the expression vector according to an embodiment of the present invention using a retroviral system to be.

FIG. 6a is a diagram showing whether CD47 is recognized by cytotoxic T cells (SIRP-α CAR-T cells) transduced with an expression vector according to an embodiment of the present invention using a lentiviral system using flow cytometry. .

FIG. 6b is a diagram showing whether natural killer cells (SIRP-α CAR-NK cells) transduced with an expression vector according to an embodiment of the present invention using a retroviral system recognize CD47 using flow cytometry.

Figure 7a shows CD47 CD47 using cytotoxic T cells (SIRP-α CAR-T cells) or cytotoxic T cells (Mock T cells) transduced with an 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 the cancer cells (Raji cells) expressing.

Figure 7b shows CD47 expression using natural killer cells (SIRP-α CAR-NK cells) or mock NK cells transduced with an empty vector as effector cells according to an embodiment of the present invention; It is a diagram showing the results of measuring the cytotoxicity to cancer cells (Raji cells).

The present invention provides an antigen-binding domain; transmembrane domain; and an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain of SIRP-α that specifically binds to CD47.

In the present invention, "chimeric antigen receptor (CAR)" binds to a desired antigen without the mediation of antigen presenting cells (APCs) or antibodies necessary for naturally activating T cells or natural killer cells, thereby producing an antigen-antibody reaction. It refers to a fusion protein for expression in T cells or natural killer cells in order to induce activation of T cells and attack cells expressing the corresponding antigen. That is, when expressed in T cells or natural killer cells, it can be considered as a protein that binds to an antigen and induces activation of these 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 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 that it has 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 (1) 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 (2) a polypeptide having substituent(s) at one or more amino acid residues, or (3) 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 (4) 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.

SIRP-α protein is an integral membrane protein belonging to the immunoglobulin (Ig) superfamily, and has three extracellular domains. 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 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 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.

In the present invention, "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. 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, but is not limited thereto.

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

In one embodiment, the transmembrane domain is selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. It may include one or more, preferably, the CD8 may be the transmembrane domain of CD8α, which may consist of SEQ ID NO: 3 or an amino acid sequence showing 95% or more homology thereto, but is limited thereto not.

In the present invention, "intracellular signaling domain" refers to a site that activates an immune response of an immune cell by binding to an antigen-binding domain. In one embodiment, the intracellular domain may be CD28, 4-1BB, CD3 zeta, or a combination thereof, but is not limited thereto.

The chimeric antigen receptor according to the present invention can exhibit a killing effect on cancer cells, particularly cancer cells expressing CD47, with high activity by using CD28, 4-1BB, and CD3 zeta as intracellular signaling domains.

The CD28 is SEQ ID NO: 4 or 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more sequence homology thereto, and the amino acid sequence represented by SEQ ID NO: 4 and may consist of an amino acid sequence exhibiting a substantially equivalent function, and 4-1BB (CD137) is SEQ ID NO: 5 or 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% thereof Having the above sequence homology, it may consist of an amino acid sequence exhibiting a function substantially equivalent to the amino acid sequence shown in SEQ ID NO: 5, CD3 zeta functions as an NK cell activation domain, and SEQ ID NO: 6 or 70 thereof % or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and an amino acid sequence that exhibits a substantially equivalent function to the amino acid sequence represented by SEQ ID NO: 6. can be done

The antigen-binding domain may include a CD8α signal peptide, and the CD8α signal peptide may consist of SEQ ID NO: 7 or an amino acid sequence exhibiting 95% or more homology thereto, but is not limited thereto.

In another aspect according to the present invention, there is provided a polynucleotide encoding the chimeric antigen receptor protein.

The polynucleotide encoding the antigen receptor of the present invention changes the amino acid sequence of the antigen receptor expressed from the coding region due to codon degeneracy or in consideration of the codon preferred in the organism to express the antigen receptor. Various modifications may be made to the coding region within the range that does not occur, and various modifications or modifications may be made within the range that does not affect the expression of the gene in parts other than the coding region, and such modified genes are also within the scope of the present invention. It will be well understood by those skilled in the art. That is, as long as the polynucleotide of the present invention encodes a protein having an equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.

In addition, as another aspect according to the present invention, there is provided a vector comprising the polynucleotide, and a cell transformed with the vector.

The vector used in the present invention may use a variety of vectors known in the art, and may include a promoter, a terminator, an enhancer, etc., depending on the type of host cell to produce the antigen receptor. 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.

As a preferred embodiment of the present invention, a vector for a lenti-virus or a vector for a retro-virus can be used. pLNCX2 (retro-virus vector) was used, but is not limited thereto.

In one embodiment, a cell can be transformed by introducing a chimeric antigen receptor that specifically binds to CD47 into the cell through the vector. The cells may be T cells, tumor-infiltrating lymphocytes, B cells, natural killer cells, or NKT cells, preferably cytotoxic T cells or natural killer cells. The cell may be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells or umbilical cord blood, and the cell may be a human cell, but is not limited thereto.

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 T cells (hereinafter abbreviated as 'CAR-T cells')" or "chimeric antigen receptor NK cells (hereinafter referred to as chimeric antigen receptor NK cells)" 'CAR-NK cells') is a chimeric antigen receptor that specifically responds to cancer cells other than the original T cell receptor or NK cell receptor by transducing normal T cells or natural killer cells, etc. refers to T cells or NK cells expressing T cells or NK cells having this receptor induce apoptosis of target cells and exhibit cytotoxicity.

In the present invention, in particular, CAR-T cells or CAR-NK cells may be cytotoxic T cells or cells in which the chimeric antigen receptor of the present invention is introduced into NK cells. The cells have the advantage of anticancer-specific targeted therapy, which is the existing advantage of CAR-T therapeutics. In particular, the CAR-T cells or CAR-NK cells of the present invention can recognize and effectively destroy CD47-expressing cancer cells.

Therefore, as another aspect, the present invention provides a cell therapeutic agent comprising the cell or a pharmaceutical composition for the treatment of cancer comprising the same as an active ingredient.

In the present invention, "cell therapeutic" refers to cells and tissues prepared through isolation, culture, and special manipulation from an individual, and as pharmaceuticals used for therapeutic purposes, living autologous, allogeneic, or xenogeneic cells or tissues are restored to function. It refers to a drug used for therapeutic purposes through a series of actions, such as in vitro proliferation and selection or changing the biological properties of cells in other ways.

As used herein, the term “treatment” refers to any action in which symptoms of cancer 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 treatment of CD47-expressing 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 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 cancer to be treated include ovarian cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, and 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 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. .

Example 1. Chimeric antigen receptor by gene synthesis method cloning of genes

In order to prepare the chimeric antigen receptor of the present invention, a pair of domain 1 of the extracellular domain of SIRP-α is linked to the human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region), respectively, an antigen recognition site and CD8α The protein coding sequence of each intracellular domain of the transmembrane domain of CD28, 4-1BB, and CD3 zeta was identified in the gene database.

Thereafter, the remaining sequences except for the stop codon portion of the nucleotide sequence constituting the domains were joined together to perform gene synthesis. The accuracy of gene synthesis was confirmed through sequencing after a protein expression plasmid was prepared. A schematic diagram showing the cDNA region of each domain expressing the chimeric antigen receptor is shown in FIG. 1 .

Example 2. Chimeric Antigen Receptor Preparation of protein expression plasmids

In order to recognize CD47 and express the recombinant protein in which the human immunoglobulin G ₁ heavy chain constant region and the signaling protein domain are fused to cytotoxic T cells and natural killer cells, the gene is expressed in pCDH-CMV, a lentivirus-derived expression vector. -MCS-EF1-copGFP (System Biosciences) or a retrovirus-derived expression vector pLNCX2 (Addgene) was cloned.

First, for cloning into the pCDH-CMV-MCS-EF1-copGFP vector, a primer that creates a cleavage site sequence of restriction enzyme XbaI at the 5' end was synthesized, and a restriction enzyme cleavage site was created by polymerase chain reaction, and also restricted at the 3' end. Restriction enzyme cleavage site was created by polymerase chain reaction by synthesizing a primer making the cleavage site sequence of the enzyme NotI. After that, the 5' end of the gene having a restriction enzyme cleavage site was treated with XbaI, and the 3' end was treated with NotI due to the polymerase chain reaction. Then, the multicloning site of the expression vector was treated with XbaI and NotI to allow the gene to be inserted. After mixing the restriction enzyme-treated gene with the expression vector, it was ligated by treatment with a ligase.

Next, for cloning into the pLNCX2 vector, a primer that creates a cleavage site sequence of restriction enzyme Bgl II at the 5' end was synthesized to create a restriction enzyme cleavage site by polymerase chain reaction, and the cleavage site sequence of restriction enzyme NotI was also synthesized at the 3' end. A restriction enzyme cleavage site was created by synthesizing the primers to be made and by polymerase chain reaction. After that, the 5' end of the gene having a restriction enzyme cleavage site was treated with BglII, and the 3' end was treated with NotI due to the polymerase chain reaction. And the multicloning site of the expression vector was treated with BglII and NotI to allow the gene to be inserted. After mixing the restriction enzyme-treated gene with the expression vector, it was ligated by treatment with a ligase.

As a result, the recombinant vector CD47.CAR-T.pCDH-CMV-MCS-EF1- that recognizes CD47 and expresses a protein in which a human immunoglobulin G ₁ heavy chain constant region and a signaling protein domain are fused (see FIG. 3 ). copGFP and CD47.CAR-NK.pLNCX2 were completed (see Figures 2a and 2b).

Example 3. Screening of human cell lines expressing CD47

In order to screen human cell lines expressing CD47 on the cell surface, the B cell lymphoma-derived Raji (ATCC) cell line and the acute T cell leukemia-derived cell line Jurkat (ATCC) cell line CD47 expression was confirmed. 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 strongly expressed in the Raji cell line and weakly expressed in the Jurkat cell line (see FIG. 4 ).

Based on the above results, the Raji cell line strongly expressing CD47 among the two cells was used as a target cell, and the Jurkat cell line weakly expressing CD47 was used as a negative control.

Example 4. Cytotoxic T Cell Isolation and Activation

To prepare T cells expressing a chimeric antigen receptor according to an embodiment of the present invention, cytotoxic T cells were first isolated from human peripheral blood mononuclear cells. After purchasing human peripheral blood mononuclear cells (Medilab Korea), magnetic-activated cell sorting was used to isolate cytotoxic T cells. Peripheral blood mononuclear cells were combined with an antibody (CD8 ⁺ T cell biotin-conjugated antibody cocktail, Miltenyi Biotec) capable of binding to other immune cells except for cytotoxic T cells, and then the antibodies were re-conjugated with magnetic microbeads (anti- biotin microbead) (Miltenyi Biotec). The microbeads, antibodies and cells attached thereto were passed through a magnetic seperation column (Miltenyi Biotec) to obtain cytotoxic T cells not labeled with the antibody. In order to confirm the purity of the isolated cytotoxic T cells, flow cytometry using CD8 and CD3ε, which are cell surface factors of cytotoxic T cells, was performed, and as a result, the purity was greater than 95%.

For activation of isolated cytotoxic T cells, 1Х10 ⁶ pieces/ml of magnetic beads coated with human CD3 and CD28 antibodies (Thermo Fisher Scientific), and 10% calf serum supplemented with 100 U/μl recombinant human IL-2 Cells were reconstituted in RPMI (Welgene) at a density of 1.5Х10 ⁶ pieces/ml and cultured for 24 hours after inoculation in a 24-well cell culture dish.

Example 5. Construction of chimeric antigen receptor-expressing cytotoxic T cells

In order to introduce the recombinant vector prepared in Example 2 into cytotoxic T cells, a lentiviral system using 293FT cells (Thermo Fisher Scientific) 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 60-70% of the dish, 20 µg of SIRP-α.CAR-T.pCDH-CMV-MCS-EF1-copGFP vector DNA is mixed with 10 µg of psPAX2 (Addgene) and 3 µg pMD2.G (Addgene) vector was crystallized using calcium phosphate and Hepes-buffered solution, and then introduced into 293FT cells. Thereafter, the culture supernatant containing the 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. After the concentrated virus was mixed with 4 μg/ml of polybrene and added to the culture of activated cytotoxic T cells, the transduction was performed by centrifugation at 1,800 g for 75 minutes using a centrifuge. After centrifugation, the cytotoxic T cells were further cultured for 4 hours, and then replaced with an RPMI culture medium containing 10% calf serum. After 48 hours, some of the transduced cytotoxic T cells were used to measure the transduction efficiency. The transduction efficiency was measured by using flow cytometry to measure the expression level of GFP inside the cell, and cytotoxic T cells (Mock) transduced with a virus prepared by using a blank vector of transduced cytotoxic T cells (SIRP-α CAR-T cells). /T cell) is shown in Figure 5a the result compared with the transduction ratio.

Example 6. Preparation of chimeric antigen receptor-expressing natural killer cells

In order to introduce the recombinant vector prepared in Example 2 into NK cells, a retroviral system using 293GPG cells was used.

First, 3Х10 ⁶ 293GPG cells were dissolved in 10 ml of 10 ml of 10% calf serum-containing DMEM culture medium and inoculated in a 100π cell culture dish and then cultured for 24 hours. 20 μg of the previously prepared recombinant vector (SIRP-α.CAR-NK.pLNCX2 vector) was crystallized using calcium phosphate and HEPES-buffered solution, and then added to the culture medium of the previously cultured 293GPG cells. Thereafter, the culture medium was changed over 72 hours at 24 hour intervals, and the culture supernatant of 293GPG cells containing retrovirus was collected and stored. The collected retrovirus-containing supernatant was centrifuged at 21,000 rpm for 2 hours using an ultra-high speed centrifuge, and then reconstituted in Myelocult H5100 culture medium (STEMCELL) containing 10% calf serum to be concentrated 100 times compared to before concentration. In order to transduce the concentrated retrovirus into NK92MI cells (American type culture collection, ATCC), NK92MI cells were inoculated into a 24 well cell culture dish at a density of 5Х10 ⁵ /ml. Then, a mixed solution of 8 ug/ml of polybrene added to the concentrated retrovirus was added to the culture medium of NK92MI cells in culture and cultured for 24 hours. After culturing, the culture medium was replaced with a new culture medium, and from 24 hours after the culture medium replacement for selection of the cells into which the gene was introduced, the NK92MI cells treated with retrovirus were cultured using the Myelocult H5100 culture medium supplemented with neomycin antibiotic (600 μg/ml). After 14 days of neomycin selection, NK92MI cells were transferred to fresh culture medium and proliferated for 1 week.

After proliferation, SIRP-α expression levels of NK92MI cells were measured using flow cytometry. NK92MI cells introduced with SIRP-α.CAR-NK.pLNCX2 (SIRP-α CAR-NK cells) and NK92MI cells introduced with empty vector pLNCX2. The results of comparing the SIRP-α expression level of (Mock CAR-NK cells) using a flow cytometer using an anti-human SIRP-α antibody (Invitrogen) are shown in FIG. 5B .

Example 7. chimeric Affinity determination of antigen receptors and human cell lines expressing CD47

In order to confirm whether the prepared chimeric antigen receptor has affinity with a human cell line expressing CD47, the SIRP-α CAR-T cells and SIRP-α CAR-NK cells prepared in Examples 6 and 7 were used for CD47. In order to measure the binding to SIRP-α CAR-NK cells, soluble recombinant human CD47 IgG (R&D system) was treated with SIRP-α CAR-T cells and confirmed by flow cytometry. As a result, soluble recombinant human CD47 IgG did not bind to mock CAR-T cells, but bound to SIRP-α CAR-T cells (see Fig. 6a), and soluble recombinant human CD47 IgG did not bind to mock CAR-NK cells. However, it was bound to SIRP-α CAR-NK cells (see FIG. 6b ).

Based on the above results, SIRP-α CAR-T cells expressing chimeric antigen receptor and SIRP-α CAR-NK cells were used as effector cells, and the Raji cell line expressing CD47 was used as a target cell. Thus, the CD47-specific cytotoxicity of SIRP-α CAR-T cells and SIRP-α CAR-NK cells was verified.

Example 8. CD47+ cell-specific cytotoxicity verification of CAR-T cells

To confirm that the prepared chimeric antigen receptor-expressing cytotoxic T cell specifically recognizes the CD47-expressing cell and shows toxicity, the cell expressing CD47 (CD47 positive cell) is selectively toxic For this purpose, Raji cells, which are the target cells selected in Example 3, and Jurkat cells, which are negative control cells, are chimeric antigen receptor-expressing cytotoxic T cells (SIRP-α CAR-T cells) or cytotoxic T cells introduced with an empty vector. Cells (Mock T cells) and co-cultured for 6 hours. A non-radioactive cytotoxicity assay that measures the degree of cytotoxicity of cytotoxic T cells to target cells by the amount of lactate dehydrogenase present in the supernatant after co-culture was performed. was used.

First, put cytotoxic T cells (1x10 ⁵ cells) and target cells (1x10 ⁴ cells) into the wells of a 96-well cell culture dish, set the effector: target ratio to 10:1, and inoculate so that the volume per well becomes 100μl Centrifuge for 4 minutes at 250 g conditions using a centrifuge to close the intercellular space. After culturing for 6 hours, 50 μl of the supernatant from each well is removed, transferred to a transparent 96-well dish for absorbance measurement, and treated with an analysis solution and 1M hydrochloric acid solution to proceed and stop the enzymatic reaction. After stopping the enzymatic reaction, the absorbance in the 490 nm wavelength band was measured and numerically converted using a fluorescence/luminescence/absorption meter (multi-detection plate reader) to quantify the degree of cytotoxicity of cytotoxic T cells in each well.

As a result, as shown in Figure 7a, the prepared chimeric antigen receptor-expressing cytotoxic T cells (SIRP-α CAR-T cells) showed high toxicity of over 35% to the Raji cell line, whereas the toxicity was 3.5% to the Jurkat cell line. was shown (p<0.01). On the other hand, the cytotoxic T cells introduced with the empty vector as a control showed 11% toxicity to the Raji cell line, but 4.2% to the Jurkat cell line.

Through the above results, the chimeric antigen recognition receptor-expressing cytotoxic T cells containing the extracellular domain of SIRP-α showed cytotoxicity specific to the CD47 protein, so that the treatment of related cancer cells using the CAR-T cells was was able to confirm that it was possible.

Example 9. CD47+ cell-specific cytotoxicity verification of CAR-NK cells

In order to confirm whether the prepared chimeric antigen receptor-expressing NK cells specifically recognize CD47-expressing cells and show toxicity, Raji cells, which are target cells selected in Example 3, and Jurkat cells, which are negative control cells, were used. It was co-cultured with chimeric antigen receptor-expressing NK cells (SIRP-α CAR-NK cells) or NK cells introduced with an empty vector (mock NK cells).

A non-radioactive cytotoxicity assay was used to measure the degree of cytotoxicity of NK cells to target cells with the amount of lactate dehydrogenase present in the supernatant after co-culture. .

First, put natural killer cells (1x10 ⁵ cells) and target cells (1x10 ⁴ cells) into the wells of a 96-well cell culture dish, set the effector: target ratio to 10:1, and inoculate it so that the volume per well becomes 100μl and centrifuge Centrifuge for 4 minutes at 250 g using a separator to close the intercellular space. After culturing for 6 hours, 50 μl of the supernatant from each well is removed and transferred to a transparent 96-well dish for absorbance measurement. After stopping the enzymatic reaction, the absorbance of the 490 nm wavelength band was measured and numerically converted using a fluorescence/luminescence/absorption meter (multi-detection plate reader) to quantify the degree of cytotoxicity of NK cells in each well.

As a result, as shown in FIG. 7b, the prepared chimeric antigen receptor-expressing natural killer cells (SIRP-α CAR-NK cells) showed a high toxicity of 34% or more to the Raji cell line, whereas the toxicity was 4.1% to the Jurkat cell line. was shown (p<0.01). On the other hand, the cytotoxic T cells introduced with the empty vector as a control showed 8.1% toxicity to the Raji cell line, but 0.0% to the Jurkat cell line.

Through the above results, it was found that chimeric antigen-recognizing receptor-expressing NK cells containing the extracellular domain of SIRP-α showed cytotoxicity specific to CD47 protein, so that it is possible to treat related cancer cells using the CAR-NK cells. could check

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 may 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.

Claims (16)

1. antigen binding domain;

   transmembrane domain; and

   A chimeric antigen receptor (CAR) comprising an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain of SIRP-α that specifically binds to CD47.
2. According to claim 1,

   The antigen binding domain is a pair of extracellular domains of SIRP-α in the form of a dimer, respectively, in the form of a human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) Chimeric antigen receptor, characterized in that connected to.
3. According to claim 1,

   The extracellular domain of the SIRP-α is domain 1 (domain 1) of the extracellular region, the chimeric antigen receptor.
4. 4. The method of claim 3,

   The extracellular domain 1 (domain 1) of the SIRP-α will include the amino acid sequence shown in SEQ ID NO: 1, a chimeric antigen receptor.
5. 3. The method of claim 2,

   Human immunoglobulin G ₁ heavy chain constant region (IgG ₁ heavy chain constant region) is a chimeric antigen receptor comprising the amino acid sequence represented by SEQ ID NO: 2.
6. According to claim 1,

   The transmembrane domain is CD8α, the chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO: 3.
7. According to claim 1,

   The intracellular signaling domain is a chimeric antigen receptor comprising one consisting of CD28, 4-1BB and CD3-zeta or a combination thereof.
8. 8. The method of claim 7,

   The CD28 is SEQ ID NO: 4; 4-1BB is SEQ ID NO: 5; And CD3-zeta is a chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO: 6.
9. According to claim 1,

   The antigen-binding domain comprises a CD8α signal peptide, and the amino acid sequence shown in SEQ ID NO: 7, the chimeric antigen receptor.
10. A polynucleotide encoding the chimeric antigen receptor protein of any one of claims 1 to 9.
11. A vector comprising the polynucleotide of claim 10 .
12. A cell transformed with the vector of claim 11 .
13. 13. The method of claim 12,

    The cell, characterized in that the cell is a cytotoxic T cell or NK cell.
14. A pharmaceutical composition for the treatment of cancer expressing CD47 comprising the cell of claim 13 as an active ingredient.
15. 15. The method of claim 14,

    The CD47-expressing cancer is ovarian cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, glioblastoma, liver cancer (hepatocellular carcinoma) , squamous cell carcinoma, leukemia, cancer stem cell (cancer stem cell) and non-Hodgkin's lymphoma (non-Hodgkin's lymphoma), characterized in that the composition is selected from the group consisting of.
16. A cell therapeutic agent comprising the cell of claim 12 .

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