KR20230032557A - Recombinant protein which contains BAFF extracellular domain and the composition comprising the same for treating cancer - Google Patents

Abstract

The present invention relates to a recombinant protein which is toxic to cells expressing a B-cell activating factor (BAFF) receptor, B cell maturation antigen (BCMA), or transmembrane activator and CAML interactor (TACI) and, more specifically, to a recombinant protein including a BAFF protein with high affinity for BAFF receptor, BCMA, or TACI, and a pharmaceutical composition using the same for preventing or treating cancer expressing the BAFF receptor, BCMA, or TACI. According to the present invention, among the recombinant proteins of the present invention, the extracellular domain of BAFF recognizes the BAFF receptor, BCMA, or TACI to bind specific cancer cells expressing the BAFF receptor, BCMA, or TACI and systemic lupus erythematosus B lymphocytes, and the human immunoglobulin G_1 heavy chain constant region of the recombinant protein binds to the IgG receptor (Fc gamma RIIIa, CD16a) of natural killer cells to transfer an activation signal into the natural killer cells, thereby having cytotoxicity against cells expressing the BAFF receptor, BCMA, or TACI and being used by binding a drug conjugate to the recombinant protein. Therefore, the recombinant protein is useful as a therapeutic agent with specific cytotoxicity to cells expressing BAFF receptor, BCMA, or TACI.

Description

Recombinant protein which contains BAFF extracellular domain and the composition comprising the same for treating cancer}

The present invention relates to a recombinant protein containing a BAFF extracellular domain, and specifically, includes a BAFF protein having high affinity for BAFF receptor, B cell maturation antigen (BCMA) or transmembrane activator and CAML interactor (TACI). It relates to a recombinant protein and a pharmaceutical composition for treating cancer expressing a BAFF receptor, BCMA or TACI using the same.

B-cell activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor (TNF) ligand, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK (TNF homologue that activates apoptosis, nuclear factor-κB and c-Jun NH2-terminal kinase), and is called TNFSF13B (TNFsuperfamily member 13B) or zTNF4 (Mackay and Browning, 2002). BAFF is a type 2 transmembrane protein originally composed of 285 amino acids, which is expressed on the cell surface as a homotrimer, and then the transmembrane portion is cleaved and secreted to the outside of the cell (Mackay and Browning, 2002). BAFF is secreted from macrophages, monocytes, and dendritic cells (Mackay and Browning, 2002). Receptors that bind to BAFF include the BAFF receptor, B-cell maturation antigen (BCMA), and Transmembrane activator and CAML interactor (TACI) (Mackay and Browning, 2002). BAFF receptors are expressed on most human B cells, except for plasma cells in the bone marrow, and BCMA is mainly expressed on antibody-secreting human cells (Mackay and Schneider, 2009). TACI is also expressed in most peripheral B cells (Mackay and Schneider, 2009). Therefore, signal transduction by the interaction between BAFF and three receptors that bind to BAFF, the BAFF receptor, BCMA, and TACI, is considered to be very important for B cell activity (Mackay and Schneider, 2009).

The BAFF receptor, BCMA, and TACI, three receptors that bind to BAFF, are also overexpressed in various malignant cells. In particular, BCMA is a marker for multiple myeloma (Carpenter et al., 2013), breast cancer, T cell lymphoma, and B cell chronic lymphocytic leukemia (B -CLL) and non-small cell lung cancer (Haiat et al., 2006; Dou et al., 2016; Kampa et al., 2020). In addition, the BAFF receptor is a B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, B cell It is expressed in various lymphomas such as B-cell non-Hodgkin lymphoma, and also in leukemias such as acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL). manifest In addition, BAFF receptors are also overexpressed in B cells of autoimmune diseases such as systemic lupus erythematosus (SLE) (Haiat et al., 2006; Parameswaran et al., 2010; Takahata et al., 2010; Yang et al., 2010). al., 2014). Meanwhile, TACI is found in multiple myeloma, breast cancer, thyroid carcinoma, and non-small cell lung cancer (Moreaux et al., 2009; Dou et al., 2016; Kampa et al., 2020).

It has been reported that natural killer cells (NK cells) that directly target cancer cells are important for effective cancer treatment, and to induce antibody dependent cellular cytotoxicity (ADCC) as a major mechanism. (Lu et al., 2020). In addition, an attempt to induce cytotoxicity by an antibody-drug conjugate that directly kills cancer recognized by the antibody by binding a compound that acts as a toxin to the antibody is being studied (Alley et al., 2010).

Currently, CAR-T T cells recognizing BCMA or BAFF receptors as tumor antigens (US Patent Publication No. 2020-0283534) or water-soluble TACI immunoglobulin compositions and methods of use (US Patent Registration No. 08524232) have been disclosed. A recombinant protein containing a BAFF extracellular domain shows cytotoxicity against cancer cells expressing BAFF receptors, BCMA, and TACI or B cells in systemic lupus erythematosus, thereby using the recombinant protein as an immunocancer drug or autoimmune disease treatment. no bar

Under this background, the present inventors identified or induced expression of the BAFF receptor, BCMA or TACI in various cancer cells, and the fact that the BAFF and the BAFF receptor, BCMA or TACI can be bound together, to treat the extracellular domain of BAFF. The present invention was completed by preparing a recombinant protein having specific cytotoxicity to cancer cells or systemic lupus erythematosus B cells expressing the used BAFF receptor, BCMA or TACI.

US Patent Publication No. 2020-0283534 (2020. 09. 10) US Patent No. 08524232 (2013. 09. 03)

Accordingly, an object of the present invention is to provide a recombinant protein that specifically binds to the BAFF receptor, BCMA or TACI.

Another object of the present invention is to provide a polynucleotide encoding the recombinant protein, a vector containing the polynucleotide, and a cell transformed from the vector.

Another object of the present invention is to provide a pharmaceutical composition for treating cancer and systemic lupus erythematosus expressing BAFF receptor, BCMA or TACI, including the recombinant protein as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention is an antigen binding site, the extracellular domain of BAFF; Ig kappa (Ig kappa) leader peptide (reader peptide); and a human immunoglobulin G1 heavy chain constant region.

In addition, the present invention provides a polynucleotide encoding the recombinant protein, a vector always containing the polynucleotide, and a cell transformed with the vector.

In addition, the present invention provides a pharmaceutical composition for treating cancer cells and systemic lupus erythematosus in which the recombinant protein expresses BAFF receptor, BCMA or TACI.

Among the recombinant proteins of the present invention, the extracellular domain of BAFF recognizes the BAFF receptor, BCMA or TACI and binds to specific cells expressing the BAFF receptor, BCMA or TACI, and the human immunoglobulin G1 heavy chain constant region of the recombinant protein ( human IgG1 heavy chain constant region) binds to natural killer cell IgG receptors (FcγRIIIa, CD16a) and transmits an activation signal into natural killer cells, thereby specifically targeting carcinomas and autoimmune diseases that express BAFF receptors, BCMA or TACI. Since it has cytotoxicity, it can be usefully used as an immune cell therapy for the treatment of cancer and systemic lupus erythematosus.

1 is a schematic diagram showing cDNA regions of each domain expressing a recombinant BAFF fusion protein according to an embodiment of the present invention.
2 is a schematic diagram of an expression vector (retroviral vector) according to an embodiment of the present invention.
3 is a schematic diagram of a recombinant BAFF fusion protein according to an embodiment of the present invention.
4a is a schematic diagram of inserting BAFF receptor cDNA into a retroviral vector to express BAFF receptor, which is a target of the recombinant BAFF fusion protein provided in the present invention, in Chinese hamster ovary (CHO) cells.
FIG. 4B is a schematic diagram of inserting BCMA cDNA into a retroviral vector to express BCMA, the target of the recombinant BAFF fusion protein provided in the present invention, in Chinese hamster ovary (CHO) cells.
FIG. 4c is a schematic diagram showing TACI cDNA inserted into a retroviral vector to express TACI, the target of the recombinant BAFF fusion protein provided in the present invention, in Chinese hamster ovary (CHO) cells.
5a is a diagram showing the results of measuring the expression level of the BAFF receptor in Chinese hamster ovary (CHO) cells expressing the BAFF receptor, which is the target of the recombinant BAFF fusion protein provided in the present invention, using flow cytometry.
Figure 5b is a diagram showing the results of measuring the expression level of BCMA using flow cytometry in CHO cells expressing BCMA, which is the target of the recombinant BAFF fusion protein provided in the present invention.
5C is a diagram showing the results of measuring the expression level of TACI in CHO cells expressing TACI, which is the target of the recombinant BAFF fusion protein provided in the present invention, using flow cytometry.
6a is a diagram showing the results of purification by affinity chromatography using a protein A column after expressing the recombinant protein provided in the present invention in Chinese hamster ovary (CHO) cells.
Figure 6b is a view showing the results of Western blotting using an antibody (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody) recognizing the human IgG heavy chain constant region of the recombinant protein provided in the present invention. .
7a is a diagram showing whether a recombinant BAFF fusion protein made according to an embodiment of the present invention recognizes a BAFF receptor using flow cytometry.
7B is a diagram showing whether a recombinant BAFF fusion protein prepared according to an embodiment of the present invention recognizes BCMA using flow cytometry.
7c is a diagram showing whether a recombinant BAFF fusion protein made according to an embodiment of the present invention recognizes TACI using flow cytometry.
8 is a schematic diagram of a CD16a expression vector (retroviral vector) according to an embodiment of the present invention.
Figure 9 shows the expression ratio of CD16a in natural killer cells (CD16a.NK92.MI) transduced with a CD16a expression vector using a retroviral system according to an embodiment of the present invention. It is a diagram showing the results compared with the natural killer cells (Mock.NK92.MI).
10 is a non-radioactive cytotoxicity assay (non-radioactive cytotoxicity assay) to determine whether a recombinant protein according to an embodiment of the present invention induces antibody dependent cellular cytotoxicity (ADCC) in cancer cells expressing a BAFF receptor, BCMA or TACI. It is a diagram showing the results measured by radioactive cytotoxicity assay).

The present invention, as one aspect, the extracellular domain of BAFF; Ig kappa (Ig kappa) leader peptide (reader peptide); and _a human immunoglobulin G ₁ heavy chain constant region.

Among the "recombinant proteins" provided in the present invention, the extracellular domain of BAFF recognizes the BAFF receptor, BCMA, and TACI, binds to specific cells expressing the BAFF receptor, BCMA, and TACI, and human immunoglobulin G ₁ of the recombinant protein The heavy chain constant region (human IgG ₁ heavy chain constant region) binds to natural killer cell IgG receptors (FcγRIIIa, CD16a) and transmits an activation signal into natural killer cells. Ultimately, these signals activate natural killer cells, allowing them to have cytotoxicity against cells expressing BAFF receptors, BCMA, and TACI. In addition, by binding a drug conjugate to the recombinant protein, the recombinant protein binds to a specific cell in which the BAFF receptor, BCMA, and TACI are expressed, and the BAFF receptor, BCMA, and TACI are expressed by the drug conjugate bound to the recombinant protein. cytotoxicity to cells.

In the present invention, "BAFF receptor", "BCMA", and "TACI" are observed in various malignant cells and autoimmune diseases, preferably systemic lupus erythematosus (SLE). So far, carcinomas expressing "BAFF receptor", "BCMA", and "TACI" are acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and follicular lymphoma (follicular lymphoma). lymphoma), mantle cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), B-cell non-Hodgkin lymphoma lymphoma), multiple myeloma, T cell lymphoma, breast cancer, thyroid cancer, and non-small cell lung cancer. , but not limited thereto.

The recombinant protein of the present invention is characterized in that the extracellular domain of BAFF binds specifically to the BAFF receptor, BCMA, and TACI, and the "BAFF" consists of SEQ ID NO: 1 or an amino acid sequence showing 95% or more homology thereto. It may be, but is not limited thereto. In addition, the scope of the recombinant protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 1 and functional equivalents of the protein. 'Functional equivalent' means at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably It refers to a protein that has 95% or more sequence homology and exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 1. 'Substantially homogeneous physiological activity' means having an activity that can specifically bind to BAFF receptors, BCMA, and TACI.

An Igκ (Ig kappa) reader peptide is linked to the N' end of the BAFF receptor, BCMA, TACI binding site located in the extracellular domain of the BAFF, and to the C' end of the BAFF receptor, BCMA, TACI binding site. Human immunoglobulin G ₁ heavy chain constant region (human IgG ₁ heavy chain constant region) may be linked, but is not limited thereto. The Ig kappa (Ig kappa) leader peptide includes the amino acid sequence represented by SEQ ID NO: 2, _{and the human immunoglobulin G 1 heavy} chain constant region is represented by SEQ ID NO: 3 It may include an amino acid sequence that is, at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 It refers to a protein having a homology and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 2 or SEQ ID NO: 3.

In addition, another aspect of the present invention is a polynucleotide capable of encoding (encoding) the above-described 'recombinant protein that specifically binds to the BAFF receptor, BCMA, and TACI'. The polynucleotide encoding the recombinant protein 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 codons preferred in organisms intended to express the antigen receptor. Various modifications may be made to the coding region within a range not specified, and various modifications or modifications may be made to a part other than the coding region within a range that does not affect gene expression, 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 activity equivalent thereto, 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.

Another aspect of the present invention is a vector containing the polynucleotide and a cell transformed with the vector.

Vectors used in the present invention may use various vectors known in the art, and depending on the type of host cell to produce the recombinant protein, such as a promoter, terminator, enhancer, etc. Expression control sequences, sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways depending on the purpose. Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose.

As used herein, the term "treatment" refers to all activities that improve or beneficially change symptoms caused by cancer or autoimmune disease expressing BAFF receptor, BCMA or TACI by administration of the composition.

The composition may include a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not inhibit the biological activity and properties of the compound to be injected without irritating living organisms. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can 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 containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, and lactose in the compound. , can be prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions for internal use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The composition can 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 type and severity of the subject, age, sex, infected virus type, and drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitantly used drugs and other factors well known in the medical arts.

The administration means introducing the composition of the present invention to a 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, intranasal administration may be administered, 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 2 to 3 times. The number of administrations when the two active ingredients are each a single agent may be the same or may be different. In addition, the composition of the present invention can be used alone or in combination with other drug treatments for the prevention or treatment of blood cancer. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.

The subject refers to humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or animals that have or may develop cancer expressing BAFF receptors, BCMA, or TACI. All animals, including guinea pigs. The type of subject is included without limitation as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.

Diseases to be treated in the present invention include cancer and autoimmune diseases. The autoimmune disease is preferably systemic lupus erythematosus (SLE), but is not limited thereto. Types of the cancer include acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, B B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), B-cell non-Hodgkin lymphoma, multiple myeloma, T-cell lymphoma It may be selected from the group consisting of (T cell lymphoma), breast cancer, thyroid cancer, and non-small cell lung cancer, but is not limited thereto.

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

Example 1. Cloning of a water-soluble recombinant BAFF fusion protein gene by gene synthesis method

In order to prepare the water-soluble recombinant BAFF fusion protein of the present invention, the protein coding sequence in which each of the extracellular domains of BAFF was linked to the human immunoglobulin G1 heavy chain constant region was prepared in a gene database. confirmed in In addition, gene synthesis was performed by connecting the remaining sequences except for the stop codon portion of the base sequence constituting the domains into one. The accuracy of gene synthesis was confirmed through sequencing after constructing a protein expression plasmid. A schematic diagram showing the cDNA region of each domain expressing the recombinant protein is shown in FIG. 1 .

Example 2. Preparation of water soluble recombinant BAFF fusion protein expression plasmid

In order to express the recombinant protein fused with the human BAFF receptor, BCMA or TACI extracellular domain and the human immunoglobulin G ₁ heavy chain constant region in Chinese hamster ovary (CHO) cells, the corresponding gene is expressed from a retrovirus. It was cloned into the vector pLNCX2 (Addgene).

First, in order to clone into the pLNCX2 vector, a primer that creates a restriction enzyme Xho I cleavage site sequence is synthesized at the 5' end, a restriction enzyme cleavage site is created by polymerase chain reaction (PCR), and restriction is also made at the 3' end. A restriction enzyme cleavage site was prepared by synthesizing a primer that creates the cleavage site sequence of the enzyme Not I and polymerase chain reaction. After that, the 5' end of the gene having a restriction enzyme cleavage site due to polymerase chain reaction was treated with Xho I, and the 3' end was treated with Not I. In addition, the multicloning site of the expression vector was treated with xho I and Not I so that the gene could be inserted. After mixing the restriction enzyme-treated gene and the expression vector, they were ligated by treatment with a ligase.

As a result, a recombinant vector BAFF-IgG.pLNCX2 expressing a protein obtained by fusing the human BAFF receptor, BCMA or TACI-recognizing BAFF extracellular domain with the human immunoglobulin G ₁ heavy chain constant region (see FIG. 3) was completed ( see Figure 2).

Example 3. Production of cell lines expressing BAFF receptors, BCMA, and TACI

In order to prepare a cell line expressing the BAFF receptor, BCMA, and TACI on the cell surface, each of the BAFF receptor, BCMA, and TACI genes had a restriction enzyme Bgl II cleavage site sequence at the 5' end and a restriction enzyme Hind III cleavage site at the 3' end. It was cloned into the pLNCX2 vector using the sequence (see FIGS. 4a, 4b and 4c).

Thereafter, a retroviral system using 293GPG cells was used to introduce the pLNCX2 vector into CHO cells (ATCC) into which the BAFF receptor, BCMA, and TACI genes had been inserted. First, ⁶ 3Х10 293GPG cells were dissolved in 10 ml of 10 ml of DMEM culture medium containing 10% calf serum, inoculated into a 100π cell culture dish, and cultured for 24 hours. The previously prepared recombinant vectors (BAFF-R.pLNCX2 vector, BCMA.pLNCX2 vector, TACI.pLNCX2 vector) and empty pLNCX2 vector (20 μg) were crystallized using calcium phosphate and HEPES-buffered solution, respectively. It was added to the culture medium of 293GPG cells previously cultured. Thereafter, the culture medium was replaced at 24 hour intervals over 72 hours, and the culture supernatant of 293GPG cells containing the 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 DMEM (welgene) containing 10% calf serum to be concentrated 100 times compared to before concentration. To transduce the concentrated retrovirus into CHO cells, the CHO cells were inoculated in a 24-well cell culture dish at a density of 5Х10 ⁵ /ml. Then, a mixture obtained by adding 8 μg/ml of polybrene to the concentrated retrovirus was added to the culture medium of CHO cells, and cultured for 24 hours. After culturing, it was replaced with a new culture medium, and retrovirus was treated using DMEM culture medium containing 10% calf serum supplemented with neomycin antibiotic (600 μg/ml) 24 hours after the culture medium change for selection of cells into which the gene was introduced. was cultured. CHO cells that completed the neomycin selection process for 14 days were transferred to a fresh culture medium and grown for one week.

After proliferation, CHO cells introduced with recombinant vectors (BAFF-R.pLNCX2 vector, BCMA.pLNCX2 vector, TACI.pLNCX2 vector) and CHO cells introduced with empty vector (empty pLNCX2 vector) were analyzed by flow cytometry (Mock transduced CHO cell) BAFF receptor, BCMA, and TACI cell surface expression levels were analyzed. At this time, each cell was stained with anti-human BAFF receptor antibody (Biolegend), anti-human BCMA receptor antibody (Biolegend), and anti-human TACI receptor antibody (Biolegend), and cell surface expression levels of BAFF receptor, BCMA, and TACI were compared. Results are shown in Figures 5a to 5c.

Based on the above results, CHO cell lines that strongly express BAFF receptor, BCMA, and TACI were used as target cells, and mock transduced CHO cell lines introduced with empty pLNCX2 vector were used as target cells. (target cell) was used as a negative control.

Example 4. Affinity measurement of human cell lines expressing the soluble recombinant BAFF fusion protein and the BAFF receptor, BCMA, and TACI

To confirm the affinity of the prepared soluble recombinant BAFF fusion protein with human cell lines expressing the BAFF receptor, BCMA, and TACI, after expressing the soluble recombinant BAFF fusion protein in CHO cells, affinity chromatography was performed using a protein A column. It was purified by (affinity chromatography) (GE healthcare) (see FIG. 6a).

In addition, the purified water-soluble chimeric antigen receptor was confirmed by Western blotting using an antibody (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody, abcam) recognizing the human IgG heavy chain constant region (Fig. see 6b).

Then, the purified water-soluble recombinant BAFF fusion protein was treated with a CHO cell line expressing the BAFF receptor, BCMA, and TACI, respectively, and it was confirmed through flow cytometry whether the water-soluble recombinant BAFF fusion protein could bind to the BAFF receptor, BCMA, and TACI, respectively. is shown in Figures 7a to 7c.

As shown in FIGS. 7a to 7c, CHO cells expressing the BAFF receptor, BCMA, and TACI, respectively, bind to the soluble fusion protein (BAFF-Ig), but the control Ig (human IgG) binds to the BAFF receptor, BCMA, and TACI. confirmed that it does not.

As confirmed in the above results, it was confirmed that the prepared water-soluble fusion protein (BAFF-Ig) had high affinity with the BAFF receptor, BCMA, and TACI, and this was confirmed by the empty vector and CHO cells expressing the BAFF receptor, BCMA, and TACI, respectively. This means that CHO cells into which the (empty pLNCX2 vector) was introduced can be used to verify the BAFF receptor, BCMA, and TACI protein-specific cytotoxicity of the prepared water-soluble fusion protein (BAFF-Ig).

Example 5. Expression of IgG receptors (FcγRIIIa, CD16a) in natural killer cells

In order to test whether the prepared water-soluble recombinant BAFF fusion protein induces antibody dependent cellular cytotoxicity (ADCC) in cells expressing the BAFF receptor, BCMA or TACI, natural killer cells (NK cells) IgG receptors (FcγRIIIa, CD16a) were expressed in human NK92.MI cells (ATCC).

First, gene synthesis of human CD16a was performed, and the accuracy of gene synthesis was confirmed by sequencing after constructing a protein expression plasmid (GenBank bumber: NM_000569.7). Then, in order to express human CD16a in NK92.MI cells, human CD16a.pLNCX2 was cloned into pLNCX2 (Addgene), a retrovirus-derived expression vector, using restriction enzymes Hind III and Not I cleavage sites ( see Figure 8). Then, after expressing human CD16a in NK92.MI cells, using an antibody and flow cytometry, the expression of human CD16a in NK92.MI cells was measured by anti-human CD16a antibody (BD biosciences) and flow cytometry. It was confirmed by flow cytometry. At this time, as a negative control, cells (Mock.NK92.MI) in which only the empty vector (empty pLNCX2) was expressed in NK92.MI cells were used (see FIG. 9).

As confirmed in the above results, the BAFF receptor, BCMA, and TACI protein-specific antibody-dependent cytotoxicity (ADCC, It was confirmed that it can be used for antibody dependent cellular cytotoxicity (ADCC) verification.

Example 6. Verification of BAFF receptor, BCMA, and TACI expressing cell-specific antibody-dependent cytotoxicity of soluble recombinant BAFF fusion protein

In order to test whether the prepared water-soluble recombinant BAFF fusion protein induces antibody dependent cellular cytotoxicity (ADCC) in cells expressing the BAFF receptor, BCMA, and TACI, the target cells selected in Examples 3 and 4 ( CHO cells expressing BAFF receptor, BCMA, and TACI as target cells, respectively, and CHO cells into which an empty vector (empty pLNCX2 vector) was introduced as a negative control were compared to NK92.MI cells expressing human CD16a prepared in Example 5 above ( A water-soluble recombinant BAFF fusion protein (1 μg/ml) prepared using CD16a.NK cells as effectors was added to the culture medium and co-cultured for 6 hours each. At this time, control Ig (human IgG, 1 μg/ml) was used as a negative control for the fusion protein. A non-radioactive cytotoxicity assay that measures the degree of cytotoxicity of each natural killer cell (CD16a.NK cell or mock.NK cell) to target cells by the amount of lactate dehydrogenase present in the supernatant after co-culture (non-radioactive cytotoxicity assay) was used.

First, effector cells (1x10 ⁵ cell) and target cells (1x10 ⁴ cell) were put into wells of a 96-well cell culture dish, respectively, and the effector: target ratio was adjusted to 10:1, and the volume per well was inoculated to be 100 μl. Thereafter, the prepared water-soluble recombinant BAFF fusion protein (1 μg/ml) or control Ig (human IgG, 1 μg/ml) was added, and centrifuged at 250 g for 4 minutes using a centrifuge to close the cell spacing. After 6 hours of incubation, 50 μl of the supernatant from each well is removed and transferred to a transparent 96-well dish for measuring absorbance, and then the enzyme reaction is progressed and stopped by treatment with analysis solution and 1M hydrochloric acid solution. After stopping the enzyme reaction, the absorbance in the 490 nm wavelength band was measured and converted into numbers using a fluorescence/luminescence/absorption meter (multi-detection plate reader) to quantify the degree of cytotoxicity of target cells by effector cells in each well. .

As a result, as shown in FIG. 10, when the prepared water-soluble recombinant BAFF fusion protein was added and CHO cells expressing the BAFF receptor, BCMA, and TACI were co-cultured as target cells, the BAFF receptor was expressed. CHO cells showed 35% toxicity, BCMA-expressing CHO cells showed 31% toxicity, and TACI-expressing CHO cells showed 30% toxicity. On the other hand, when control Ig (human IgG) was added and CHO cells expressing BAFF receptors, BCMA, and TACI were co-cultured as target cells, CHO cells expressing BAFF receptors exhibited 2% toxicity. , BCMA-expressing CHO cells showed 4% toxicity and TACI-expressing CHO cells showed 7% toxicity.

In addition, when the prepared water-soluble recombinant BAFF fusion protein was added and CHO cells introduced with an empty vector (empty pLNCX2 vector) were co-cultured as target cells, toxicity was 2%. When control Ig (human IgG) was added and CHO cells introduced with an empty vector (empty pLNCX2 vector) were co-cultured as target cells, toxicity was as low as 5%.

Through the above results, it was confirmed that the prepared water-soluble recombinant BAFF fusion protein induces antibody dependent cellular cytotoxicity (ADCC) specific to the BAFF receptor, BCMA, and TACI proteins, respectively. It was confirmed that the treatment of cancer or autoimmune diseases is possible.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to 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 should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

<110> IMMUNOLOGICAL DESIGNINGLAB CO., LTD <120> Recombinant protein which contains BAFF extracellular domain and the composition comprising the same for treating cancer <130> KP21-0043-ILDL <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 218 <212> PRT <213> artificial sequence <220> <223> BAFF extracellular domain <400> 1 Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu Leu 1 5 10 15 Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly Ala Pro Lys 20 25 30 Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile Phe 35 40 45 Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn Ser Arg Asn 50 55 60 Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys Leu 65 70 75 80 Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr 85 90 95 Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu 100 105 110 Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile 115 120 125 Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu 130 135 140 Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val 145 150 155 160 Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn 165 170 175 Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu 180 185 190 Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp 195 200 205 Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 210 215 <210> 2 <211> 21 <212> PRT <213> artificial sequence <220> <223> Human Ig kappa leader peptide <400> 2 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp 20 <210> 3 <211> 234 <212> PRT <213> artificial sequence <220> <223> IgG1 heavy chain constant region <400> 3 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> 4 <211> 654 <212> DNA <213> artificial sequence <220> <223> BAFF extracellular domain <400> 4 caggtggccg ccctgcaagg ggacctggcc agcctccggg cagagctgca gggccaccac 60 gcggagaagc tgccagcagg agcaggagcc cccaaggccg gcctggagga agctccagct 120 gtcaccgcgg gactgaaaat ctttgaacca ccagctccag gagaaggcaa ctccagtcag 180 aacagcagaa ataagcgtgc cgttcagggt ccagaagaaa cagtcactca agactgcttg 240 caactgattg cagacagtga aacaccaact atacaaaaag gatcttacac atttgttcca 300 tggcttctca gctttaaaag gggaagtgcc ctagaagaaa aagagaataa aatattggtc 360 aaagaaactg gttacttttt tatatatggt caggttttat atactgataa gacctacgcc 420 atgggacatc taattcagag gaagaaggtc catgtctttg gggatgaatt gagtctggtg 480 actttgtttc gatgtattca aaatatgcct gaaacactac ccaataattc ctgctattca 540 gctggcattg caaaactgga agaaggagat gaactccaac ttgcaatacc aagagaaaat 600 gcacaaatat cactggatgg agatgtcaca ttttttggtg cattgaaact gctg 654 <210> 5 <211> 63 <212> DNA <213> artificial sequence <220> <223> Human Ig kappa leader peptide <400> 5 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gac 63 <210> 6 <211> 705 <212> DNA <213> artificial sequence <220> <223> IgG1 heavy chain constant region <400> 6 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 gccgcggggag 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 aatga 705 <210> 7 <211> 765 <212> DNA <213> artificial sequence <220> <223> Human CD16a <400> 7 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660 gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720 aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga 765

Claims (9)

an antigen binding domain of BAFF that specifically binds to the BAFF receptor, BCMA or TACI; Ig kappa (Ig kappa) leader peptide (reader peptide); and _a human immunoglobulin G ₁ heavy chain constant region. According to claim 1,
The antigen-binding domain is a recombinant protein _, characterized in that a pair of extracellular domains of BAFF are linked to a human immunoglobulin G ₁ heavy chain constant region, respectively, in the form of a dimer. According to claim 1,
The recombinant protein, wherein the extracellular domain of the BAFF comprises the amino acid sequence represented by SEQ ID NO: 1. According to claim 1,
The Ig kappa (Ig kappa) leader peptide includes the amino _{acid sequence represented by SEQ ID NO: 2, and the human immunoglobulin G 1 heavy} chain constant region is represented by SEQ ID NO: 3 A recombinant protein comprising an amino acid sequence. A polynucleotide encoding the recombinant protein according to any one of claims 1 to 4. A vector comprising a polynucleotide encoding the recombinant protein according to any one of claims 1 to 4. A cell transformed with the vector of claim 6. A pharmaceutical for preventing or treating cancer or systemic lupus erythematosus (SLE) expressing BAFF receptor, BCMA or TACI, comprising the recombinant protein according to any one of claims 1 to 4 as an active ingredient enemy composition. According to claim 8,
Cancers expressing the BAFF receptor, BCMA or TACI are acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), follicular lymphoma, mantle cell lymphoma ( mantle cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), B-cell non-Hodgkin lymphoma, multiple myeloma myeloma), T cell lymphoma, breast cancer, thyroid cancer, and non-small cell lung cancer.

Images