Novel recombinant Fc receptor and cell comprising same

Abstract

The present invention relates to: a recombinant Fc receptor which has ADAM17 cleavage resistance by modifying an amino acid at an ADAM17 cleavage site of a CD16 polypeptide, and which can improve binding affinity with an antibody Fc region by replacing all or a part of an extracellular binding domain of CD64;

Description

Technical Field

[0001] The present invention relates to novel recombinant Fc receptors and cells comprising the same. Background Art

[0002] Immunotherapy, which treats diseases by activating or suppressing the immune system, is a rapidly developing field with increasing clinical importance. In particular, the development of chimeric antigen receptor (CAR) T cells has led to significant progress in adoptive cell transfer (ACT).

[0003] CAR is a recombinant receptor for activating immune cells, which contains one or more intracellular signaling domains and antigen binding domains. By manipulating CAR to express on the surface, immune cells (CAR-X) such as cytotoxic T cells and natural killer cells (NK cells) will be targeted by the antigen bound by the antigen binding domain and activated after binding to the antigen, thereby triggering an immune response against cells expressing the antigen.

[0004] In the past 10 years, a variety of CARs targeting various cell surface antigens have been reported. In addition to targeting antigens, compared with the first-generation CAR design consisting of an antigen binding domain and an intracellular signaling domain and its connected transmembrane domain, a variety of improvements have been made for the addition of signaling domains derived from immune co-stimulatory molecules and the combination of overall domain structures, cell proliferation, cytotoxicity, cytokine secretion, in vivo survival, immune compatibility and safety, especially in certain blood cancers. Significant results have been achieved.

[0005] However, existing CARs are only specific for specific antigens, so there are many limitations. Cell therapy agents containing individual CARs need to go through multiple steps such as the discovery of specific target antigens targeting specific disease cells (dis eased cells), design / derive the antigen-specific binding sites, design CARs containing them and their intracellular introduction, and need to be verified for a long time such as preclinical studies and clinical trials, as well as derive appropriate administration methods and compositions. In addition, most autologous CAR-Xs currently require additional costs and time for each patient during the preparation process. In addition, cancers in particular often show continuous variation and heterogeneity between different patients and even between cells from the same patient, which reduces the effect of CAR-X with fixed target antigens in cancer treatment. In addition, existing CAR-X cells have also reported risks such as cytokine release syndrome (cytokine release syndrome) caused by excessive activation of the cells, including neurotoxicity and toxicity to normal tissues of the targeted antigen (on-target, off-tumor effect). Furthermore, especially in solid tumors, immune cells have reduced accessibility and despite successful infiltration into the tumor, they often fail to activate normally due to the immunosuppressive effects of the tumor microenvironment.

[0006] The present invention aims to transcend the limitations of existing fixed target CARs and provide a new chimeric receptor, namely a recombinant Fc receptor, which is versatile and can enhance the activity of immune cells. The recombinant Fc receptor and the transgenic mammalian cells containing the same according to the present invention have improved effects in immunotherapy, and because they do not have fixed target molecules, they can be used to treat various diseases. The recombinant Fc receptor is resistant to ADA M17 cleavage that occurs after the activation of existing CD16, thereby avoiding the reduction of the receptor immune activation effect caused by cleavage. Summary of the invention

[0007] Technical issues

[0008] The present invention aims to solve the above problems, and its purpose is to provide a recombinant Fc receptor that can be used as a therapeutic agent for cancer or infectious diseases.

[0009] Furthermore, another object of the present invention is to provide a nucleic acid molecule encoding the recombinant Fc receptor.

[0010] Furthermore, another object of the present invention is to provide an expression vector comprising the nucleic acid molecule.

[0011] Furthermore, another object of the present invention is to provide a host cell transformed by the expression vector.

[0012] Furthermore, another object of the present invention is to provide a pharmaceutical composition for treating cancer or a pharmaceutical composition for treating or preventing viral infections comprising the host cell.

[0013] The technical problems to be solved by the present invention are not limited to the above-mentioned technical problems. Other technical problems not mentioned will be clearly understood by ordinary technicians in this field according to the following description.

[0014] Means of solving the problem

[0015] In order to achieve the technical problem, one embodiment of the present invention provides a recombinant Fc receptor, which comprises at least a portion of a CD16 polypeptide, and the CD16 polypeptide comprises an ADAM17 cleavage site sequence corresponding to positions 196 to 198 of SEQID No.1, which comprises: i) at least one amino acid modification of a replacement in which the serine at position 197 is replaced by proline, and a replacement in which the threonine at position 198 is replaced by serine; and ii) all or a portion of the extracellular binding domain of the CD16 polypeptide is replaced by at least one of the extracellular binding domains of CD64.

[0016] In an embodiment of the present invention, the CD16 polypeptide comprises the amino acid sequence of SEQ ID No.1, and can achieve the following: the serine at position 197 is replaced by proline, and the threonine at position 198 is replaced by serine, and at least one of the amino acid modifications, and the amino acid at position 176 of SEQ ID No.1 is substituted by valine.

[0017] In an embodiment of the present invention, the serine at the amino acid site 197 can be substituted with proline, and all or part of the extracellular binding domain of the CD16 polypeptide can be replaced with the extracellular binding domain of CD64.

[0018] In an embodiment of the present invention, the serine at the amino acid site at position 197 can be substituted with proline and the threonine at the amino acid site at position 198 can be substituted with serine, and all or part of the extracellular binding domain of the CD16 polypeptide can be replaced with the extracellular binding domain of CD64.

[0019] In an embodiment of the present invention, all or part of the extracellular binding domain of the CD16 polypeptide may be replaced by the extracellular binding domain of CD64.

[0020] In an embodiment of the present invention, the extracellular binding domain (ectodoma in) of CD64 may comprise an amino acid sequence of SEQ ID No. 2 or 3.

[0021] In an embodiment of the present invention, the recombinant Fc receptor may comprise any one of the amino acid sequences of SEQ ID No. 4 to 8.

[0022] Other embodiments of the present invention provide nucleic acid molecules encoding the Fc receptor.

[0023] Other embodiments of the present invention provide expression vectors comprising the nucleic acid molecule.

[0024] Other embodiments of the present invention provide host cells transformed by the expression vector.

[0025] In an embodiment of the present invention, the host cell may be a mammalian immune cell, a stem cell, or a cell differentiated from a stem cell.

[0026] Another embodiment of the present invention provides a pharmaceutical composition for treating cancer comprising the host cell.

[0027] Other embodiments of the present invention provide a pharmaceutical composition for treating viral infections comprising the host cells.

[0028] The means for solving the problem are only examples and should not be interpreted as limiting the present invention. In addition to the exemplary embodiments described, there may be other embodiments in the drawings and the content of the invention.

[0029] Effects of the Invention

[0030] The recombinant Fc receptor according to the embodiment of the present invention can be combined with an antibody to give the cell expressing the recombinant Fc receptor the specificity of the desired antigen. Based on this characteristic, the cell expressing the recombinant Fc receptor in the embodiment of the present invention can be used to treat a variety of diseases according to the type of combined antibodies, and can also effectively respond to diseases such as cancer that continuously mutate and show intercellular heterogeneity by changing the type of combined antibodies.

[0031] Furthermore, the recombinant Fc receptor according to one embodiment of the present invention has resistance to ADAM17 cleavage that occurs after antibody binding, and may have a higher affinity for the Fc domain of the antibody compared to CD16, and therefore, cells expressing the recombinant Fc receptor can induce more effective effector responses (e.g., antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), degranulation, cytokine secretion, etc.).

[0032] Furthermore, the recombinant Fc receptor of one embodiment of the present invention also allows cytotoxic effector cells (eg, cytotoxic T lymphocytes (CTL)) that originally function independently of antibodies to activate their effector functions after the introduction of antibodies.

[0033] Furthermore, the Fc receptor contained in the cell of one embodiment of the present invention is designed to be regulated by the regulatory sequence of endogenous CD16. This can reproduce the expression pattern in cells expressing CD16 (such as NK cells) and prevent the expression of consumable proteins before differentiation when inducing these cells from stem cells.

[0034] The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be derived from the inventive concept described in the description or claims of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The results show that NK92MI cells were transfected with a vector containing an engineered receptor by electrofection, followed by sorting and antibiotic selection, and then flow cytometric analysis was used to measure the expression of CD16 or CD64 receptors on the surface of the cells.

[0036] Figure 2 The results of Western blot analysis of the expression of CD16 receptor in the intracellular domain of NK92MI cells expressing engineered receptors. Both CD16 full form and CD64 / 16 chimeric form receptors expressed CD16 receptors in the inner layer, which can be confirmed by bands. In contrast, no bands appeared in the NK92MI experimental group that did not express the receptor.

[0037] FIG. 3 shows the results of flow cytometric analysis of the shedding effect of ADAM17 when strong stimulation with PMA / Ionomycin was applied to NK92MI cells expressing the engineered receptor. Figure 3a It represents the relative MFI value of receptor expression with 100 as the benchmark when unstimulated. The control group (hCD16) was cleaved and the expression was reduced under the three stimulation concentrations. On the contrary, the Candidate, D2, D5, and F4 cell lines were not cleaved, but the expression increased or remained stable. Figure 3b The graph measures receptor expression at 5 concentrations of PMA / Ionomycin. The control group showed downregulation of expression when activated compared to the unstimulated experimental group, while D5 and F4 showed upregulation or stability of expression when activated and supplemented with Figure 3a result. Figure 3c To represent the histogram Figure 3a Resulting graph.

[0038] Figure 4 shows the results of measuring the ADCC activity of NK92MI cells expressing the engineered receptor in various cancer cell lines by lactate dehydrogenase release (LDH release). LDH is a cytoplasmic enzyme released into the culture supernatant after cell membrane damage during cell death and is often used as an indicator of cytotoxicity. Figure 4a and Figure 4bThe results showed that in MDA-MB-231 and MDA-MB-453 cell lines, when antigen-targeting antibodies were used, Candidate, D2, and D5 showed higher cytotoxicity than NK92MI or the control group. Figure 4c ACC results of AsPC-1 cell line resistant to lysis by NK92 cells expressing CD16 compared to other cell lines.

[0039] Figure 5 shows the results of measuring the long-term ADCC activity of NK92MI cells expressing the engineered receptor in various cancer cell lines using the Incucyte real-time imaging device and Caspase-3 / 7 red dye. Green fluorescence can be confirmed in D2, D5, and F4 NK92MI cell lines into which the GFP-containing vector has entered, while red fluorescence can be confirmed in dead target cells. Figure 5a and Figure 5b It shows the apoptosis counts (Apoptosis counts) changing with time during ADCC in MDA-MB-231 and MDA-MB-453 cell lines. Figure 5c The images of each experimental group are shown 1.5 hours after the maximum apoptosis. Figure 5d Images showing changes in each experimental group over time.

[0040] FIG6 shows a universal receptor vector prepared for expressing the recombinant Fc receptor of the present invention. Figure 6a A vector for expressing Candidate (CD16 full form). Figure 6b This is a vector used to express D2, D5, and F4 (CD64 / 16 chimera). DETAILED DESCRIPTION

[0041] Hereinafter, the present invention will be described in detail.

[0042] In this specification, the term "domain" refers to a unit having functional or structural characteristics. The domain according to the present invention is preferably a polypeptide. Such a polypeptide may be a single polypeptide chain or a module formed by the aggregation of parts of one or more polypeptide chains. The polypeptide may contain proteinaceous parts and non-proteinaceous parts, in which case the non-proteinaceous parts may be chemical cross-linkers such as glutaraldehyde or chemical linkers.

[0043] The amino acid sequences included in this specification may include sequences having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more homology to the sequence if sequence differences occur while maintaining the characteristics of each domain of the present invention. The changes may be one or more selected from the addition, substitution or deletion of a portion of amino acid residues.

[0044] As used herein, "amino acid substitution" or "substitution" refers to a situation in which the amino acid at a specific position of a parent polypeptide sequence is replaced by a different amino acid.

[0045] The present invention relates to recombinant Fc receptors.

[0046] The present invention relates to a recombinant Fc receptor, at least a portion of a CD16 polypeptide thereof, wherein the CD16 polypeptide comprises an ADA M17 cleavage site sequence corresponding to positions 196 to 198 of SEQ ID No. 1, and the recombinant Fc receptor comprises: i) at least one of the amino acid modifications of substitution in which the serine at position 197 is substituted with proline and the threonine at position 198 is substituted with serine; and ii) at least one of the substitutions in which all or a portion of the extracellular binding domain of the CD16 polypeptide is replaced with the extracellular binding domain of CD64.

[0047] CD16 is expressed in two different forms, namely two different but highly homologous gene products CD16a and CD16b. CD16a is a protein anchored by a polypeptide, while CD16b is a protein anchored by glycosylphosphatidylinositol. As used herein, CD16 refers to these two forms of the protein that are clearly known to those of ordinary skill.

[0048] The CD16 polypeptide may be any CD16 polypeptide sequence comprising an ADAM17 cleavage site sequence, for example, a sequence known in databases such as NCBI.

[0049] The recombinant Fc receptor of the present invention may comprise at least a portion of the CD16 polypeptide having the amino acid sequence of SEQ ID No. 1, for example.

[0050] ADAM17 is a protease that, when activated by CD16 signal transduction, cleaves the extracellular binding domain (ectodomain) of CD16, inhibiting ADCC activation signal transduction. The ADAM17 cleavage site sequence may include, for example, a sequence corresponding to positions 196 to 198 of SEQ ID No. 1, but is not limited thereto.

[0051] The recombinant Fc receptor of the present invention comprises: i) at least one of the amino acid modifications of substitution in which the serine at position 197 is replaced by proline, and substitution in which the threonine at position 198 is replaced by serine; and ii) at least one of the substitutions in which all or a part of the extracellular binding domain of the CD16 polypeptide is replaced by the extracellular binding domain of CD64.

[0052] If the recombinant Fc receptor of the present invention comprises at least one of the amino acid modifications of substitution in which the serine at position 197 is replaced by proline, and substitution in which the threonine at position 198 is replaced by serine, specifically, the ADAM17 cleavage site sequence corresponding to positions 196 to 198 of SEQ ID No. 1 of the CD16 polypeptide can be as follows: position 196 is valine, position 197 is proline, and position 198 is threonine; or position 196 is valine, position 197 is proline, and position 198 is serine; or position 196 is valine, and positions 197 and 198 are serine.

[0053] The recombinant Fc receptor of the present invention comprises the above amino acid modifications, and thus has resistance to cleavage by ADA M17.

[0054] The recombinant Fc receptor of the present invention may comprise a replacement of the whole or part of the extracellular binding domain (ectodomain) of the CD16 polypeptide with the extracellular binding domain of CD64.

[0055] Fc receptors include an extracellular binding domain, a transmembrane domain, and an intracellular binding domain. The present invention may include a portion or all of the extracellular binding domain of CD64. Both CD64 and CD16 bind to the Fc region of an antibody, but CD64 has an affinity of up to about 100 to 1000 times. CD64 includes a glycoprotein α chain consisting of three immunoglobulin domains responsible for antibody binding by the extracellular binding domain, and the presence of the three immunoglobulin domains has been shown to be essential for high-affinity interactions with antibodies. In contrast, the extracellular binding domain of CD16 has only two immunoglobulin-like domains. The presence of an additional immunoglobulin-like folding structure in CD64 may be a strong determinant of high Fc affinity. The recombinant Fc receptor of the present invention can also obtain high affinity for antibodies by inserting this additional immunoglobulin-like folding structure or a mutant version thereof in CD64 into the extracellular binding domain of CD16.

[0056] The present invention can fuse the extracellular binding domain of CD64 with any appropriate transmembrane domain and intracellular binding domain of CD16. In addition, the three immunoglobulin domains (EC1, EC2, EC3) of CD64 and a part of the hinge domain, a part of the hinge of CD16, the transmembrane domain and the intracellular binding domain can be fused, but it is not limited to this. In order to improve the affinity of the CD16 polypeptide to the Fc region of the antibody, the extracellular binding domain of CD64 can be replaced at an appropriate position corresponding to all or part of the extracellular binding domain (ectodomain) of the CD16 polypeptide.

[0057] The extracellular binding domain of CD64 may comprise, for example, the amino acid sequence of SEQ ID No. 2 or 3.

[0058] In the present invention, for example, the CD16 polypeptide may comprise the amino acid sequence of SEQ ID No.1, and may be implemented as follows: at least one amino acid modification of the substitution of the serine at the 197th amino acid site being substituted with proline, and the threonine at the 198th amino acid site being substituted with serine, and the substitution of the 176th amino acid of SEQ ID No.1 being substituted with valine. More specifically, the recombinant Fc receptor of the present invention may comprise the amino acid sequence of SEQ ID No.4.

[0059] Furthermore, in the present invention, for example, the serine at the amino acid position 197 may be substituted with proline, and all or part of the extracellular binding domain of the CD16 polypeptide may be replaced with the extracellular binding domain of CD64. More specifically, the recombinant Fc receptor of the present invention may comprise the amino acid sequence of SEQ ID No.5.

[0060] Furthermore, in the present invention, for example, the serine at the 197th amino acid site can be substituted with proline, the threonine at the 198th amino acid site can be substituted with serine, and all or part of the extracellular binding domain of the CD16 polypeptide is replaced with the extracellular binding domain of CD64. More specifically, the recombinant Fc receptor of the present invention can comprise the amino acid sequence of SEQ ID No. 6 or 7.

[0061] In the present invention, for example, all or part of the extracellular binding domain of the CD16 polypeptide can be replaced with the extracellular binding domain of CD64. More specifically, the recombinant Fc receptor of the present invention can comprise the amino acid sequence of SEQ ID No.8.

[0062] The present invention relates to a nucleic acid molecule encoding said Fc receptor.

[0063] The nucleic acid molecules include DNA (gDNA and cDNA) and RNA molecules. The basic constituent units of nucleic acid molecules, namely nucleotides, can be not only natural nucleotides, but also analogs with modified sugar or base sites (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)). The sequence of the nucleic acid molecule encoding the recombinant Fc receptor of the present invention can be modified. The modification can include the addition, deletion or non-conservative substitution or conservative substitution of nucleotides. According to the well-known content in the relevant technical field, the nucleic acid molecules of the present invention can be mixed into the expression vector of the host cell used to produce the recombinant Fc receptor of the present invention.

[0064] The present invention relates to an expression vector comprising the nucleic acid molecule.

[0065] The expression vector may comprise an expression control sequence activatably linked to the nucleic acid molecule.

[0066] "Vector" used in the present invention refers to a nucleic acid molecule that can carry other nucleic acids bound thereto. For example, the vector can be a plasmid, i.e., a circular double-stranded DNA fragment to which an additional DNA fragment can be connected. Furthermore, the vector can also be a viral vector, i.e., a vector to which an additional DNA fragment can be connected to a viral genome. The vector can replicate autonomously in a host cell into which the vector is introduced. For example, it can be a bacterial vector and an episomal mammalian vector with a bacterial replication origin. Furthermore, after the vector (e.g., a non-episomal mammalian vector) is introduced into a host cell, it can be integrated into the genome of the host cell, thereby replicating together with the host genome. Furthermore, any vector can express a gene that can be activated to be connected to the vector.

[0067] The "expression control sequence" used in the present invention refers to a polynucleotide sequence connected to a coding sequence to facilitate the expression and processing of the coding sequence. The expression control sequence may include appropriate transcription initiators, terminators, promoters, enhancer sequences, effective RNA processing signals, such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that improve translation efficiency (i.e., Kozak consensus sequences) and sequences that enhance protein stability, and may also include sequences that promote protein secretion when necessary. The properties of such expression control sequences vary depending on the host organism: for prokaryotes, the expression control sequence generally includes a promoter, a ribosome binding site, and a transcription termination sequence; for eukaryotes, the expression control sequence generally includes a promoter and a transcription termination sequence. In the present invention, the expression control sequence at least includes all the components that are essential in the expression and processing process, and its presence may be a beneficial additional component, such as a leader sequence and a fusion partner sequence.

[0068] The present invention relates to a host cell transformed by the expression vector.

[0069] The term "host cell" used in the present invention refers to a nucleic acid, such as a cell that can be used to express the nucleic acid of the present invention. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.

[0070] The host cell of the present invention may be, for example, a mammalian immune cell, a stem cell, or a cell differentiated from a stem cell.

[0071] The immune cell may be a NK cell, a T cell, a B cell, a NKT cell, a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a dendritic cell or a macrophage. In addition, the immune cell may be a cytotoxic effector cell. Among them, the cytotoxic effector cell may be a NK cell, a cytotoxic T cell, a macrophage, a monocyte, a neutrophil and an eosinophil, etc. The cell may preferably be a NK cell or a cytotoxic T cell. Among them, T cells have the characteristics of interacting with the major histocompatibility complex (MHC) and the peptides loaded on the MHC through the T cell receptor (T cell receptor, referred to as TCR), and at this time, when the TCR recognizes the foreign MHC, it attacks the cells expressing the MHC. On the contrary, NK cells do not contain TCR, so when administered to the subject for therapeutic purposes, the transplanted cells do not attack the cells of the subject, thereby not causing graft-versus-host disease (GVHD), which makes it more advantageous.

[0072] The stem cells may be, for example, embryonic stem cells, adult stem cells or induced stem cells. Since immune cells are generally difficult to amplify, when stem cells with high proliferation capacity are transformed, there may be an advantage in terms of monoclonal amplification.

[0073] The host cell can be any of autologous, allogeneic or xenogeneic origins. If it is a xenogeneic source, it can preferably be from a mammal. Preferably, in order to reduce the possibility of immune rejection, the host cell can be selected from: 1) cells of autologous origin, or 2) cells of allogeneic origin that have been modified with genes related to immune response regulation. The genes related to the immune response regulation may include genes related to the attack of transplanted cells on host cells and / or the attack of host cells on transplanted cells. The genes related to the attack of the transplanted cells on the host cells can be, for example, TCR that recognizes MHC, and its expression can be reduced or removed. In addition, promoting the expression of non-classical MHC such as HLA-G or HLA-E can have the effect of protecting transplanted cells from host cell attacks.

[0074] The expression of genes related to MHC expression of the host cell, such as TAP1, TAP2, TAPBP, NLRC5, CIITA, RFXANK, RFX5, RFXAP, etc., can be reduced or eliminated.

[0075] Specifically, the host cell can be transformed into a landing pad by knocking out the gene related to cellular MHC-1 antigen presentation and at the same time safely inserting a recombinase recognition sequence into the gene region of the cell, but is not limited thereto.

[0076] The gene region includes not only the exon region, but also all regions including enhancers, promoters, introns, etc. related to the expression regulation of the gene.

[0077] By reducing or eliminating the expression of the gene, the expression of MHC that can recognize non-self molecules through the host TCR can be reduced or eliminated.

[0078] Compared with autologous cells that need to be transformed, expanded, screened, and tested for activity each time for a new patient, cells modified with the above immune response regulating genes can be prepared in advance in an off-the-shelf form, thus having advantages in terms of time and cost.

[0079] The present invention can effectively treat cancer or viral infection by administering the host cell and the antibody to a subject simultaneously or sequentially.

[0080] The antibody may have specificity for one or more antigens, and may be selected from a monoclonal antibody of more than one combination, or from the combination. The antibody may be selected, for example, from the following: i) an immunoglobulin selected from IgG1, IgG2, IgG3 and IgG4; ii) a native antibody fragment, such as Fv, Fab, Fab', F(ab')2, VHH, VNAR, etc.; iii) an engineered antibody, such as scFv, dsFv, ds-scFv, (scFv)2, diabody, triabody, tetrabody, pentabody, etc. In this case, the antibody may preferably contain a protein fragment derived from IgG related to the Fc binding domain and IgG binding of CD16 and CD64, such as an Fc domain or a portion thereof, and a protein fragment selectively containing a hinge domain or a portion thereof. The protein fragment derived from IgG may preferably be from IgG1, IgG3 or IgG4, and may more preferably be from IgG1 or IgG3. It is known that the binding affinity of Fcγ receptors to Fc is IgG3>IgG1>IgG4, and IgG1 and IgG3 in particular are considered to be potent activators of antibody-dependent effector functions. Although IgG3 can most strongly activate these effector functions, its half-life in vivo is short, so IgG1 is considered to be more preferred when inducing therapeutic cytotoxicity. The antibody may contain one antigen binding domain (monovalent) or one or more antigen binding domains (multivalent). The antibody may be monoclonal or polyclonal. The antibody may be a natural source antibody isolated or purified from animals such as mice, rabbits, goats, horses, chickens, hamsters, and humans, or a synthetic antibody designed and genetically synthesized with reference to the sequence characteristics of natural source antibodies. The antibody may be a modified antibody such as a humanized antibody or a chimeric antibody. The antibody may be in monomeric form or in polymeric form. The antibody may comprise one or more chemical modifications, such as addition of disulfide bonds, glycosylation, side chain modified amino acids, methionine oxidation, asparagine or glutamine deamidation, γ-carboxylation, β-hydroxylation, sulfation, etc. The antibody may have any level of affinity or avidity for the target antigen. The antibody may be any one selected from mouse, rabbit, human or humanized or chimeric proteins.

[0081] The subject may be an animal, preferably a mammal, especially a human, or a cell, tissue, organ, etc. derived from an animal. Furthermore, the subject may be a patient who needs the treatment.

[0082] The present invention relates to a pharmaceutical composition for treating cancer or viral infection diseases comprising the host cell.

[0083] The cancer is not particularly limited, and may be, for example, adenocarcinoma, breast cancer, pancreatic cancer, blood cancer, ovarian cancer, colon cancer, bladder cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, prostate cancer, uterine cancer, cervical cancer, melanoma, colorectal cancer, renal cancer, or metastatic pleural tumor, etc.

[0084] The virus is not limited to a specific form of virus, and can be a dsDNA virus, ssDNA virus or RNA virus. Specifically, it can be applicable to adenovirus, herpes virus, mama virus, parvovirus, respiratory virus, rotavirus or retrovirus, etc. The composition of the present invention can show antiviral activity and is not particularly limited.

[0085] As used in the present invention, "treatment" refers to an activity that improves or positively changes the symptoms caused by cancer or viral infection.

[0086] The composition may include a pharmaceutically acceptable carrier.

[0087] The pharmaceutically acceptable carrier refers to any physiologically miscible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. Examples of pharmaceutically acceptable carriers include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and mixtures thereof. For a plurality of, it is preferred to include isotonic agents such as sugars, glycols (e.g., mannitol, sorbitol or sodium chloride) in the composition.

[0088] Compositions of the present invention can have various forms, such as liquid, semisolid and solid administration types, such as the form of liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Preferred forms depend on the expected mode of administration and therapeutic methods. Compositions of the present invention can adopt the form of emulsions in suspensions, solutions or oily or aqueous carriers, and can include formulations such as suspending agents, stabilizers and / or dispersants. Alternatively, active ingredients can exist in powder form, so as to be used with suitable carriers, for example, sterilized pyrogen-free materials before use.

[0089] The pharmaceutical composition of the present invention can be prepared into the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injections according to conventional methods. Specifically, for dosage forms, commonly used diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, surfactants, etc. can be used for formulation. Orally administered solid preparations can include tablets, pills, powders, granules, capsules, etc., but are not limited thereto. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talcum powder can also be used. In addition to oral liquid preparations and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can be added for preparation. Non-oral preparations can include sterilized aqueous solutions, non-water-soluble solvents, suspensions, emulsions, freeze-dried preparations and suppositories. Non-water-soluble solvents and suspensions can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. The base of the suppository can be semi-synthetic fatty acid ester (witepsol), polyethylene glycol (macrogol), Tween 61, cocoa butter, trilaurin, glyceryl gelatin and the like.

[0090] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, disease severity, drug form, and time, but can be appropriately selected by a person skilled in the art.

[0091] The composition may further comprise antibodies, and the contents regarding the antibodies are as described above.

[0092] Hereinafter, in order to specifically illustrate the present invention, examples will be given and described in detail.

[0093] Preparation Example: Preparation of Universal Fc Receptor NK Cells Binding to the Fc Portion of Antibodies

[0094] 1. Preparation of expression vector

[0095] Order the construct of the universal Fc receptor in the puC vector from Bionics, clone the sequence, and express the candidate drug (SEQ ID No. 4) in the pcDNA3.1(+) vector (see Figure 6a ), D2 (SEQ ID No.6), D5 (SEQ ID No.5), and F4 (SEQ ID No.8) were expressed in the pcDNA3.1(+)IRES GFP vector ( Figure 6b). Candidate's vector contains sequence changes of F176V, S197P, and T198S in the human CD16a sequence. In D2, D5, and F4, S1, S2, EC1, EC2, EC3, and a portion of Hinge are CD64 receptor sequences, and the remaining Hinge, TM, and IC contain the sequence of hCD16a. The sequences of D2, D5, and F4 contain EGFP so that expression can be confirmed.

[0096] As a control group, hCD16 (SEQ ID No. 9) containing the F176V sequence change of human hCD16 was used.

[0097] 2. Vector transfection and cell line construction

[0098] The prepared vector was transformed into NK92MI cells derived from NK cell non-Ho dgkin's lymphoma to confirm expression. Transformation was performed by electroporation, and the Neon transfection system kit of Ther mo Fisher was used as a kit. After optimizing the transformation conditions of the NK92MI cell line provided by Thermo, (1200V, 20ms / 3pulses) was used, and the culture medium was added at 37°C and 5% CO2 after the experiment. After a stable period of 2-3 weeks, the cells expressing CD16 were initially screened by BD, FACS AriaⅢ, Candidate using FITC anti-human CD16 antibody (FITC anti-human CD16antibody) (Biol egend), and D2, D5, and F4 screened cells expressing GFP. After a stabilization period of 2-3 weeks, secondary screening was performed in the same manner, or G418 slufate (Enzo) was used to treat for 2 weeks to construct a universal NK cell line.

[0099] Experimental Example 1: Confirmation of vector expression (flow cytometry analysis)

[0100] Using the cells of the preparation example, the expression of receptors in the EC domain was confirmed by flow cytometry. Each cell was treated with mouse IgG1 kappa isotype control, APC (Invitrogen), anti-CD16-APC, and anti-CD64-APC antibodies for staining, then washed with FACS buffer after 45 minutes and analyzed with BD FACS lyric. In this experiment, the expression of CD16 receptors in the EC domain in the control group (hCD16) and Candidate, and the expression of CD64 receptors in D2, D5, and F4 ( Figure 1 ).

[0101] Experimental Example 2: Confirmation of vector expression (Western blot)

[0102] Using the cells of the preparation example, the expression of CD16 receptor in the IC domain was confirmed by Western blot. After the cells were lysed in RIPA buffer for 1 hour, the proteins were collected by centrifugation at 14000 rpm, and then the concentration was confirmed by BCA protein concentration determination method (BCA assay) (Pierce, Thermo Fisher). After confirming the concentration, about 10 μg of protein was transferred to a PVDF membrane after SDS-PAGE and blocked with 5% skim milk for 1 hour at room temperature (RT). Anti-CD16 antibody (anti-CD16-antibody) (abca m) binding to the CD16 C-terminus was reacted at a ratio of 1:1000 at 4°C overnight. After the reaction, the band was confirmed by Image Quant 800. The results showed that both CD16 full form NK cells (hCD16, Candidate) and CD64 / 16 chimeric form NK cells (D2, D5, F4) expressed CD16 receptors in the inner layer and could be confirmed by bands. In contrast, no bands appeared in the NK92MI experimental group that did not express the receptor ( Figure 2 ).

[0103] Experimental Example 3: Confirmation of receptor cleavage by ADAM17 by flow cytometry analysis

[0104] Using the cells of the preparation example, an experiment was conducted on receptor cleavage caused by ADAM17. First, in order to confirm by flow cytometry whether the receptor would be reduced in expression due to shedding or cleavage of ADAM17 when strong PMA / Ionomycin stimulation was applied to Universal-NK cells, the expression level of CD16 or CD64 was observed. Each cell was treated with a PM A / Ionomycin mixture (Biolegend) at three different concentrations, harvested 4 hours later, stained with antibodies recognizing CD16 and CD64 (Biolegend), respectively, and then analyzed using BD FACS lyric. The control group was cleaved and the expression was reduced under the three stimulation concentrations. In contrast, the Candidate, D2, D5, and F4 cell lines were not cleaved, but the expression increased or remained stable ( Figure 3a and 3c ). Further, the expression of receptors was determined by flow cytometry at 5 concentration intervals of PMA / Ionomycin. The control group showed downregulation of expression when activated compared with the unstimulated experimental group, while D5 and F4 showed upregulation or stability of expression when activated ( Figure 3b ).

[0105] Experimental Example 4: Confirmation of the efficacy of Universal Fc receptor

[0106] After confirming the expression of the vector, ADCC (Antibody Dependent Cell Cytotoxicity) experiments were performed using MDA-MB-231, MDA-MB-453, and AsPC-1 cell lines to confirm the efficacy of Universal-NK cells.

[0107] 4-1. Confirmation of ADCC activity by LDH release

[0108] Using CytoTox Non-radioactive cytotoxicity assay kit (CytoTox Non-RadioactiveCytotoxicity Assay kit) (Promega) was used to confirm the target cell LDH release caused by the ADCC mechanism after the target cell (Target Cell) bound to the antibody (Antibody) and Universal-NK cells. The target cells used were MDA-MB-231, MDA-MB-453, and AsPC-1 cell lines. The target cells were seeded in a 96-well plate. After one day of stabilization, Universal-NK cells were added as antibodies at a concentration of 1μg / ml and at three ratios of E:T=2:1, 1:1, and 0.5:1. After 4 hours, the 96-well plate was centrifuged, and then only the cell supernatant was collected and placed in a new 96-well plate according to different experimental groups. After the experiment was carried out according to the experimental protocol of the analysis kit (Assay kit), the absorbance was measured at 490nm using a multireader. The calculation method for % cytotoxicity (Cytotoxicity) is as follows:

[0109]

[0110] The experimental results showed that among all cell lines, when using antibodies against antigens, NK92MI cells or the control group showed high cytotoxicity of Candidate, D2, and D5 compared with the control IgG experimental group ( Figures 4a to 4c ).

[0111] 4-2. Confirmation of long-term ADCC activity by Caspase-3 / 7 staining

[0112] In order to observe the target cell apoptosis caused by the ADCC mechanism after the target cell is combined with the antibody and Universal-NK cells, the Incucyte real time imaging device and Caspase-3 / 7 apoptosis red dye (Sartorius) are used. The red dye binds to the motif of the DEVD peptide recognized by caspase 3 / 7 in the target cell. The target cells used are MDA-MB-231 and MDA-MB-453 cell lines. Although healthy cells do not change, in apoptotic cells, activated Caspase cuts the DEVD recognition motif and enters the cell nucleus, causing DNA to emit red fluorescence, thereby enabling real-time quantification of dead cells. Target cells were seeded in a 96-well plate. After stabilization for one day, antibodies and red dye were treated and Universal-NK cells were added at a ratio of E:T = 2:1. Green fluorescence was confirmed in D2, D5, and F4 NK92MI cell lines into which the vector containing GFP entered, while red fluorescence was confirmed in dead target cells. The experimental results showed that in both cell lines, D2 and D5 showed high cytotoxicity compared with Candidate ( Figures 5a to 5d ).

[0113] The foregoing description of the present invention is only an example, and a person skilled in the art of the present invention can understand that it can be easily transformed into other specific modes without changing the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as being exemplary in all aspects, rather than restrictive. For example, each structural element described in a single type can also be implemented in a dispersed manner; similarly, the structural elements described as dispersed can also be implemented in a combined manner.

[0114] The scope of the present invention is indicated by the appended claims. The meaning and scope of the claims and all changes or modifications derived from their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A recombinant Fc receptor comprising at least a portion of a CD16 polypeptide, wherein the CD16 polypeptide comprises an ADAM17 cleavage site sequence corresponding to positions 196 to 198 of SEQ ID No. 1, wherein the recombinant Fc receptor comprises: i) at least one amino acid modification in which the serine at position 197 is substituted with proline, and the threonine at position 198 is substituted with serine; and ii) All or part of the extracellular binding domain of the CD16 polypeptide is replaced with at least one of the extracellular binding domains of CD64.

2. The recombinant Fc receptor according to claim 1, characterized in that The CD16 polypeptide comprises the amino acid sequence of SEQ ID No.1, The method is implemented as follows: at least one of the amino acid modifications of the serine at the amino acid position 197 is substituted with proline, and the threonine at the amino acid position 198 is substituted with serine, and The amino acid at position 176 of SEQ ID No. 1 is substituted with valine.

3. The recombinant Fc receptor according to claim 1, characterized in that The serine at the amino acid position 197 is substituted with proline, All or part of the extracellular binding domain of the CD16 polypeptide is replaced with the extracellular binding domain of CD64.

4. The recombinant Fc receptor according to claim 1, characterized in that The serine at the amino acid position 197 is substituted with proline and the threonine at the amino acid position 198 is substituted with serine, All or part of the extracellular binding domain of the CD16 polypeptide is replaced by the extracellular binding domain of CD64.

5. The recombinant Fc receptor according to claim 1, characterized in that All or part of the extracellular binding domain of the CD16 polypeptide is replaced by the extracellular binding domain of CD64.

6. The recombinant Fc receptor according to claim 1, characterized in that The extracellular binding domain of CD64 comprises the amino acid sequence of SEQ ID No. 2 or 3.

7. The recombinant Fc receptor according to claim 1, characterized in that The recombinant Fc receptor comprises any one of the amino acid sequences of SEQ ID No. 4 to 8.

8. A nucleic acid molecule, characterized in that Encodes the Fc receptor according to claim 1.

9. An expression vector, characterized in that: Comprising the nucleic acid molecule according to claim 8.

10. A host cell, characterized in that Transformed by the expression vector according to claim 9.

11. The host cell according to claim 10, characterized in that The host cell is a mammalian immune cell, a stem cell, or a cell differentiated from a stem cell.

12. A pharmaceutical composition for cancer treatment, characterized in that: Comprising the host cell according to claim 10.

13. A pharmaceutical composition for treating viral infections, characterized in that: Comprising the host cell according to claim 10.

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